Methods for transiently modifying soybean reproductive behavior

Abstract

Provided is a method for accelerating the development of genome edited and transgenic soybean plants by transiently modifying soybean reproductive behavior using silencing nucleic acid. Also provided is a method for accelerated trait introgression by transiently modifying soybean reproductive behavior using silencing nucleic acid and a method for temporarily increasing soybean plant yield or enabling wide maturity crosses.

Description

METHODS FOR TRANSIENTLY MODIFYING SOYBEAN REPRODUCTIVEBEHAVIORREFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0001] The official copy of the sequence listing is submitted electronically via Patent Center as an XML formatted sequence listing with a file named 9675_SequenceListing created on May 24, 2023, and having a size of 411,228 bytes and is filed concurrently with the specification. The sequence listing comprised in this XML formatted document is part of the specification and is herein incorporated by reference in its entirety.BACKGROUND

[0002] Soybean is a photoperiod sensitive plant that will generally flower and mature faster under short day-lengths. Through selective breeding for naturally occurring native alleles of genes that modify photoperiod sensitivity, soybean breeders have been able to create productive varieties within geographies beyond the original range of soybean cultivation. Soybean products are classified and marketed within 13 maturity groups (MG), ranging from MG 000 in high northern latitudes to MG X in tropical latitudes. More than ten loci, including the El-El l, and j genes, are known to regulate flowering and / or maturity highlighting the complexity of the genetic regulation of these traits. Naturally occurring null alleles and induced knockout mutations of El, El-like-a, El-like-b have been shown to confer earlier flowering and maturity times in soybean.

[0003] Current methods for genome editing, trait development, and trait introgression are time intensive as they often require plants to be grown over multiple generations. Moreover, biotechnology pipelines often rely on small number of representative cultivars to maximize transformation process efficiency. This creates complexity downstream as newly developed traits or gene edits are introgressed into diverse germplasm that may have disparate relative maturities. Accordingly, methods for accelerating the development of genome edited and transgenic plants across a diverse range of germplasm are needed.SUMMARY

[0004] Provided is a method for accelerating genome edited soybean development comprising introducing into a regenerable soybean cell an editing nucleic acid and a silencing nucleic acid, the editing nucleic acid introducing a modification in an endogenous gene of interest and the silencing nucleic acid decreasing expression of at least one endogenous gene that inhibits flowering time and / or maturity, growing a first soybean plant from the regenerable plant cell and collecting the seed therefrom, self-pollinating a soybean plant grown from the collected seed or crossing a soybean plant grown from the collected seed with a second soybean plant to produce a progeny soybean plant; and selecting progeny seed comprising the introduced modification in the endogenous gene of interest from the progeny soybean plant.

[0005] Also provided is a method for accelerating transgenic soybean development comprising introducing into a regenerable soybean cell a silencing nucleic acid and a nucleic acid construct, the nucleic acid construct comprising gene of interest operably linked to a regulatory element, optionally, a heterologous regulatory element, the silencing nucleic acid decreasing expression of at least one endogenous gene that inhibits flowering time and / or maturity, growing a first soybean plant from the regenerable plant cell and collecting the seed therefrom, self-pollinating a soybean plant grown from the collected seed or crossing a soybean plant grown from the collected seed with a second soybean plant to produce a progeny soybean plant, and selecting progeny seed comprising the nucleic acid construct from the progeny soybean plant.

[0006] Further provided is a method for trait introgression comprising crossing a first soybean plant with a second soybean plant comprising a trait of interest and a silencing nucleic acid, the silencing nucleic acid decreasing expression of at least one endogenous gene that inhibits flowering time and / or maturity, collecting the seed comprising the trait of interest and the silencing nucleic acid produced therefrom, backcrossing a soybean plant grown from the collected seed to the first soybean plant to produce a backcross progeny plant, and selecting backcross progeny seed comprising the trait of interest from the backcross progeny soybean plant.

[0007] Also provided is a method of increasing seed production comprising introducing into a regenerable soybean cell a silencing nucleic acid, the silencing nucleic acid decreasing expression of at least one endogenous gene that promotes flowering time and / or maturity, growing a first soybean plant from the regenerable plant cell and collecting the seed therefrom,self-pollinating a soybean plant grown from the collected seed or crossing a soybean plant grown from the collected seed with a second soybean plant to produce a progeny soybean plant, and selecting progeny seed comprising the transgene from the progeny soybean plant.BRIEF DESCRIPTION OF THE DRAWINGS AND THE SEQUENCE LISTING

[0008] The disclosure can be more fully understood from the following detailed description and the accompanying drawings and Sequence Listing, which form a part of this application. The sequence descriptions and sequence listing attached hereto comply with the rules governing nucleotide and amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §§1 .831-1 .835.

[0009] Fig. 1 is an illustration of an RNAi construct that inhibits flowering resulting in plants having an accelerated flowering time.

[0010] Fig. 2 is an illustration of a nucleic acid construct for combining RNAi for flowering time and CRISPR / Cas9 editing.

[0011] Fig. 3 is an illustration of a nucleic acid construct for combining RNAi for flowering time and introducing a gene of interest.

[0012] Fig. 4 is an illustration of a nucleic acid construct for combining an excisable RNAi construct with a gene that is targeted for germplasm introgression.

[0013] Fig. 5 is an illustration of a nucleic acid construct that promotes flowering resulting in plants having a delayed flowering time.DETAILED DESCRIPTION

[0014] The process of developing genome edited soybean plants, transgenic soybean plants or the introgression of desired traits into a soybean plant requires growing plants over multiple generations. Thus, shortening the reproductive period to enable faster cycling of soybean generations over a diverse set of soybean germplasm would improve efficiency and productivity across a range of biotechnology process such as, for example, trait development and characterization, trait introgression, and gene editing. Accordingly, provided herein are methods for accelerating the development of genome edited and transgenic soybean plants by expressing in a soybean plant a gene of interest or an editing nucleic acid and a silencing nucleic acid targeting an endogenous flowering time and / or maturity gene in a plant, and breeding the plant asufficient number of generations to produce a plant comprising the gene of interest or gene edit and not comprising the silencing nucleic acid.

[0015] Provided are methods for accelerating genome edited soybean development comprising introducing into a regenerable soybean cell an editing nucleic acid and a silencing nucleic acid, growing a first soybean plant from the regenerable plant cell and collecting the seed produced therefrom, self-pollinating a soybean plant grown from the collected seed or crossing a soybean plant grown from the collected seed with a second soybean plant to produce a progeny soybean plant, and selecting progeny seed comprising the introduced modification in the endogenous gene of interest from the progeny soybean plant. In certain embodiments, the editing nucleic acid introduces a modification in an endogenous gene of interest. In certain embodiments, the first soybean plant has an earlier flowering time, early maturity or both an earlier flowering time and maturity as compared to a plant grown from the selected progeny seed. In certain embodiments, the progeny seed does not comprise the silencing nucleic acid. In certain embodiments, the method further comprises growing and self-pollinating or crossing the progeny seed at least one (e.g., 1, 2, 3, 4, 5, 6, 7 or more) generation to produce seed not comprising the silencing construct.

[0016] As used herein, a “regenerable plant cell” is a plant cell capable of forming a fully functional fertile plant. Regenerable plant cells capable of forming a fully functional fertile plant include but are not limited to, embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, flowers, seeds, pods, or stems. In certain embodiments of the methods described herein, the regenerable plant cells are used in a tissue culture that is capable of regenerating plants having the physiological and morphological characteristics of the plant from which the cell was isolated and of regenerating plants having substantially the same genotype as the plant from which the cell was isolated. In certain embodiments of the methods described herein, the regenerable plant cell is isolated or derived from an elite line.

[0017] As used herein, “elite line”, “elite variety” or the like refers to any line that has resulted from breeding and selection for superior agronomic performance that allows a producer to harvest a product of commercial significance. Numerous elite lines are available and known to those of skill in the art of plant breeding. An “elite population” is an assortment of elite individuals or lines that can be used to represent the state of the art in terms of agronomically superior genotypes of a given crop species, such as soybean.

[0001] As used herein “editing nucleic acid” refers to a nucleic acid sequence used to introduce a modification in an endogenous gene of interest. In certain embodiments, the editing nucleic acid comprises a nuclease, endonuclease, or a deaminase enzyme. In certain embodiments, the editing nucleic acid comprises a guide polynucleotide. In certain embodiments, the editing nucleic acid comprises a polynucleotide modification template. In certain embodiments, the editing nucleic acid comprises a donor DNA. In certain embodiments, the editing nucleic acid comprises two or more of an endonuclease, a guide RNA, a polynucleotide modification template, or a donor DNA. In certain embodiments, the editing nucleic acid comprises an endonuclease, a guide RNA, and a polynucleotide modification template. In certain embodiments, the editing nucleic acid comprises an endonuclease, a guide RNA, and a donor DNA.

[0002] A double-strand-break-inducing agent, such as a guided Cas endonuclease can recognize, bind to a DNA target sequence, and introduce a single strand (nick) or double-strand break. Once a single or double-strand break is induced in the DNA, the cell’s DNA repair mechanism is activated to repair the break, for example via nonhomologous end-joining (NHEJ) or a Homology-Directed Repair (HDR) processes which can lead to modifications at the target site.

[0018] Modification of an endogenous gene of interest includes any one or more of the following: insertion of at least one nucleotide, deletion of at least one nucleotide, chemical alteration of at least one nucleotide, replacement of at least one nucleotide, or mutation of at least one nucleotide. In certain embodiments, the DNA repair mechanism creates an imperfect repair of the double-strand break, resulting in a change of a nucleotide at the break site. In certain embodiments, a polynucleotide template may be provided to the break site, wherein the repair results in a template-directed repair of the break. In certain embodiments, a donor polynucleotide may be provided to the break site, wherein the repair results in the incorporation of the donor polynucleotide into the break site.

[0019] In certain embodiments, the nuclease, endonuclease, or deaminase is selected from the group consisting of a polynucleotide-guided endonuclease, CRISPR-Cas endonucleases, base editing deaminases, zinc finger nuclease, a transcription activator-like effector nuclease (TALEN) or engineered site-specific meganuclease. In certain embodiments, the editing nucleic acid comprises a CRISPR-Cas endonucleases. The Cas endonuclease for use in the methods described herein is not particularly limited and may be any Cas endonuclease known in the art. In certain embodiments, the Cas endonuclease is a Class 2 Type II. In Class 2 Type II systems,the Cas endonuclease acts in complex with a guide RNA (gRNA) that directs the Cas endonuclease to cleave the DNA target to enable target recognition, binding, and cleavage by the Cas endonuclease. The gRNA comprises a Cas endonuclease recognition (CER) domain that interacts with the Cas endonuclease, and a Variable Targeting (VT) domain that hybridizes to a nucleotide sequence in a target DNA. In certain embodiments, the Cas endonuclease is selected from the group consisting of Cas9, Cpfl and Casl2f (also known in the art as Cas-alpha 10).

[0020] As used herein, the term “guide polynucleotide”, relates to a polynucleotide sequence that can form a complex with a Cas endonuclease, including the Cas endonuclease described herein, and enables the Cas endonuclease to recognize, optionally bind to, and optionally cleave a DNA target site. The guide polynucleotide sequence can be an RNA sequence, a DNA sequence, or a combination thereof (a RNA-DNA combination sequence). In certain embodiments, the guide polynucleotide is a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA) to guide the Cas endonuclease to its DNA target. The crRNA comprises a spacer region complementary to one strand of the double strand DNA target and a region that base pairs with the tracrRNA, forming an RNA duplex. In certain embodiments, the gRNA is a “single guide RNA” (sgRNA) that comprises a synthetic fusion of crRNA and tracrRNA.

[0021] The term “polynucleotide modification template” includes a polynucleotide that comprises at least one nucleotide modification when compared to the nucleotide sequence to be edited. A nucleotide modification can be at least one nucleotide substitution, insertion, or deletion. Optionally, the polynucleotide modification template can further comprise homologous nucleotide sequences flanking the at least one nucleotide modification, wherein the flanking homologous nucleotide sequences provide sufficient homology to the desired nucleotide sequence to be edited.

[0022] As used herein, “donor DNA” is a DNA construct that comprises a polynucleotide of interest to be inserted into the target site of a Cas endonuclease. The donor DNA construct further comprises a first and a second region of homology that flank the polynucleotide of interest. The first and second regions of homology of the donor DNA share homology to a first and a second genomic region, respectively, present in or flanking the target site of the cell or organism genome. The donor DNA can be tethered to the guide polynucleotide. Tethered donor DNAs can allow for co-localizing target and donor DNA, useful in genome editing, geneinsertion, and targeted genome regulation, and can also be useful in targeting post-mitotic cells where function of endogenous HR machinery is expected to be highly diminished (Mali et al., 2013, Nature Methods Vol. 10: 957-963). The amount of homology or sequence identity shared by a target and a donor polynucleotide can vary and includes total lengths and / or regions.

[0023] The type of edit is not particularly limited in the methods described herein and may be any type of edit in a gene of interest, or an edit to introduce a gene of interest, to result in a desired phenotype and / or genotype.

[0024] As used herein, a “gene of interest” refers to any nucleotide sequence encoding a protein or polypeptide that improves desirability of crops, e.g., a trait of agronomic interest. Polynucleotides of interest include, but are not limited to, polynucleotides encoding important traits for agronomics (e.g., yield, drought tolerance, nitrogen use efficiency, stature), insecticidal resistance, disease resistance, nematode resistance, herbicide resistance, microbial resistance, fungal resistance, viral resistance, fertility or sterility, grain characteristics, commercial products, phenotypic marker, or any other trait of agronomic or commercial importance. A gene of interest may additionally be utilized in either the sense or anti-sense orientation. Further, more than one polynucleotide of interest may be utilized together, or “stacked”, to provide additional benefit.

[0025] As used herein a “silencing nucleic acid” refers to a nucleic acid sequence which is capable of suppressing, reducing, or eliminating the level or expression of a target polynucleotide or the polypeptide encoded thereby. The silencing nucleic acid employed can reduce or eliminate the expression level of the target sequence by influencing the level of the target RNA transcript or, alternatively, by influencing translation and thereby affecting the level of the encoded polypeptide. Silencing nucleic acids of the methods described herein may include, but are not limited to, a sense suppression element, an antisense suppression element, a double stranded RNA, a siRNA, an amiRNA, a miRNA, or a hairpin suppression element.

[0026] In certain embodiments of the methods described herein, the silencing nucleic acid results in activity or amount of protein encoded by the target polynucleotide that is at least 0%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45% or 50% and less than 90%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% of the activity or amount of the protein in a wild-type control plant.

[0027] In certain embodiments of the compositions and methods described herein, the silencing nucleic acid results in expression or amount of mRNA encoded by the target polynucleotide that is at least 0%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45% or 50% and less than 90%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% of the expression or amount of mRNA in a wild-type control plant or seed.

[0028] As used herein, a “sense suppression element” comprises a polynucleotide designed to express an RNA molecule corresponding to at least a part of a target messenger RNA in the "sense" orientation. Expression of the RNA molecule comprising the sense suppression element reduces or eliminates the level of the target polynucleotide or the polypeptide encoded thereby. The polynucleotide comprising the sense suppression element may correspond to all or part of the sequence of the target polynucleotide, all or part of the 5' and / or 3' untranslated region of the target polynucleotide, all or part of the coding sequence of the target polynucleotide, or all or part of both the coding sequence and the untranslated regions of the target polynucleotide. Typically, a sense suppression element has substantial sequence identity to the target polynucleotide, typically greater than about 65% sequence identity, greater than about 85% sequence identity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. The sense suppression element of the methods described herein can be any length so long as it allows for the suppression of the flowering time and / or maturity gene sequence in which decreased expression or activity is desired. In certain embodiments, the sense suppression element is at least 15, 16, 17, 18, 19, 20, 22, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 900, 1000, 1100, 1200, 1300 nucleotides or longer of the target polynucleotides set forth in any of SEQ ID NOs: 1-44 or 47-80. In other embodiments, the sense suppression element can be, for example, about 15-25, 19-35, 19-50, 25-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1050, 1050-1100, 1100-1200, 1200-1300, 1300- 1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800 nucleotides or longer of the target polynucleotides set forth in any of SEQ ID NO: 1-44 or 47-80.

[0029] As used herein, an “antisense suppression element” comprises a polynucleotide which is designed to express an RNA molecule complementary to all or part of a target messenger RNA. Expression of the antisense RNA suppression element reduces or eliminates the level of thetarget polynucleotide. The polynucleotide for use in antisense suppression element may correspond to all or part of the complement of the sequence encoding the target polynucleotide, all or part of the complement of the 5' and / or 3' untranslated region of the target polynucleotide, all or part of the complement of the coding sequence of the target polynucleotide, or all or part of the complement of both the coding sequence and the untranslated regions of the target polynucleotide. In addition, the antisense suppression element may be fully complementary (i.e., 100% identical to the complement of the target sequence) or partially complementary (i.e., less than 100% identical to the complement of the target sequence) to the target polynucleotide. In certain embodiments, the antisense suppression element comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence complementarity to the target polynucleotide. Antisense suppression may be used to inhibit the expression of multiple proteins in the same plant. Furthermore, the antisense suppression element can be complementary to a portion of the target polynucleotide. Generally, sequences of at least 15, 16, 17, 18, 19, 20, 22, 25, 50, 100, 200, 300, 400, 450 nucleotides or greater of the sequence set forth in any of SEQ ID NO: 1-44 or 47-80 may be used.

[0030] A “double stranded RNA” or “dsRNA” refers to a polyribonucleotide structure formed either by a single self-complementary RNA molecule or a polyribonucleotide structure formed by the expression of least two distinct RNA strands. The dsRNA molecule(s) employed in the methods and compositions described herein mediate the reduction of expression of a target sequence, for example, by mediating RNA interference “RNAi” or gene silencing in a sequencespecific manner. In the context of the present disclosure, the dsRNA is capable of reducing or eliminating the level or expression of a target polynucleotide or the polypeptide encoded thereby in a plant.

[0031] The dsRNA can reduce or eliminate the expression level of the target sequence by influencing the level of the target RNA transcript, by influencing translation and thereby affecting the level of the encoded polypeptide, or by influencing expression at the pre- transcriptional level (i.e., via the modulation of chromatin structure, methylation pattern, etc., to alter gene expression). Accordingly, as used herein, the term “dsRNA” is meant to encompass other terms used to describe nucleic acid molecules that are capable of mediating RNA interference or gene silencing, including, for example, short-interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), hairpin RNA, short hairpin RNA (shRNA), post-transcriptional gene silencing RNA (ptgsRNA), and others.

[0032] In certain embodiments, at least one strand of the duplex or double-stranded region of the dsRNA shares sufficient sequence identity or sequence complementarity to the target polynucleotide to allow for the dsRNA to reduce the level of expression of the target sequence.

[0033] In certain embodiments, the dsRNA comprises a hairpin RNA. A hairpin RNA comprises an RNA molecule that is capable of folding back onto itself to form a double stranded structure. Multiple structures can be employed as hairpin elements. In certain embodiments, the dsRNA suppression element comprises a hairpin element which comprises in the following order, a first segment, a second segment, and a third segment, where the first and the third segment share sufficient complementarity to allow the transcribed RNA to form a double-stranded stem-loop structure. The “second segment” of the hairpin comprises a "loop" or a "loop region.” These terms are used synonymously herein and are to be construed broadly to comprise any nucleotide sequence that confers enough flexibility to allow self-pairing to occur between complementary regions of a polynucleotide (i.e., segments 1 and 3 which form the stem of the hairpin). For example, in certain embodiments, the loop region may be substantially single stranded and act as a spacer between the self-complementary regions of the hairpin stem-loop. In some embodiments, the loop region can comprise a random or nonsense nucleotide sequence and thus not share sequence identity to a target polynucleotide. In other embodiments, the loop region comprises a sense or an antisense RNA sequence or fragment thereof that shares identity to a target polynucleotide. In specific embodiments, the loop region can be optimized to be as short as possible while still providing enough intramolecular flexibility to allow the formation of the base-paired stem region. Accordingly, the loop sequence is generally less than 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 25, 20, 19, 18, 17, 16, 15, 10 nucleotides or less.

[0034] Any region of the target polynucleotide can be used to design the domain of the silencing element that shares sufficient sequence identity to allow expression of the hairpin transcript to decrease the level of the target polynucleotide. For instance, the domain can be designed to share sequence identity to the 5' untranslated region of the target polynucleotide(s), the 3' untranslated region of the target polynucleotide(s), exonic regions of the target polynucleotide(s), intronic regions of the target polynucleotide(s), and any combination thereof. In specific embodiments, a domain of the silencing element shares sufficient homology to at least about 15, 16, 17, 18, 19,20, 22, 25 or 30 consecutive nucleotides from about nucleotides 1 -50, 25-75, 75-125, 50-100, 125-175, 175-225, 100-150, 150-200, 200-250, 225-275, 275-325, 250-300, 325-375, 375-425, 300-350, 350-400, 425-475, 400-450, 475-525, 450-500, 525-575, 575-625, 550-600, 625-675,675-725, 600-650, 625-675, 675-725, 650-700, 725-825, 825-875, 750-800, 875-925, 925-975,850-900, 925-975, 975-1025, 950-1000, 1000-1050, 1025-1075, 1075-1125, 1050-1100, 1125-1175, 1100-1200, 1175-1225, 1225-1275, 1200-1300, 1325-1375, 1375-1425, 1300-1400, 1425- 1475, 1475-1525, 1400-1500, 1525-1575, 1575-1625, 1625-1675, 1675-1725, 1725-1775, 1775- 1825, 1825-1875, 1875-1925, 1925-1975, 1975-2025, 2025-2075, 2075-2125, 2125-2175, 2175- 2225, 1500-1600, 1600-1700, 1700-1800, 1800-1900, 1900-2000 of the target sequence, such as, for example, the flowering time and / or maturity genes set forth in SEQ ID NOs: 1-44 or 47-80. In certain embodiments, to optimize the siRNA sequences employed in the hairpin, the synthetic oligodeoxyribonucleotide / RNAse H method can be used to determine sites on the target mRNA that are in a conformation that is susceptible to RNA silencing.

[0035] The hairpin silencing element may also be designed such that the sense sequence or the antisense sequence do not correspond to a target polynucleotide. In this embodiment, the sense and antisense sequence flank a loop sequence that comprises a nucleotide sequence corresponding to all or part of the target polynucleotide. Thus, it is the loop region that determines the specificity of the RNA interference.

[0036] In addition, transcriptional gene silencing (TGS) may be accomplished through use of a hairpin suppression element where the inverted repeat of the hairpin shares sequence identity with the promoter region of a target polynucleotide to be silenced.

[0037] In other embodiments, the dsRNA can comprise a small RNA (sRNA). sRNAs can comprise both micro RNA (miRNA) and short-interfering RNA (siRNA) miRNAs are regulatory agents comprising about 19 ribonucleotides which are highly efficient at inhibiting the expression of target polynucleotides. For miRNA interference, the silencing element can be designed to express a dsRNA molecule that forms a hairpin structure containing a 19-nucleotide sequence that is complementary to the target polynucleotide of interest. The miRNA can be synthetically made, or transcribed as a longer RNA which is subsequently cleaved to produce the active miRNA. Specifically, the miRNA can comprise 19 nucleotides of the sequence having homology to a target polynucleotide in sense orientation and 19 nucleotides of a corresponding antisense sequence that is complementary to the sense sequence.

[0038] When expressing an miRNA, it is recognized that various forms of an miRNA can be transcribed including, for example, the primary transcript (termed the “pri-miRNA”) which is processed through various nucleolytic steps to a shorter precursor miRNA (termed the “pre- miRNA”); the pre-miRNA; or the final (mature) miRNA is present in a duplex, the two strands being referred to as the miRNA (the strand that will eventually basepair with the target) and miRNA*. The pre-miRNA is a substrate for a form of dicer that removes the miRNA / miRNA* duplex from the precursor, after which, similarly to siRNAs, the duplex can be taken into the RISC complex.

[0039] The methods and compositions of the disclosure employ silencing elements that when transcribed “form” a dsRNA molecule. Accordingly, the heterologous polynucleotide being expressed need not form the dsRNA by itself but can interact with other sequences in the plant cell to allow the formation of the dsRNA. For example, a chimeric polynucleotide that can selectively silence the target polynucleotide can be generated by expressing a chimeric construct comprising the target sequence for a miRNA or siRNA to a sequence corresponding to all or part of the gene or genes to be silenced. In this embodiment, the dsRNA is “formed” when the target for the miRNA or siRNA interacts with the miRNA present in the cell. The resulting dsRNA can then reduce the level of expression of the gene or genes to be silenced. The construct can be designed to have a target for an endogenous miRNA or alternatively, a target for a heterologous and / or synthetic miRNA can be employed in the construct. If a heterologous and / or synthetic miRNA is employed, it can be introduced into the cell on the same nucleotide construct as the chimeric polynucleotide or on a separate construct. As discussed elsewhere herein, any method can be used to introduce the construct comprising the heterologous miRNA.

[0040] In certain embodiments of the methods described herein, the silencing nucleic acid targets an endogenous flowering time or maturity gene. In certain embodiments, the silencing nucleic acid targets a flowering time or maturity gene whereby decreased expression results in an earlier flowering or maturity, such genes are referred to herein as flowering time inhibitors, genes that inhibit flowering time, maturity inhibitors, or genes that inhibit maturity. In certain embodiments, the silencing nucleic acid targets at least one gene comprising a nucleic acid sequence that is at least, or at least about, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 1-44. In certain embodiments, the silencing nucleic acid targetsan El gene comprising a nucleic acid sequence that is at least, or at least about, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2, an El-like-a gene comprising a nucleic acid sequence that is at least, or at least about, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 4, or an El-like-b gene comprising a nucleic acid sequence that is at least, or at least about, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 6, or any combination thereof. In certain embodiments, the silencing nucleic acid targets an E2 gene comprising a nucleic acid sequence that is at least, or at least about, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 8, an E2-like-a gene comprising a nucleic acid sequence that is at least, or at least about, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 10, or an E2-like-b gene comprising a nucleic acid sequence that is at least, or at least about, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 12, or any combination thereof. In certain embodiments, the silencing nucleic acid comprises a nucleotide sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% to SEQ ID NO: 45 or 46.

[0041] As used herein, "reproductive growth stage" “reproductive growth phenotype” or "reproductive stage" is a description of the characteristics associated with various phases of reproductive growth. "Rl" is the first reproductive growth stage when soybean begins to bloom by producing the first flower. Thus, as used herein flowering refers to the number of days to the initiation of flowering (Rl), such that an early flowering time refers to a decrease in the number of days (fewer days) between planting and the Rl stage, whereas a late flowering phenotype refers to an increase in the number of days (more days) between planting and the Rl stage. Similarly, when comparing two plants, the soybean plant or soybean variety that reaches the Rl stage first is considered to have an earlier flowering time or earlier flowering phenotype, whereas the plant or variety that reaches the Rl stage second is considered to have a later flowering time or later flowering phenotype. "R7" is the seventh reproductive growth stage when a soybeanbegins maturity. A soybean plant is identified as beginning maturity when it has one mature pod. "R8" is the eighth and final reproductive growth stage when a soybean is fully mature. A soybean plant is identified as fully mature when 95% of the pods are mature. Thus, as used herein maturity phenotype refers to the number of days to full maturity (R8), such that an early maturity phenotype refers to a decrease in the number of days (fewer days) between planting and the R8 stage, whereas a late maturity phenotype refers to an increase in the number of days (more days) between planting and the R8 stage. Similarly, when comparing two plants, the plant or variety that reaches the R8 stage first is considered to have an earlier maturity or earlier maturity phenotype, whereas the plant or variety that reaches the R8 stage second is considered to have a later maturity or later maturity phenotype.

[0042] As used herein "percent (%) sequence identity" with respect to a reference sequence (subject) is determined as the percentage of amino acid residues or nucleotides in a candidate sequence (query) that are identical with the respective amino acid residues or nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any amino acid conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (e.g., percent identity of query sequence = number of identical positions between query and subject sequences / total number of positions of query sequence * 100).

[0043] Unless otherwise stated, sequence identity / similarity values provided herein refer to the value obtained using the BLAST 2.0 suite of programs using default parameters (Altschul, et al., (1997) Nucleic Acids Res. 25:3389-402).

[0044] The term “crossed” or “cross” or “crossing” or the like as used herein refers to the fusion of gametes via pollination to produce progeny (i.e., cells, seeds, or plants). The term encompasses both sexual crosses (the pollination of one plant by another) and selfing (self- pollination, i.e., when the pollen and ovule (or microspores and megaspores) are from the same plant or genetically identical plants). As used herein, a “backcross” refers to crossing theoffspring of any filial generation of a parental cross to one of the parental lines or an individual genetically like its parent to obtain progeny with a genetic identity closer to that of the parent.

[0045] In certain embodiments, the development of the genome edited soybean (e.g., length of time from the introduction into the regenerable soybean cell to the selection of progeny seed) is decreased by at least about 10 days, 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 120 days, 140 days or 150 days as compared to a control (e.g., the development of a genome edited soybean using the same process but lacking the introduction of the silencing element).

[0046] In certain embodiments, the yield of the plant comprising the editing nucleic acid and the silencing nucleic acid is equal to or within at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% as compared to the yield of a control plant, such as, for example, the plant from which the regenerable plant cell was isolated or a plant grown from the regenerable plant cell not comprising the silencing construct. As used herein, “yield” refers to the amount of agricultural production harvested per unit of land and may include reference to bushels per acre or kilograms per hectare of a crop at harvest, as adjusted for grain moisture. Grain moisture is measured in the grain at harvest. The adjusted test weight of grain is determined to be the weight in pounds per bushel or kilogram, adjusted for grain moisture level at harvest.

[0047] As used herein, “introduced”, “introducing” or the like is intended to mean presenting to the host cell the polynucleotide described herein (e.g., silencing nucleic acid, editing nucleic acid, gene of interest) or resulting polypeptide in such a manner that the sequence gains access to the interior of the cell. The methods of the disclosure do not depend on a particular method for introducing a sequence into a host cell, only that the polynucleotide or polypeptide gains access to the interior of the cell. In certain embodiments, the introducing step comprises transforming the regenerable plant cell with the editing nucleic acid and the silencing nucleic acid. Transformation protocols as well as protocols for introducing polypeptides or polynucleotide sequences into host cells may include but are not limited to microinjection, electroporation, Agro bacteriu -mediated transformation, Ochrobacterium-mediated transformation, direct gene transfer, viral mediated, ballistic particle acceleration, heat shock, and lipid-mediated transfection.

[0048] In certain embodiments, the transformation method may involve the construction of a nucleic acid construct comprising the editing nucleic acid or the silencing nucleic acid or both the editing nucleic acid and silencing nucleic acid. Accordingly, in certain embodiments, the introducing step comprises transforming the regenerable plant cell with a nucleic acid construct comprising the editing nucleic acid and the silencing nucleic acid. In certain embodiments, the editing nucleic acid and the silencing nucleic acid are introduced using a single nucleic acid construct (e.g., single construct comprises both an editing nucleic acid and a silencing nucleic acid). In certain embodiments, the editing nucleic acid and the silencing nucleic acid are introduced using separate nucleic acid constructs (e.g., a construct comprising the editing nucleic acid and a construct comprising the silencing nucleic acid). In certain embodiments, the nucleic acid construct comprising the editing nucleic acid and / or the silencing nucleic acid further comprises a selectable marker gene for the selection of transformed cells. In certain embodiments, the nucleic acid construct comprises an editing nucleic acid comprising a Cas endonuclease and gRNA. In certain embodiments, the nucleic acid construct comprises an editing nucleic acid comprising a Cas endonuclease and gRNA, a silencing nucleic acid, and a selectable marker.

[0049] In certain embodiments, the nucleic acid construct further comprises a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glyphosate, glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D). The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used in the present disclosure.

[0050] The use of the term “nucleic acid construct”, "nucleotide constructs" or the like herein is not intended to limit the embodiments to nucleotide constructs comprising DNA. Those of ordinary skill in the art will recognize that nucleotide constructs, particularly polynucleotides and oligonucleotides composed of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides, may also be employed in the methods disclosed herein. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments additionally encompass all complementary forms of such constructs, molecules, and sequences. Further, the nucleotideconstructs, nucleotide molecules, and nucleotide sequences of the embodiments encompass all nucleotide constructs, molecules, and sequences which can be employed in the methods of the embodiments for transforming plants including, but not limited to, those comprised of deoxyribonucleotides, ribonucleotides, and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The nucleotide constructs, nucleic acids, and nucleotide sequences of the embodiments also encompass all forms of nucleotide constructs including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures and the like.

[0051] In certain embodiments of the methods described herein, the editing nucleic acid, the silencing nucleic acid or both the editing nucleic acid and silencing nucleic acid are operably linked to at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7 or more) regulatory element. In certain embodiments, the regulatory element is a promoter. In certain embodiments, the promoter is a leaf-specific promoter or a constitutive promoter. In certain embodiments, the silencing nucleic acid is operably linked to a tissue-preferred or a leaf-specific promoter and the editing nucleic acid is operably linked to a constitutive promoter. In certain embodiments, the regulatory element is a heterologous regulatory element (e.g., heterologous promoter). In certain embodiments the heterologous regulatory element is heterologous to the editing nucleic acid and / or the silencing nucleic acid. In certain embodiments, the heterologous regulatory element operably linked to the editing nucleic acid and / or the silencing nucleic acid is heterologous to the host cell (e.g., non-soybean sequence).

[0052] As used herein, “heterologous” in reference to a sequence is a sequence that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. For example, a promoter operably linked to a heterologous polynucleotide that is from a species different from the species from which the polynucleotide was derived, or, if from the same / analogous species, one or both are substantially modified from their original form and / or genomic locus, or the promoter is not the native promoter for the operably linked polynucleotide.

[0053] As used herein "operably linked" is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional link that allows for expression of the polynucleotide of interest. Operably linked elements may be contiguous or non-contiguous.When used to refer to the joining of two protein coding regions, operably linked is intended that the coding regions are in the same reading frame.

[0054] As used herein “regulatory element” generally refers to a transcriptional regulatory element involved in regulating the transcription of a nucleic acid molecule such as a gene or a target gene. The regulatory element is a nucleic acid and may include a promoter, an enhancer, an intron, expression modulating elements (EMEs), a 5 ’-untranslated region (5’-UTR, also known as a leader sequence), or a 3’-UTR or a combination thereof. A regulatory element may act in "cis" or "trans", and generally it acts in "cis", i.e., it activates expression of genes located on the same nucleic acid molecule, e.g., a chromosome, where the regulatory element is located.

[0055] An “enhancer” element is any nucleic acid molecule that increases transcription of a nucleic acid molecule when functionally linked to a promoter regardless of its relative position. Various enhancers are known in the art including for example, introns with gene expression enhancing properties in plants, the ubiquitin intron (i.e., the maize ubiquitin intron 1 (see, for example, NCBI sequence S94464)), the omega enhancer or the omega prime enhancer (Gallie, et al., (1989) Molecular Biology ofRNA ed. Cech (Liss, New York) 237-256 and Gallie, et al., (1987) Gene 60:217-25), the CaMV 35S enhancer (see, e.g., Benfey, et al., (1990) EMBO J. 9: 1685-96) and the enhancers of US Patent Number 7,803,992 may also be used. The above list of transcriptional enhancers is not meant to be limiting. Any appropriate transcriptional enhancer can be used in the embodiments described herein.

[0056] A “repressor” (also sometimes called herein silencer) is defined as any nucleic acid molecule which inhibits the transcription when functionally linked to a promoter regardless of relative position. The term "cis-element" generally refers to transcriptional regulatory element that affects or modulates expression of an operably linked transcribable polynucleotide, where the transcribable polynucleotide is present in the same DNA sequence. A cis-element may function to bind transcription factors, which are trans-acting polypeptides that regulate transcription.

[0057] An “intron” is an intervening sequence in a gene that is transcribed into RNA but is then excised in the process of generating the mature mRNA. The term is also used for the excised RNA sequences. An “exon” is a portion of the sequence of a gene that is transcribed and is found in the mature mRNA derived from the gene but is not necessarily a part of the sequence that encodes the final gene product. The 5' untranslated region (5’UTR) (also known as atranslational leader sequence or leader RNA) is the region of an mRNA that is directly upstream from the initiation codon. This region is involved in the regulation of translation of a transcript by differing mechanisms in viruses, prokaryotes and eukaryotes. The “3' non-coding sequences” refer to DNA sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.

[0058] As used herein “promoter” refers to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells. In certain embodiments, the polynucleotides described herein are operably linked to a promoter that drives expression in a plant cell. Any promoter known in the art can be used in the methods of the present disclosure including, but not limited to, constitutive promoters, pathogeninducible promoters, wound-inducible promoters, tissue-preferred promoters, and chemical- regulated promoters. The choice of promoter may depend on the desired timing and location of expression in the transformed plant as well as other factors, which are known to those of skill in the art. Such constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99 / 43838 and U.S. Patent No.6,072,050; the core CaMV 35S promoter; rice actin; ubiquitin; pEMU; MAS; ALS; and the like. Other constitutive promoters include, for example, those disclosed in U.S. Patent Nos.5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6,177,611, which are known in the art, and can be contemplated for use in the present disclosure.

[0059] Generally, it can be beneficial to express the gene from an inducible promoter, particularly from a pathogen-inducible promoter. Such promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen, e.g., PR proteins, SAR proteins, beta-1,3 -glucanase, chitinase, etc.

[0060] Of interest are promoters that are expressed locally at or near the site of pathogen infection. Additionally, as pathogens find entry into plants through wounds or insect damage, a wound-inducible promoter can be used in the constructions of the disclosure. Such woundinducible promoters include potato proteinase inhibitor (pin II) gene, wunl and wun2, winl and win2, systemin, WIP1, MPI gene, and the like.

[0061] Chemi cal -regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter can be a chemical-inducible promoter, where application of the chemical induces gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical -inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-la promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (e.g., the glucocorticoid-inducible promoter, and tetracycline-inducible and tetracycline-repressible promoters).

[0062] Tissue-preferred promoters can be utilized to target enhanced expression of the target genes or proteins within a particular plant tissue. Such tissue-preferred promoters include, but are not limited to, leaf-preferred promoters, root-preferred promoters, seed-preferred promoters, and stem-preferred promoters. Tissue-preferred promoters include Yamamoto et al. (1997) Plant J. 12(2): 255 -265; Kawamata et al. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol. Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 112(3): 1331-1341; Van Camp et al. (1996) Plant Physiol.112(2): 525-535; Canevascini et al. (1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ. 20: 181-196; Orozco et al. (1993) Plant Mol Biol. 23(6): 1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters can be modified.

[0063] Leaf-specific promoters are known in the art. See, for example, Yamamoto et al. (1997) Plant J. 12(2)255-265; Kwon et al. (1994) Plant Physiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23(6): 1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

[0064] "Seed-preferred" promoters include both "seed-specific" promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as "seedgerminating" promoters (those promoters active during seed germination). Such seed-preferredpromoters include, but are not limited to, Ciml (cytokinin-induced message), cZ19Bl (maize 19 kDa zein), milps (myo-inositol- 1 -phosphate synthase), and celA (cellulose synthase) (see WO 00 / 11177, herein incorporated by reference). Gama-zein is a preferred endosperm-specific promoter. Glob-1 is a preferred embryo-specific promoter. For dicots, seed-specific promoters include, but are not limited to, bean P-phaseolin, napin, -conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also WO 00 / 12733, where seed-preferred promoters from endl and end2 genes are disclosed; herein incorporated by reference.

[0065] Also provided is a method for accelerating transgenic soybean development comprising introducing into a regenerable soybean cell a silencing nucleic acid and a nucleic acid construct comprising a gene of interest operably linked to a regulatory element, growing a first soybean plant from the regenerable plant cell and collecting the seed therefrom, self-pollinating a soybean plant grown from the collected seed or crossing a soybean plant grown from the collected seed with a second soybean plant to produce a progeny soybean plant, and selecting progeny seed comprising the gene of interest from the progeny soybean plant. In certain embodiments, the first soybean plant has an earlier flowering time, early maturity or both an earlier flowering time and maturity as compared to a plant grown from the selected progeny seed. In certain embodiments, the progeny seed does not comprise the silencing nucleic acid. In certain embodiments, the method further comprises growing and self-pollinating or crossing the progeny seed at least one (e.g., 1, 2, 3, 4, 5, 6, 7 or more) generation to produce seed not comprising the silencing construct. The gene of interest may be any gene of interest that provides a desirable characteristic to the plant such as those described herein or known in that art. In certain embodiments, the regulatory element is a promoter. In certain embodiments, the gene of interest is operably linked to a heterologous regulatory element. In certain embodiments, the heterologous regulatory element is a heterologous promoter.

[0066] In certain embodiments, the development of the transgenic soybean (e.g., length of time from the introduction into the regenerable soybean cell to the selection of progeny seed) is decreased by at least about 10 days, 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 120 days, 140 days or 150 days as compared to a control (e.g., thedevelopment of a transgenic soybean using the same process but lacking the introduction of the silencing element).

[0067] In certain embodiments, the yield of the plant comprising the introduced gene of interest and the silencing nucleic acid is equal to or within at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% as compared to the yield of a control plant, such as, for example, the plant from which the regenerable plant cell was isolated or a plant grown from the regenerable plant cell not comprising the silencing construct.

[0068] In certain embodiments, the introducing step comprises transforming the regenerable plant cell with a nucleic acid construct comprising the gene of interest operably linked to the regulatory element (e.g., heterologous promoter) and the silencing nucleic acid. In certain embodiments, the gene of interest operably linked to the regulatory element (e.g., heterologous promoter) and the silencing nucleic acid are introduced using a single nucleic acid construct (e.g., single construct comprises both the gene of interest and the silencing nucleic acid). In certain embodiments, the gene of interest operably linked to the regulatory element (e.g., heterologous promoter) and the silencing nucleic acid are introduced using separate nucleic acid constructs (e.g., a construct comprising the gene of interest and a construct comprising the silencing element). In certain embodiments, the silencing nucleic acid is operably linked to a regulatory element. The regulatory element operably linked to the gene of interest and / or the silencing element may be any regulatory element described herein or known in the art. In certain embodiments, the regulatory element is a promoter (e.g., heterologous promoter). In certain embodiments, the promoter operably linked to the silencing nucleic acid is a tissue-preferred or a leaf-specific promoter. In certain embodiments, the nucleic acid construct comprising the gene of interest and / or silencing element further comprises a selectable marker gene for the selection of transformed cells.

[0069] In certain embodiments of the methods described herein, the silencing nucleic acid is flanked by site-specific excision sites, such that in certain embodiments, the nucleic acid construct further comprises site-specific excision sites flanking the silencing nucleic acid construct. In certain embodiments, the method further comprises expressing a recombinase enzyme that targets the site-specific excision sites in a plant comprising the silencing nucleic acid construct such as, for example, a progeny plant, a backcross progeny plant, or a plant grownfrom the backcross progeny seed, thereby resulting in a plant comprising the gene of interest or introduced gene modification and not comprising the silencing nucleic acid.

[0070] As used herein a site-specific excision site refers to a nucleic acid sequence that is specifically targeted by a recombinase enzyme which are enzymes that catalyze site-specific recombination events within DNA. The site-specific excision site is not particularly limited and is dependent on the type of recombinase used. Examples of recombinases that can be used in the methods and constructs disclosed herein include, but are not limited to, FLP, FLPe, KD, Cre, SSV1, lambda Int, phi C31 Int, HK022, R, B2, B3, Gin, Tnl721, CinH, ParA, Tn5O53, Bxbl, TP907-1, or U153. In certain embodiments, the Cre / Lox system is used in the methods described herein. When a site-specific recombinase is used to control the expression of the silencing nucleic acid, the nucleic acid construct further comprises appropriate site-specific excision sites flanking the nucleotide sequences to be excised. In certain embodiments, the nucleic acid construct comprising the silencing nucleic acid further comprises a nucleotide sequence encoding a site-specific recombinase. Alternatively, the site-specific recombinase does not need to be co-located on the same expression construct comprising the silencing nucleic acid and, instead, the recombinase can be expressed from a different construct.

[0071] Further provide is a method for trait introgression comprising crossing a first soybean plant with a second soybean plant, the second soybean plant comprising a desired allele of a genetic locus, the first soybean plant, the second soybean plant, or both comprising a silencing nucleic acid and collecting the seed comprising the trait of interest and the silencing nucleic acid produced therefrom, the silencing nucleic acid decreasing expression of at least one endogenous flowering time or maturity gene, backcrossing a soybean plant grown from the collected seed to the first soybean plant to produce a backcross progeny plant, selecting backcross progeny seed comprising the desired allele from the backcross progeny soybean plant. In certain embodiments, the method further comprises backcrossing a plant grown from the selected backcross progeny seed to the first soybean plant at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) additional time to generate a population of plants comprising the desired allele and otherwise comprising all or essentially all of the phenotypic characteristics, or all or essentially all of the morphological and physiological characteristics of the first soybean plant, and / or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to the first soybean plant as determined by molecular markers, such as SNP markers. In certain embodiments, the backcrossprogeny seed, the population of plants or both the backcross progeny seed and the population of plants do not comprise the silencing nucleic acid. By essentially all of the phenotypic characteristics or morphological and physiological characteristics, it is meant that all of the characteristics of a plant are recovered that are otherwise present when compared in the same environment, other than an occasional variant trait that might arise during backcrossing or direct introduction of a transgene or specific genetic modification.

[0072] As used herein, “percent identity” with respect to a soybean plant refers to the comparison of the homozygous alleles of two soybean varieties. Percent identity is determined by comparing a statistically significant number of the homozygous alleles of two developed varieties. For example, a percent identity of 90% between soybean variety 1 and soybean variety 2 means that the two varieties have the same allele at 90% of the loci used in the comparison.

[0073] The term “introgression” refers to the transmission of a desired allele of a genetic locus from one genetic background to another. For example, introgression of a desired allele at a specified locus can be transmitted to at least one progeny plant via a sexual cross between two parent plants, where at least one of the parent plants has the desired allele within its genome.

[0074] The desired allele can be, e.g., a transgene, a modified (mutated or edited) native allele, a native allele, or a selected allele of a marker or QTL. As used herein an “allele” is one of several alternative forms of a gene occupying a given locus on a chromosome. The term “quantitative trait locus” or “QTL” refers to a region of DNA that is associated with the differential expression of a quantitative phenotypic trait in at least one genetic background, e.g., in at least one breeding population. The region of the QTL encompasses or is closely linked to the gene or genes that affect the trait in question. An “allele of a QTL” can comprise multiple genes or other genetic factors within a contiguous genomic region or linkage group, such as a haplotype. An allele of a QTL can denote a haplotype within a specified window wherein said window is a contiguous genomic region that can be defined, and tracked, with a set of one or more polymorphic markers.

[0075] In certain embodiments, prior to crossing the first and second soybean plant the method comprises introducing in the second soybean plant a nucleic acid construct comprising the desired allele operably linked to a heterologous regulatory element and the silencing nucleic acid. The nucleic acid construct may be any nucleic acid construct described herein comprising a desired allele (e.g., gene of interest) and a silencing construct. In certain embodiments, prior to crossing the first and second soybean plant the method comprises introducing in the secondsoybean plant a nucleic acid construct comprising an editing nucleic acid, the editing nucleic acid introducing a modification in an endogenous gene to produce the desired allele and the silencing nucleic acid. The nucleic acid construct may be any nucleic acid construct described herein comprising an editing nucleic acid and a silencing construct. In certain embodiments, prior to crossing the first and second soybean plant the method comprises introducing in the first soybean plant, the second soybean plant, or both the first and second soybean plant a nucleic acid construct comprising the silencing nucleic acid. The silencing nucleic acid may be operably linked to a regulatory element, such as, for example, a promoter.

[0076] Also provided herein are methods for increasing breeding efficiency comprising crossing a first soybean plant with a second soybean plant, the first soybean plant, the second soybean plant or both comprising a silencing nucleic acid and collecting the seed comprising the silencing nucleic acid produced therefrom, crossing a soybean plant grown from the collected seed to produce a progeny plant and selecting the progeny seed produced therefrom that does not comprise the silencing nucleic acid. In certain embodiments, the silencing nucleic acid targets a gene promoting flowering time and / or maturity in which silencing of the gene results in delayed or later flowering and / or delayed or later maturity as compared to a control plant not comprising the silencing nucleic acid. In certain embodiments, the silencing nucleic acid targets a gene having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any one of SEQ ID NOs: 47-80. In certain embodiments, the silencing nucleic acid targets a gene having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 52. In certain embodiments, the silencing nucleic acid targets a flowering time and / or maturity gene in which silencing of the gene results in early flowering and / or early maturity as compared to a control plant not comprising the silencing nucleic acid. In certain embodiments, the silencing nucleic acid targets a gene having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any one of SEQ ID NOs: 1-44. In certain embodiments, the crossing step is repeated at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) times to produce the progeny seed not comprising the silencing nucleic acid. In certain embodiments, the crossing step comprises self-pollinating a soybean plant grown from the collected seed from the cross of the first and second soybean plant. In certain embodiments, the crossing step comprisesbackcrossing a soybean plant grown from the collected seed from the cross of the first and second soybean plant to the first soybean plant. In certain embodiments, the progeny seed not comprising the silencing nucleic acid produced by backcrossing to the first soybean plant comprises all or essentially all of the phenotypic characteristics, or all or essentially all of the morphological and physiological characteristics of the first soybean plant, and / or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity to the first soybean plant as determined by molecular markers, such as SNP markers. In certain embodiments, the crossing step comprises crossing a soybean plant grown from the collected seed from the cross of the first and second soybean plant to a third soybean plant. In certain embodiments, prior to crossing the first soybean plant and the second soybean plant the method further comprises introducing into the second soybean plant a silencing nucleic acid. In certain embodiments, the difference in relative maturity between the first soybean plant and the second soybean plant prior to the introduction of the silencing nucleic acid is at least 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 10, 11, 12, or 13.

[0077] Also provided is a method of increasing seed production comprising introducing into a regenerable soybean cell a silencing nucleic acid, the silencing nucleic acid decreasing expression of at least one endogenous gene that promotes flowering time and / or maturity, growing a first soybean plant from the regenerable plant cell and collecting the seed therefrom, self-pollinating a soybean plant grown from the collected seed or crossing a soybean plant grown from the collected seed with a second soybean plant to produce a progeny soybean plant, and selecting progeny seed comprising the transgene from the progeny soybean plant. In certain embodiments, the silencing nucleic acid targets a gene promoting flowering time and / or maturity in which silencing of the gene results in delayed or later flowering and / or delayed or later maturity as compared to a control plant not comprising the silencing nucleic acid. In certain embodiments, the silencing nucleic acid targets a gene having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to any one of SEQ ID NOs: 47-80. In certain embodiments, the silencing nucleic acid targets a flowering time and / or maturity gene in which silencing of the gene results in early flowering and / or early maturity as compared to a control plant not comprising the silencing nucleic acid. In certain embodiments, the silencing nucleic acid targets a gene having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 52. In certain embodiments, the crossing or self-pollinating step is repeated at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) times to produce the progeny seed not comprising the silencing nucleic acid. In certain embodiments, the crossing step comprises self-pollinating a soybean plant grown from the collected seed from the cross of the first and second soybean plant. In certain embodiments, the progeny seed does not comprise the silencing nucleic acid. In certain embodiments, the first soybean plant has a later flowering time, later maturity or both as compared to a plant grown from the selected progeny seed.

[0078] In certain embodiments, the method further comprises introducing into the regenerable soybean cell a gene of interest operably linked to a heterologous regulatory element and / or an editing nucleic acid, the editing nucleic acid introducing a modification in an endogenous gene of interest. The gene of interest and / or editing nucleic acid may be any gene of interest or editing nucleic acid described herein.

[0079] In certain embodiments, the yield of the plant comprising the silencing nucleic acid decreasing expression of at least one endogenous gene that promotes flowering time and / or maturity is increased by at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% as compared to the yield of a control plant, such as, for example, the plant from which the regenerable plant cell was isolated or a plant grown from the regenerable plant cell not comprising the silencing construct.

[0080] In certain embodiments, the plant from which the regenerable plant cell was isolated or a plant grown from the regenerable plant cell not comprising the silencing construct is from a maturity group of at least group 0.00, 0.05, 0.1, 0.5, 1.0, 1.5, 2, and 2.5 and less than 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, 0.5, 0.1, 0.05.

[0081] Further provided herein is a screening method for determining gene editing efficiency comprising introducing into a regenerable soybean cell an editing nucleic acid, the editing nucleic acid introducing a modification in an endogenous gene affecting flower, pubescence, seed and / or hilum color, generating a plant from the plant cell, and determining editing efficiency based on the number of plants with an altered flower, pubescence, seed and / or hilum color as compared to a control plant. In certain embodiments, the modification is in an endogenous gene having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 84. Incertain embodiments, the genetic modification is introduced into an endogenous gene having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 84 and results in a non-functional flavonoid 3'-hydroxylase gene. In certain embodiments, plants comprising the modification resulting in a non-functional flavonoid 3'-hydroxylase gene have buff or tan hilum and gray pubescence such that editing efficiency is determined from the hilum and / or pubescence color.

[0082] The following are examples of specific embodiments of some aspects of the invention. The examples are offered for illustrative purposes only and are not intended to limit the scope of the invention in any way.EXAMPLE 1

[0083] This example demonstrates how the silencing of El and El homologs can be used to expedite industrial agricultural processes.

[0084] The El-like-a (SEQ ID NO: 4) and El-like-b (SEQ ID NO: 6) genes share approximately 94% identity to the El coding nucleotide sequence (SEQ ID NO: 2). A construct referred to as Eli comprising an El (SEQ ID NO: 45) fragment with homology to the El-like-a, El-like-b, and El gene expressed under a constitutive promoter in forward and reverse orientations was created. An example construct is shown in Fig. 1. The construct can be used to generate plants with accelerated growth cycles. Briefly, the procedure to generate plants with accelerated growth cycles is as follows: the vector comprising the El fragment was introduced in the Agrobacterium tumefaciens strain via electroporation, a regenerable plant cell from a soybean variety was treated with the A. tumefaciens containing the Eli vector to produce transformed TO plants, the TO plants are grown on a selective media and surviving plants are transplanted to soil and grown in greenhouse conditions under a standard light cycle. The plants are analyzed from the timing of transplant to soil to the appearance of the first flower which is measured as R1 and for the timing from the transplant to soil to the when 95% of the pods have reached a mature pod color (e.g., brown or tan) which is measured as R8.

[0085] Plants comprising the Eli vector reach R1 and R8 earlier than control plants not containing the vector.EXAMPLE 2

[0086] This example demonstrates how the silencing of other flowering time or maturity genes expedite industrial agricultural processes.

[0087] The soybean E2 maturity gene is an ortholog of the Arabidopsis GIGANTEA gene, which facilitates circadian clock maintenance and participates in flowering time control. The E2 maturity gene and two closely related E2 homologs, E2-like-a (E21a) and E2-like-b (E21b), are predicted to collectively inhibit flowering time by promoting El expression. The predicted E2 coding sequence shares 96.3% and 83.9% nucleotide similarity to the coding regions of E21a and E21b, respectively. Thus, an RNAi construct can be created to simultaneously suppress expression of the three genes following the method of Example 1 . E2i is an example E2 fragment (Seq ID NO: 46) that when expressed in forward and reverse orientations will initiate silencing of the three E2 homologs. Following RNAi triggered suppression of the E2 genes, plants will flower and mature earlier than wild-type counterparts. These plants can then be used to expedite industrial processes such as population development, inbred development, and gene introgression.

[0088] Additional candidate genes for which an RNAi construct may be created to suppress expression and promote an earlier flowering time or maturity are found in Table 1. The RNAi construct or constructs may be created and used as described in Example 1.Table 1: Candidate targets for accelerating time to maturity and flowering using RNAi.EXAMPLE 3

[0089] This example demonstrates how a DNA editing pipeline is accelerated using RNAi silencing of genes that inhibit flowering and maturity in soybean.

[0090] Gene editing technologies, such as those enabled by CRISPR / Cas9, allow for precise manipulation of DNA target sequences. Often, the editing machinery is introduced early in plant life cycle to produce edits that are then propagated to all cells during development. The induced edits are then inherited in subsequent generations.

[0091] The construct of Example 1 is designed to contain in tandem editing machinery that would be used to create permanent DNA edits while simultaneously accelerating the time to flowering and maturity. An example construct is shown in Fig. 2.

[0092] In one example, a CRISPR / Cas9 system is placed in a construct containing the El RNAi components listed in Example 1. In particular, a construct comprising the El fragment and CRISPR / Cas9 components designed to edit an endogenous Sweet39 (SWT39) gene or an endogenous F3H gene was introduced in the Agrobacterium tumefaciens strain via electroporation, a regenerable plant cell from a soybean variety was treated with the A. tumefaciens containing the Eli vector to produce transformed TO plants, the TO plants weregrown on a selective media and surviving plants are transplanted to soil and grown in greenhouse conditions under a standard light cycle. T1 seed was harvested 3 months after planting and the T1 plants showed the expected segregation of the construct. Growth of plants that contained the construct were stunted compared to that of nulls. Seed set was on average 15 seeds for plant containing the construct, compared to an average of 317 seeds harvested from nulls. T2 Seed was harvested about 1 week earlier for plants that contained RNAi constructs, compared with nulls. However, because plants were kept on the same table, watering and fertilizer was likely applied to the plants containing the construct longer than desired and this may have decreased the difference in harvest time between plants containing the RNAi construct and nulls.

[0093] To test for changes in gene expression, total nucleic acids were isolated from leaf tissue and expression levels of El, Ellike, vegetative and reproductive growth genes were determined. As shown in Table 2, expression of El, ElLa and EILb genes were very low, and almost undetectable, in T1 material that contained the RNAi construct, whereas expression in T1 material that was negative for the RNAi construct was higher, relative to T1 positive material. Expression of the FT4 vegetative growth gene was also lower in T1 positive material, compared to that in T1 negative material and the reproductive growth genes, FT2a and FT5a, had a higher level of expression in the T1 positive material compared to T1 negative material. Additionally, the presence of the genome edit in either the SWT39 or F3H gene was confirmed by sequencing.Table 2: Average relative expression levels of El, ElLa, EILb, FT4, FT2a and FT5aExpression level is relative to the expression level of EIF4aEXAMPLE 4

[0094] This example demonstrates how a trait introgression pipeline can be accelerated using RNAi silencing of genes that inhibit flowering and maturity in soybean.

[0095] A common practice to introduce genetic loci into new plant material is to cross a donor parent containing a gene of interest by a recurrent parent and then conduct 2 or more generations of backcrossing to the recurrent parent paired with phenotypic or marker assisted selection of the target donor locus. This process produces a line that is nearly identical to the recurrent parent but also contains the locus of interest.

[0096] To accelerate the backcrossing process, the RNAi construct of Example 1 is introduced into the donor parent alongside an exogenous gene of interest. An example construct is shown in Fig. 3. This construct is transformed into donor plants, and then, the transformed donor plants are crossed to recurrent parents as described above. Due to the RNAi based silencing of El and El homologs, each generation is accelerated compared to wild-type plants. Therefore, a trait introgression scheme is completed faster allowing breeders or researchers earlier access to desired plant material. Alternatively, the RNAi construct without the gene of interest could be placed either randomly into a donor line or adjacent to a specific native locus using genome modification strategies like SDN3. Selection of this construct and the native locus from the donor line during backcrossing similarly enables faster trait introgression.EXAMPLE 5

[0097] This example demonstrates how RNAi components can be removed to return plants to a desired maturity.

[0098] To return a plant to a desired maturity following accelerated cycling, the RNAi El components must be removed. This can be accomplished through genetic segregation following self-fertilization or backcrossing where plants are selected that do not carry the construct encoding RNAi El using phenotypic or molecular approaches. Alternatively, an inducible excision system can be deployed to remove the RNAi construct components from the genome. In one example, an inducible CRE system is deployed where the CRE recombinase, an inducible heat shock promoter, and LoxP sites are added as part of the construct containing the RNAi components of Example 1. An example construct is shown in Fig. 4. After heat shock induction early in plant development, the CRE recombinase initiates excision of the CRE containingsequences and the RNAi components. Following removal of the RNAi components, El and the El homologs are no longer repressed, and the plant returns to the expected flowering and maturity behavior.EXAMPLE 6

[0099] This example demonstrates how RNAi of genes that promote flowering is used to enable breeding and biotechnology operations.

[0100] Several genes have been described that promote flowering (Luo, X. et al. 2022.Molecular genetic understanding of photoperiodic regulation of flowering time in Arabidopsis and soybean. International Journal of Molecular Sciences, 23(1), 466). Natural mutations that abolish or disrupt function of these genes lead to flowering delays and have been used to create cultivars with wide relative maturities. To assist crossing of cultivars with disparate relative maturities, it may be desirable to delay flowering in lines that otherwise flower early in nonadapted environments. To accomplish this, transient silencing of genes, such as those found in Table 2, that promote flowering using RNAi is invoked to delay flowering. An example RNAi construct targeting the GmFT5a gene (Gyma.16G044100), which promotes flowering, is shown in Fig. 5. Plants that have the GmFT5a silencing construct containing the GmFT5a fragment (sequence) have delayed flowering compared to wild-type controls due to reduced expression of GmFT5a. It is also possible that the GmFT5a fragment (sequence) may lead to silencing of the FT (Flowering Locus T) homologs listed in Table 3, which may exacerbate the flowering delay.

[0101] The RNAi system targeting FT5a or other genes that promote flowering could be co- opted to increase seed yield during plant grow-outs. Late flowering and maturity are associated with increases in plant yield as more flowers and pods are created during prolonged vegetative growth (Xu, Kun, et al. "Fine-tuning florigen increases field yield through improving photosynthesis in soybean." Frontiers in Plant Science 12 (2021): 710754.). Thus, delaying flowering by silencing FT5a could benefit seed production operations where seed counts are constrained by resource, environmental, or biological limitations.

[0102] Precise regulation of flowering and maturity as described here and in previous examples further enables centralized population development or technology centers that handle material from various geographies. Normalizing reproductive behaviors allows researchers to be moreefficient with planning and cycling of diverse material for crossing, seed productions, or trait introgression.Table 3: Candidate targets for delaying time to maturity and flowering using RNAi.EXAMPLE 7

[0103] This example demonstrates optimization of the RNAi system described in Examples 1-6.

[0104] Accelerating maturity by suppressing key genetic regulators of reproductive behavior can result in a reduction in plant seed yield due to early stem and flowering termination.

[0105] To modify cycle time without compromising yield, optimization steps to the method of Examples 1-6 can be performed. For example, alternative promoters that increase or decrease expression levels of the RNAi construct can be operably linked to the RNAi construct to condition the expression changes at native maturity gene targets. In particular, tissue specific promoters or conditionally activated promoters are linked to the RNAi construct to optimize expression of the construct for a desired phenotype. Alternatively, other gene silencing methods could be invoked to transiently modify reproductive behavior and improve seed yields. These include, but are not limited to, both transcriptional and post-transcriptional gene silencing methods, like sense suppression, anti-sense suppression, and targeted methylation.

[0106] Additionally, the methods can be optimized by simultaneously modifying the expression of different combinations of genes described in Examples 1-6, or in combination with any known genetic regulators of maturation in soybean. For example, temporarily silencing the expression of the El and E2 genes, while not silencing their respective homologs, may lead to more desirable final plant phenotypes.

[0107] Testing or screening can be done to evaluate optimized gene knockdown methods in conjunction with a genome editing approach. In one example, a guide RNA targeting the classical T-Locus (Glyma.06G202300), which encodes a Flavonoid 3 '-hydroxylase, is used to create frameshift mutations that renders the gene non-functional. Natural frameshift mutations in Glyma.06G202300 have been shown to result in an altered hilum and pubescence color. Plants with the intact T allele have seed with a black hilum and brown pubescence; however, seed with natural homozygous deletions of a single base pair in the third exon (t allele) can have buff or tan hilum and gray pubescence (Toda K, Yang D, Yamanaka N, Watanabe S, Harada K, Takahashi R. A single-base deletion in soybean flavonoid 3 ’-hydroxylase gene is associated with gray pubescence color. Plant Mol Biol. 2002;50: 187-96). Therefore, in certain genetic backgrounds when the T allele is disrupted by gene editing, non-functional alleles are created which results in a phenotype that is visually screened. This screening method allows for quick assessment of gene editing efficiency as the pubescence color change can be observed within the TO directly, or hilum color changes can be observed in the T1 seed. When desired, the RNAi and Cas9 construct can be removed via genetic segregation or through outcrossing.

[0108] Other genes that function in morphological or biochemical processes can be evaluated for use in the editing system described above. Genes with known roles in flower, pubescence, seed,and hilum color are desirable targets as they produce easily screened visual phenotypes that are not detrimental to plant development or yield. For example, the R locus (Glyma.09g235100) encodes a R2R3 MYB transcription factor that when disrupted results in brown seed coat and hilum; thus, it could serve as an alternate target to the T locus (Gillman, J. D., Tetlow, A., Lee, J. D., Shannon, J. G., & Bilyeu, K. (2011).

[0109] Loss-of-function mutations affecting a specific Glycine max R2R3 MYB transcription factor result in brown hilum and brown seed coats. BMC plant biology, 11(1), 1-12).

[0110] All publications and patent applications in this specification are indicative of the level of ordinary skill in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated by reference.

[0111] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative only and not limiting.

[0112] Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0113] Units, prefixes and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5’ to 3’ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges are inclusive of the numbers defining the range. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

Claims

We claim:

1. A method for accelerating genome edited soybean development, the method comprising: a. introducing into a regenerable soybean cell an editing nucleic acid and a silencing nucleic acid, the editing nucleic acid introducing a modification in an endogenous gene of interest and the silencing nucleic acid decreasing expression of at least one endogenous gene that inhibits flowering time and / or maturity; b. growing a first soybean plant from the regenerable plant cell and collecting the seed produced therefrom; c. self-pollinating a soybean plant grown from the collected seed or crossing a soybean plant grown from the collected seed with a second soybean plant to produce a progeny soybean plant; and d. selecting progeny seed comprising the introduced modification in the endogenous gene of interest from the progeny soybean plant.

2. The method of claim 1, wherein the first soybean plant has an earlier flowering time, early maturity or both as compared to a plant grown from the selected progeny seed.

3. The method of claim 1 or 2, wherein the progeny seed does not comprise the silencing nucleic acid.

4. The method of any one of claims 1-3, wherein the editing nucleic acid comprises at least one of an endonuclease, a guide RNA, or a polynucleotide modification template, or any combination thereof.

5. The method of claim 4, wherein the endonuclease is a Cas endonuclease.

6. The method of any one of claims 1-5, wherein the silencing nucleic acid targets at least one gene comprising a nucleic acid sequence having at least 95% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-44.

7. The method of any one of claims 1-6, wherein the silencing nucleic acid targets an El gene comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 2, an El-like-a gene comprising a nucleic acid sequence having at least 95% sequence identityto SEQ ID NO: 4, or an El-like-b gene comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 6, or any combination thereof.

8. The method of any one of claims 1-6, wherein the silencing nucleic acid targets an E2 gene comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 8, an E2-like-a gene comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 10, or an E2-like-b gene comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 12, or any combination thereof.

9. The method of any one of claims 1-8, wherein the introducing step comprises transforming the regenerable plant cell with a nucleic acid construct comprising the editing nucleic acid and the silencing nucleic acid.

10. The method of claim 9, wherein the editing nucleic acid is operably linked to a first regulatory element.

11. The method of claim 10, wherein the first regulatory element comprises a promoter sequence.

12. The method of any one of claims 9-11, wherein the silencing nucleic acid is operably linked to a second regulatory element.

13. The method of claim 12, wherein the second regulatory element comprises a promoter sequence.

14. The method of claim 13, wherein the second regulatory element is a leaf-preferred promoter sequence.

15. A method for accelerating transgenic soybean development, the method comprising: a. introducing into a regenerable soybean cell a nucleic acid construct and a silencing nucleic acid, the nucleic acid construct comprising a gene of interest operably linked to a heterologous regulatory element and the silencing nucleic acid decreasing expression of at least one endogenous gene that inhibits flowering time and / or maturity; b. growing a first soybean plant from the regenerable plant cell and collecting the seed therefrom;c. self-pollinating a soybean plant grown from the collected seed or crossing a soybean plant grown from the collected seed with a second soybean plant to produce a progeny soybean plant; and d. selecting progeny seed comprising the introduced gene of interest from the progeny soybean plant.

16. The method of claim 15, wherein the first soybean plant has an earlier flowering time, early maturity or both as compared to a plant grown from the selected progeny seed.

17. The method of claim 15 or 16, wherein the progeny seed does not comprise the silencing nucleic acid.

18. The method of any one of claims 15-17, wherein the silencing nucleic acid targets at least one gene comprising a nucleic acid sequence having at least 95% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-44.

19. The method of any one of claims 15-18, wherein the silencing nucleic acid targets an El gene comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 2, an El-like-a gene comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 4, or an El-like-b gene comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 6, or any combination thereof.

20. The method of any one of claims 15-18, wherein the silencing nucleic acid targets an E2 gene comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 8, an E2-like-a gene comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 10, or an E2-like-b gene comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 12, or any combination thereof.

21. The method of any one of claims 15-20, wherein the introducing step comprises transforming the regenerable plant cell with a nucleic acid construct comprising the gene of interest operably linked to the heterologous regulatory element and the silencing nucleic acid.

22. The method of claim 21, wherein the heterologous regulatory element operably linked to the gene of interest comprises a promoter.

23. The method of any one of claims 21-22, wherein the silencing nucleic acid is operably linked to a regulatory element.

24. The method of claim 23, wherein the regulatory element operably linked to the silencing nucleic acid comprises a promoter.

25. The method of claim 24, wherein the promoter is a leaf-preferred promoter.

26. The method of any one of claims 21-25, wherein the nucleic acid construct further comprises an inducible excision sequence flanking the silencing nucleic acid construct.

27. The method of claim 26, wherein the method further comprises expressing an inducer that targets the inducible excision sequence in the backcross progeny plant or a plant grown from the backcross progeny seed to excise the silencing nucleic acid.

28. A method for trait introgression, the method comprising: a. crossing a first soybean plant with a second soybean plant comprising a trait of interest and a silencing nucleic acid, the silencing nucleic acid decreasing expression of at least one endogenous gene that inhibits flowering time and / or maturity; b. collecting the seed comprising the trait of interest and the silencing nucleic acid produced therefrom; c. backcrossing a soybean plant grown from the collected seed to the first soybean plant to produce a backcross progeny plant; and d. selecting backcross progeny seed comprising the trait of interest from the backcross progeny soybean plant.

29. The method of claim 28, wherein the method further comprises backcrossing a plant grown from the selected backcross progeny seed to the first soybean plant at least one additional time.

30. The method of claim 28 or 29, wherein the backcross progeny seed, the population of plants or both do not comprise the silencing nucleic acid.31 . The method of any one of claims 28-30, wherein the silencing nucleic acid targets at least one gene comprising a nucleic acid sequence having at least 95% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-44.

32. The method of any one of claims 28-31, wherein the silencing nucleic acid targets an El gene comprising a nucleic acid sequence having at least 95% sequence identity to SEQ IDNO: 2, an El-like-a gene comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 4, or an El-like-b gene comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 6, or any combination thereof.

33. The method of any one of claims 28-31, wherein the silencing nucleic acid targets an E2 gene comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 8, an E2-like-a gene comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 10, or an E2-like-b gene comprising a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 12, or any combination thereof.

34. The method of any one of claims 28-33, wherein prior to crossing the first and second soybean plant the method comprises introducing in the second soybean plant a nucleic acid construct comprising the trait of interest operably linked to a heterologous regulatory element and the silencing nucleic acid.

35. The method of claim 34, wherein the heterologous regulatory element operably linked to the gene of interest comprises a promoter.

36. The method of claim 34 or 35, wherein the silencing nucleic acid is operably linked to a regulatory element.

37. The method of claim 36, wherein the regulatory element operably linked to the silencing nucleic acid comprises a promoter.

38. The method of claim 37, wherein the promoter is a leaf-preferred promoter.

39. The method of any one of claims 34-38, wherein the nucleic acid construct further comprises an inducible excision sequence flanking the silencing nucleic acid construct.

40. The method of claim 39, wherein the method further comprises expressing an inducer that targets the inducible excision sequence in the backcross progeny plant or population of plants to excise the silencing nucleic acid.41 . The method of any one of claims 28-33, wherein prior to crossing the first and second soybean plant the method comprises introducing in the second soybean plant a nucleic acid construct comprising an editing nucleic acid, the editing nucleic acid introducing a modification in an endogenous gene to produce the trait of interest and the silencing nucleic acid.

42. The method of claim 41, wherein the editing nucleic acid comprises at least one of an endonuclease, a guide RNA, or a polynucleotide modification template, or any combination thereof.

43. The method of claim 42, wherein the endonuclease is a Cas endonuclease.

44. The method of any one of claims 41-43, wherein the editing nucleic acid is operably linked to a first regulatory element.

45. The method of claim 44, wherein the first regulatory element comprises a promoter sequence.

46. The method of any one of claims 41-45, wherein the silencing nucleic acid is operably linked to a regulatory element.

47. The method of claim 46, wherein the regulatory element operably linked to the silencing nucleic acid comprises a promoter.

48. The method of claim 47, wherein the promoter is a leaf-preferred promoter.

49. A method of increasing seed production, the method comprising: a. introducing into a regenerable soybean cell a silencing nucleic acid, the silencing nucleic acid decreasing expression of at least one endogenous gene that promotes flowering time and / or maturity; b. growing a first soybean plant from the regenerable plant cell and collecting the seed therefrom; c. self-pollinating a soybean plant grown from the collected seed or crossing a soybean plant grown from the collected seed with a second soybean plant to produce a progeny soybean plant; and d. selecting progeny seed.

50. The method of claim 49, wherein the method further comprises introducing into the regenerable soybean cell a gene of interest operably linked to a heterologous regulatory element and / or an editing nucleic acid, the editing nucleic acid introducing a modification in an endogenous gene of interest.51 . The method of claim 49 or 50, wherein the first soybean plant has a later flowering time, later maturity or both as compared to a plant grown from the selected progeny seed.

52. The method of any one of claims 49-51, wherein the progeny seed does not comprise the silencing nucleic acid.

53. The method of claim 50, wherein the editing nucleic acid comprises at least one of an endonuclease, a guide RNA, or a polynucleotide modification template, or any combination thereof.

54. The method of claim 53, wherein the endonuclease is a Cas endonuclease.

55. The method of any one of claims 49-54, wherein the silencing nucleic acid targets at least one gene comprising a nucleic acid sequence having at least 95% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 47-80.

56. The method of any one of claims 49-55, wherein the silencing nucleic acid targets a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 52.

57. The method of any one of claims 49-56, wherein the introducing step comprises transforming the regenerable plant cell with a nucleic acid construct comprising the silencing nucleic acid.

58. The method of claim 57, wherein the silencing nucleic acid is operably linked to a regulatory element.

59. The method of claim 58, wherein the regulatory element comprises a promoter sequence.

60. The method of claim 59, wherein the regulatory element is a leaf-preferred promoter sequence.

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