Compositions and methods for targeting donor polynucleotides in soybean genomic loci

Abstract

Compositions and methods for soybean genome integration sites are provided. These methods and compositions for producing soybean plants include targeting soybean genomic integration sites with a site-specific nuclease. The use of a site-specific nuclease for creating a double-strand break target site may be done with a zinc finger endonuclease, an engineered endonuclease, a wide range nuclease, a TALEN, and / or a CRISPRCas endonuclease. Soybean genomic integration sites may comprise at least one genomic locus of interest, such as a trait cassette, a transgene, a mutant gene, a native gene, an edited gene, or a site-specific integration (SSI) target site.

Description

[0001] Incorporated by Reference

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 346,112, filed on May 26, 2022, the disclosure of which is hereby incorporated by reference in its entirety.

[0003] References to sequence listings

[0004] The official copy of the sequence listing is submitted electronically via EFS-Web as a sequence listing in XML format, with the file name "9215-WO-PCT.xml", created on May 11, 2023, and with a size of 2.10 Mb, and is submitted simultaneously with this specification. The sequence listing contained in the XML format file is part of this specification and is incorporated herein by reference in its entirety. Background Art

[0005] In the early 1990s, the genome of soybean plants was successfully transformed with transgenics. Over the past three decades, many methods have been developed for transforming the genome of soybean plants, in which transgenics are stably integrated into the genome of soybean plants. This evolution of transformation methods has enabled the successful introduction of transgenics containing agronomic traits into the genome of soybean plants. In the late 1990s, the introduction of insect resistance and herbicide tolerance traits in soybean plants provided producers with new and convenient technological innovations for controlling insects and a wide spectrum of weeds, which is unparalleled in cultivation farming methods. Currently, transgenic plants are commercially available worldwide, and new transgenic products (such as Enlist TM Soybean) offers an improved solution to the growing weed challenge. Without the development and improvement of transformation methods, genetically modified soybean plants would not be possible to be utilized in modern agronomic practices.

[0006] However, current transformation methods rely on the random insertion of transgenes into the genome of soybean plants. There are several disadvantages to relying on the random insertion of genes into the genome. Transgenic events can be randomly integrated within gene transcription sequences, thereby interrupting the expression of endogenous traits and altering the growth and development of soybean plants. In addition, transgenic events may be indiscriminately integrated into genomic positions susceptible to gene silencing, ultimately leading to a reduction or complete inhibition of transgenic expression in the first or subsequent generations of transgenic soybean plants. Finally, the random integration of transgenes in the soybean plant genome requires considerable effort and cost to identify the position of transgenic events and select transgenic events that are designed to be carried out and have no agronomic impact on soybean plants. New assays must be continuously developed to determine the precise position of the integrated transgene for each transgenic event. The randomness of plant transformation methods leads to a "position effect" of integrated transgenes, which hinders the effectiveness and efficiency of transformation methods.

[0007] Targeted genome modification of soybean plants has always been a long-term and difficult to achieve goal for application and basic research. Targeting genes and gene stacking to specific locations in the soybean plant genome will improve the quality of transgenic events, reduce the cost associated with the generation of transgenic events, and provide a novel method for preparing transgenic soybean plant products, such as sequential gene stacking. In general, it may be commercially beneficial to target transgenic to specific genomic sites. In the past few years, significant progress has been made in developing methods and compositions to induce targeted mutagenesis, induce targeted deletion of cell DNA sequences and promote targeted recombination of exogenous donor DNA polynucleotides in predetermined genomic loci, these methods and compositions are used to target and cut genomic DNA by site-specific nucleases (e.g., zinc finger nucleases (ZFNs), meganucleases, transcription activator-like effector nucleases (TALENs) and clustered regularly spaced short palindromic repeats / CRISPR-associated nucleases (CRISPR Cas) with engineered crRNA / tracr RNA). See, e.g., U.S. Patent Publication Nos. 20030232410; 20050208489; 20050026157; 20050064474; and 20060188987, and International Patent Publication No. WO 2007 / 014275, the disclosures of which are incorporated by reference in their entirety for all purposes. U.S. Patent Publication No. 20080182332 describes the use of non-classical zinc finger nucleases (ZFNs) for targeted modification of plant genomes, and U.S. Patent Publication No. 20090205083 describes ZFN-mediated targeted modification of plant EPSP genomic loci. Current methods for targeted insertion of exogenous DNA typically involve co-transformation of plant tissues with a donor DNA polynucleotide containing at least one transgene and a site-specific nuclease (e.g., CRISPR) that is designed to bind to and cleave a specific genomic locus of an actively transcribed coding sequence. This results in stable insertion of the donor DNA polynucleotide into the cleaved genomic locus, leading to targeted gene addition at a specific genomic locus containing actively transcribed coding sequences.

[0008] An alternative method is to target transgenics to preselected genomic integration sites in the soybean plant genome. In recent years, several technologies have been developed and applied to soybean plant cells for targeted delivery of transgenics in the genome of soybean plants. However, little is known about the properties of genomic sites suitable for targeting. Historically, non-essential genes and pathogen (virus) integration sites in the genome have been used as targeted loci. The quantity of such sites in the genome is quite limited, so it is necessary to identify and characterize the targetable optimal genomic loci that can be used for targeting donor polynucleotide sequences. Except being suitable for targeting, it is expected that the genomic integration site is a neutral site that can support transgenic expression and soybean breeding applications. It is necessary to define the composition and method of the standard for the genomic integration site for targeted transgenic integration in the soybean plant genome. Summary of the invention

[0009] Disclosed herein are sequences, constructs, and methods for soybean genomic integration sites. In some aspects, the size of the integration site is greater than 5Kb. In other aspects, the genetic diversity of the integration site is low, wherein the low genetic diversity comprises a haplotype with a frequency greater than 80%. In further aspects, the expected recombination frequency of the integration site is high, wherein the recombination rate is greater than 0.7cM / 1Mb. In other aspects, the integration site is closely adjacent to the telomere, wherein the close proximity is less than 20cM or less than 4.7Mb from the end of the chromosome. In further aspects, the genomic integration site comprises SEQ ID NO:206 or SEQ ID NO:207. In some aspects, the genomic integration site comprises SEQ ID NO:1-109 or SEQ ID NO:264-334. In additional aspects, the genetic size of the low diversity genomic region is less than 10cM. In other aspects, the physical size of the low diversity genomic region is less than 2.6Mb. In one aspect, the integration site comprises euchromatin. In further aspects, the distance of the integration site from heterochromatin is greater than 10cM or 1.05Mb. In other aspects, the haplotype frequency range of the integration site is 80% to 100%. In some aspects, the integration site is greater than 5cM from the QTL. In further aspects, the integration site does not occur in the region containing structural variations that inhibit genome recombination. In other aspects, the integration site comprises a gene expression cassette. For example, the gene expression cassette comprises an insecticide resistance gene, a herbicide tolerance gene, a nitrogen use efficiency gene, a water use efficiency gene, a nutritional quality gene, a DNA binding gene, and a selectable marker gene. In other aspects, the insertion site comprises at least one target site. Therefore, the target site is cut by a site-specific nuclease. For example, the nuclease is selected from the group consisting of zinc finger nuclease, CRISPR nuclease, TALEN, homing endonuclease or a meganuclease. In other aspects, during the insertion of the donor DNA into the insertion site sequence, the insertion site sequence is modified.

[0010] Disclosed herein are soybean plants, soybean plant parts or soybean plant cells comprising recombinant sequences. In some aspects, soybean plants, soybean plant parts or soybean plant cells comprise soybean genomic integration sites. In some aspects, the size of the integration sites of soybean plants, soybean plant parts or soybean plant cells is greater than 5Kb. In other aspects, the genetic diversity of the integration sites of soybean plants, soybean plant parts or soybean plant cells is low, wherein low genetic diversity comprises haplotypes with a frequency greater than 80%. In further aspects, the expected recombination frequency of the integration sites of soybean plants, soybean plant parts or soybean plant cells is high, wherein the recombination rate is greater than 0.7cM / 1Mb. In other aspects, the integration sites of soybean plants, soybean plant parts or soybean plant cells are closely close to telomeres, wherein the close proximity is less than 20cM or less than 4.7Mb from the end of the chromosome. In further aspects, the genomic integration sites of soybean plants, soybean plant parts or soybean plant cells comprise SEQ ID NO:206 or SEQ ID NO:207. In some aspects, the genomic integration site of soybean plants, soybean plant parts or soybean plant cells comprises SEQ ID NO: 1-109 or SEQ ID NO: 264-334. In other aspects, the genetic size of the low diversity genomic region is less than 10cM. In other aspects, the physical size of the low diversity genomic region is less than 2.6Mb. In one aspect, the integration site of soybean plants, soybean plant parts or soybean plant cells comprises euchromatin. In further aspects, the distance between the integration site of soybean plants, soybean plant parts or soybean plant cells and heterochromatin is greater than 10cM or 1.05Mb. In other aspects, the haplotype frequency range of the integration site of soybean plants, soybean plant parts or soybean plant cells is 80% to 100%. In some aspects, the integration site of soybean plants, soybean plant parts or soybean plant cells is greater than 5cM from QTL. In further aspects, the integration site of soybean plants, soybean plant parts or soybean plant cells does not occur in a region containing structural variations that inhibit genomic recombination. In other aspects, the integration site of soybean plants, soybean plant parts or soybean plant cells comprises a gene expression cassette. In some embodiments, the gene expression cassette comprises an insecticide resistance gene, a herbicide tolerance gene, a nitrogen use efficiency gene, a water use efficiency gene, a nutritional quality gene, a DNA binding gene, and a selectable marker gene. In other aspects, the integration site of a soybean plant, a soybean plant part, or a soybean plant cell comprises at least one target site. Therefore, the target site is cut by a site-specific nuclease. For example, the nuclease is selected from the group consisting of zinc finger nuclease, CRISPR nuclease, TALEN, homing endonuclease, or a meganuclease. In other aspects, during the insertion of donor DNA into the insertion site sequence of a soybean plant, a soybean plant part, or a soybean plant cell, the insertion site sequence of a soybean plant, a soybean plant part, or a soybean plant cell is modified.

[0011] Disclosed herein are sequences, constructs and methods for preparing transgenic soybean plant cells comprising donor DNA targeting a genomic integration site. In some aspects, the method for preparing transgenic soybean plant cells includes selecting a target site within a genomic integration site. In other aspects, the method for preparing transgenic soybean plant cells includes introducing a site-specific nuclease into a plant cell, wherein the site-specific nuclease cuts the target site. In further aspects, the method for preparing transgenic soybean plant cells includes introducing donor DNA into a plant cell. In some aspects, the method for preparing transgenic soybean plant cells includes targeting donor DNA to the target site of a genomic integration site, wherein the cutting of the target site promotes the integration of donor DNA into the target site. In other aspects, the method for preparing transgenic soybean plant cells includes selecting a transgenic plant cell comprising a donor DNA targeting the target site of a genomic integration site. In further aspects, the method for preparing transgenic soybean plant cells includes donor DNA, which includes a gene expression cassette. For example, the gene expression cassette includes an insecticide resistance gene, a herbicide tolerance gene, a nitrogen use efficiency gene, a water use efficiency gene, a nutritional quality gene, a DNA binding gene and a selectable marker gene. In a further aspect, the method for preparing transgenic soybean plant cells includes a site-specific nuclease selected from the group consisting of zinc finger nucleases, CRISPR nucleases, TALENs, homing endonucleases or meganucleases. In another aspect, the method for preparing transgenic soybean plant cells includes donor DNA, which is integrated into the target site via a homology-directed repair integration method. In a further aspect, the method for preparing transgenic soybean plant cells includes donor DNA, which is integrated into the target site via a non-homologous end-joining integration method. In some aspects, the method for preparing transgenic soybean plant cells includes a genomic site greater than 1cM in size. In other aspects, the method for preparing transgenic soybean plant cells includes a genomic site with low genetic diversity, wherein the low genetic diversity includes a haplotype with a frequency greater than 80%. In a further aspect, the method for preparing transgenic soybean plant cells includes a genomic site with a high expected recombination frequency, wherein the recombination rate is greater than 0.7cM / 1Mb. In additional aspects, the method for making a transgenic soybean plant cell comprises a genomic site in close proximity to a telomere, wherein close proximity is less than 20 cM or less than 4.7 Mb from the end of a chromosome. In some aspects, the method for making a transgenic soybean plant cell comprises a genomic site of SEQ ID NO: 206 or SEQ ID NO: 207. In other aspects, the method for making a transgenic soybean plant cell comprises a genomic site of SEQ ID NO: 1-109 or SEQ ID NO: 264-334.In a further aspect, the method for preparing a transgenic soybean plant cell comprises a genomic site having a low diversity genomic region less than 10 cM and ranging from 1 cM to 10 cM. ​​In an additional aspect, the method for preparing a transgenic soybean plant cell comprises a genomic site having a low diversity genomic region less than 2.6 Mb in physical size and ranging from 785,899 bp to 954,789 bp. In some aspects, the method for preparing a transgenic soybean plant cell comprises a genomic integration site comprising euchromatin. In other aspects, the method for preparing a transgenic soybean plant cell comprises a genomic site having a distance from heterochromatin greater than 10 cM or 1.05 Mb. In a further aspect, the method for preparing a transgenic soybean plant cell comprises a genomic site wherein the haplotype frequency ranges from 80% to 100%. In an additional aspect, the method for preparing a transgenic soybean plant cell comprises a genomic site wherein the integration site is greater than 5 cM from the QTL. In some aspects, the method for preparing a transgenic soybean plant cell comprises a genomic site that does not occur in a region containing structural variations that inhibit genomic recombination. In a further aspect, the gene expression cassette expresses the gene. In some aspects, the gene is expressed at a concentration of 1 to 100,000 ppm. In a further aspect, the gene is expressed at a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500 0, 7000, 7500, 8000, 8500, 9000, 9500, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, 55000, 60000, 65000, 70000, 75000, 80000, 85000, 90000, 95000 or 10000 ppm of expression. In other aspects, the transgenic soybean plant cells are propagated into transgenic soybean plants. The transgenic soybean plants are used for breeding with another soybean plant to produce offspring plants.In some aspects, the following method is used for breeding: identifying transgenic soybean plant cells containing donor DNA targeted to a genomic integration site; selecting soybean plants having the donor DNA at the genomic integration site; crossing the selected soybean plants to produce offspring; and selecting offspring having a desired trait associated with the genomic integration site.

[0012] Disclosed herein are sequences, constructs, and methods for identifying soybean genomic integration sites, which are used for site-specific nuclease-mediated integration of donor polynucleotides. In some aspects, the method for identifying soybean genomic integration sites includes obtaining a soybean genomic DNA sample from at least one soybean variety, which is used for site-specific nuclease-mediated integration of donor polynucleotides. In other aspects, the method for identifying soybean genomic integration sites includes genotyping the soybean genomic DNA sample, which is used for site-specific nuclease-mediated integration of donor polynucleotides. In further aspects, the method for identifying soybean genomic integration sites includes identifying the haplotype of the soybean genomic DNA sample, which is used for site-specific nuclease-mediated integration of donor polynucleotides. In other aspects, the method for identifying soybean genomic integration sites includes assessing the genetic diversity of haplotypes to identify haplotypes with low genetic diversity, which is used for site-specific nuclease-mediated integration of donor polynucleotides. In a further aspect, the method of identifying a soybean genomic integration site comprises selecting a haplotype with low genetic diversity, the soybean genomic integration site is used for site-specific nuclease-mediated integration of a donor polynucleotide, the haplotype comprising the following characteristics: 1. Size greater than 1 cM; 2. Low genetic diversity, wherein the low genetic diversity comprises haplotypes with a frequency greater than 80%; 3. High expected recombination frequency, wherein these recombination frequencies are greater than 0.7 cM / 1 Mb; and, 4. Close proximity to telomeres, wherein the close proximity is less than 20 cM or less than 4.7 Mb from the end of the chromosome. In an additional aspect, the method of identifying a soybean genomic integration site comprises targeting the soybean genomic integration site with a site-specific nuclease to integrate the donor polynucleotide within the soybean genomic integration site, the soybean genomic integration site is used for site-specific nuclease-mediated integration of the donor polynucleotide. In other aspects, the method of identifying a soybean genomic integration site comprises a gene expression cassette, the soybean genomic integration site is used for site-specific nuclease-mediated integration of a donor polynucleotide. In aspects of the method of identifying a soybean genomic integration site, the gene expression cassette comprises an insecticide resistance gene, an herbicide tolerance gene, a nitrogen use efficiency gene, a water use efficiency gene, a nutritional quality gene, a DNA binding gene and / or a selectable marker gene, and the soybean genomic integration site is used for site-specific nuclease-mediated integration. In further aspects of the method of identifying a soybean genomic integration site, the site-specific nuclease is selected from the group consisting of a zinc finger nuclease, a CRISPR nuclease, a TALEN, a homing endonuclease or a meganuclease, and the soybean genomic integration site is used for site-specific nuclease-mediated integration.In further aspects of the method for identifying a soybean genomic integration site, the donor DNA is integrated into the target site via a homology directed repair integration method, and the soybean genomic integration site is used for site-specific nuclease-mediated integration. In some aspects of the method for identifying a soybean genomic integration site, the donor DNA is integrated into the target site via a non-homologous end joining integration method, and the soybean genomic integration site is used for site-specific nuclease-mediated integration. In other aspects of the method for identifying a soybean genomic integration site, the genomic integration site comprises SEQ ID NO: 206 or SEQ ID NO: 207, and the soybean genomic integration site is used for site-specific nuclease-mediated integration. In further aspects of the method for identifying a soybean genomic integration site, the genomic integration site comprises SEQ ID NO: 1-109 or SEQ ID NO: 264-334, and the soybean genomic integration site is used for site-specific nuclease-mediated integration. In further aspects of the method for identifying a soybean genomic integration site, the genetic size of the low diversity genomic region is less than 10 cM and ranges from 1 cM to 10 cM, and the soybean genomic integration site is used for site-specific nuclease-mediated integration. In other aspects of the method for identifying a soybean genomic integration site, the physical size of the low-diversity genomic region is less than 2.6 Mb and ranges from 785,899 bp to 954,789 bp, and the soybean genomic integration site is used for site-specific nuclease-mediated integration. In some aspects of the method for identifying a soybean genomic integration site, the integration site comprises euchromatin, and the soybean genomic integration site is used for site-specific nuclease-mediated integration. In other aspects of the method for identifying a soybean genomic integration site, the distance between the integration site and heterochromatin is greater than 10 cM or 1.05 Mb, and the soybean genomic integration site is used for site-specific nuclease-mediated integration. In further aspects of the method for identifying a soybean genomic integration site, the haplotype frequency ranges from 80% to 100%, and the soybean genomic integration site is used for site-specific nuclease-mediated integration. In some aspects of the method for identifying a soybean genomic integration site, the integration site is greater than 5 cM from the QTL, and the soybean genomic integration site is used for site-specific nuclease-mediated integration. In another aspect of the method for identifying a soybean genomic integration site, the integration site does not occur in a region containing structural variations that inhibit genomic recombination, and the soybean genomic integration site is used for site-specific nuclease-mediated integration. In a further aspect of the method for identifying a soybean genomic integration site, genotyping of a soybean genomic DNA sample is accomplished by sequencing or analyzing SNP markers, and the soybean genomic integration site is used for site-specific nuclease-mediated integration.

[0013] Disclosed herein are soybean plants, cells, plant parts, and seeds comprising a transgenic expression cassette inserted into a chromosomal locus in the soybean genome, wherein the chromosomal locus is located at SEQ ID NO: 1-109, 206, 207, or 264-334 on chromosome 1 or chromosome 2, and wherein the transgene is expressed in the soybean plant.

[0014] Sequence Listing

[0015] As defined in 37 CFR § 1.822, the nucleic acid sequences listed in the attached sequence listing are shown using standard letter abbreviations for nucleotide bases. Only one strand of each nucleic acid sequence is shown, but any reference to the displayed strand is understood to include the complementary strand and the reverse complementary strand. Because the complementary sequence and the reverse complementary sequence of a primary nucleic acid sequence are necessarily disclosed by the primary sequence, any reference to a nucleic acid sequence includes the complementary sequence and the reverse complementary sequence of the nucleic acid sequence, unless otherwise explicitly indicated (or otherwise clearly apparent from the context in which the sequence appears). DETAILED DESCRIPTION

[0016] definition

[0017] Unless otherwise defined, all technical terms and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present disclosure relates. In the event of a conflict, the present application including the definitions shall prevail. Unless the context otherwise requires, singular terms shall include plural terms, and plural terms shall include the singular. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes, as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference, unless only a specific portion of a patent or patent publication is indicated to be incorporated by reference.

[0018] To further clarify the present disclosure, the following terms, abbreviations, and definitions are provided.

[0019] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contain," or "containing," or any other variation thereof, are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus comprising a list of elements is not necessarily limited to those elements, but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. In addition, unless expressly stated to the contrary, "or" refers to an inclusive "or" and not to an exclusive "or." For example, a condition A or B is satisfied by any of the following: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), and both A and B are true (or present).

[0020] Likewise, the indefinite article "a / an" before the elements or components of the embodiments of the present disclosure is intended to be non-restrictive with respect to the number of examples (i.e., the occurrence of the element or component). Therefore, "a" or "an" should be understood to include one / a kind or at least one / at least one, and the singular word form of an element or component also includes the plural, unless the number obviously means the singular.

[0021] As used herein, the term "invention" or "present invention" is a non-limiting term and is not intended to refer to any single embodiment of a particular invention but encompasses all possible embodiments as disclosed in the present application.

[0022] As used herein, the term "isolated" means removed from its natural environment, or from other compounds that were present when the compound was first formed. The term "isolated" encompasses material isolated from a natural source as well as material recovered following preparation by recombinant expression in a host cell (e.g., nucleic acids and proteins), or chemically synthesized compounds such as nucleic acid molecules, proteins, and peptides.

[0023] As used herein, the term "purified" refers to the isolation of a molecule or compound in a form that is substantially free of contaminants (usually associated with the molecule or compound in its native or natural environment), or highly enriched in concentration relative to other compounds present when the compound was originally formed, and means that the purity has been increased due to separation from other components of the original composition. The term "purified nucleic acid" is used herein to describe a nucleic acid sequence that has been separated, produced separately, or purified from other biological compounds, including but not limited to polypeptides, lipids, and carbohydrates, while affecting chemical or functional changes to the components (e.g., nucleic acids can be purified from chromosomes by removing protein contaminants and breaking the chemical bonds that connect the nucleic acid to the rest of the DNA in the chromosome).

[0024] As used herein, the term "synthetic" refers to polynucleotide (i.e., DNA or RNA) molecules produced via chemical synthesis as an in vitro process. For example, TM Synthetic DNA is produced during the reaction in the tube so that the synthetic DNA is enzymatically produced by the natural chain of DNA or RNA. Other laboratory methods can be used to synthesize polynucleotide sequences. Oligonucleotides can be chemically synthesized on an oligonucleotide synthesizer via solid phase synthesis using phosphoramidites. Synthetic oligonucleotides can be annealed to each other as complexes, thereby producing "synthetic" polynucleotides. Other methods for chemically synthesizing polynucleotides are known in the art and can be easily implemented for use in the present disclosure.

[0025] As used herein, the term "about" means greater than or less than ten percent of the stated value or range of values, but is not intended to specify any value or range of values ​​only for this broader definition. Each value or range of values ​​following the term "about" is also intended to cover embodiments of the stated absolute value or range of values.

[0026] For purposes of this disclosure, a "gene" includes a DNA region encoding a gene product (see below), as well as all DNA regions that regulate the production of that gene product, whether or not such regulatory sequences are adjacent to coding and / or transcribed sequences. Thus, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translation regulatory sequences (such as ribosome binding sites and internal ribosome entry sites), enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, introns, and locus control regions.

[0027] As used herein, the term "native" or "natural" defines a naturally occurring condition. A "native DNA sequence" is a DNA sequence found in nature that is produced by natural means or traditional breeding techniques, but not by genetic engineering (e.g., using molecular biology / transformation techniques).

[0028] As used herein, "transgenic" is defined as a nucleic acid sequence encoding a gene product, including, for example, but not limited to, mRNA. In one embodiment, the transgenic / heterologous coding sequence is an exogenous nucleic acid, wherein the transgenic / heterologous coding sequence is introduced into a host cell (or its progeny) by genetic engineering, wherein the transgenic / heterologous coding sequence is generally not found. In one example, the transgenic / heterologous coding sequence encodes an industrially or pharmaceutically useful compound, or a gene encoding a desired agronomic trait (e.g., a herbicide resistance gene). In another example, the transgenic / heterologous coding sequence is an antisense nucleic acid sequence, wherein the expression of the antisense nucleic acid sequence inhibits the expression of a target nucleic acid sequence. In one embodiment, the transgenic / heterologous coding sequence is an endogenous nucleic acid, wherein it is desired to be an additional genomic copy of the endogenous nucleic acid, or a nucleic acid in an antisense direction relative to the sequence of the target nucleic acid in the host organism.

[0029] As used herein, the term "non-GmPSID2 transgene" or "non-GmPSID2 gene" is any transgenic / heterologous coding sequence that has less than 80% sequence identity to the GmPSID2 gene coding sequence.

[0030] As used herein, "heterologous DNA coding sequence" means any coding sequence other than the coding sequence of the naturally encoding GmPSID2 gene, or any homolog of the expressed GmPSID2 protein. In the context of the present invention, the term "heterologous" is used for any combination of nucleic acid sequences that are not normally found in close association in nature.

[0031] As defined herein, "gene product" is any product produced by a gene. For example, a gene product can be a direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, interfering RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by mRNA translation. Gene products also include RNA modified by methods such as capping, polyadenylation, methylation and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation and glycosylation. Gene expression may be affected by external signals (e.g., cells, tissues or organisms are exposed to agents that increase or decrease gene expression). Gene expression can also be regulated anywhere in the pathway from DNA to RNA to protein. Regulation of gene expression, for example, by transcription, translation, RNA transport and processing, control of the degradation of intermediate molecules (e.g., mRNA) or by activation, inactivation, compartmentalization or degradation or by a combination thereof after manufacturing a specific protein molecule. Gene expression can be measured at the RNA level or at the protein level by any method known in the art, including but not limited to Northern blot, RT-PCR, Western blot, or one or more in vitro, in situ, or in vivo protein activity assays.

[0032] As used herein, the term "gene expression" refers to a process by which the coded information of a nucleic acid transcription unit (including, for example, genomic DNA) is converted into an operable, inoperable, or structural part of a cell, often including the synthesis of a protein. Gene expression can be affected by external signals; for example, cells, tissues, or organisms are exposed to agents that increase or decrease gene expression. Gene expression can also be regulated anywhere in the pathway from DNA to RNA to protein. Regulation of gene expression occurs, for example, by controlling transcription, translation, RNA transport and processing, degradation of intermediate molecules (such as mRNA), or by activating, inactivating, compartmentalizing, or degrading a specific protein molecule after it is manufactured, or by a combination thereof. Gene expression can be measured at the RNA level or protein level by any method known in the art (including, but not limited to, Northern blot, RT-PCR, Western blot, or one or more in vitro, in situ, or in vivo protein activity assays).

[0033] As used herein, "homology-based gene silencing" (HBGS) is a general term that includes transcriptional gene silencing and post-transcriptional gene silencing. Transcriptional repression (transcriptional gene silencing; TGS) or mRNA degradation (post-transcriptional gene silencing; PTGS) can result in the silencing of an unlinked silencing locus to a target locus due to the production of double-stranded RNA (dsRNA) corresponding to a promoter or transcriptional sequence. The involvement of different cellular components in each method suggests that dsRNA-induced TGS and PTGS may be the result of the diversification of an ancient common mechanism. However, it is difficult to make a strict comparison between TGS and PTGS because it generally relies on the analysis of different silencing loci. In some cases, a single transgenic locus can trigger TGS and PTGS due to the production of dsRNA corresponding to the promoters and transcriptional sequences of different target genes. Mourrain et al. (2007) Planta 225:365-79. siRNA may be the actual molecule that triggers TGS and PTGS on homologous sequences: these siRNAs would initiate the silencing and methylation of cis- and trans-homologous sequences in this model by spreading the methylation of the transgenic sequence into the endogenous promoter.

[0034] As used herein, the term "nucleic acid molecule" (or "nucleic acid" or "polynucleotide") can refer to a polymeric form of nucleotides that can include RNA, cDNA, genomic DNA, and both sense and antisense strands of the above synthetic forms and hybrid polymers. Nucleotides can refer to ribonucleotides, deoxyribonucleotides, or modified forms of either type of nucleotide. As used herein, "nucleic acid molecule" is synonymous with "nucleic acid" and "polynucleotide". Unless otherwise specified, the length of a nucleic acid molecule is typically at least 10 bases. The term can refer to RNA or DNA molecules of indeterminate length. The term includes single-stranded and double-stranded forms of DNA. Nucleic acid molecules can include one or both of naturally occurring nucleotides and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide bonds.

[0035] As will be readily understood by those skilled in the art, nucleic acid molecules can be chemically or biochemically modified or can contain non-natural or derivatized nucleobases. Such modifications include, for example, labeling, methylation, substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications (e.g., uncharged linkages: e.g., methylphosphonates, phosphotriesters, phosphoramidites, carbamates, etc.; charged linkages: e.g., phosphorothioates, dithiophosphates, etc.; pendant moieties: e.g., peptides; intercalators: e.g., acridine, psoralen, etc.; chelators; alkylating agents; and modified linkages: e.g., α-anomeric nucleic acids, etc.). The term "nucleic acid molecule" also includes any topological conformation, including single-stranded, double-stranded, partially double-stranded, triple-stranded, hairpin, circular, and padlock configurations.

[0036] Transcription proceeds in a 5' to 3' fashion along the DNA chain. This means that RNA is made by the sequential addition of ribonucleotide 5'-triphosphates (with the necessary elimination of pyrophosphate) to the 3' end of the growing chain. In a linear or circular nucleic acid molecule, discrete elements (e.g., specific nucleotide sequences) may be said to be located "upstream" or "5'" relative to another element if they bind or will bind to the same nucleic acid in the 5' direction from that element. Similarly, a discrete element may be "downstream" or "3'" relative to another element if it is or will bind to the same nucleic acid in the 3' direction from that element.

[0037] As used herein, a base "position" refers to the position of a given base or nucleotide residue within a specified nucleic acid. A specified nucleic acid can be defined by alignment with a reference nucleic acid (see below).

[0038] Hybridization involves the binding of two polynucleotide chains via hydrogen bonds. Oligonucleotides and their analogs are hybridized by hydrogen bonding between complementary bases, including Watson-Crick, Hoogsteen or reverse Hoogsteen hydrogen bonding. Typically, nucleic acid molecules are composed of nitrogenous bases, which are pyrimidines (cytosine (C), uracil (U) and thymine (T)) or purines (adenine (A) and guanine (G)). These nitrogenous bases form hydrogen bonds between pyrimidines and purines, and the bonding of pyrimidines to purines is called "base pairing". More specifically, A will hydrogen bond with T or U, and G will bind to C. "Complementarity" refers to the base pairing that occurs between two different nucleic acid sequences or two different regions of the same nucleic acid sequence.

[0039] "Specifically hybridizable" and "specifically complementary" are terms that represent a sufficient degree of complementarity so that stable and specific binding occurs between an oligonucleotide and a DNA or RNA target. An oligonucleotide need not be 100% complementary to its target sequence to specifically hybridize. When the binding of an oligonucleotide to a target DNA or RNA molecule interferes with the normal function of the target DNA or RNA, the oligonucleotide can specifically hybridize and has a sufficient degree of complementarity to avoid nonspecific binding of the oligonucleotide to non-target sequences under conditions where specific binding is desired (e.g., under physiological conditions in the case of an in vivo assay or system). This binding is referred to as specific hybridization.

[0040] The hybridization conditions that lead to a specific degree of stringency will vary according to the composition and length of the selected hybridization method and the hybridization nucleic acid sequence. Typically, the ionic strength (especially Na+ and / or Mg2+ concentration) of the hybridization temperature and hybridization buffer will contribute to the stringency of the hybridization, although the wash time will also affect the stringency. Calculations about obtaining the hybridization conditions required for a specific stringency are discussed in Sambrook et al. (editor), Molecular Cloning: A Laboratory Manual, 2nd edition, Vol. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, Chapters 9 and 11.

[0041] As used herein, "stringent conditions" encompass conditions under which hybridization occurs only when the mismatch between the hybridizing molecule and the DNA target is less than 50%. "Stringent conditions" include further stringent specific levels. Thus, as used herein, "medium stringency" conditions refer to conditions under which molecules with a sequence mismatch rate of more than 50% will not hybridize; "high stringency" conditions refer to conditions under which sequences with a mismatch rate of more than 20% will not hybridize; and "very high stringency" conditions refer to conditions under which sequences with a mismatch rate of more than 10% will not hybridize.

[0042] In a specific embodiment, stringent conditions may include hybridization at 65°C, followed by a wash in 0.1 x SSC / 0.1% SDS at 65°C for 40 minutes.

[0043] The following are representative, non-limiting hybridization conditions:

[0044] Very high stringency: hybridization in 5x SSC buffer at 65°C for 16 hours; wash twice in 2x SSC buffer at room temperature for 15 minutes each; and wash twice in 0.5x SSC buffer at 65°C for 20 minutes each.

[0045] High stringency: hybridization in 5x-6x SSC buffer at 65°C-70°C for 16-20 hours; wash twice in 2xSSC buffer at room temperature for 5-20 minutes each; and wash twice in 1x SSC buffer at 55°C-70°C for 30 minutes each.

[0046] Medium stringency: hybridize in 6x SSC buffer at room temperature to 55°C for 16-20 hours; wash at least twice in 2x-3x SSC buffer at room temperature to 55°C for 20-30 minutes each.

[0047] In a specific embodiment, the nucleic acid molecules of specific hybridization can keep binding under very high stringency hybridization conditions. In these and further embodiments, the nucleic acid molecules of specific hybridization can keep binding under high stringency hybridization conditions. In these and further embodiments, the nucleic acid molecules of specific hybridization can keep binding under medium stringency hybridization conditions.

[0048] As used herein, the term "oligonucleotide" refers to a short nucleic acid polymer. Oligonucleotides can be formed by cutting longer nucleic acid segments or by polymerizing single nucleotide precursors. Automated synthesizers allow the synthesis of oligonucleotides up to hundreds of base pairs in length. Because oligonucleotides can be combined with complementary nucleotide sequences, they can be used as probes for detecting DNA or RNA. The oligonucleotides (oligodeoxyribonucleotides) composed of DNA can be used for PCR (technology for amplifying small DNA sequences). In PCR, oligonucleotides are typically referred to as "primers", which allow DNA polymerase to extend oligonucleotides and replicate complementary chains.

[0049] The terms "percent sequence identity" or "percent identity" or "identity" are used interchangeably and refer to a sequence comparison based on identical matches between corresponding identical positions in the sequences being compared between two or more amino acid or nucleotide sequences. Percent identity refers to the degree to which two optimally aligned polynucleotide or peptide sequences are invariant over the comparison window of components, such as nucleotides or amino acids. Hybridization experiments and mathematical algorithms known in the art can be used to determine percent identity. There are many mathematical algorithms as sequence alignment computer programs known in the art that calculate percent identity. These programs can be classified as global sequence alignment programs or local sequence alignment programs.

[0050] Global sequence alignment programs calculate the percent identity of two sequences by comparing the sequences end-to-end to find exact matches, dividing the number of exact matches by the length of the shorter sequence and then multiplying by 100. Basically, the percentage of nucleotides that are identical when the linear polynucleotide sequence of a reference ("query") polynucleotide molecule is compared to a test ("subject") polynucleotide molecule when the two sequences are optimally aligned (with appropriate nucleotide insertions, deletions or gaps).

[0051] Local sequence alignment programs are similar in calculation, but only compare aligned fragments of sequences, rather than using end-to-end analysis. Local sequence alignment programs such as BLAST can be used to compare specific regions of two sequences. The BLAST comparison of two sequences will produce an E value or expected value, which represents the number of different alignments with a score equal to or better than the original alignment score (S), which may occur accidentally in a database search. The lower the E value, the more significant the match. Because database size is an element in the E value calculation, for any given query / entry match, the E value obtained by BLASTing for public databases (such as GENBANK) usually increases over time. When setting the confidence standard for polypeptide function prediction, a "high" BLAST match is considered herein to have an E value of less than 1E-30 for the highest BLAST hit; a medium BLASTX E value of 1E-30 to 1E-8; and a low BLASTX E value of greater than 1E-8. A combination of E value, percent identity, query coverage, and hit coverage is used to determine the protein function assignment in the present invention. Query coverage refers to the percentage of the query sequence represented by BLAST alignment. Hit coverage refers to the percentage of database entries represented by BLAST alignments. In one embodiment of the present invention, the function of the query polypeptide is inferred from the function of the protein homolog, where (1) hit_p<1e-30 or % identity>35% and query_coverage>50% and hit_coverage>50%, or (2) hit_p<1e-8 and query_coverage>70% and hit_coverage>70%. The following abbreviations are generated during BLAST analysis of sequences.

[0052] SEQ_NUM provides the SEQ ID NO of the listed recombinant polynucleotide sequence.

[0053] CONTIG_ID provides an arbitrary sequence name taken from the name of the clone from which the cDNA sequence was obtained.

[0054] PROTEIN_NUM provides the SEQ ID NO of the recombinant polypeptide sequence

[0055] NCBI_GI provides the GenBank ID number of the top BLAST hit for a sequence. The top BLAST hit is indicated by the National Center for Biotechnology Information GenBank identifier number.

[0056] NCBI_GI_DESCRIPTION refers to the description of the GenBank top BLAST hit for the sequence.

[0057] E_VALUE provides the expected value of the highest BLAST match.

[0058] MATCH_LENGTH The length of the sequence that provides the highest BLAST match.

[0059] TOP_HIT_PCT_IDENT refers to the percentage of identically matched nucleotides (or residues) present along the length of that portion of the aligned sequence in the top BLAST hit.

[0060] CAT_TYPE indicates the classification scheme used to classify the sequence. GO_BP = Gene Ontology Consortium - Biological Process; GO_CC = Gene Ontology Consortium - Cellular Component; GO_MF = Gene Ontology Consortium - Molecular Function; KEGG = KEGG Functional Hierarchy (KEGG = Kyoto Encyclopedia of Genes and Genomes); EC = Enzyme Classification in ENZYME Database Version 25.0; POI = Pathway of Interest.

[0061] CAT_DESC Provides the classification scheme subcategory to which the query sequence is assigned.

[0062] PRODUCT_CAT_DESC provides the FunCAT annotation category to which the query sequence is assigned.

[0063] PRODUCT_HIT_DESC provides a description of the BLAST hit that resulted in the assignment of the sequence to the functional category provided in the cat_desc column.

[0064] HIT_E provides E-values ​​for BLAST hits in the hit_desc column.

[0065] PCT_IDENT refers to the percentage of identically matched nucleotides (or residues) present along the length of that portion of the sequence aligned in the BLAST match provided in hit_desc.

[0066] QRY_RANGE Lists the range of query sequences that align to the hits.

[0067] HIT_RANGE lists the range of hit sequences that align to the query.

[0068] QRY_CVRG provides the percentage of the query sequence length that matches the hit (NCBI) sequence in the BLAST match (%qrycvrg = (match length / total query length) x 100).

[0069] HIT_CVRG provides the percentage of hit sequence lengths that match the query sequence among the matches generated using BLAST (%hitcvrg = (match length / total length of hits) x 100).

[0070] Methods of sequence alignment for comparison are well known in the art. Various programs and alignment algorithms are described. In embodiments, the present disclosure relates to calculating the percent identity between two polynucleotide or amino acid sequences using the AlignX alignment program of the Vector NTI suite (Invitrogen, Carlsbad, CA). The AlignX alignment program is a global sequence alignment program for polynucleotides or proteins. In embodiments, the present disclosure relates to calculating the percent identity between two polynucleotide or amino acid sequences using the MegAlign program of the LASERGENE bioinformatics computing suite (MegAlign TM ( 1993-2016). DNASTAR Inc. Madison, Wisconsin). The MegAlign program is a global sequence alignment program for polynucleotides or proteins. In embodiments, the present disclosure relates to calculating the percent identity between two polynucleotide or amino acid sequences using the Clustal suite of alignment programs, including but not limited to ClustalW and ClustalV (Higgins and Sharp (1988) Gene [Gene]. Dec 15; 73(1):237-44; Higgins and Sharp (1989) CABIOS [Computer Applications Bioscience] 5:151-3; Higgins et al. (1992) Comput. Appl. Biosci. [Computer Applications Bioscience] 8:189-91). In embodiments, the present disclosure relates to calculating the percent identity between two polynucleotide or amino acid sequences using the GCG suite of programs (Wisconsin Software Package Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin). In embodiments, the present disclosure relates to calculating the percent identity between two polynucleotide or amino acid sequences using the BLAST suite of alignment programs (e.g., including but not limited to BLASTP, BLASTN, BLASTX, etc.) (Altschul et al. (1990) J. Mol. Biol. [Journal of Molecular Biology] 215: 403-10). In embodiments, the present disclosure relates to calculating the percent identity between two polynucleotide or amino acid sequences using the FASTA suite of alignment programs (including but not limited to FASTA, TFASTX, TFASTY, SSEARCH, LALIGN, etc.) (Pearson (1994) Comput. Methods Genome Res. [Computational Methods in Genome Research] [Proc. Int. Symp.], Conference Date 1992 (Suhai and Sandor, eds.), Plenum: New York City, New York, pp. 111-20). In an embodiment, the present disclosure relates to the use of the T-Coffee alignment program to calculate the percent identity between two polynucleotide or amino acid sequences (Notredame, et al. (2000) J. Mol. Biol. 302, 205-17).In embodiments, the present disclosure relates to calculating the percent identity between two polynucleotide or amino acid sequences using the DIALIGN suite of alignment programs (including but not limited to DIALIGN, CHAOS, DIALIGN-TX, DIALIGN-T, etc.) (Al Ait, et al. (2013) DIALIGN at GOBICS Nuc. Acids Research 41, W3-W7). In embodiments, the present disclosure relates to calculating the percent identity between two polynucleotide or amino acid sequences using the MUSCLE suite of alignment programs (Edgar (2004) Nucleic Acids Res. 32(5): 1792-1797). In embodiments, the present disclosure relates to calculating the percent identity between two polynucleotide or amino acid sequences using the MAFFT alignment program (Katoh et al. (2002) Nucleic Acids Research 30(14): 3059-3066). In embodiments, the present disclosure relates to using the Genoogle program to calculate the percent identity between two polynucleotides or amino acid sequences (Albrecht, Felipe. arXiv130702987v1 [cs.DC] July 10, 2015). In embodiments, the present disclosure relates to using the HMMER suite of programs to calculate the percent identity between two polynucleotides or amino acid sequences (Eddy. (1998) Bioinformatics [Bioinformatics], 14: 755-63). In embodiments, the present disclosure relates to using the PLAST suite of alignment programs (including but not limited to TPLASTN, PLASTP, KLAST, and PLASTX) to calculate the percent identity between two polynucleotides or amino acid sequences (Nguyen and Lavenier. (2009) BMC Bioinformatics [BMC Bioinformatics], 10: 329). In embodiments, the present disclosure relates to using the USEARCH alignment program to calculate the percent identity between two polynucleotide or amino acid sequences (Edgar (2010) Bioinformatics 26(19), 2460-61). In embodiments, the present disclosure relates to using the SAM suite of alignment programs to calculate the percent identity between two polynucleotide or amino acid sequences (Hughey and Krogh (January 1995) Technical Report UCSCOCRL-95-7, University of California, Santa Cruz).In an embodiment, the present disclosure relates to using an IDF retriever to calculate the percent identity between two polynucleotide or amino acid sequences (O'Kane, KC, The Effect of Inverse Document Frequency Weights on Indexed Sequence Retrieval, Online Journal of Bioinformatics, Vol. 6 (2) 162-173, 2005). In an embodiment, the present disclosure relates to using the Parasail alignment program to calculate the percent identity between two polynucleotide or amino acid sequences (Daily, Jeff. Parasail: SIMD C library for global, semi-global, and local pairwise sequence alignments. BMC Bioinformatics. 17: 18. February 10, 2016). In an embodiment, the present disclosure relates to using the ScalaBLAST alignment program to calculate the percent identity between two polynucleotide or amino acid sequences (Oehmen C, Nieplocha J. "ScalaBLAST: A scalable implementation of BLAST for high-performance data-intensive bioinformatics analysis." IEEE Transactions on Parallel & Distributed Systems 17(8):740-749 August 2006). In an embodiment, the present disclosure relates to using the SWIPE alignment program to calculate the percent identity between two polynucleotide or amino acid sequences (Rognes, T. Faster Smilth-Waterman database searches with inter-sequence SIMD parallelization. BMC Bioiinformatics 12, 221 (2011)).In an embodiment, the present disclosure relates to the use of the ACANA alignment program to calculate the percent identity between two polynucleotide or amino acid sequences (Weichun Huang, David M. Umbach and Leping Li, Accurate anchoring alignment of divergent sequences. Bioinformatics 22:29-34, January 1, 2006). In an embodiment, the present disclosure relates to the use of the DOTLET alignment program to calculate the percent identity between two polynucleotide or amino acid sequences (Junier, T. and Pagni, M. DOTLET: diagonal plots in a web browser. Bioinformatics 16(2):178-9 February 2000). In an embodiment, the present disclosure relates to using the G-PAS alignment program to calculate the percent identity between two polynucleotide or amino acid sequences (Frohmberg, W. et al. G-PAS2.0—an improved version of protein alignment tool with an efficient backtracking routine on multiple GPUs. Bulletin of the Polish Academy of Sciences Technical Sciences, Vol. 60, 491 November 2012). In an embodiment, the present disclosure relates to using the GapMis alignment program to calculate the percent identity between two polynucleotide or amino acid sequences (Flouri, T. et al., Gap Mis: A tool for pairwise sequence alignment with a single gap. Recent Pat DNA Gene Seq. 7(2): 84-95 August 2013).In an embodiment, the present disclosure relates to calculating the percent identity between two polynucleotide or amino acid sequences using the EMBOSS suite of alignment programs, including but not limited to Matcher, Needle, Streter, Water, Wordmatch, etc. (Rice, P., Longden, I. and Bleasby, A. EMBOSS: The European Molecular Biology Open Software Suite. [EMBOSS: European Molecular Biology Open Software Suite] Trends in Genetics [Genetics Trends] 16 (6) 276-77 (2000)). In an embodiment, the present disclosure relates to calculating the percent identity between two polynucleotide or amino acid sequences using the Ngila alignment program (Cartwright, R. Ngila: global pairwise alignments with logarithmic and affinegap costs. [Ngila: global pairwise alignments with logarithmic and affine gap costs] Bioinformatics. [Bioinformatics] 23 (11): 1427-28. June 1, 2007). In an embodiment, the present disclosure relates to the use of the probA (also known as propA) alignment program to calculate the percent identity between two polynucleotide or amino acid sequences (Mückstein, U., Hofacker, IL and Stadler, PF. Stochastic pairwise alignments. Bioinformatics 18 Suppl 2: S153-60. 2002). In an embodiment, the present disclosure relates to the use of the SEQALN suite of alignment programs to calculate the percent identity between two polynucleotide or amino acid sequences (Hardy, P. and Waterman, M. The Sequence Alignment Software Library at USC. 1997). In an embodiment, the present disclosure relates to calculating the percent identity between two polynucleotide or amino acid sequences using the SIM suite of alignment programs (including but not limited to GAP, NAP, LAP, etc.) (Huang, X and Miller, WA Time-Efficient, Linear-Space Local Similarity Algorithm. Advances in Applied Mathematics, Vol. 12 (1991) 337-57).In an embodiment, the present disclosure relates to using the UGENE alignment program to calculate the percent identity between two polynucleotides or amino acid sequences (Okonechnikov, K., Golosova, O. and Fursov, M. Unipro UGENE: a unified bioinformatics toolkit. [UniproUGENE: unified bioinformatics toolkit] Bioinformatics. [Bioinformatics] 2012 28: 1166-67). In an embodiment, the present disclosure relates to using the BAli-Phy alignment program to calculate the percent identity between two polynucleotides or amino acid sequences (Suchard, MA and Redelings, BD. BAli-Phy: simultaneous Bayesian inference of alignment and phylogeny [BAli-Phy: Bayesian simultaneous inference of alignment and phylogeny]. Bioinformatics. [Bioinformatics] 22: 2047-48. 2006). In an embodiment, the present disclosure relates to using a Base-By-Base alignment program to calculate the percentage identity between two polynucleotides or amino acid sequences (Brodie, R., et al. Base-By-Base: Single nucleotide-level analysis of whole viral genome alignments, BMC Bioinformatics, 5, 96, 2004). In an embodiment, the present disclosure relates to using a DECIPHER alignment program to calculate the percentage identity between two polynucleotides or amino acid sequences (ES Wright (2015) "DECIPHER: harnessing local sequence context to improve protein multiple sequence alignment. DECIPHER: using local sequence context to improve protein multiple sequence alignment" BMC Bioinformatics, doi: 10.1186 / s12859-015-0749-z.).In an embodiment, the present disclosure relates to using the FSA alignment program to calculate the percent identity between two polynucleotide or amino acid sequences (Bradley, RK et al. (2009) Fast Statistical Alignment. PLoS Computational Biology. 5: e1000392). In an embodiment, the present disclosure relates to using the Geneious alignment program to calculate the percent identity between two polynucleotide or amino acid sequences (Kearse, M., et al. (2012). Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics, 28(12), 1647-49). In an embodiment, the present disclosure relates to using the Kalign alignment program to calculate the percent identity between two polynucleotide or amino acid sequences (Lassmann, T. and Sonnhammer, E. Kalign—an accurate and fast multiple sequence alignment algorithm. BMC Bioinformatics 2005 6:298). In an embodiment, the present disclosure relates to using the MAVID alignment program to calculate the percent identity between two polynucleotide or amino acid sequences (Bray, N. and Pachter, L. MAVID: Constrained Ancestral Alignment of Multiple Sequences. Genome Res. 2004 April; 14(4):693-99). In embodiments, the disclosure relates to calculating the percent identity between two polynucleotide or amino acid sequences using the MSA alignment program (Lipman, DJ, et al. A tool for multiple sequence alignment. Proc. Nat'l Acad. Sci. USA. 1989; 86:4412-15).In embodiments, the present disclosure relates to the use of the MultAlin alignment program to calculate the percent identity between two polynucleotide or amino acid sequences (Corpet, F., Multiple sequence alignment with hierarchial clustering. Nucl. Acids Res., 1988, 16(22), 10881-90). In embodiments, the present disclosure relates to the use of the LAGAN or MLAGAN alignment program to calculate the percent identity between two polynucleotide or amino acid sequences (Brudno, et al. LAGAN and Multi-LAGAN: efficient tools for large-scale multiple alignment of genomic DNA. Genome Research 2003 April; 13(4): 721-31). In an embodiment, the present disclosure relates to using the Opal alignment program to calculate the percent identity between two polynucleotide or amino acid sequences (Wheeler, TJ and Kececiouglu, JD Multiple alignment by aligning alignments. Proceedings of the 15. thISCB conference on Intelligent Systems for Molecular Biology [Proceedings of the 15th ISCB Molecular Biology Intelligent Systems Conference]. Bioinformatics [Bioinformatics]. 23, i559-68, 2007). In an embodiment, the present disclosure relates to using the PicXAA suite of programs (including but not limited to PicXAA, PicXAA-R, PicXAA-Web, etc.) to calculate the percent identity between two polynucleotide or amino acid sequences (Mohammad, S., Sahraeian, E. and Yoon, B. PicXAA: greedy probabilistic construction of maximum expected accuracy alignment of multiple sequences [greedy probabilistic construction of maximum expected accuracy alignment of multiple sequences]. Nucleic Acids Research [Nucleic Acids Research]. 38 (15): 4917-28. 2010). In embodiments, the present disclosure relates to the use of the PSAlign alignment program to calculate the percent identity between two polynucleotide or amino acid sequences (SZE, S.-H., Lu, Y., and Yang, Q. (2006) A polynomial time solvable formulation of multiple sequence alignment Journal of Computational Biology, 13, 309-19). In embodiments, the present disclosure relates to the use of the StatAlign alignment program to calculate the percent identity between two polynucleotide or amino acid sequences (Novák, et al. (2008) StatAlign: an extendable software package for joint Bayesian estimation of alignments and evolutionary trees. Bioinformatics, 24(20): 2403-04). In an embodiment, the disclosure relates to calculating the percent identity between two polynucleotide or amino acid sequences using the Gap alignment program of Needleman and Wunsch (Needleman and Wunsch, Journal of Molecular Biology 48: 443-453, 1970). In embodiments, the disclosure relates to the use of the BestFit alignment program of Smith and Waterman to calculate the percent identity between two polynucleotide or amino acid sequences (Smith and Waterman, Advances in Applied Mathematics, 2:482-489, 1981, Smith et al., Nucleic Acids Research 11:2205-2220, 1983). These programs generate biologically meaningful multiple sequence alignments of divergent sequences. The best match alignments calculated for the selected sequences are aligned so that identities, similarities, and differences can be seen.

[0071] The term "similarity" refers to a comparison between amino acid sequences, and not only the identical amino acids at corresponding positions are considered, but also the functionally similar amino acids at corresponding positions are considered. Therefore, in addition to sequence similarity, the similarity between polypeptide sequences also indicates functional similarity.

[0072] The term "homology" is sometimes used to refer to the level of similarity between two or more nucleic acid or amino acid sequences, expressed as a percentage of positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of evolutionary relatedness, often demonstrated by similar functional properties between different nucleic acids or proteins that share similar sequences.

[0073] As used herein, the term "variant" refers to substantially similar sequences. For nucleotide sequences, naturally occurring variants can be identified using well-known molecular biology techniques, such as, for example, polymerase chain reaction (PCR) and hybridization techniques as outlined herein.

[0074] For nucleotide sequences, variants are included in the deletion and / or addition of one or more nucleotides at one or more internal sites in natural polynucleotides, and / or the replacement of one or more nucleotides at one or more sites in natural polynucleotides. As used herein, "natural" nucleotide sequences include naturally occurring nucleotide sequences. With respect to nucleotide sequences, well-known molecular biology techniques can be used to identify naturally occurring variants, for example, using the polymerase chain reaction (PCR) and hybridization techniques outlined below to identify. Nucleotide sequence variants also include synthetically obtained nucleotide sequences, such as those produced by site-directed mutagenesis. Typically, as determined by the sequence alignment programs and parameters described elsewhere herein, the variants of the specific nucleotide sequences of the present invention will have at least about 40%, 45%, 50%>, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with the specific nucleotide sequence. Biologically active variants of the disclosed nucleotide sequences may differ from the sequence by only 1 to 15 nucleic acid residues, only 1 to 10, such as 6-10, only 5, only 4, 3, 2, or even only 1 nucleic acid residue.

[0075] As used herein, the term "operably linked" refers to a first nucleic acid sequence being operably linked to a second nucleic acid sequence when the first nucleic acid sequence has a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence when it affects the transcription or expression of the coding sequence. When produced recombinantly, operably linked nucleic acid sequences are generally contiguous and, in the case of two protein coding regions that need to be linked, are in the same reading frame. However, elements do not have to be contiguous to be operably linked.

[0076] As used herein, the term "promoter" refers to a DNA region that is usually located upstream of a gene (towards the 5' region of a gene), and is required for starting and driving gene transcription. A promoter can allow for the proper activation or inhibition of the gene it controls. A promoter can contain a specific sequence that is recognized by a transcription factor. These factors may bind to the promoter DNA sequence, leading to the recruitment of RNA polymerase, which is an enzyme that synthesizes RNA from a gene coding region. A promoter generally refers to all gene regulatory elements located upstream of a gene, including an upstream promoter, a 5'UTR, an intron, and a leader sequence.

[0077] As used herein, the term "upstream promoter" refers to a continuous polynucleotide sequence sufficient to guide transcription initiation. As used herein, upstream-promoter covers transcription initiation sites with several sequence motifs, including TATA Box, promoter sequence, TFIIB recognition element and other promoter motifs (Jennifer, EF et al., (2002) Genes & Dev. [gene and development], 16: 2583-2592). Upstream promoter provides an action site for RNA polymerase II, which is a multi-subunit enzyme with basic or general transcription factors (such as TFIIA, B, D, E, F and H). These factors are assembled into a pre-transcription initiation complex, which catalyzes the synthesis of RNA from a DNA template before transcription.

[0078] Upstream promoter activation is completed by various proteins binding and then interacting with the transcription initiation complex to activate the additional sequence of the regulatory DNA sequence element of gene expression. These gene regulatory element sequences interact with specific DNA binding factors. These sequence motifs can sometimes be referred to as cis elements. Such cis elements, bound to its tissue-specific or development-specific transcription factors, can determine the spatiotemporal expression pattern of the promoter at the transcriptional level, individually or in combination. The control types imposed by these cis elements on operably connected genes vary greatly. The effect of some elements is to increase the transcriptional response environmental response (for example, temperature, humidity and injury) of operably connected genes. Other cis elements may respond to developmental clues (for example, germination, seed maturity and flowering) or spatial information (for example, tissue specificity). See, for example, Langridge et al., (1989) Proc.Natl.Acad.Sci.USA [Proceedings of the National Academy of Sciences of the United States] 86:3219-23. These cis-elements are located at varying distances from the start of transcription, with some cis-elements (called proximal elements) adjacent to the minimal core promoter region, while others may be located several kilobases upstream or downstream of the promoter (enhancers).

[0079] As used herein, the term "5' untranslated region" or "5'UTR" is defined as an untranslated segment in the 5' end of a pre-mRNA or mature mRNA. For example, on a mature mRNA, a 5'UTR typically carries a 7-methylguanosine cap at its 5' end and is involved in many processes such as splicing, polyadenylation, export of mRNA to the cytoplasm, identification of the 5' end of the mRNA by the translation machinery, and protection of the mRNA from degradation.

[0080] As used herein, the term "intron" refers to any nucleic acid sequence contained in a transcribed but untranslated gene (or expressed polynucleotide sequence of interest). Introns include untranslated nucleic acid sequences within the expressed DNA sequence, and corresponding sequences in RNA molecules transcribed therefrom. Constructs described herein may also contain sequences that enhance translation and / or mRNA stability, such as introns. An example of such an intron is the first intron of gene II of Arabidopsis thaliana histone H3 variant or any other commonly known intron sequence. Introns can be used in combination with promoter sequences to enhance translation and / or mRNA stability.

[0081] As used herein, the term "transcription terminator" or "terminator" is defined as a transcribed segment in the 3' end of a pre-mRNA or mature mRNA. For example, a longer segment of DNA beyond the "polyadenylation signal" site is transcribed into a pre-mRNA. This DNA sequence usually contains a transcription termination signal for correct processing of the pre-mRNA into a mature mRNA.

[0082] As used herein, the term "3' untranslated region" or "3'UTR" is defined as an untranslated segment in the 3' end of a pre-mRNA or mature mRNA. For example, on a mature mRNA, this region carries a poly-(A) tail and is known to have many roles in mRNA stability, translation initiation, and mRNA export. In addition, the 3'UTR is believed to include a polyadenylation signal and a transcription terminator.

[0083] As used herein, the term "polyadenylation signal" refers to a nucleic acid sequence present in an mRNA transcript that, when a poly-(A) polymerase is present, allows the transcript to be polyadenylated at a polyadenylation site, e.g., 10 to 30 bases downstream of the poly-(A) signal. Many polyadenylation signals are known in the art and can be used in the present invention. Exemplary sequences include AAUAAA and variants thereof, as described in Loke J. et al., (2005) Plant Physiology 138(3); 1457-1468.

[0084] A "DNA binding transgene" is a polynucleotide coding sequence that encodes a DNA binding protein. The DNA binding protein is then capable of binding to another molecule. The binding protein can bind to, for example, a DNA molecule (DNA binding protein), an RNA molecule (RNA binding protein), and / or a protein molecule (protein binding protein). If it is a protein binding protein, it can bind to itself (to form homodimers, homotrimers, etc.) and / or to one or more molecules of different proteins. The binding protein can have more than one type of binding activity. For example, zinc finger proteins have DNA binding, RNA binding, and protein binding activities.

[0085] Examples of DNA binding proteins include; meganucleases, zinc fingers, CRISPR, and TALEN binding domains that can be "engineered" to bind to a predetermined nucleotide sequence. Typically, engineered DNA binding proteins (e.g., zinc fingers, CRISPR, or TALEN) are non-naturally occurring proteins. Non-limiting examples of methods for designing and selecting engineered DNA binding proteins. The designed DNA binding protein is a protein that does not exist in nature, and its design / composition is mainly derived from reasonable criteria. Reasonable criteria for design include applying substitution rules and computer algorithms to process information stored in databases for existing ZFP, CRISPR and / or TALEN design information and binding data. See, e.g., U.S. Patents 6,140,081; 6,453,242; and 6,534,261; see also WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536 and WO 03 / 016496 and U.S. Publication Nos. 20110301073, 20110239315, and 20119145940.

[0086] A "zinc finger DNA binding protein" (or binding domain) is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized by the coordination of zinc ions. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP. Zinc finger binding domains can be "engineered" to bind to a predetermined nucleotide sequence. Non-limiting examples of methods for designing and selecting engineered zinc finger proteins. Designed zinc finger proteins are proteins that do not exist in nature, and their design / composition is primarily derived from rational criteria. Rational criteria for design include the application of substitution rules and computer algorithms to process information stored in databases for existing ZFP design information and binding data. See, e.g., U.S. Patent Nos. 6,140,081; 6,453,242; 6,534,261 and 6,794,136; see also WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536 and WO 03 / 016496.

[0087] In other examples, the DNA binding domain of one or more nucleases comprises a naturally occurring or engineered (non-naturally occurring) TAL effector DNA binding domain. See, e.g., U.S. Patent Publication No. 20110301073, which is incorporated herein by reference in its entirety. Plant pathogenic bacteria of the genus Xanthomonas are known to cause many diseases in important crop plants. The pathogenicity of Xanthomonas depends on a conserved type III secretion (T3S) system that injects more than different effector proteins into plant cells. Among these injected proteins are transcription activator-like (TALEN) effectors that mimic plant transcription activators and manipulate plant transcriptomes (see Kay et al., (2007) Science [Science] 318: 648-651). These proteins contain a DNA binding domain and a transcription activation domain. One of the best characterized TAL effectors is AvrBs3 from Xanthomonas campestgrispv. Vesicatoria (see Bonas et al. (1989) Mol Gen Genet 218: 127-136 and WO 2010079430). TAL effectors contain a concentrated domain of tandem repeats, each containing approximately 34 amino acids, which is key to the DNA binding specificity of these proteins. In addition, they contain a nuclear localization sequence and an acidic transcriptional activation domain (for review, see Schornack S et al. (2006) J Plant Physiol 163(3): 256-272). Additionally, in the plant pathogenic bacterium Ralstonia solanacearum, two genes, designated brg11 and hpx17 have been found to be homologous to the Xanthomonas AvrBs3 family of Ralstonia solanacearum biovariant strain GMI1000 and biovariant 4 strain RS1000 (see Heuer et al., (2007) Appl and Enviro Micro 73(13):4379-4384). These genes are 98.9% identical to each other in nucleotide sequence, but differ by a 1,575 bp deletion in the repeat domain of hpx17. However, both gene products have less than 40% sequence identity with the AvrBs3 family proteins of Xanthomonas. See, e.g., U.S. Patent Publication No. 20110301073, which is incorporated by reference in its entirety.

[0088] The specificity of these TAL effectors depends on the sequences found in the tandem repeats. The repeats contain approximately 102 bp, and the repeats are typically 91%-100% homologous to each other (Bonas et al., supra). Polymorphisms in the repeats are usually located at positions 12 and 13, and there appears to be a one-to-one correspondence between the identities of the hypervariable di-residues at positions 12 and 13 and the identities of consecutive nucleotides in the target sequence of the TAL effectors (see Moscou and Bogdanove, (2009) Science 326:1501 and Boch et al. (2009) Science 326:1509-1512). Experimentally, the natural code for DNA recognition by these TAL effectors has determined that the HD sequences at positions 12 and 13 result in binding to cytosine (C), NG to T, NI to A, C, G or T, NN to A or G, and ING to T. These DNA binding repeat sequences have been assembled into proteins with new combinations and numbers of repeat sequences to make artificial transcription factors that can interact with new sequences and activate expression of non-endogenous reporter genes in plant cells (Boch et al., supra). Engineered TAL proteins have been linked to FokI cleavage half-domains to generate TAL effector domain nuclease fusion proteins (TALENs) that are active in yeast reporter gene assays (plasmid-based targets).

[0089] The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / Cas (CRISPR-associated) nuclease system is a recently engineered nuclease system based on a bacterial system that can be used for genome engineering. It is based on a part of the adaptive immune response of many bacteria and archaea. When a virus or plasmid invades bacteria, the invader's DNA segment is converted into CRISPR RNA (crRNA) through an "immune" response. This crRNA then binds to another RNA called tracrRNA through a partially complementary region to guide the Cas9 or Cas12f1 nuclease to a region homologous to the crRNA in the target DNA, called the "pre-spacer sequence". Cas9 or Cas12f1 cuts DNA to produce a blunt end at the end of a double-strand break (DSB) at a site specified by a 20-nucleotide guide sequence contained in the crRNA transcript. Cas9 or Cas12f1 requires crRNA and tracrRNA for site-specific DNA recognition and cutting. The system has now been engineered so that crRNA and tracrRNA can be combined into one molecule ("single guide RNA"), and the crRNA equivalent of a single guide RNA can be engineered to direct Cas9 or Cas12f1 nucleases to target any desired sequence (see Jinek et al., (2012) Science 337, pp. 816-821, Jinek et al., (2013), eLife 2: e00471 and David Segal, (2013) eLife 2: e00563). In other examples, crRNA is combined with tracrRNA to guide Cpf1 nuclease to a region homologous to the crRNA to cleave DNA with staggered ends (see Zetsche, Bernd et al., Cell 163.3 (2015): 759-771.). Thus, CRISPR / Cas systems can be engineered to generate DSBs at desired targets in the genome, and repair of DSBs can be affected by using repair inhibitors, resulting in increased error-prone repair.

[0090] In other examples, the DNA binding transgene / heterologous coding sequence is a site-specific nuclease, which comprises an engineered (non-naturally occurring) meganuclease (also known as a homing endonuclease). The recognition sequences of homing endonucleases or meganucleases, such as I-Scel, I-CeuI, PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-Crel, I-TevI, I-TevII and I-TevIII are known. See also U.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252; Belfort et al., (1997) Nucleic Acids Res. 25:3379–30 3388; Dujon et al., (1989) Gene 82:115-118; Perler et al., (1994) Nucleic Acids Res. 22, 11127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al., (1996) J. Mol. Biol. 263:163–180; Argast et al., (1998) J. Mol. Biol. 280:345-353 and the New England Biolabs catalog. In addition, the DNA binding specificity of homing endonucleases and meganucleases can be engineered to bind to non-natural target sites. See, e.g., Chevalier et al. (2002) Molec. Cell 10:895-905; Epinat et al., (2003) Nucleic Acids Res. 5 31:2952-2962; Ashworth et al., (2006) Nature 441:656-659; Paques et al., (2007) Current Gene Therapy 7:49-66; U.S. Patent Publication No. 20070117128. The DNA binding domain of homing endonucleases and meganucleases can be altered in the context of the entire nuclease (i.e., such that the nuclease includes a homologous cleavage domain), or can be fused to a heterologous cleavage domain.

[0091] As used herein, the term "transformation" encompasses all techniques by which a nucleic acid molecule can be introduced into such a cell. Examples include, but are not limited to: transfection with viral vectors; transformation with plasmid vectors; electroporation; lipofection; microinjection (Mueller et al., (1978) Cell 15:579-85); Agrobacterium-mediated transfer; direct DNA uptake; WHISKERS TM mediated transformation; and microprojectile bombardment. These techniques can be used for stable transformation and transient transformation of plant cells. "Stable transformation" refers to the introduction of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Once stably transformed, the nucleic acid fragment is stably integrated into the genome of the host organism and any progeny. Host organisms containing the transformed nucleic acid fragment are referred to as "transgenic" organisms. "Transient transformation" refers to the introduction of a nucleic acid fragment into the nucleus or DNA-containing organelle of a host organism, resulting in gene expression that is not genetically stable.

[0092] Exogenous nucleic acid sequence. In one example, the transgenic / heterologous coding sequence is a gene sequence (e.g., a herbicide resistance gene), a gene encoding an industrially or pharmaceutically useful compound, or a gene encoding a desired agronomic trait. In another example, the transgenic / heterologous coding sequence is an antisense nucleic acid sequence, wherein the expression of the antisense nucleic acid sequence inhibits the expression of the target nucleic acid sequence. The transgenic / heterologous coding sequence can contain a regulatory sequence operably connected to the transgenic / heterologous coding sequence (e.g., a promoter). In certain embodiments, the purpose polynucleotide sequence is a transgenic. However, in other embodiments, the purpose polynucleotide sequence is an endogenous nucleic acid sequence, wherein it is desired to be another genomic copy of the endogenous nucleic acid sequence, or a nucleic acid sequence in the antisense direction relative to the sequence of the target nucleic acid molecule in the host organism.

[0093] As used herein, the term transgenic "event" is generated by transforming plant cells with heterologous DNA, which is a nucleic acid construct that includes a transgene / heterologous coding sequence of interest; regenerating a population of plants resulting from the insertion of the transgene / heterologous coding sequence into the genome of the plant; and selecting specific plants characterized as being inserted at a specific genomic location. The term "event" refers to the original transformant that includes the heterologous DNA and the progeny of the transformant. The term "event" also refers to the progeny produced by a sexual cross between a transformant and another variety that includes the genomic / transgenic DNA. Even after repeated backcrossing with the recurrent parent, the inserted transgene / heterologous coding sequence DNA and the flanking genomic DNA (genomic / transgenic DNA) from the transformed parent are present at the same chromosomal location in the progeny of the cross. The term "event" also refers to DNA from the original transformant and its progeny, which progeny contain the inserted DNA and flanking genomic sequences immediately adjacent to the inserted DNA, which inserted DNA is expected to be transferred to progeny that receive the inserted DNA including the transgene / heterologous coding sequence of interest, resulting from a sexual cross between a parental line that includes the inserted DNA (e.g., the original transformant and progeny produced by selfing) and a parental line that does not contain the inserted DNA.

[0094] As used herein, the term "polymerase chain reaction" or "PCR" defines a procedure or technique in which trace amounts of nucleic acids, RNA and / or DNA are amplified as described in U.S. Patent No. 4,683,195, issued July 28, 1987. Typically, sequence information is required from the ends of the region of interest or beyond so that oligonucleotide primers can be designed; these primers are identical or similar in sequence to the opposite strands of the template to be amplified. The 5' terminal nucleotides of the two primers can be consistent with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, phage or plasmid sequences, etc. See generally Mullis et al., Cold Spring Harbor Symp. Quant. Biol. [Cold Spring Harbor Quantitative Biology Symposium], 51: 263 (1987); Erlich, ed., PCR Technology [PCR Technology], (Stockton Press, New York, 1989).

[0095] As used herein, the term "primer" refers to an oligonucleotide that can serve as a starting point for synthesis along a complementary strand when conditions are suitable for the synthesis of a primer extension product. Synthesis conditions include the presence of four different deoxyribonucleotide triphosphates and at least one polymerization inducing agent, such as reverse transcriptase or DNA polymerase. They are present in a suitable buffer, which may include a component as a cofactor or affect a component such as pH at various suitable temperatures. Primers are preferably single-stranded sequences so that amplification efficiency is optimized, but double-stranded sequences may be utilized.

[0096] As used herein, the term "probe" refers to an oligonucleotide that hybridizes to a target sequence. or In a method of detecting a target of a certain type, the probe hybridizes to a portion of the target located between the annealing sites of the two primers. The probe comprises about eight nucleotides, about ten nucleotides, about fifteen nucleotides, about twenty nucleotides, about thirty nucleotides, about forty nucleotides, or about fifty nucleotides. In some embodiments, the probe comprises about eight nucleotides to about fifteen nucleotides. The probe may also include a detectable label, such as a fluorophore ( fluorescein isothiocyanate, etc.). The detectable label can be covalently attached directly to the probe oligonucleotide, for example, at the 5' end of the probe or at the 3' end of the probe. The probe including the fluorophore can further include a quencher, such as a Black Hole Quencher TM , Iowa Black TM wait.

[0097] As used herein, the terms "restriction endonucleases" and "restriction enzymes" refer to bacterial enzymes, each of which cuts double-stranded DNA at or near a specific nucleotide sequence. Type 2 restriction enzymes recognize and cut DNA at the same site, including but not limited to XbaI, BamHI, HindIII, EcoRI, XhoI, SalI, KpnI, AvaI, PstI, and SmaI.

[0098] As used herein, the term "vector" is used interchangeably with the term "construct", "cloning vector" and "expression vector", and means a vehicle that can introduce a DNA or RNA sequence (e.g., an exogenous gene) into a host cell to transform the host and promote the expression (e.g., transcription and translation) of the introduced sequence. "Non-viral vector" is intended to mean any vector that does not contain a virus or retrovirus. In some embodiments, a "vector" is a DNA sequence that includes at least one DNA replication origin and at least one selectable marker gene. Examples include, but are not limited to, plasmids, cosmids, bacteriophages, bacterial artificial chromosomes (BACs) or viruses that bring exogenous DNA into cells. The vector may also include one or more genes, antisense molecules and / or selectable marker genes and other genetic elements known in the art. The vector may transduce, transform or infect cells so that the cells express nucleic acid molecules and / or proteins encoded by the vector.

[0099] The term "plasmid" defines a circular chain of nucleic acid capable of autosomal replication in a prokaryotic or eukaryotic host cell. The term includes nucleic acids that may be DNA or RNA and may be single-stranded or double-stranded. The defined plasmid may also include a sequence corresponding to a bacterial origin of replication.

[0100] As used herein, the term "selectable marker gene" as used herein defines a gene or other expression cassette encoding a protein that helps identify cells into which a selectable marker gene has been inserted. For example, "selectable marker gene" encompasses reporter genes and genes for plant transformation to, for example, protect plant cells from a selection agent or provide resistance / tolerance to a selection agent. In one embodiment, only those cells or plants that receive a functional selectable marker can divide or grow under conditions with a selection agent. The phrase "marker positive" refers to a plant that has been converted to include a selectable marker gene.

[0101] As used herein, the term "detectable label" refers to a label that can be detected, such as a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator or an enzyme. Examples of detectable labels include, but are not limited to, the following: fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzyme labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescence, biotinyl, a predetermined polypeptide epitope recognized by a secondary reporter molecule (e.g., a leucine zipper pair sequence, a binding site for a secondary antibody, a metal binding domain, an epitope tag). In an embodiment, the detectable label can be attached by spacer arms of various lengths to reduce potential steric hindrance.

[0102] As used herein, the terms "cassette", "expression cassette" and "gene expression cassette" refer to a DNA segment that can be inserted into a nucleic acid or polynucleotide at a specific restriction site or by homologous recombination. As used herein, the segment of DNA contains a polynucleotide encoding a polypeptide of interest, and the cassette and restriction sites are designed to ensure that the cassette is inserted into an appropriate reading frame for transcription and translation. In an embodiment, the expression cassette may include a polynucleotide encoding a polypeptide of interest, and may have elements in addition to the polynucleotides that promote transformation of a specific host cell. In an embodiment, the gene expression cassette may also include elements that allow enhanced expression of the polynucleotide encoding the polypeptide of interest in a host cell. These elements may include, but are not limited to, promoters, minimal promoters, enhancers, response elements, terminator sequences, polyadenylation sequences, and the like.

[0103] As used herein, "joint" or "spacer" is a key, molecule or group of molecules that bind two separate entities to each other.Joints and spacers can provide the best spacing for two entities, or can further provide an unstable connection that allows two entities to be separated from each other.Unstable connections include photocleavable groups, acid-labile parts, base-labile parts and enzyme-cleavable groups.As used herein, the term "polylinker" or "multiple cloning site" defines a cluster of three or more type 2 restriction enzyme sites within 10 nucleotides of each other on a nucleic acid sequence.In other cases, as used herein, the term "polylinker" refers to a seamless cloning method (i.e., Gibson cloning) that is used to clone a nucleic acid sequence. NEBuilderHiFID NA Golden Gate Assembly, The construct containing the polylinker is used for the insertion and / or excision of nucleic acid sequences (such as the coding region of a gene).

[0104] A "centimorgan" (cM) or "map distance unit" is the distance between two linked genes, markers, target sites, genomic loci of interest, loci, or any pair thereof, at which 1% of the products of meiosis are recombined. Thus, one centimorgan is equivalent to a distance between two linked genes, markers, target sites, loci, genomic loci of interest, or any pair thereof that is equal to 1% average recombination frequency.

[0105] As used herein, the term "control" refers to a sample used for comparison purposes in an analytical procedure. A control can be "positive" or "negative." For example, where the purpose of the analytical procedure is to detect differentially expressed transcripts or polypeptides in cells or tissues, it is generally preferred to include a positive control (e.g., a sample from a known plant that exhibits the desired expression) and a negative control (e.g., a sample from a known plant that lacks the desired expression).

[0106] As used herein, the term "plant" includes the whole plant and any progeny, cells, tissues or parts of the plant. Plant species that can be used in the present invention generally include higher and lower plants suitable for variation, including angiosperms (monocots and dicots), gymnosperms, ferns and multicellular algae. Therefore, "plants" include dicots and monocots. The term "plant part" includes any part of a plant, including, for example and not limited to: seeds (including mature seeds and immature seeds); plant segments; plant cells; plant cell cultures; plant organs (such as pollen, embryos, flowers, fruits, branches, leaves, roots, stems and explants). Plant tissues or plant organs can be seeds, protoplasts, callus or any other group of plant cells organized into structural or functional units. Plant cells or tissue cultures may be able to regenerate plants with physiological and morphological characteristics of the plant from which the cells or tissues are obtained, and can regenerate plants with substantially the same genotype as the plant. In contrast, some plant cells cannot be regenerated to produce plants. The regenerable cell in plant cell or tissue culture can be an embryo, protoplast, meristem cell, callus tissue, pollen, leaf, anther, root, root tip, filament, flower, kernel, ear, cob, bract or stalk.

[0107] Plant parts include harvestable parts and parts that can be used for propagation of future generations of plants. Plant parts that can be used for propagation include, for example, but are not limited to: seeds; fruits; cuttings; seedlings; tubers; and rhizomes. The harvestable parts of a plant can be any useful part of a plant, including, for example, but not limited to: flowers; pollen; seedlings; tubers; leaves; stems; fruits; seeds; and roots.

[0108] Plant cell is the structural and physiological unit of plant, comprises protoplast and cell wall.Plant cell can be in the form of separated single cell or cell aggregate (for example, fragile callus and cultured cell), and can be the part of higher organized unit (for example, plant tissue, plant organ and plant).Therefore, plant cell can be protoplast, produce gamete cell or can be regenerated into the cell or cell collection of whole plant.Like this, comprise multiple plant cells and can be regenerated into the seed of whole plant in the embodiment of this paper and be considered as " plant cell ".

[0109] As used herein, the term "small RNA" refers to several classes of non-coding ribonucleic acids (ncRNA). The term small RNA describes short chains of ncRNA produced in bacterial cells, animals, plants, and fungi. These short chains of ncRNA can be produced naturally in cells or by introducing exogenous sequences expressing the short chains or ncRNA. Small RNA sequences do not directly encode proteins and are functionally different from other RNAs because small RNA sequences are only transcribed but not translated. Small RNA sequences are involved in other cellular functions, including gene expression and modification. Small RNA molecules are generally composed of about 20 to 30 nucleotides. Small RNA sequences can be derived from longer precursors. Precursors form structures that fold over each other in self-complementary regions; they are then processed by nuclease Dicer in animals or DCL1 in plants.

[0110] Many types of small RNA exist naturally or are artificially produced, including microRNA (miRNA), short interfering RNA (siRNA), antisense RNA, short hairpin RNA (shRNA), and small nucleolar RNA (snoRNA). Certain types of small RNA, such as microRNA and siRNA, are important in gene silencing and RNA interference (RNAi). Gene silencing is a process of genetic regulation in which a gene that would normally be expressed is "turned off" by an intracellular element (in this case, a small RNA). The protein that would normally be formed by the genetic information cannot be formed due to the interference, and the information encoded in the gene is prevented from being expressed.

[0111] As used herein, the term "small RNA" encompasses RNA molecules described in the literature as "tiny RNA" (Storz, (2002) Science 296:1260-3; Illangasekare et al., (1999) RNA 5:1482-1489); prokaryotic "small RNA" (sRNA) (Wassarman et al., (1999) Trends Microbiol. 7:37-45); eukaryotic "non-coding RNA (ncRNA)"; "microRNA (miRNA)"; "small non-mRNA (snmRNA)"; "functional RNA (fRNA)"; "transfer RNA (tRNA)"; "catalytic RNA" [e.g., ribozymes, including self-acylating ribozymes (Illangaskare et al., (1999) RNA 5:1482-1489); "small nucleolar RNA (snoRNA)", "tmRNA" (also known as "10S RNA," Muto et al., (1998) Trends Biochem Sci. [Biological Chemical Science Trends] 23:25-29; and Gillet et al., (2001) Mol Microbiol. [Molecular Microbiology] 42:879-885); RNAi molecules include, but are not limited to, "small interfering RNA (siRNA)", "endo-ribonuclease-made siRNA (e-siRNA)", "short hairpin RNA (shRNA)", and "small sequential RNA (stRNA)", "cut siRNA (d-siRNA)", and aptamers, oligonucleotides and other synthetic nucleic acids containing at least one uracil base.

[0112] Unless otherwise specifically explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Definitions of commonly used terms in molecular biology can be found, for example, in Lewin, Genes V, Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Meyers (ed.), Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0113] Example

[0114] In some embodiments, the present disclosure relates to soybean plant genomic integration sites.Aspects of the present disclosure include compositions and methods.

[0115] In an embodiment, the soybean plant genome integration site comprises a polynucleotide having a length of at least 1 centimorgan (cM). In the aspect of this embodiment, the genome integration site can be 1cM, 2cM, 3cM, 4cM, 5cM, 6cM, 7cM, 8cM, 9cM, 10cM or larger. In other aspects, the soybean plant genome integration site can comprise various components. Such components can include a target site. In some aspects, the soybean plant genome integration site can comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more target sites. The soybean plant genome integration site can be cut by site-specific nuclease at the target site. The resulting double-stranded break can be repaired to incorporate a donor polynucleotide, which is incorporated into the genome of the soybean plant.

[0116] In an embodiment, the soybean plant genome integration site comprises low genetic diversity. The genetic diversity of soybean plants can be determined by methods known in the art. The region of the soybean plant genome can be classified as having high genetic diversity or low genetic diversity. In order to determine which regions of the genome are classified as high genetic diversity or low genetic diversity, those skilled in the art can compare the homologous genomic regions between multiple soybean plant varieties. The region with high-level genetic diversity in the genome will include more variability in the genome sequence. For example, compared with other soybean plant varieties, soybean plant varieties have many single nucleotide polymorphisms (SNPs) in the genome region. On the contrary, the region with low-level genetic diversity in the genome will include less variability in the genome sequence. When comparing the genomic regions of multiple soybean plant varieties, these regions are almost identical. Typically, the low genetic diversity region has a dominant haplotype, and may have one or more other minor haplotypes. In an embodiment, the region of low genetic diversity comprises at least one major haplotype in the breeding group having a haplotype frequency of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In further embodiments, the region of low genetic diversity comprises at least one major haplotype in the maturity group having a haplotype frequency of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In another embodiment, the region of low genetic diversity comprises at least one major haplotype in a haplotype and maturity group having a haplotype frequency of 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0117] In order to evaluate the level of genetic diversity in the soybean plant genome region, a large number of soybean plant varieties are sequenced. The resulting soybean plant genome is then divided into boxes based on genetic and physical coordinates. Other genomic features are included in the box to distinguish the region. For example, SNPs can be identified in the box. Next, based on the SNP allele spectrum in the box, the box is classified into haplotypes. Boxes with similar genotypes across varieties without a large amount of differentiation (e.g., low or no SNP occurrence) are considered to be the same haplotype. In certain embodiments, boxes with a major haplotype with a frequency of 80%-100% across defined germplasms include low diversity regions. The technician will understand that haplotype frequencies may be different in different germplasm pools. The germplasm pools of these genomic regions in soybean plant varieties may vary due to maturity differences, heterosis grouping, geographic distribution, etc.

[0118] In an embodiment, the soybean plant genome integration site comprises a high recombination frequency. In one aspect, the soybean plant genome integration site is greater than 10 cM from a region known to be difficult to recombine. For example, the pericentromeric region is not a region in the soybean plant genome known to comprise a high recombination frequency. In a further aspect, the recombination rate of the region with a high recombination frequency is greater than 0.7 cM / 1 Mb.

[0119] In an embodiment, the soybean plant genome integration site comprises a polynucleotide that is closely close to the telomere. In some aspects, the soybean plant genome site is closely close to less than 20cM from the chromosome end. In other aspects, the soybean plant genome site is closely close to less than 20cM, 19cM, 18cM, 17cM, 16cM, 15cM, 14cM, 13cM, 12cM, 11cM, 10cM, 9cM, 8cM, 7cM, 6cM, 5cM, 4cM, 3cM, 2cM or 1cM from the chromosome end. In a further aspect, the soybean plant genome site is closely close to less than 4.7Mb from the chromosome end. In various aspects, close proximity to a soybean plant genomic locus is less than 4.7 Mb, 4.6 Mb, 4.5 Mb, 4.4 Mb, 4.3 Mb, 4.2 Mb, 4.1 Mb, 4.0 Mb, 3.9 Mb, 3.8 Mb, 3.7 Mb, 3.6 Mb, 3.5 Mb, 3.4 Mb, 3.3 Mb, 3.2 Mb, 3.1 Mb, 3.0 Mb, 2.9 Mb, 2.8 Mb, 2.7 Mb, 2.6 Mb, 2.5 Mb, 2.4 Mb, 2.3 Mb, 2.2 Mb, 2.1 Mb, 2.0 Mb, 1.9 Mb, 1.8 Mb, 1.7 Mb, 1.6 Mb, 1.5 Mb, 1.4 Mb, 1.3 Mb, 1.2 Mb, 1.1 Mb or 1.0 Mb from the end of a chromosome.

[0120] In an embodiment, the soybean plant genome integration site comprises a polynucleotide without structural variation. On the one hand, the soybean plant genome integration site does not contain translocation. In other aspects, the soybean plant genome integration site does not contain inversion. In a further aspect, the soybean plant genome integration site does not contain deletion. It will be appreciated by those skilled in the art that these types of structural variants can suppress or reduce the combination in the target region, or prevent the target segment from effectively infiltrating another genome. For example, the present disclosure compares the genomes of 26 public soybean genome assemblies and 10 internal soybean assemblies in the target low diversity region. Check the structural variation of these low diversity regions, including inversions, insertions / deletions and translocations greater than 200kb within 10cM of the target low diversity region. The example of identifying such structural variation is further provided in Liu, Yucheng, et al. "Pan-genome of wild and cultivated soybeans. [Pan-genome of wild and cultivated soybeans]" Cell [Cell] 182.1 (2020): 162-176.

[0121] In an embodiment, the soybean plant genome integration site comprises a polynucleotide having a genetic size of less than 10 cM. ​​In some aspects, the genetic size is less than 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM or 1 cM. It will be appreciated by those skilled in the art that the genetic size is based on a genetic map associated with the population used to create the genetic map. In some aspects, a diversity analysis is performed based on the genetic map using 1 cM bins, and the resulting genetic size is determined from the analysis.

[0122] In an embodiment, the plant genome integration site comprises a polynucleotide having a genetic size less than 10 cM. ​​In some aspects, the genetic size is less than 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM or 1 cM. It will be appreciated by those skilled in the art that the genetic size is based on a genetic map associated with a population used to create a genetic map. In some aspects, a diversity analysis is performed based on a genetic map using 1 cM bins to determine the resulting genetic size from the analysis.

[0123] In an embodiment, the plant genome integration site comprises a polynucleotide with a physical size of less than 2.6 Mb. In one aspect, the soybean plant genome integration site comprises a polynucleotide with a physical size of less than 2.6 Mb, 2.5 Mb, 2.4 Mb, 2.3 Mb, 2.2 Mb, 2.1 Mb, 2.0 Mb, 1.9 Mb, 1.8 Mb, 1.7 Mb, 1.6 Mb, 1.5 Mb, 1.4 Mb, 1.3 Mb, 1.2 Mb, 1.1 Mb or 1.0 Mb. In other embodiments, the soybean plant genome integration site comprises a polynucleotide with a physical size ranging from 785,899 bp to 954,789 bp. In further embodiments, the soybean plant genomic integration site comprises a physical size of 785,899 bp, 790,000 bp, 795,000 bp, 800,000 bp, 805,000 bp, 810,000 bp, 815,000 bp, 820,000 bp, 825,000 bp, 830,000 bp, 835,000 bp, 840,000 bp, 845,000 bp, 850,000 bp, 855,000 bp, 860,000 bp, 865 ,000bp, 870,000bp, 875,000bp, 880,000bp, 885,000bp, 890,000bp, 895,000bp, 900,000bp, 905,000bp, 910,000bp, 915,000bp, 920,000bp, 925,000bp, 930,000bp, 935,000bp, 940,000bp, 945,000bp, 950,000bp or 954,789bp. It will be understood by those skilled in the art that each chromosome has a physical "length" measured in base pairs (bp) starting from the first nucleotide in the genome assembly and extending to the last nucleotide. In certain embodiments, the physical size of the low diversity region is the approximate number of nucleotides that make up the corresponding low diversity genetic box.

[0124] In an embodiment, the soybean plant genomic integration site comprises euchromatin. Euchromatin is a loosely packed protein-bound DNA that is relatively loose during the cell cycle. Those skilled in the art will appreciate that, based on the genetic and physical relationships across chromosomes, genomic regions comprising euchromatin experience higher recombination rates. In some aspects, a target site of a soybean plant genomic integration site within euchromatin is desirable for efficient recombination and site-specific integration (SSI) of a donor polynucleotide.

[0125] In an embodiment, the soybean plant genomic integration site comprises a polynucleotide greater than 10 cM from heterochromatin. In other embodiments, the soybean plant genomic integration site comprises a polynucleotide greater than 1.05 Mb from heterochromatin. It will be appreciated by those skilled in the art that heterochromatin is a tightly packed protein-bound DNA that remains tightly packed during the cell cycle. Heterochromatin typically undergoes a lower recombination rate during meiosis. In addition, heterochromatin typically contains centromere and pericentromere regions. In some aspects, the target site of the soybean plant genomic integration site outside the heterochromatin region is desirable for effective recombination and site-specific integration (SSI) of the donor polynucleotide.

[0126] Table 1 provides the locations of euchromatin and heterochromatin disclosed in Song, Qijian, et al. "Construction of high resolution genetic linkage maps to improve the soybean genome sequence assembly Glyma1.01." BMC genomics 17.1 (2016): 1-11.

[0127] Table 1: Soybean genomic regions containing heterochromatic and euchromatic regions.

[0128]

[0129]

[0130] In an embodiment, the soybean plant genome integration site comprises a polynucleotide near a quantitative trait locus (QTL). In some aspects, the soybean plant genome integration site is "genetically linked" to the QTL. In some aspects, when the QTL and the soybean plant genome integration site are at least 50cM each other, the soybean plant genome integration site is "genetically linked" to the QTL. In other aspects, the soybean plant genome integration site is "tightly genetically linked" to the QTL. In some aspects, when the QTL and the plant genome integration site are at least 10cM each other, the soybean plant genome integration site is "tightly genetically linked" to the QTL. In other aspects, the soybean plant genome integration site is "genetically linked" to the QTL, wherein the QTL and the soybean plant genome integration site are within 1cM, 2cM, 3cM, 4cM, 5cM, 6cM, 7cM, 8cM, 9cM, 10cM, 15cM, 20cM, 25cM, 30cM, 35cM, 40cM, 45cM or 50cM each other. In an embodiment, the soybean plant genome integration site comprises a polynucleotide that does not contain an endogenous gene. In some aspects, the plant genome integration site comprises a polynucleotide that does not encode a coding sequence that is translated into a protein. As used herein, the terms "quantitative trait locus" and "QTL" refer to a genomic region that affects the phenotypic variation of continuously varying traits such as yield or resistance. QTLs can even be included in multiple genes or other genetic factors in a continuous genomic region or linkage group.

[0131] Traits used for penetration

[0132] The transgene of interest may be expressed in the genomic sequence of the present disclosure. Exemplary transgenes of interest suitable for use in the constructs of the present disclosure include, but are not limited to, coding sequences that confer: (1) pest resistance or disease resistance, (2) tolerance to herbicides, (3) added value of agronomic traits such as; increased yield, nitrogen use efficiency, water use efficiency and nutritional quality, (4) protein binding to DNA in a site-specific manner, (5) expression of small RNAs; and (6) selectable markers. According to one embodiment, a transgenic / heterologous coding sequence encoding a selectable marker or a gene product that confers insecticide resistance, herbicide tolerance, small RNA expression, nitrogen use efficiency, water use efficiency, or nutritional quality is targeted within the genomic sequence of the present disclosure.

[0133] 1. Insect resistance

[0134] Various insect resistance genes can be targeted for insertion into the genomic sequences of the present disclosure. Regulatory elements can be engineered into gene expression cassettes containing insect resistance genes. The operably linked sequences can then be incorporated into a selected vector to allow identification and selection of transformed plants ("transformants"). Exemplary insect resistance coding sequences are known in the art. As examples of insect resistance coding sequences that can be operably linked to the regulatory elements of the present disclosure, the following traits are provided. Coding sequences providing exemplary lepidopteran insect resistance include: cry1A; cry1A.105; cry1Ab; cry1Ab (truncated); cry1Ab-Ac (fusion protein); cry1Ac (as sales); cry1C; cry1F (as (sold); cry1Fa2; cry2Ab2; cry2Ae; cry9C; mocry1F; pinII (proteinase inhibitor protein); vip3A(a); and vip3Aa20. Coding sequences providing exemplary coleopteran insect resistance include: cry34Ab1 (as sales); cry35Ab1 (as (sold); cry3A; cry3Bb1; dvsnf7; and mcry3A. Exemplary coding sequences providing multiple insect resistance include ecry31.Ab. The above list of insect resistance genes is not meant to be limiting. The present disclosure encompasses any insect resistance gene.

[0135] 2. Herbicide tolerance

[0136] Various herbicide tolerance genes can be targeted to be inserted into the genomic sequence of the present disclosure. Regulatory elements can be engineered into gene expression cassettes containing herbicide tolerance genes. The operably connected sequence can then be incorporated into the selected vector to allow identification and selection of transformed plants ("transformants"). Exemplary herbicide tolerance coding sequences are known in the art. As embodiments of herbicide tolerance coding sequences that can be operably connected to the regulatory elements of the present disclosure, the following traits are provided. Glyphosate herbicides contain a mode of action by inhibiting the EPSPS enzyme (5-enolpyruvyl shikimate-3-phosphate synthase). The enzyme participates in the biosynthesis of aromatic amino acids that are essential for the growth and development of plants. A variety of enzymatic mechanisms known in the art can be used to inhibit the enzyme. Genes encoding such enzymes can be operably connected to the gene regulatory elements of the present disclosure. In an embodiment, selectable marker genes include, but are not limited to, genes encoding glyphosate resistance genes, including: mutated EPSPS genes, such as 2mEPSPS genes, cp4 EPSPS genes, mEPSPS genes, dgt-28 genes; aroA genes; and glyphosate degradation genes, such as glyphosate acetyltransferase genes (gat) and glyphosate oxidase genes (gox). These traits are currently used as Gly-Tol TM , GT and Roundup sales. Resistance genes for glufosinate and / or bialaphos compounds include dsm-2, bar and pat genes. The bar and pat traits are currently used as Also included are tolerance genes that provide resistance to 2,4-D, such as the aad-1 gene (note that the aad-1 gene has further activity against aryloxyphenoxypropionate herbicides) and the aad-12 gene (note that the aad-12 gene has further activity against acetoxyacetate to synthesize auxins). These traits are Crop Protection Technology. Resistance genes to ALS inhibitors (sulfonylureas, imidazolinones, triazolopyrimidines, pyrimidinylthiobenzoates, and sulfonylamino-carbonyl-triazolinones) are known in the art. These resistance genes are most often caused by point mutations to the ALS encoding gene sequence. Other ALS inhibitor resistance genes include the hra gene, csrl-2 gene, Sr-HrA gene, and surB gene. Some of these traits are marketed under the trade name Sale. Herbicides that inhibit HPPD include pyrazolones, such as benzylpyrazone, pyraclostrobin and pyraclostrobin; triketones, such as mesotrione, sulfotrione, cyclosulfone, benzobicyclonone; and diketonitriles, such as isoxaflutole. Known traits can tolerate these exemplary HPPD herbicides. Examples of HPPD inhibitors include the hppdPF_W336 gene (for resistance to isoxaflutole) and the avhppd-03 gene (for resistance to mesotrione). Examples of oxini herbicide tolerance traits include the bxn gene, which has been shown to be resistant to the herbicide / antibiotic bromoxynil. Resistance genes for dicamba include dicamba monooxygenase genes (dmo), as disclosed in International PCT Publication No. WO 2008 / 105890. Resistance genes for PPO or PROTOX inhibitor herbicides (e.g., acifluorfen, fluazifop-butyl, fluazifop-butyl, pentothiopyralid, mesotrione, isopropylpyraclostrobin, pyraclostrobin, benitobar-butyl, azofopyralid, fluazifop-butyl, fluazifop-butyl, fluazifop-butyl, oxazolidinone, oxazolidinone, fluazifop-butyl, fluazifop-butyl, oxazolidinone, oxyfluorfen, lactofen, fomesulfuron, fluazifop-butyl, and sulfentrazone) are known in the art.Exemplary genes that confer resistance to PPO include overexpression of wild-type Arabidopsis PPO enzyme (Lermontova I and Grimm B, (2000) Overexpression of plastidic protoporphyrinogen IX oxidase leads to resistance to the diphenyl-ether herbicide acifluorfen. Plant Physiol 122:75–83.), B. subtilis PPO gene (Li, X. and Nicholl D. 2005. Development of PPO inhibitor-resistant cultures and crops. Pest Manag. Sci. 61:277-285 and Choi KW, Han O, Lee HJ, Yun YC, Moon YH, Kim MK, Kuk YI, Han SU and Guh JO, (1998) Generation of resistance to the diphenyl ether herbicide, oxyfluorfen, via expression of the Bacillus subtilis protoporphyrinogen oxidase gene in transgenic tobacco plants. [By expressing the Bacillus subtilis protoporphyrinogen oxidase gene in transgenic tobacco plants to generate resistance to the diphenyl ether herbicide oxyfluorfen-butyl] Biosci Biotechnol Biochem 62: 558–560). Resistance genes for pyridyloxy or phenoxypropionic acid and cyclohexanone include genes encoding ACCase inhibitors (e.g., Acc1-S1, Acc1-S2, and Acc1-S3). Exemplary genes that confer resistance to cyclohexanedione and / or aryloxyphenoxypropionic acid include haloxyfop-butyl, diclofenac, fenoxaprop-butyl acid, haloxyfop-butyl, and quizalofop-butyl. Finally, herbicides that can inhibit photosynthesis include triazines or benzonitrile, with tolerance provided by the psbA gene (tolerance to triazines), the ls+ gene (tolerance to triazines), and the nitrilase gene (tolerance to benzonitrile). The above list of herbicide tolerance genes is not meant to be limiting. The present disclosure encompasses any herbicide tolerance gene.

[0137] 3. Agronomic traits

[0138] Various agronomic trait genes can be targeted to be inserted into the genomic sequence of the present disclosure. Regulatory elements can be engineered into gene expression cassettes containing agronomic trait genes. The operably connected sequence can then be incorporated into the selected vector to allow identification and selection of transformed plants ("transformants"). Exemplary agronomic trait coding sequences are known in the art. As embodiments of agronomic trait coding sequences that can be operably connected to the regulatory elements of the present disclosure, the following traits are provided. The delayed fruit softening provided by the pg gene inhibits the production of polygalacturonase that causes the decomposition of pectin molecules in the cell wall, thereby causing delayed softening of the fruit. In addition, delayed acc gene fruit ripening / senescence inhibits the normal expression of the natural acc synthase gene, resulting in reduced ethylene production and delayed fruit ripening. The accd gene metabolizes the precursor of the fruit ripening hormone ethylene, resulting in delayed fruit ripening. Alternatively, the sam-k gene causes delayed ripening by reducing S-adenosylmethionine (SAM), a substrate for ethylene production. The drought stress tolerance phenotype provided by the cspB gene maintains normal cell function under water stress conditions by maintaining RNA stability and translation. Another example includes the EcBetA gene, which catalyzes the production of the osmotic protectant compound glycine betaine, which confers tolerance to water stress. In addition, the RmBetA gene catalyzes the production of the osmotic protectant compound glycine betaine, which confers tolerance to water stress. The bbx32 gene provides photosynthesis and yield increase, and the gene expresses a protein that interacts with one or more endogenous transcription factors to regulate the day / night physiological processes of the plant. Ethanol production can be increased by expressing the amy797E gene encoding a thermostable alpha-amylase, which can enhance the production of bioethanol by increasing the thermostability of the amylase used to degrade starch. Finally, the modified amino acid composition can be produced by the expression of the cordapA gene encoding a dihydropicolinate synthase, which increases the production of the amino acid lysine. The above list of agronomic trait coding sequences is not intended to be restrictive. The present disclosure encompasses any agronomic trait coding sequence.

[0139] 4. DNA Binding Proteins

[0140] Various DNA binding transgenic / heterologous coding sequences can be targeted to be inserted into the genomic sequence of the present disclosure. Regulatory elements can be engineered into gene expression cassettes containing DNA binding genes. The operably connected sequence can then be incorporated into the selected vector to allow identification and selection of transformed plants ("transformants"). Exemplary DNA binding protein coding sequences are known in the art. As embodiments of DNA binding protein coding sequences that can be operably connected to the regulatory elements of the present disclosure, the following types of DNA binding proteins may include: zinc fingers, TALEN, CRISPR and meganucleases. The list of the above DNA binding protein coding sequences is not intended to be restrictive. The present disclosure encompasses any DNA binding protein coding sequence.

[0141] 5. Small RNA

[0142] Various small RNA sequences can be targeted to be inserted into the genomic sequence of the present disclosure. Regulatory elements can be engineered into gene expression cassettes containing small RNA sequences. The operably linked sequence can then be incorporated into a selected vector to allow identification and selection of transformed plants ("transformants"). Exemplary small RNA traits are known in the art. As embodiments of small RNA coding sequences that can be operably linked to the regulatory elements of the present disclosure, the following traits are provided. For example, by inhibiting the production of ethylene via the ACO gene encoding the ethylene-forming enzyme, the delayed fruit ripening / senescence of the anti-efe small RNA delays ripening. By inhibiting endogenous S-adenosyl-L-methionine, the lignin production of the ccomt small RNA is changed, thereby reducing the content of guanidino (G) lignin: trans-caffeoyloxy CoA 3-O-methyltransferase (CCOMT gene). In addition, black spot bruise tolerance in Solanum verrucosum can be reduced by Ppo5 small RNA, which triggers the degradation of Ppo5 transcripts, thereby preventing the development of black spot bruises. Also included is the dvsnf7 small RNA, whose dsRNA contains a 240bp fragment of the Snf7 gene of the Western Corn Rootworm, which can inhibit the Western Corn Rootworm. Modified starch / carbohydrates can be produced by small RNAs, such as pPhL small RNA (degrading PhL transcripts to limit the formation of reducing sugars by starch degradation) and pR1 small RNA (degrading R1 transcripts to limit the formation of reducing sugars by starch degradation). In addition, the benefits also include a reduction in acrylamide content caused by asn1 small RNA, which triggers Asn1 degradation to impair the formation of asparagine and reduce polyacrylamide content. Finally, the non-brown phenotype of the pgas ppo suppression small RNA leads to the suppression of PPO to produce apples with a non-brown phenotype. The above list of small RNAs is not meant to be limiting. The present disclosure encompasses any small RNA coding sequence.

[0143] 6. Selectable markers

[0144] Various selectable markers, also described as reporter genes, can be targeted for insertion into the genomic sequence of the present disclosure. Regulatory elements can be engineered into gene expression cassettes containing reporter genes. The operably linked sequence can then be incorporated into the selected vector to allow identification and selection of transformed plants ("transformants"). There are many methods for confirming the expression of selectable markers in transformed plants, including, for example, DNA sequencing and PCR (polymerase chain reaction), Southern blotting, Northern blotting, immunological methods for detecting proteins expressed from vectors. However, reporter genes are usually observed by visual observation of proteins that produce colored products when expressed. Exemplary reporter genes are known in the art and encode β-glucuronidase (GUS), luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP, Phi-YFP), red fluorescent protein (DsRFP, RFP, etc.), β-galactosidase, etc. (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, New York, 2001, the contents of which are incorporated herein by reference in their entirety).

[0145] Transformed cells or tissues are selected using selectable marker genes. Selectable marker genes include genes encoding antibiotic resistance, such as genes encoding neomycin phosphotransferase II (NEO), spectinomycin / streptomycin resistance (AAD) and hygromycin phosphotransferase (HPT or HGR), and genes conferring resistance to herbicidal compounds. Herbicide resistance genes usually encode modified target proteins that are insensitive to herbicides, or encode enzymes that can degrade herbicides in plants or detoxify them before they work. For example, resistance to glyphosate has been obtained by using genes encoding mutant target enzymes 5-enolpyruvate shikimate-3-phosphate synthase (EPSPS). The genes and mutants of EPSPS are well known and are further described below. Resistance to glufosinate, bromoxynil and 2,4-dichlorophenoxyacetic acid (2,4-D) has been obtained by using bacterial genes encoding PAT or DSM-2, nitrilase, AAD-1 or AAD-12 (these are examples of proteins that detoxify their corresponding herbicides).

[0146] In an embodiment, herbicides can inhibit growing points or meristems, including imidazolinone or sulfonylurea, and acetohydroxy acid synthase (AHAS) and acetolactate synthase (ALS) resistance / tolerance genes for these herbicides are well known. Glyphosate resistance genes include mutant 5-enolpyruvate shikimate-3-phosphate synthase (EPSP) and dgt-28 genes (via introducing recombinant nucleic acid and / or various in vivo mutagenesis of natural EPSP genes), aroA gene and glyphosate acetyltransferase (GAT) gene, respectively. Other phosphono compounds resistance genes include bar and pat genes from streptomyces species, including Streptomyces hygroscopicus and Streptomyces viridichromogenes, and pyridyloxy or phenoxy propionic acid and cyclohexanone (genes encoding ACCase inhibitors). Exemplary genes that confer resistance to cyclohexanedione and / or aryloxyphenoxypropionic acid (including haloxyfop-butyl, diclofop-butyl, fenoxaprop-butyl acid, haloxyfop-butyl and quizalofop-butyl) include genes for acetyl-CoA carboxylase (ACCase); Acc1-S1, Acc1-S2 and Acc1-S3. In embodiments, herbicides may inhibit photosynthesis, including triazines (psbA and 1s+ genes) or benzonitrile (nitrilase gene). In addition, such selectable markers may include positive selection markers, such as phosphomannose isomerase (PMI) enzymes.

[0147] In embodiments, selectable marker genes include, but are not limited to, genes encoding: 2,4-D; neomycin phosphotransferase II; cyanamide hydratase; aspartate kinase; dihydropicolinate synthase; tryptophan decarboxylase; dihydropicolinate synthase and desensitized aspartate kinase; bar gene; tryptophan decarboxylase; neomycin phosphotransferase (NEO); hygromycin phosphotransferase (HPT or HYG); dihydrofolate reductase (DHFR); phosphinothricin acetyltransferase; 2,2-dichloropropionate dehalogenase; acetohydroxyacid synthetase; 5-enolpyruvate-shikimate-phosphate synthase (aroA); halogenated aryl nitrile hydrolase; acetyl-CoA carboxylase; dihydropterin synthase (sul I); and 32kD photosystem II polypeptide (psbA). One embodiment also includes a selectable marker gene encoding resistance to: chloramphenicol; methotrexate; hygromycin; spectinomycin; bromoxynil; glyphosate; and phosphinothricin. The above list of selectable marker genes is not intended to be limiting. The present disclosure encompasses any reporter gene or selectable marker gene.

[0148] In certain embodiments, synthetic coding sequence is to be optimally expressed in soybean plant.For example, in an embodiment, the coding sequence of gene has been modified by codon optimization to enhance expression in soybean plant.Insecticide resistance transgenic, herbicide tolerance transgenic, nitrogen use efficiency transgenic, water use efficiency transgenic, nutritional quality transgenic, DNA combined transgenic or selectable marker transgenic / heterologous coding sequence can be optimized to express in specific plant species, or alternatively above-mentioned transgenic / heterologous coding sequence can be modified to be optimally expressed in dicotyledonous or monocotyledonous plants.Plant preference codon can be determined from the codon with the highest frequency in the protein expressed in maximum amount in specific purpose plant species.In an embodiment, coding sequence, gene, heterologous coding sequence or transgenic / heterologous coding sequence are designed to be expressed with higher level in soybean plant, thereby cause higher transformation efficiency.The method of plant gene optimization is well known. Guidance on the optimization and generation of synthetic DNA sequences can be found, for example, in WO2013016546, WO 2011146524, WO 1997013402, US Pat. No. 6166302, and US Pat. No. 5380831 (incorporated herein by reference).

[0149] Molecular confirmation

[0150] Methods for confirming the presence of a gene expression cassette in the soybean plant genome are known in the art. For example, the gene expression cassette in the soybean plant genome can be detected, for example, by polymerase chain reaction (PCR). PCR detection is performed by using two oligonucleotide primers flanking the polymorphic polymorphic segment of the polymorphism, followed by DNA amplification. This step includes thermal denaturation of the DNA, followed by annealing the primers to their complementary sequences at low temperatures, and repeated cycles of extending the annealed primers with a DNA polymerase. Size separation of DNA fragments on agarose or polyacrylamide gels after amplification is an essential part of the method. Such selection and screening methods are well known to those skilled in the art. Molecular confirmation methods that can be used to identify plants are known to those skilled in the art. Several exemplary methods are further described below.

[0151] Molecular beacons for use in sequence detection have been described. In short, a FRET oligonucleotide probe is designed that overlaps the flanking genomic and insert DNA. The unique structure of the FRET probe causes it to contain a secondary structure that keeps the fluorescent and quenching moieties in close proximity. The FRET probe and PCR primers (one primer in the insert DNA sequence and one in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. After successful PCR amplification, hybridization of one or more FRET probes to the target sequence results in removal of the probe secondary structure and spatial separation of the fluorescent and quenching moieties. The fluorescent signal indicates the presence of the flanking genomic / transgenic insert sequence due to successful amplification and hybridization. Such a molecular beacon assay for detecting an amplification reaction is an embodiment of the present disclosure.

[0152] Hydrolysis probe assay, or (Life Technologies, Foster City, CA), is a method for detecting and quantifying the presence of a DNA sequence. Briefly, a FRET oligonucleotide probe is designed with one portion of the oligonucleotide within the transgene and the other portion in the flanking genomic sequence for event-specific detection. The FRET probe and PCR primers (one primer in the insert DNA sequence and one in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. During PCR amplification, the 5'-3' exonuclease activity of the Taq polymerase results in the hydrolysis of the FRET probe bound to the target sequence, thereby releasing the quenched fluorophore and allowing detection of a fluorescent signal. The fluorescent signal indicates the presence of the flanking / transgene insert sequence due to successful amplification and hybridization. Such a hydrolysis probe assay for detecting an amplification reaction is an embodiment of the present disclosure.

[0153] The assay is a method for detecting and quantifying the presence of a DNA sequence. Briefly, a genomic DNA sample containing a polynucleotide that has integrated a gene expression cassette is assayed using a polymerase chain reaction (PCR)-based assay (known as The method used in the practice of the present disclosure is as follows: The assay can utilize a PCR assay mixture. The primers used in the PCR assay mixture may include at least one forward primer and at least one reverse primer. The forward primer contains a sequence corresponding to a specific region of the DNA polynucleotide, and the reverse primer contains a sequence corresponding to a specific region of the genomic sequence. In addition, the primers used in the PCR assay mixture may include at least one forward primer and at least one reverse primer. For example, The PCR assay mixture may use two forward primers corresponding to two different alleles and one reverse primer. One of the forward primers contains a sequence corresponding to a specific region of the endogenous genomic sequence. The second forward primer contains a sequence corresponding to a specific region of the DNA polynucleotide. The reverse primer contains a sequence corresponding to a specific region of the genomic sequence. This method for detecting the amplification reaction is described in detail in detail below. Assay is one embodiment of the present disclosure.

[0154] In some embodiments, the fluorescent signal or fluorescent dye is selected from the group consisting of: HEX fluorescent dye, FAM fluorescent dye, JOE fluorescent dye, TET fluorescent dye, Cy3 fluorescent dye, Cy 3.5 fluorescent dye, Cy 5 fluorescent dye, Cy 5.5 fluorescent dye, Cy7 fluorescent dye, and ROX fluorescent dye.

[0155] In other embodiments, the amplification reaction is performed using a suitable second fluorescent DNA dye that can stain cellular DNA in a concentration range detectable by flow cytometry and has a fluorescence emission spectrum detectable by a real-time thermal cycler. It should be understood by those of ordinary skill in the art that other nucleic acid dyes are known and are being identified. Any suitable nucleic acid dye with a suitable excitation and emission spectrum can be used, such as SYTOX SYBR Green and

[0156] In a further embodiment, next generation sequencing (NGS) can be used for detection. As described in Brautigma et al., 2010, DNA sequence analysis can be used to determine the nucleotide sequence of the isolated and amplified fragments. The amplified fragments can be separated and subcloned into a vector and sequenced using a chain terminator method (also known as Sanger sequencing) or dye terminator sequencing. In addition, the amplicon can be sequenced using next generation sequencing. NGS technology does not require a subcloning step and can complete multiple sequencing reads in a single reaction. Three NGS platforms are commercially available, the genome sequencer FLX from 454 Life Sciences / Roche and the FLX 100 from Roche. TM , Illumina Genome Analyser from Solexa TM and Applied Biosystems’ SOLiD TM (Abbreviation: "sequencing by oligonucleotide ligation and detection"). In addition, two single-molecule sequencing methods are available. These includeTM True Single Molecule Sequencing (tSMS) from , and Single Molecule RealTime from Pacific Biosciences TM Sequencing (SMRT).

[0157] The FLX genome sequencer sold by 454 Life Sciences / Roche TM It is a long-read NGS that uses emulsion PCR and pyrosequencing to generate sequencing reads. DNA fragments of 300-800 bp or libraries containing 3-20 kb fragments can be used. The reaction can produce more than one million reads of approximately 250 to 400 bases per run, with a total output of 250 to 400 megabases. Compared with other NGS technologies, this technology can produce the longest reads, but the total sequence output per run is lower.

[0158] Solexa TM Illumina Genome Analyser for sale TM It is a short-read NGS that uses fluorescent dye-labeled reversible terminator nucleotides by synthesis method and based on solid phase bridge PCR sequencing. The construction of paired-end sequencing libraries containing up to 10kb DNA fragments can be used. The reaction produces more than 100 million short reads with a length of 35-76 bases. Each run can generate 3-6 billion bases of data.

[0159] Applied Biosystems TM The marketed sequencing oligonucleotide ligation and detection (SOLiD) system is a short-read technology. This NGS technology uses fragments of double-stranded DNA up to 10 kb. The system performs sequencing by ligating dye-labeled oligonucleotide primers and emulsion PCR to generate 1 billion short reads, with a total sequence output of up to 30 billion bases per run.

[0160] Helicos Bioscience TM tSMS and Pacific Biosciences TM SMRT takes a different approach, using a single DNA molecule to perform the sequence reaction. TM The system can generate up to 800 million short reads, totaling 21 billion bases per run. These reactions are completed using virtual terminator nucleotides labeled with fluorescent dyes, a method known as "synthesis sequencing."

[0161] Pacific Biosciences TMThe SMRT Next Generation Sequencing System is marketed using real-time sequencing by synthesis. This technology can generate read lengths up to 1,000 bp, as it is not limited by reversible terminators. This technology can generate a raw read throughput equivalent to double the coverage of the diploid human genome per day.

[0162] plant

[0163] In an embodiment, the soybean plant, plant tissue, or plant cell comprises a donor polynucleotide integrated within a genomic sequence of the present disclosure. In an embodiment, the soybean plant, plant tissue, plant part, or plant cell comprises a genomic sequence of the present disclosure or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to a sequence selected from SEQ ID NO: 206 or SEQ ID NO: 207. In an embodiment, the soybean plant, plant tissue, plant part, or plant cell comprises a donor polynucleotide integrated within a genomic sequence of the present disclosure. In one embodiment, the soybean plant, plant tissue, plant part or plant cell comprises a genomic sequence of the present disclosure, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to a sequence selected from SEQ ID NOs: 1-109 or SEQ ID NOs: 264-334. In embodiments, the soybean plant, plant tissue, plant part, or plant cell comprises a gene expression cassette integrated within a genomic sequence of the present disclosure, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% sequence identity to a sequence selected from SEQ ID NO: 206 or SEQ ID NO: 207, wherein the donor polynucleotide is operably linked to a transgene or a heterologous coding sequence that is an insecticide resistance transgene, an herbicide tolerance transgene, a nitrogen use efficiency transgene, a water use efficiency transgene, a nutritional quality transgene, a DNA binding transgene, a selectable marker transgene, or a combination thereof.In embodiments, the soybean plant, plant tissue, or plant cell comprises a gene expression cassette integrated within a genomic sequence of the present disclosure, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to a sequence selected from SEQ ID NOs: 1-109 or SEQ ID NOs: 264-334, wherein the donor polynucleotide is operably linked to a transgene or a heterologous coding sequence that is an insecticide resistance transgene, an herbicide tolerance transgene, a nitrogen use efficiency transgene, a water use efficiency transgene, a nutritional quality transgene, a DNA binding transgene, a selectable marker transgene, or a combination thereof.

[0164] Those skilled in the art will recognize that after the exogenous donor polynucleotide is stably incorporated into the genomic sequence of the present disclosure or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity with a sequence selected from SEQ ID NO: 206 or SEQ ID NO: 207 of a plant and confirmed to be operable, it can be introduced into other soybean plants by sexual crossing. In further embodiments, those skilled in the art will recognize that after the exogenous donor polynucleotide is stably incorporated into the genomic sequence of the present disclosure or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity with a sequence selected from SEQ ID NO: 1-109 or SEQ ID NO: 264-334 of a plant and confirmed to be operable, it can be introduced into other soybean plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending on the species to be crossed.

[0165] The present disclosure also encompasses seeds of the above-mentioned soybean plants, wherein the seeds have a transgenic / heterologous coding sequence or genetic construct containing the gene regulatory elements of the present disclosure. The present disclosure further encompasses progeny, clones, cell lines or cells of the above-mentioned soybean plants, wherein the progeny, clones, cell lines or cells have a transgenic / heterologous coding sequence or genetic construct containing the gene regulatory elements of the present disclosure.

[0166] The present disclosure also encompasses the cultivation of the above soybean plants, wherein the soybean plants have a transgenic / heterologous coding sequence or genetic construct containing a donor polynucleotide integrated within a genomic sequence of the present disclosure, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to a sequence selected from SEQ ID NO: 206 or SEQ ID NO: 207. In another embodiment, the present disclosure also encompasses the cultivation of the above soybean plants, wherein the soybean plants have a transgenic / heterologous coding sequence or genetic construct containing a donor polynucleotide integrated within a genomic sequence of the present disclosure, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity with a sequence selected from SEQ ID NO: 1-109 or SEQ ID NO: 264-334. Thus, such soybean plants can be engineered to have, among other things, one or more desired traits or events containing gene regulatory elements by transformation with a nucleic acid molecule according to the present invention, and can be tailored or cultivated by any method known to those skilled in the art.

[0167] Methods of expressing genes

[0168] In embodiments, a method of expressing at least one transgene / heterologous coding sequence in a soybean plant comprises growing a soybean plant comprising a donor polynucleotide integrated within a genomic sequence of the present disclosure, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to a sequence selected from SEQ ID NO: 206 or SEQ ID NO: 207. In embodiments, a method of expressing at least one transgene / heterologous coding sequence in a soybean plant comprises growing a soybean plant comprising a donor polynucleotide integrated within a genomic sequence of the present disclosure, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to a sequence selected from SEQ ID NOs: 1-109 or SEQ ID NOs: 264-334. In an embodiment, the method of expressing at least one transgene / heterologous coding sequence in a soybean plant tissue or soybean plant cell comprises culturing a soybean plant tissue or soybean plant cell comprising a transgene / heterologous coding sequence integrated as a donor polynucleotide within a genomic sequence of the present disclosure, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to a sequence selected from SEQ ID NO: 206 or SEQ ID NO: 207. In an embodiment, the method of expressing at least one transgene / heterologous coding sequence in a soybean plant tissue or soybean plant cell comprises culturing a soybean plant tissue or soybean plant cell comprising a transgene / heterologous coding sequence integrated as a donor polynucleotide within a genomic sequence of the present disclosure, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to a sequence selected from SEQ ID NOs: 1-109 or SEQ ID NOs: 264-334.

[0169] All references cited herein (including publications, patents, and patent applications) are hereby incorporated by reference to the extent not inconsistent with the explicit details of the present disclosure, and are incorporated to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and set forth herein in its entirety. The references discussed herein are provided only for their disclosures prior to the filing date of the present application. Nothing herein should be construed as an admission that the inventors are not entitled to antedate such disclosures by virtue of prior invention.

[0170] The embodiments of the present disclosure are further illustrated in the following examples. It should be understood that these examples are given by way of illustration only. From the above-described embodiments and the following examples, those skilled in the art can determine the essential characteristics of the present disclosure, and without departing from the spirit and scope of the present disclosure, various changes and modifications of the embodiments of the present disclosure can be made to adapt them to various uses and conditions. Therefore, from the above description, in addition to those shown and described herein, various modifications of the embodiments of the present disclosure are obvious to those skilled in the art. Such modifications are also intended to fall within the scope of the appended claims. The following content is provided by way of illustration and is not intended to limit the scope of the present invention.

[0171] In an embodiment, the soybean plant, plant tissue or plant cell comprises a donor polynucleotide integrated within a genomic sequence of the present disclosure. In an embodiment, the soybean plant, plant tissue or plant cell comprises a genomic sequence of the present disclosure or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity to a sequence selected from SEQ ID NO: 206 or SEQ ID NO: 207.

[0172] Examples

[0173] Example 1: Identification of genomic integration sites

[0174] Traditional transformation methods using random integration of exogenous DNA will produce materials with transgenes at genomic sites that are not optimal for downstream plant breeding applications. During the subsequent infiltration of these transgenes into elite germplasms, undesirable linkage DNA from the donor is introduced (e.g., linkage drag), which may have a negative impact on agriculturally important traits such as yield and biomass (Peng, T., Sun, X. and Mumm, RH Optimized breeding strategies for multiple trait integration: I. Minimizing linkage drag in single event introgression. [Optimized breeding strategies for multiple trait integration: I. Minimizing linkage drag in single event introgression] Mol Breeding [molecular breeding] 33, 89–104 (2014)). In order to mitigate this risk and improve breeding efficiency, when infiltrating transgenes and surrounding genomic regions into different elite germplasms, it is desirable to place the transgene in an area that has the least impact on performance.

[0175] The present disclosure provides a site-specific integration strategy for placing a transgene into a favorable genomic region, wherein the genomic integration site comprises the following features:

[0176] 1. Low genetic diversity across target germplasm,

[0177] 2. High expected recombination frequency,

[0178] 3. There is no large structural variation in the immediate vicinity, and

[0179] 4. Close to telomeres and away from pericentromeric regions.

[0180] Once a desired genomic integration site is selected, the target site can be identified and refined based on the proximity of the specific transformation target site to known genes and regulatory regions within the genomic region and the presence of sequence features required for integration.

[0181] At the genome level, genetic diversity is assessed by the presence and frequency of genome variants (e.g., single nucleotide polymorphisms (SNPs)) in individuals within a population. Although some regions of the genome are necessarily variable and drive phenotypic variation, there are other regions where diversity is limited or does not exist. Inferredly, when moving between two genetically different individual plants, the region where the dna sequence is identical will have the least impact on phenotype and genotype. These regions are desirable as genome integration sites because they will minimize the impact of linkage drag during infiltration.

[0182] To simplify genetic diversity assessment, the genome of a plant variety can be divided into bins based on genetic or physical coordinates and SNP groups. Typically, these bins are greater than or equal to 1 cM. Next, the bins can be analyzed and compared to bins of other plant varieties to be assigned as haplotypes. Bins that share similar SNP spectra within a bin are considered to have the same haplotype. The frequency of each haplotype across the target germplasm can then be calculated; those bins with no haplotype differentiation or bins with a high frequency of a single haplotype can be identified as low diversity regions.

[0183] In order to promote an effective transgenic infiltration strategy, it is desirable to select low diversity regions of large genetic size (e.g., >1 cM), while avoiding genomic segments that are difficult to reorganize, such as near-centromere regions that are relatively large in physical and genetic distance ratios. In addition, it is desirable to avoid regions where large structural variants (described structural variations include translocations, inversions, and deletions) in the genome may affect reorganization near the target site. The target region can be further refined based on its proximity to known QTLs, gene density in the region, and methylation levels. These features constitute the required genomic sequence features for site-specific integration.

[0184] Transgenics integrated in the genomic integration site with the above characteristics allow researchers to formulate effective infiltration strategies to minimize negative donor linkage drag. These regions are also ideal for strategies designed to stack multiple transgenic loci in a single region to form complex trait loci (Gao, H., Mutti, J., Young, JK, Yang, M., Schroder, M., Lenderts, B., and Feigenbutz, L. (2020). Complex Trait Loci in Maize Enabled by CRISPR-Cas9 Mediated Gene Insertion. [CRISPR-Cas9 mediated gene insertion realizes complex trait loci in maize] Frontiers in Plant Science [Plant Science Frontiers], 11, 535.).

[0185] Example 2: Genomic integration site criteria

[0186] The following methods were used to identify and obtain specific genomic integration sites.

[0187] Step 1: Genotype plant varieties using resequencing or by a high-density SNP genotyping platform to genotype varieties that represent the diversity within a closed breeding program.

[0188] Step 2: Using the genotyping information obtained, create a haplotype framework based on 1 cM genetic bins spanning the entire genome (Coffman, SM, Hufford, MB, Andorf, CM, and Lübberstedt, T. (2020). Haplotype structure in commercial maize breeding programs in relation to key founder lines. Theoretical and Applied Genetics, 133 (2), 547-561). Depending on the application, the genetic bins can be of various sizes, ranging from 5775 bp to 20,616,638 bp.

[0189] Step 3: Genetic diversity is assessed in the whole genome in the germplasm bank. For example, the haplotype frequency of the predefined haplotype created based on the high-density SNP spectrum in the 1 cM genetic box is used to assess genetic diversity. Other reliable diversity metrics include-nucleotide diversity (Nei and Lei, October 1, 1979 "Mathematical Model for Studying Genetic Variation in Terms of Restriction Endonucleases [Mathematical Model for Studying Genetic Variation in Terms of Restriction Endonucleases]". PNAS. [Proceedings of the National Academy of Sciences of the United States] 76 (10): 5269–73.) and haplotype diversity (Nei M (1987) Molecular evolutionary genetics. [Molecular evolutionary genetics] Columbia University Press [Columbia University Press], Columbia).

[0190] Step 4: Next, the genome is analyzed to identify regions with low diversity, hereafter referred to as low-diversity regions. These types of regions are 1 cM or longer in length, with a frequency of the major haplotype of 80%-100%. Low-diversity regions range in size from 5 kb to 2100 kb.

[0191] Step 5: Evaluate the favorable breeding characteristics of low-diversity regions. For example, low-diversity regions are ideally located in genomic segments where gene density is low and gene expression is stereotyped. In addition, such low-diversity regions will not be co-located or linked with large structural variants (such as translocations, deletions or inversions). These types of large structural variants can inhibit or reduce recombination within the target region, or prevent the target segment from effectively infiltrating another genome. In addition, low-diversity regions are greater than 10cM from genomic regions that are difficult to recombinant. Such regions that are difficult to recombinant are usually marked by low genetic and physical distance ratios. These types of genomic regions are associated with near-centromere regions and tend to have high repeat content. In contrast, low-diversity regions will appear near the telomeres of chromosomes, where the recombination rate tends to be higher and trait introgression can be simplified. Exemplary regions of low diversity include Chr02:4-9 cM (1,169,882-2,124,671 bp) and Chr01:13-20 cM (1,691,001-2,476,900) of the Williams82 genome assembly w82.a2.v1.

[0192] These genomic integration sites are novel sequences that have excellent qualities as regions for targeted insertion of polynucleotide fragments.

[0193] Example 3: Ideal target site analysis

[0194] According to the standard sequencing protocol, whole genome shotgun sequencing libraries of 45 soybean varieties were prepared. The libraries were sequenced on an Illumina HiSeq 2000 instrument to a depth of 15-41x sequencing depth. The sequence reads were aligned with the soybean genome assembly w82.a2.v1 by bowtie2, and compressed binary alignment / map (bam) files were generated from the alignment by SAMtools. Only unique read alignments were considered to identify SNPs, where the SNP sites needed to have coverage of at least three reads for >=50% of the sequencing lines, with an average purity of >=0.98. Further screening of SNP sites was performed by discarding those with high levels of missing genotypes to produce a core set of SNPs.

[0195] Another 1,693 soybean varieties were sequenced using low-pass Illumina sequencing, and genotypes were scored by checking the read alignment of core SNP sites. Thereafter, missing genotypes in all lines were estimated using FILLIN (Bradbury et al., 2007 TASSEL: software for association mapping of complex traits in diverse samples [TASSEL: software for complex traits association mapping in different samples], Bioinformatics [bioinformatics], Vol. 23, No. 19, October 1, 2007, pp. 2633–2635). Haplotypes of all samples in 1 cM boxes were identified and scored according to the method described by Coffman et al. 2020 Haplotype structure in commercial maize breeding programs inrelation to key founder lines. [The relationship between haplotype structure and key founder lines in commercial maize breeding programs] Theoretical and Applied Genetics [theory and applied genetics], 133 (2), 547-561. Centimorgan distances were calculated from a consensus genetic map adapted from Song et al. 2016 Construction of high resolution genetic linkage maps to improve the soybean genome sequence assembly Glyma1.01. BMC genomics, 17(1), 1-11.

[0196] Haplotype frequencies were calculated for each 1 cM bin for 1,738 soybean varieties. Bins with a major haplotype frequency greater than 80% were identified and annotated as low diversity bins. Low diversity bins were evaluated using the criteria listed in Example 1, and low diversity regions with ideal characteristics for site-specific integration were identified.

[0197] Example 4: Ideal target site, Chr01: 13-20cM (1,691,001-2,476,900) SEQ ID NO: 206

[0198] The region (13-20 cM) on Chr1 extending from approximately 1,691,001 to 2,476,900 bp has ideal characteristics for exogenous DNA insertion. The major haplotype frequencies across the 8 cM region range from 80% to 95%. In addition, this region is not linked to the major breeding target gene sequences, is located within a high recombination region, is greater than 20 cM from the putative heterochromatin region, and is not linked to known major structural variations based on 26 reference soybean assemblies (Liu et al. 2020 Pan-genome of wild and cultivated soybeans. Cell, 182 (1), 162-176). Table 2 provides a description of the characteristics and physical properties of Chr01:13-20.

[0199] Table 2: Physical properties of Chr01:13-20.

[0200]

[0201]

[0202] Example 5: Ideal target site, Chr02: 4-9cM (1,169,882-2,124,671bp) SEQ ID NO: 207

[0203] A region (4-9 cM) on Chr2 extending from approximately 1,169,882 to 2,124,671 bp also has ideal characteristics for exogenous DNA insertion. The major haplotype frequencies across the 6 cM region range from 92% to 97%. In addition, this region is not linked to the major breeding target gene sequences, is located in a high recombination region, is greater than 70 cM from the presumed heterochromatin region, is within 10 cM of the chr02 telomere, and is not linked to known major structural variations based on 26 reference soybean assemblies (Liu et al. 2020 Pan-genome of wild and cultivated soybeans. Cell, 182 (1), 162-176). Table 3 provides a description of the characteristics and physical properties of Chr02:4-9.

[0204] Table 3: Physical properties of Chr02:4-9.

[0205] area Chr02:4-9 Genetic size 6cM Physical size 954,789bp Near telomeres (<20 cM) yes Euchromatin yes Near heterochromatin (<10cM) No (>70cM) Haplotype (low) 92% frequency Haplotype (high) 97% frequency Near major QTL no Structural variation no

[0206] Example 6: Non-ideal target site, Chr01:48-54 (7,681,992-35,921,482 bp)

[0207] The region on Chr1 extending from 7,681,992 to 35,921,482 bp (48-54 cM) lacks the ideal characteristics for foreign DNA insertion. This region contains the centromeric and pericentromeric regions of Chr1 (Schmutz et al. 2010 Genome sequence of the palaeopolyploid soybean. Nature, 463(7278), 178-183). It is a region of low recombination rate marked by an average of 4,034,213 bp / 1 cM. Therefore, introgression of transgenes from this region into different genetic backgrounds usually results in offspring carrying large segments of the transgene donor's flanking DNA. In addition, pericentromeric regions tend to have lower levels of gene expression and contain a large number of repetitive elements (Du et al. 2012Pericentromeric effects shape the patterns of divergence, retention, and expression of duplicated genes in the paleopolyploid soybean. The Plant Cell, 24(1), 21-32), so the expression of genes inserted into this region may be inhibited or reduced.

[0208] In the germplasm studied, this region did not meet the "low diversity" criterion because the major haplotype frequencies ranged from 58% to 62% - a feature that would limit haplotype matching between donors and non-donors in this region and increase the chance of negative linkage drag during breeding. Finally, when the parents used in the cross are polymorphic for the variant locus, large structural variation was found in this genomic region on chromosome 1, which may reduce local recombination and thus reduce the effective introgression of transgenes located nearby (Liu et al. 2020 Pan-genome of wild and cultivated soybeans. Cell, 182(1), 162-176).

[0209] Example 7: Selection of soybean genomic windows for introduction of SSI target sites through guide RNA / Cas endonuclease system and complex trait locus development

[0210] In order to develop complex trait loci in soybean genome, a method for introducing transgenic SSI (site-specific integration) target sites in target soybean genomic loci using guide RNA / Cas9 or Cas12f1 nuclease systems was developed. First, a genomic window was identified in which adjacent multiple SSI target sites (CRISPR-Cas9 or Cas12f1 sites) could be introduced. Several chromosomal regions were identified, and after the diversity analysis described in the previous example, two soybean genomic regions (also referred to as genomic windows) were selected to produce complex trait loci. The first soybean genomic window for developing complex trait loci (CTL) spanned Chr01:1,730,000 to Chr01:2,513,000 on chromosome 1. Streptococcus pyogenes Cas endonuclease target sites (52 sites) within the genomic window were identified as being at least 2-2.5 kb from the transcription start site of any known gene and at least 500 bp from repetitive sequences, preferably more than 1 kb or 2 kb sites. Table 4 shows the physical and genetic map locations of these loci (Ganal, M. et al. (2011) PloS one [Public Administration Library 1], DOI: 10.1371).

[0211] Table 4: Genomic window on chromosome 1 of soybean containing the complex trait locus (CTL1).

[0212]

[0213]

[0214]

[0215] The second soybean genomic window selected for the development of complex trait loci (CTL) spans Chr02:1,117,000 to Chr02:2,129,999 on chromosome 2. The Streptococcus pyogenes Cas endonuclease target sites (58 sites) within the genomic window were identified as sites at least 2-2.5 kb from the transcription start site of any known gene and at least 500 bp, preferably 1 kb or 2 kb from the repeat sequence. Table 5 shows the physical and genetic map locations of these sites (Ganal, M. et al. (2011) PloS one [Public Administration Library Comprehensive], DOI: 10.1371).

[0216] Table 5: Genomic window on chromosome 2 of soybean containing complex trait locus 2.

[0217]

[0218]

[0219]

[0220] Example 8: DNA constructs of the guide RNA / Cas endonuclease system for genome modification in soybean

[0221] In order to achieve optimal expression and highest activity of the guide RNA / Cas nuclease system in soybean, the Cas9 gene (SEQ ID NO: 110) from Streptococcus pyogenes M1 GAS (SF370) was optimized for soybean codons according to standard techniques known in the art, and the potato ST-LS1 intron was introduced to eliminate the expression of the gene in Escherichia coli (E. coli) and Agrobacterium. In order to promote nuclear localization of the Cas9 protein in soybean cells, the Arabidopsis nuclear localization signal AT-NLS (SEQ ID NO: 111) and the carboxyl terminal nuclear localization signal of the dichotomous VirD2 T-DNA border nuclease of Agrobacterium tumefaciens (SEQ ID NO: 112) were incorporated into the amino and carboxyl termini of the Cas9 open reading frame, respectively. The soybean optimized Cas9 gene was operably linked to the soybean elongation factor (EF1A2) promoter by standard molecular biology techniques. The soybean U6-13.1 polymerase III promoter is used to express guide RNAs that guide the Cas9 nuclease to a specified genomic site (U.S. Patent Application 20180230476). The guide RNA coding sequence is 77bp long and contains a 20bp variable targeting domain from a soybean genomic target site selected on the 5' end soybean U6 polymerase III terminator (U.S. Patent Application 20180230476). Synthetic variable targeting (GenScript USA Inc., 860 Memorial Avenue, Piscataway, NJ 08854). As described in the previous examples, the guide RNA boxes for different target sites differ only in the variable target sequence. The variable target sequence in the guide RNA box always starts with G, and if G is not present in the first position in the soybean genome, G is used instead. Example 9: Vector cloning of guide RNA expression cassettes, Cas9 endonuclease expression cassettes, and donor DNA for introducing transgenic SSI target sites in soybean genomic windows

[0222] The soybean U6-13.1 polymerase III promoter (SEQ ID NO: 113) was used to express guide RNAs that directed the Cas9 nuclease to the specified genomic target sites (Tables 4 and 5). The soybean codon-optimized Cas9 endonuclease expression cassette, guide RNA expression cassette, and donor DNA (repair DNA) cassette were cloned into a T-DNA backbone containing a transformation selection marker via the Gateway system; the T-DNA was then electroporated into an AGL1 or LBA4404 thy- Agrobacterium strain. The donor DNA contained the FRT1 / FRT6 (87, 12) site flanked by the AT-UBQ10 promoter: PMI: UBQ14 terminator, which created a FRT1 / FRT6 (87, 12) target line for SSI technology application after gene integration by homologous recombination using the Cas9 / gRNA system. The CRISPR-Cas9 target site sequence from the corresponding soybean genomic site was placed outside the homology arm to improve insertion efficiency.

[0223] Tables 6 and 7 list the sequences of guide RNA / Cas9 DNA and donor DNA boxes targeting various soybean genomic sites constructed for the introduction of transgenic SSI sites into Cas endonuclease target sites by homologous recombination. All guide RNA / Cas9 constructs differ only in the 20 bp guide RNA variable targeting domain corresponding to the soybean genomic target site. All donor DNA constructs differ in two homology regions, such as arm 1 (HDR1) and arm 2 (HDR2), and CRISPR-Cas9 target sites. These guide RNA / Cas9 DNA constructs and donor DNA were delivered to the elite soybean genome 93Y21 by the Agrobacterium transformation procedure described in the previous example.

[0224] Table 6: Guide RNA / Cas9 and donor DNA for complex trait loci on Chr01 used in soybean transformation.

[0225]

[0226]

[0227] Table 7: Guide RNA / Cas9 and donor DNA for complex trait loci on Chr02 used in soybean transformation.

[0228]

[0229]

[0230] Example 10: Delivery of the Guide RNA / Cas9 Endonuclease System to Soybean via Agrobacterium Transformation

[0231] CRISPR components were delivered to the soybean genome by the transformation procedure described in WO 2020 005933. As described by Di et al. ((1996) Plant Cell Rep [Plant Cell Report] 15: 746-750), chlorine gas produced by mixing 3.5mL of 12N HCl with 100mL of commercial bleach (5.25% sodium hypochlorite) was used to surface sterilize mature dry seeds from soybean lines for 16 hours. The sterilized seeds were soaked in sterile distilled water for 16 hours at room temperature (100 seeds in 25x100 mm Petri dishes) and absorbed on a semi-solid medium containing 5g / L sucrose and 6g / L agar at room temperature in the dark. After incubation overnight, the seeds were soaked in distilled water in the dark for another 3 to 4 hours at room temperature. Complete embryo axes (EA) were separated from cotyledons. As described herein and disclosed in U.S. Patent Application No. 20180216123, Agrobacterium-mediated EA transformation was performed. WO 2020 005933 summarizes the composition of various cultivation media for soybean EA transformation and plant regeneration. A 10 mL volume of Agrobacterium suspension (OD 0.50 at 600 nm) in infection medium containing 300 mM acetosyringone was added to EA. Hypocotyls were co-cultured with Agrobacterium suspension containing Agrobacterium vectors. The culture medium was covered with parafilm ("Parafilm M TM "VWR catalog number 52858) and then sonicated (Sonicator-VWR Model 50T) for 30 seconds. After sonication, approximately 90-500 hypocotyls were transferred to a monolayer of autoclaved sterile filter paper (VWR #415 / catalog number 28320-020). The plates were sealed with micropore tape (catalog number 1530-0, 3M, St. Paul, MN) and incubated at 21°C in dim light (1-2 pE / m2 / s, white cool fluorescent light) for 16 hours for 3 days. After co-cultivation, the base of each EA was embedded in shoot induction (SI) medium containing 25 mg / L spectinomycin and 500 mg / L cefotaxime. Shoot induction was performed in a Percival biological incubator or growth chamber.

[0232] Example 11: Detection of target site mutations mediated by the guide RNA / Cas9 system in transiently transformed soybean EA

[0233] First, the guide RNA corresponding to the target site at the two genomic chromosomes was evaluated by transient testing. The transformation followed the same procedure as described in the previous example, but only until the bud clusters formed on the EA. As described in the previous example, ten days after infection, the bud clusters of the EA were removed by a scalpel. Six replicates were obtained, each containing five EA bud clusters for each guide / Cas9 vector (SEQ ID NO: 114-159). Following the supplier's plant extraction procedure, Sbeadex was used. TM The plant DNA extraction kit (Catalog No. NAP41620, LGC, Biosearch Technologies, Middleton, WI 53562) was used to extract genomic DNA from the bud cluster samples. The sequence of each CRISPR-Cas9 target site was amplified, and the resulting amplicon was analyzed by next-generation sequencing, with no less than 1 million reads for each sample. The genomic primers used to amplify each target site in NGS are listed in Table 10. The mutation frequency at each target site is listed in Tables 8 and 9. The target site mutation frequency is measured by dividing the total number of mutation reads by the total number of reads. Since most bud clusters were not transformed by Agrobacterium within ten days, a mutation frequency of 0.4%-5% in the transient test was considered to be a normal range. 21 of the 23 test guides showed good mutation frequencies and were suitable for insertion via stable transformation.

[0234] Table 8: Chromosome 1 target site mutations.

[0235]

[0236] Table 9: Chromosome 2 target site mutations.

[0237]

[0238]

[0239] Example 12: Detection of transgenic SSI target sites (SSILPs) at CRISPR sites in soybean complex trait loci

[0240] A subset of target sites at two chromosomal regions was selected to introduce SSI landing pads (SSILPs) via homology-directed DNA repair to establish SSI target sites, which can then be used to integrate desired trait genes via FLP recombinase-mediated cassette exchange (Gao, et al., 2020). Transformation followed the procedures described in the previous example, and after bud formation and rooting, T0 plants were produced and grown in flat soil in a controlled environment. When each Cas9 site was targeted, approximately 500 to 750 T0 plants were produced. Leaf punches were collected from each T0 plant and DNA was extracted using the same procedure described in Example 11. Use ligation PCR and Phusion Flash High-Fidelity PCR Master Mix TM (#F-548L Thermo Scientific) detects SSILP insertions in T0 plants. Plants that are both positive for 5' and 3' junctions (hereinafter referred to as 2x HDR) are selected to be transferred to pots and grown to maturity. The insertion frequency of each site (Table 10) is measured by dividing the number of 2xHDR T0 plants by the total number of T0 plants analyzed. At each junction, one primer is on a genomic sequence other than homology to the HDR arm sequence in the donor, and the other sequence is on the donor, and the primer sequences are listed in Table 11. Next, the 2xHDR T0 plants are transferred to pots and selfed to produce T1 seeds.

[0241] T1 seeds are planted in the flat and leaf punches are collected, and DNA is processed using the same method as for T0 plants.Connection PCR is carried out to confirm that the SSILP at each target site inserts. In addition, qPCR is also carried out to T1 samples.qPCR is used to check whether T-DNA exists in T1 plants, and whether Cas9, guide, selection marker, Ds-RED are checked on T-DNA. The phosphomannose isomerase (PMI) of soybean codon optimization is included in each SSILP, and the copy number of PMI is used to identify homozygous or hemizygous insertion at CRISPR site. DNA hybridization and sequencing (Southern by Sequencing, SbS) are used to further analyze the T1 plants that insert SSILP and no T-DNA at the target site based on qPCR determination. Plants that perfectly insert SSILP at CRISPR target site and do not have any T-DNA insertion in any part of genome are referred to as SbS green plants.

[0242] Table 10: CRISPR-Cas9-mediated insertion of SSILP selection sites in soybean complex trait loci

[0243]

[0244] The PMI sequences in the donors of the following loci Chr01-TS2, Chr02-TS18, Chr01-tS31 and Chr01-TS37 sites were different from those previously provided and were codon optimized using soybean-preferred codons. The PMI sequences were used for stable transformation and are provided in SEQ ID NOs: 208, 209, 210 and 211.

[0245] Table 11: Primer sequences used to amplify the junction of SSILP to each CRISPR / Cas9 target site

[0246]

[0247]

[0248] Example 13: Detection of gene expression at SSI target sites

[0249] In order to develop useful transgenic traits, genomic sites must be able to support transgenic expression. In order to assess the impact of the insertion site on transgenic expression, the protein expression level of the phosphomannose isomerase (PMI or PMI (S01)) gene in SSILP was measured in T2 plant leaves using ELISA. 16 homozygous seeds at each site were randomly planted in the flat, and leaf samples were collected at the 4-leaf stage. Table 12 shows the expression of PMI (S01) at 3 test sites, and all of these three sites support gene expression. Completed the additional expression of subsequent generations of plants (e.g., T2, T3, T4), and confirmed that the protein expression of PMI at soybean CTL remained robust in offspring plants.

[0250] Table 12: Protein expression of PMI gene at three tested targets in soybean CTL

[0251] Target site name Physical Location Genomic location (cM) PMI(ppm) Chr02-TS11 1438275 5.46 8783+859 Chr02-TS26 1743109 8.04 5473+317 Chr02-TS45 1990238 9.15 7016+593

[0252] Example 14: Cas12f1-mediated insertion into soybean complex trait loci

[0253] The Cas endonuclease from Syntrophomonas palmitatica Cas12f1 (SpaCas12f1) can also be used to generate double-strand breaks in the genome and promote genome editing. It recognizes NTTC at 5'PAM. The palm Cas endonuclease target site (71 sites) in the second soybean genome window spanning from Chr02:1117000 to Chr02:2129999 on chromosome 2 is identified as a site at least 2-2.5kb from the transcription start site of any known gene and at least 500bp from the repeat sequence, preferably 1kb or 2kb. Table 13 shows the physical and genetic map positions of these sites (Ganal, M. et al. (2011) PloS one [Public Administration Library Comprehensive], DOI: 10.1371).

[0254] Table 13: CRISPR-Cas12f1-mediated insertion sites in soybean complex trait loci.

[0255]

[0256]

[0257]

[0258] Although the present disclosure may be susceptible to various modifications and alternative forms, specific embodiments have been described in detail herein by way of example. However, it should be understood that the present disclosure is not intended to be limited to the specific forms disclosed. Rather, the present disclosure will cover all modifications, equivalents, and alternatives that fall within the scope of the present disclosure as defined by the following appended claims and their legal equivalents.

Claims

1. A soybean genomic integration site, the integration site comprising the following features: a. The size is greater than 5Kb; b. Low genetic diversity, wherein the low genetic diversity comprises haplotypes with a frequency greater than 80%; c. a high expected recombination frequency, wherein the recombination frequency is greater than 0.7 cM / 1 Mb; and d. In close proximity to telomeres, wherein the close proximity is less than 20 cM or less than 4.7 Mb from the end of the chromosome.

2. The soybean genomic integration site of claim 1, wherein the genomic integration site comprises SEQ ID NO: 206 or SEQ ID NO:

207.

3. The soybean genomic integration site of claim 1, wherein the genomic integration site comprises SEQ ID NO: 1-109 or SEQ ID NO: 264-334.

4. The soybean genomic integration site of claim 1, wherein the genetic size of the low diversity genomic region is less than 10 cM.

5. The soybean genomic integration site of claim 1, wherein the physical size of the low diversity genomic region is less than 2.6 Mb.

6. The soybean genomic integration site of claim 1, wherein the integration site comprises euchromatin.

7. The soybean genomic integration site of claim 1, wherein the distance between the integration site and heterochromatin is greater than 10 cM or 1.05 Mb.

8. The soybean genomic integration site of claim 1, wherein the haplotype frequency ranges from 80% to 100%.

9. The soybean genomic integration site of claim 1, wherein the integration site is greater than 5 cM from the QTL.

10. The soybean genomic integration site of claim 1, wherein the integration site does not occur in a region containing structural variations that inhibit genomic recombination.

11. The soybean genomic integration site of claim 1, wherein the integration site comprises a gene expression cassette.

12. The soybean genomic integration site of claim 11, wherein the gene expression cassette comprises an insecticide resistance gene, an herbicide tolerance gene, a nitrogen use efficiency gene, a water use efficiency gene, a nutritional quality gene, a DNA binding gene and / or a selectable marker gene.

13. The soybean genomic integration site of claim 1, wherein the integration site comprises at least one target site.

14. The soybean genomic integration site of claim 13, wherein the target site is cleaved by a site-specific nuclease.

15. The soybean genomic integration site of claim 14, wherein the site-specific nuclease is selected from the group consisting of a zinc finger nuclease, a CRISPR nuclease, a TALEN, a homing endonuclease, or a meganuclease.

16. The soybean genomic integration site of claim 1, wherein the integration site sequence is modified during insertion of donor DNA into the integration site sequence.

17. A soybean plant, soybean plant part or soybean plant cell comprising the recombinant sequence of any one of claims 1-16.

18. A method for preparing a transgenic soybean plant cell comprising a donor DNA targeted to a genomic integration site, the method comprising: a. selecting a target site within the genomic integration site; b. introducing a site-specific nuclease into a plant cell, wherein the site-specific nuclease cleaves the target site; c. introducing the donor DNA into the plant cell; d. targeting the donor DNA into the target site of the genomic integration site, wherein cleavage of the target site promotes integration of the donor DNA into the target site; and e. selecting a transgenic plant cell comprising said donor DNA targeted to said target site of said genomic integration site.

19. The method for preparing transgenic soybean plant cells of claim 18, wherein the donor DNA comprises a gene expression cassette.

20. The method for preparing transgenic soybean plant cells of claim 19, wherein the gene expression cassette comprises an insecticide resistance gene, a herbicide tolerance gene, a nitrogen use efficiency gene, a water use efficiency gene, a nutritional quality gene, a DNA binding gene and / or a selectable marker gene.

21. The method for preparing a transgenic soybean plant cell of claim 18, wherein the site-specific nuclease is selected from the group consisting of a zinc finger nuclease, a CRISPR nuclease, a TALEN, a homing endonuclease, or a meganuclease.

22. The method for preparing transgenic soybean plant cells of claim 18, wherein the donor DNA is integrated into the target site via a homology-directed repair integration method.

23. The method for preparing transgenic soybean plant cells of claim 18, wherein the donor DNA is integrated into the target site via a non-homologous end joining integration method.

24. The method for preparing a transgenic soybean plant cell of claim 18, wherein the genomic locus comprises the following characteristics: a.Size larger than 1cM; b. Low genetic diversity, wherein the low genetic diversity comprises haplotypes with a frequency greater than 80%; c. a high expected recombination frequency, wherein the recombination frequency is greater than 0.7 cM / 1 Mb; and d. In close proximity to telomeres, wherein the close proximity is less than 20 cM or less than 4.7 Mb from the end of the chromosome.

25. The method for preparing a transgenic soybean plant cell of claim 18, wherein the genomic integration site comprises SEQ ID NO: 206 or SEQ ID NO:

207.

26. The method for preparing a transgenic soybean plant cell of claim 18, wherein the genomic integration site comprises SEQ ID NO: 1-109 or SEQ ID NO: 264-334.

27. The method for preparing a transgenic soybean plant cell of claim 18, wherein the genetic size of the low diversity genomic region is less than 10 cM and ranges from 1 cM to 10 cM.

28. The method of making a transgenic soybean plant cell of claim 18, wherein the physical size of the low diversity genomic region is less than 2.6 Mb and ranges from 785,899 bp to 954,789 bp.

29. The method for preparing a transgenic soybean plant cell of claim 18, wherein the integration site comprises euchromatin.

30. The method for preparing a transgenic soybean plant cell of claim 18, wherein the distance between the integration site and heterochromatin is greater than 10 cM or 1.05 Mb.

31. The method for preparing transgenic soybean plant cells of claim 18, wherein the haplotype frequency ranges from 80% to 100%.

32. The method for preparing a transgenic soybean plant cell of claim 18, wherein the integration site is greater than 5 cM from the QTL.

33. The method for preparing a transgenic soybean plant cell of claim 18, wherein the integration site does not occur in a region containing structural variations that inhibit genomic recombination.

34. The method for preparing a transgenic soybean plant cell of claim 20, wherein the gene expression cassette expresses a gene.

35. The method for preparing a transgenic soybean plant cell of claim 34, wherein the gene is expressed at a concentration of 1 to 100,000 ppm.

36. The method for preparing a transgenic soybean plant cell of claim 18, wherein the transgenic plant cell is propagated into a transgenic soybean plant.

37. The method of making a transgenic soybean plant cell of claim 18, wherein the selected transgenic plant is used for breeding with another soybean plant to produce a progeny plant, wherein the method comprises: a. Identifying transgenic soybean plant cells containing donor DNA targeted to the genomic integration site; b. selecting a soybean plant having the donor DNA at the genomic integration site; c. hybridizing the selected soybean plants to produce offspring; as well as, d. Selecting progeny having the desired trait associated with the donor DNA at the genomic integration site.

38. A method for identifying a soybean genomic integration site for site-specific nuclease-mediated integration of a donor polynucleotide, the method comprising the steps of: a. obtaining a soybean genomic DNA sample from at least one soybean variety; b. genotyping the soybean genomic DNA sample; c. identifying the haplotype of the soybean genomic DNA sample; d. evaluating the genetic diversity of the haplotypes to identify haplotypes with low genetic diversity; e. Select haplotypes with low genetic diversity, which contain the following characteristics:

1. Size greater than 1cM; 2. Low genetic diversity, wherein the low genetic diversity comprises haplotypes with a frequency greater than 80%; 3. A high expected recombination frequency, wherein the recombination frequency is greater than 0.7 cM / 1 Mb; and, 4. Closely adjacent to telomeres, wherein close proximity is less than 20 cM or less than 4.7 Mb from the end of a chromosome; The soybean genomic integration site is targeted with a site-specific nuclease to integrate the donor polynucleotide into the soybean genomic integration site.

39. The method of identifying a soybean genomic integration site of claim 38, wherein the donor polynucleotide comprises a gene expression cassette.

40. The method of identifying a soybean genomic integration site of claim 39, wherein the gene expression cassette comprises an insecticide resistance gene, an herbicide tolerance gene, a nitrogen use efficiency gene, a water use efficiency gene, a nutritional quality gene, a DNA binding gene and / or a selectable marker gene.

41. The method of identifying a soybean genomic integration site of claim 38, wherein the site-specific nuclease is selected from the group consisting of a zinc finger nuclease, a CRISPR nuclease, a TALEN, a homing endonuclease, or a meganuclease.

42. The method of identifying a soybean genomic integration site of claim 38, wherein the donor DNA is integrated into the target site via a homology directed repair integration method.

43. The method of identifying a soybean genomic integration site of claim 38, wherein the donor DNA is integrated into the target site via a non-homologous end joining integration method.

44. The method of identifying a soybean genomic integration site of claim 38, wherein the genomic integration site comprises SEQ ID NO: 206 or SEQ ID NO:

207.

45. The method of identifying a soybean genomic integration site of claim 38, wherein the genomic integration site comprises SEQ ID NO: 1-109 or SEQ ID NO: 264-334.

46. ​​The method of identifying soybean genomic integration sites of claim 38, wherein the genetic size of the low diversity genomic region is less than 10 cM and ranges from 1 cM to 10 cM.

47. The method of identifying a soybean genomic integration site of claim 38, wherein the physical size of the low diversity genomic region is less than 2.6 Mb and ranges from 785,899 bp to 954,789 bp.

48. The method of identifying a soybean genomic integration site of claim 38, wherein the integration site comprises euchromatin.

49. The method for identifying a soybean genomic integration site of claim 38, wherein the integration site is greater than 10 cM or 1.05 Mb away from heterochromatin.

50. The method for identifying a soybean genomic integration site of claim 38, wherein said haplotype frequency ranges from 80% to 100%.

51. The method of identifying a soybean genomic integration site of claim 38, wherein the integration site is greater than 5 cM from the QTL.

52. The method for identifying a soybean genomic integration site of claim 38, wherein the integration site does not occur in a region containing structural variation that inhibits genomic recombination.

53. The method for identifying soybean genomic integration sites as claimed in claim 38, wherein the genotyping of the soybean genomic DNA sample is performed by sequencing or analyzing SNP markers.

54. The method for identifying a soybean genomic integration site of claim 40, wherein the gene expression cassette expresses a gene.

55. The method of identifying a soybean genomic integration site of claim 54, wherein the gene is expressed at a concentration of 1 to 100,000 ppm.

56. A soybean plant comprising a transgenic expression cassette inserted into a chromosomal locus in the soybean genome, wherein the chromosomal locus is located at SEQ ID NO: 1-109, 206, 207 or 264-334 on chromosome 1 or chromosome 2, and wherein the transgene is expressed in the soybean plant.

Images