Genome relocation of trait locus in soybean

Abstract

Provided herein are methods for modifying the genomic structure of a soybean plant cell. The method comprises introducing a targeted endonuclease into a soybean plant cell and introducing at least two double-strand breaks (DSBs) into chromosome(s) of the soybean plant cell. The DSBs cause translocation of at least one locus of interest. Also provided herein are modified soybean plants comprising modified chromosome(s).

Description

GENOME RELOCATION OF TRAIT LOCUS IN SOYBEANFIELD

[0001] The present disclosure is related to genomic relocation of trait loci in soybean (e.g., Glycine max) plants.INCORPORATION BY REFERENCE OF THE SEQUENCE LISITING

[0002] The official copy of the sequence listing is submitted electronically as an XML file named “108743-WO-SEC-1 Sequence Listing” created on June 12, 2024. The XML file has a size of 29,424 bytes, and the sequence listing is filed concurrently with this specification. The sequence listing contained in this XML file document is part of the present specification and is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION

[0003] Recombinant DNA technology has made it possible to insert DNA sequences at targeted genomic locations and / or modify specific endogenous chromosomal sequences. Site-specific integration techniques, which employ site-specific recombination systems, as well as other types of recombination technologies, have been used to generate targeted insertions of genes of interest in a variety of organism. Genome-editing techniques such as designer zinc finger nucleases (ZFNs), transcription activator- 1 ike effector nucleases (TALENs), or homing meganucleases, are available for producing targeted genome perturbations, but these systems tend to have low specificity and employ designed nucleases that need to be redesigned for each target site, which renders them costly and time-consuming to prepare.

[0004] Newer technologies utilizing archaeal or bacterial adaptive immunity systems have been identified, called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), which comprise different domains of effector proteins that encompass a variety of activities (DNA recognition, binding, and optionally cleavage).

[0005] Genetic recombination is a main source of variability and is a foundation of conventional plant breeding. However, additional methods to increase capability in breeding practices are needed to reduce the impact of issues such as linkage drag and limitations on the ability to select for certain trait(s) without interfering with selection for other trait(s).SUMMARY OF THE INVENTION

[0006] Soybean (Glycine max) is a species of legume which produces edible beans containing agriculturally significant amounts of protein and fats / oils. Soybean breeding focuses on improving desirable traits (e.g., yield, disease resistance) either by selecting for such traits oragainst phenotypes which negatively impact the desirable traits. The | provides methods (and related modified plants) that allow for genetic loci of interest to be moved from their naturally occurring chromosomal position to another, non-natural chromosomal position. The methods and plants disclosed herein improve flexibility and options during breeding programs which were previously limited by the physical position of certain loci of interest in the genome.

[0007] Provided herein is a method for modifying the genomic structure of a soybean plant cell. The method comprises introducing a targeted endonuclease into a soybean plant cell and introducing a first double-strand break (DSB) in a first chromosome of the soybean plant cell and a second DSB in a second chromosome of the soybean plant cell. The method comprises effecting a first reciprocal translocation of two sequences, a first sequence that is distal to the first DSB and a second sequence which is distal to the second DSB, such that a portion of the first chromosome comprising the first sequence is translocated to the second chromosome, thereby creating a modified second chromosome, and a portion of the second chromosome comprising the second sequence is translocated to the first chromosome thereby creating a modified first chromosome. The translocated second sequence comprises a locus of interest. The method can optionally comprise introducing a third DSB in the modified first chromosome and a fourth DSB in the modified second chromosome to effect a second reciprocal translocation of a third sequence that is distal to the third DSB and fourth sequence that is distal to the fourth DSB. The second reciprocal translocation restores the modified first chromosome’s telomeric region, thereby generating a modified, restored first chromosome. The third DSB is distal to the translocated locus of interest and the modified, restored first chromosome retains the locus of interest.

[0008] Also provided herein is a method for modifying the genomic structure of a soybean plant cell. The method comprises introducing a targeted endonuclease into a diploid soybean plant cell comprising two copies of a first chromosome and two copies of a second chromosome and introducing a first double-strand break (DSB) into a first copy of the first chromosome and a second DSB into a first copy of the second chromosome. The method comprises effecting a first reciprocal translocation of two sequences, a first sequence that is distal to the first DSB and a second sequence which is distal to the second DSB, such that a portion of the first copy of the first chromosome is translocated to the first copy of the second chromosome thereby creating a modified second chromosome, and a portion of the first copy of the second chromosome comprising the second sequence is translocated to the first copy of the first chromosome, thereby creating a modified first chromosome. The second sequence comprises a locus of interest. The method optionally comprises introducing a third DSB in the modified first chromosome and a fourth DSB in a second homologous (e.g., wild-type (WT)) copy of the first chromosome to effect a second reciprocal translocation of a third sequencethat is distal to the third DSB and a fourth sequence that is distal to the fc restoring the modified first chromosome’s telomere to generate a modified, restored first chromosome. The third DSB is distal to the translocated locus of interest and the modified, restored first chromosome retains the locus of interest.

[0009] Regarding the above two methods, the reciprocal translocation effected by the first DSB and second DSB can be between chromosome 15 and chromosome 18. The locus of interest can comprise a soybean cyst nematode (SCN) QTL or a charcoal rot (CR) QTL. The second reciprocal translocation effected by the third DSB and fourth DSB can be between a modified chromosome 15 and a modified chromosome 18. Alternatively, the second reciprocal translocation effected by the third DSB and fourth DSB is between a modified chromosome 15 and a naturally occurring chromosome 15. The targeted endonuclease can utilize one or more guide RNA targeting one or more of the following genomic sequences: SEQ ID NOs: 1- 4. The method can comprise selecting a soybean plant cell that has undergone the first reciprocal translocation and, optionally, the second translocation and regenerating a soybean plant from the at least one selected soybean plant cell. The regenerated plant can be selffertilized to produce a segregating population. One or more molecular markers targeting a junction sequence can be used for the selection. The locus of interest, when located on the second chromosome, can be linked to a trait for which elimination of the trait via breeding is desired. The method can comprise comprising breeding the regenerated plant with another soybean plant. Breeding can result in a progeny plant comprising the modified genomic structure and lacking one or more of: gene(s) encoding the targeted endonuclease; gene(s) encoding guide RNAs; or modified chromosomes that have not been restored. The locus of interest can comprise an SCN QTL allele, and the breeding can be performed with a plant comprising a different SCN QTL allele located on chromosome 18, thereby producing a progeny plant with SCN resistance traits provided by both QTL alleles. The targeted endonuclease can comprise a CRISPR / Cas system, a zinc finger nuclease, a TALEN, or a meganuclease. The targeted endonuclease can utilize a guide RNA comprising one or more of the following sequences: SEQ ID NOs: 5-12. The targeted endonuclease can utilize a guide RNA comprising one or more of the following sequences: SEQ ID NOs: 5-8. The targeted endonuclease can utilize a guide RNA comprising one or more of the following sequences: SEQ ID NOs: 9-12. The targeted endonuclease can utilize a guide RNA comprising the spacer sequence of one or more of SEQ ID NOs: 5-8 and a tracrRNA sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the tracrRNA sequences of SEQ ID NOs: 9-12.

[0010] Also provided herein is a method for modifying the genomic structure of a soybean plant cell. The method comprises introducing a targeted endonuclease into a soybean plant cell and introducing a first double-strand break (DSB) in a first arm of a chromosome and asecond DSB in a second arm of the chromosome in the soybean plant cell, each the first and second arms into a distal portion and a centromeric portion. The method comprises effecting a first chromosomal inversion, such that the distal portion of the first arm is joined to the centromeric portion of the second arm thereby creating a modified second arm, and the distal portion of the second arm is joined to the centromeric portion of first arm thereby creating a modified first arm. The translocated distal portion of the second arm comprises a locus of interest. The method optionally comprises introducing a third DSB in the modified first arm that is distal to the translocated locus of interest and a fourth DSB in the modified second arm to effect a second chromosomal inversion such that the first arm telomeric region and second arm telomeric region are restored to generate a modified, restored first arm and a modified, restored second arm. The modified, restored first arm comprises the translocated locus of interest.

[0011] Also provided herein is a method for modifying the genomic structure of a soybean plant cell. The method comprises introducing a targeted endonuclease into a soybean plant cell and introducing a first double-strand break (DSB) in a first arm of a chromosome and a second DSB in the first arm of the chromosome. The first DSB is distal to a locus of interest, the second DSB is between the centromere and the locus of interest, and a first genomic sequence is a portion of genomic sequence between the first DSB and second DSB. The method comprises effecting a first chromosomal inversion of the first genomic sequence, thereby generating a modified chromosomal arm comprising an inversion of the first genomic sequence, which comprises the locus of interest. The method optionally comprises introducing a third DSB distal to the inverted locus of interest and a fourth DSB distal to the third DSB, effecting a second chromosomal inversion of a second genomic sequence located between the third DSB and fourth DSB, thereby restoring the chromosomal structure of the second genomic sequence on the modified chromosomal arm of the chromosome.

[0012] Regarding the above two methods, the methods can comprise selecting a soybean plant cell that has undergone the first chromosomal inversion and, optionally, the second chromosomal inversion and regenerating a soybean plant from the at least one selected soybean plant cell.

[0013] Also provided herein is a modified soybean plant. The modified soybean plant comprises a modified chromosome 15. The modified chromosome 15 comprises both a SCN QTL and a CR QTL. The SCN QTL is distal to the CR QTL.BRIEF DESCRIPTION OF DRAWINGS

[0014] Various aspects of the materials and methods disclosed and described herein can be better understood by reference to the accompanying figures. The figures are offered for illustrative purposes only and are not intended to limit the scope of the disclosure in any way.

[0015] Figure 1 shows an example a method disclosed herein. A first c soybean chromosome 15) is subjected to a first DSB and a second chromosome (e.g., soybean chromosome 18) is subjected to a second DSB. The second DSB is proximal, i.e., between the centromere and a locus of interest (e.g., an SCN QTL on Chr. 18), and the first DSB is distal, i.e., farther from the centromere than a second locus of interest (e.g., a CR QTL on Chr. 15). The first DSB and the second DSB effect a reciprocal translocation that results in a modified first chromosome comprising (a) both loci of interest and (b) a second chromosome telomeric region. A modified second chromosome comprises a telomeric region derived from the first chromosome.

[0016] Figure 2 shows a vector suitable for transformation into a soybean cell in methods disclosed herein. The vector provides a targeted endonuclease (e.g., a Cas9 or Cas12f) and at least two guide RNAs (e.g., sgRNAs or pairs of crRNAs and tracrRNAs). The endonuclease can comprise a plant-optimized polynucleotide encoding a Cas endonuclease.

[0017] Figure 3A shows an example of a method disclosed herein. A modified first chromosome (e.g., the modified chromosome 15 produced in Figure 1) is subjected to a third DSB. A modified second chromosome (e.g., the modified chromosome 18 produced in Figure 1) is subjected to a fourth DSB. The third DSB is distal to the locus of interest (e.g., the SCN QTL), and the fourth DSB is targeted to remove the translocated telomeric region from the modified second chromosome. The third DSB and the fourth DSB effect a reciprocal translocation that results in a restored, modified first chromosome comprising (a) both loci of interest and (b) the native telomeric region from the first chromosome (prior to its modification). A restored modified second chromosome comprises the native telomeric region from the second chromosome (prior to its modification).

[0018] Figure 3B shows an alternative to the portion of the method shown in Figure 3A. A modified first chromosome (e.g., that of Figure 1 , modified chromosome 15) is subjected to a third DSB. A homologous (e.g., wild-type) copy of the first chromosome (e.g., unmodified copy of chromosome 15) is subjected to a fourth DSB. The third DSB is distal to the locus of interest (e.g., the SCN QTL) on the modified first chromosome, and the fourth DSB is targeted to remove the telomeric region from the homologous chromosome. The third DSB and the fourth DSB effect a reciprocal translocation that results in a restored, modified first chromosome comprising (a) both loci of interest and (b) the first chromosome telomeric region sourced from the homologous chromosome copy. A modified homologous chromosome comprises the second chromosome telomeric region.

[0019] Figures 4 and 5 include two panels showing a method for moving two loci or QTLs of interest within the same chromosome. Specifically, Figure 4, shows a double stranded break (DSB) generated on the distal side (telomere side) of one QTL and a second DSB on the proximal side, between the centromere and the second QTL. The resulting pericentricchromosomal inversion (Inversion #1) would result in bringing both QTLs iOptionally, further steps can be taken to restore chromosomal structure by generating a second inversion (Optional inversion #2) shown in Figure 5.

[0020] Figures 6 and 7 include two panels showing a method for moving two loci of interest or QTLs within the same chromosome arm. Specifically, Figure 6 shows double stranded breaks that are generated on the telomeric side of each QTL, as shown. The resulting inversion (Inversion #1) brings the QTLs in closer proximity to each other. Optionally, further steps can be taken to restore chromosomal structure by generating the second inversion (inversion #2) as shown in Figure 7.DETAILED DESCRIPTION OF THE INVENTION

[0021] As breeding programs seek the addition of more traits and corresponding genetic loci of interest (e.g., QTLs (quantitative trait loci), specific genes, etc.), the genome of the resulting plants becomes more complicated. Desired traits and undesirable traits may be closely linked making their separation from each other via recombination unlikely (e.g., linkage drag). Additionally, multiple desirable traits may be present in such close genomic proximity that it is difficult to introduce both into a given variety if the traits are not already all located next to each other in a single parent. The present disclosure provides methods of relocating loci of interest that harbor traits for which incorporation into a breeding line (e.g., elite germplasm) is desired. In some examples, the methods co-locate traits adjacent to each other on the same chromosome. In some examples, the methods move a trait to a more advantageous position in the genome. In some examples, the methods move a first trait to allow a second trait to be selected for at or near the original locus of the first trait.

[0022] Trait stacking is a powerful approach allowing stacking multiple traits / genes in one genomic location, which is then inherited as a single genetic locus significantly simplifying breeding programs. If a desirable trait is associated with a specific and defined gene, it can be cloned into a plasmid vector, flanked with regions of homology and inserted into the desirable location of elite genotype using targeted endonuclease (e.g., CRISPR / Cas) technology through the homology directed repair (HDR) approach. However, if a trait is associated with a QTL that may span hundreds of kilobases or if the genomic locus underlying the trait is complex, its cloning becomes impractical. In some examples, the methods disclosed herein describe the application of targeted endonuclease technology for relocation of a locus of interest of any size into any desired location. This approach improves breeding efficiencies and enables new breeding methods as described below and in subsequent examples.

[0023] The locus relocation described herein - translocating a locus of interest (e.g., a QTL) from its original location to a different, non-natural location - provides several benefits. A first is to enable more efficient forward breeding, e.g., when the locus of interested is translocatedinto new chromosomal region that is adjacent or genetically linked to a seco. In this case, the traits encoded by the loci of interest can be selected for at the same time, thereby facilitating breeding plants comprising both loci of interest and saving marker and trait introgression resources. A second benefit is to free up the original location for additional breeding (e.g., without a dominant trait QTL, this locus will now be free for forward breeding, traditional breeding, or genomic selection / prediction of any other trait). For example, freeing up the original location allows for stacking not possible before if additional desirable alleles exist at the original location. A third benefit is that translocations can be designed to break any undesirable linkage relationships at the original locus. The disclosed translocation process can be done iteratively to both stack loci of interest and free up additional loci for breeding, allowing for greater diversity.

[0024] Locus relocation can be accomplished using targeted endonuclease technology through one, two, or more consecutive chromosomal translocations. Examples of this approach are schematically depicted in Figures 1 , 3A, and 3B. First, translocation(s) are performed between two non-homologous chromosomes to move a locus of interest, (e.g., a telomeric fragment comprising a QTL) to a desired location (Figure 1) on a chromosome different from the chromosome originally containing the QTL. A second translocation can be optionally performed to restore the structure of telomere(s) of the two chromosomes. The second translocation can be carried out by one of two alternative methods (Figures 3A and 3B).

[0025] Soybean cyst nematode (SCN) is an important pest in soybean production, responsible for consistent, severe losses in the US and worldwide. Charcoal rot (CR) is another disease responsible for significant yield losses. Selection of resistant alleles for these and other diseases is imperative for soy breeders to develop disease tolerant, high yielding varieties for commercial release.

[0026] The predominant SCN resistance allele, Rhg1-b, used in modern breeding and product development has nine tandemly repeated 31.2 kb segments containing four genes, three of which contribute to resistance. The entire QTL is greater than 300 kb and located in sub telomeric region of chromosome 18. The complexity of this QTL, the need to use this locus in breeding, and the desire for co-selection with other traits at different loci make the QTL an ideal candidate for use of in the methods disclosed herein. A CR resistance QTL is located in the sub telomeric region of chromosome 15. Since this QTL has not been fine mapped, it is also a good candidate for use in the methods disclosed herein. Further, as shown in the Examples, the methods disclosed herein can be used for SCN Rhg1-b locus relocation, thereby stacking this SCN trait with the CR trait on chromosome 15.

[0027] Genetic diversity can be increased at genomic regions that were previously under selection pressure after translocation of the locus of interest. Genetic diversity is essential forsustained agronomic improvement. However, breeding selection, particulcQTLs that condition large favorable effects, can decrease genetic diversity at both the selection target and across the flanking genomic region (e.g., linkage drag). Any decrease in genome-wide diversity is exacerbated by the need to select multiple genomic loci, such as the simultaneous selection for SCN and CR QTLs on chromosomes 18 and 15, respectively, mentioned above. Consolidating multiple loci to a single region using a QTL relocation approach can mitigate the impact of genomic selection on genome-wide diversity by allowing for selection of multiple traits in the same region. This eliminates pressure to select for plants that all have the same QTLs at multiple regions - which can lead to a breeding population that is fixed (contains little diversity) in these multiple genome loci. Thus, the disclosed methods enable opportunities to infuse new diversity at previously occupied (i.e., trait containing) genomic loci. For example, a line harboring a relocation of SCN resistance QTL to chromosome 15 will have the sub-telomeric portion of chromosome 18 available for new genetic diversity (whether introduced by classical breeding or introgression) as it will no longer be under intensive selection for SCN. For example, plants containing a translocated SCN resistance QTL can be crossed with plants that have one or more non-SCN haplotypes on chromosome 18 to develop progeny with alternative genetic diversity. Such alternate haplotype diversity at the chromosome 18 locus provides a foundation for yield improvement or other desirable traits via conventional breeding and / or introgression .

[0028] Terms used in the claims and specification are defined as set forth below (and throughout this specification) unless otherwise specified. It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0029] As used herein, “progeny” comprises any subsequent generation of a plant.

[0030] The term “crossed” or “cross” or “crossing” in the context of this disclosure means the fusion of gametes via pollination to produce progeny (i.e., cells, seeds, or plants). The term encompasses both sexual crosses (the pollination of one plant by another) and selfing (self- pollination, i.e., when the pollen and ovule (or microspores and megaspores) are from the same plant or genetically identical plants).

[0031] The term “introgression” refers to the transmission of a desired allele of a genetic locus from one genetic background to another. For example, introgression of a desired allele at a specified locus can be transmitted to at least one progeny plant via a sexual cross between two parent plants, where at least one of the parent plants has the desired allele within its genome. Alternatively, for example, transmission of an allele can occur by recombination between two donor genomes, e.g., in a fused protoplast, where at least one of the donor protoplasts has the desired allele in its genome. The desired allele can be, e.g., a transgene, a modified (mutated or edited) native allele, or a selected allele of a marker or QTL.

[0032] "Introducing" is intended to mean presenting to a target, such as a polynucleotide or polypeptide or polynucleotide-protein complex, in such a manner that the component(s) gains access to the interior of a cell of the organism or to the cell itself. Methods that can accomplish such introductions include bacteria- mediated transformation and biolistic particle bombardment.

[0033] A “locus of interest” includes any coding or noncoding nucleotide sequence that improves desirability of crops, i.e. a trait of agronomic interest. Loci of interest include, but are not limited to, polynucleotides encoding traits for agronomics, herbicide-resistance, insecticidal resistance, disease resistance, nematode resistance, herbicide resistance, microbial resistance, fungal resistance, viral resistance, fertility or sterility, grain characteristics, commercial products, phenotypic marker, or any other trait of agronomic or commercial importance. Loci of interest can refer to individual genes or larger nucleotide sequences (e.g., QTLs).

[0034] When larger nucleotide sequences are employed as loci of interest, the methods disclosed herein can utilize a locus of interest that is at least 100 kbp in size such as, for example, at least 300 kbp in size, at least 500 kbp in size, at least 1 ,000 kbp in size, or at least 2,000 kbp in size. Thus, loci of interest too large for manipulation via transformation or homology-directed insertion can be translocated. Further, more than one locus of interest may be utilized together, or “stacked”, to provide additional benefits.

[0035] The compositions and methods herein can provide new genomic locations for desirable traits in a plant. As used herein, a “desirable trait” may be, but is not limited to, the following: disease resistance, drought tolerance, heat tolerance, cold tolerance, salinity tolerance, metal tolerance, herbicide tolerance, improved water use efficiency, improved nitrogen utilization, improved nitrogen fixation, pest resistance, herbivore resistance, pathogen resistance, yield improvement, health enhancement, vigor improvement, growth improvement, photosynthetic capability improvement, nutrition enhancement, altered protein content, altered oil content, increased biomass, increased shoot length, increased root length, improved root architecture, modulation of a metabolite, modulation of the proteome, increased seed weight, altered seed carbohydrate composition, altered seed oil composition, altered seed protein composition, or altered seed nutrient composition, as compared to an isoline plant not comprising a modification derived from the methods or compositions herein.

[0036] I. Methods of Locus Relocation Comprising Reciprocal Chromosomal Translocations

[0037] Provided herein is a method for modifying the genomic structure of a soybean plant cell. The method comprises introducing a targeted endonuclease into a soybean plant cell. The method comprises introducing a first double-strand break (DSB) in a first chromosome of the soybean plant cell and a second DSB in a second chromosome of the soybean plant cell. The first DSB and the second DSB can be introduced into their respective chromosomes viathe targeted endonuclease. The first DSB and the second DSB car translocation of a first sequence and a second sequence, respectively, by separating these sequences from the chromosome. The first translocated sequence is distal to the first DSB, and the second translocated sequence is distal to the second DSB.

[0038] The first DSB can optionally be located distal to a locus of interest present in the first chromosome. As used herein, “distal” is used to denote that the first DSB is farther from the centromere region than the locus of interest on that chromosome arm. In Figure 1 , chromosome 15 is provided as an example of a first chromosome, the CR QTL is provided as an example of a locus of interest, and the scissors above the CR QTL represent the site of the first DSB.

[0039] While Figure 1 shows an example of the disclosed method that results in stacking two loci of interest on the first chromosome, there need not be any particular locus of interest in the first chromosome. For example, the locus of interest initially present in the second chromosome may be relocated to the first chromosome to gain any other benefit of such relocation (i.e. , the disclosed relocation need not involve stacking multiple loci of interest).

[0040] The second DSB can be located proximal to a locus of interest present in the second chromosome. (Thus, the second translocated sequence can comprise the locus of interest). As used herein, “proximal” is used to denote that the second DSB is closer to the centromere region than the locus of interest on that chromosome arm. In Figure 1 , chromosome 18 is provided as an example of a second chromosome, the SCN QTL is provided as an example of a locus of interest, and the scissors below the SCN QTL represent the site of the second DSB which is proximal relative to the SCN locus of interest.

[0041] The first DSB and the second DSB can effect a first reciprocal translocation of the two sequences that are distal to each of the first DSB and second DSB. Because the second DSB is proximal to the locus of interest present on the second chromosome, the portion of the second chromosome that is translocated to the first chromosome will comprise the locus of interest. Similarly, the first DSB can be distal to the locus of interest on the first chromosome (when present) if the method is used to make a stack of the loci of interest on the modified first chromosome.

[0042] A portion of the first chromosome is translocated to the second chromosome thereby creating a modified second chromosome, and a portion of the second chromosome is translocated to the first chromosome thereby creating a modified first chromosome. The portion of the second chromosome comprises the locus of interest.

[0043] As shown in Figure 1 , the first translocation can result in both a first chromosome and a second chromosome that comprise telomeric regions which are derived from the other respective chromosome. Depending on the desired outcome of downstream processes, this chromosomal configuration may or may not be desirable. For example, if a plant is crossed(e.g., self-crossed) such that a progeny plant comprises only pairs of modi(e.g., the chromosomes produced by the process depicted in Figure 1), the progeny plant can perform well agronomically. However, an optional, second reciprocal translocation can be performed to restore the telomeric regions of the first and second chromosome. With the naturally occurring telomeric regions restored, the modified first and second chromosomes can more easily participate in crossing over and / or recombination between homologous chromosomes provided by other plant varieties. Accordingly, the following two optional strategies for performing the second reciprocal translocation are provided.

[0044] Strategy 1

[0045] In this optional strategy, a third DSB is made via the targeted endonuclease in the modified first chromosome. A fourth DSB is made via the targeted endonuclease in the modified second chromosome. The third DSB is distal to the locus of interest, such that the locus of interest remains on the modified first chromosome. The fourth DSB can be located near the junction between the second chromosome native sequence and the first telomeric sequence in order to reduce or eliminate first chromosome sequence that remains on the modified second chromosome after the second translocation. The third DSB and the fourth DSB can each effect the translocation of a third sequence and a fourth sequence, respectively, by separating these sequences from the respective chromosomes. The third translocated sequence is distal to the third DSB, and the fourth translocated sequence is distal to the fourth DSB.

[0046] The third DSB and fourth DSB can effect a second reciprocal translocation of the third and fourth translocated sequences that are distal to each of the third DSB and fourth DSB, respectively. The second reciprocal translocation restores first chromosome’s telomeric region to the modified first chromosome, thereby generating a modified, restored first chromosome. The third DSB is distal to the locus of interest and the modified, restored first chromosome retains the locus of interest. Figure 3A shows an example of this strategy consistent with a genome in which the events of Figure 1 were performed first (or performed in a parental genome).

[0047] Strategy 2

[0048] In this optional strategy, a third DSB is made via the targeted endonuclease in the modified first chromosome. A fourth DSB is made via the targeted endonuclease in a homologous (e.g., native or wild-type) copy of the first chromosome. The third DSB is distal to the locus of interest, such that the locus of interest remains on the modified first chromosome. The fourth DSB can be located in the homologous chromosome to provide a telomeric region that substantially replaces the telomeric region initially removed from the first chromosome.

[0049] The third DSB can effect a second reciprocal translocation of a third sequence that is distal to the third DSB, and the fourth DSB can effect the second reciprocal translocation of afourth sequence that is distal to the fourth DSB. The second reciprocal trar the first chromosome’s telomeric region to the modified first chromosome, thereby generating a modified, restored first chromosome. The modified, restored first chromosome retains the locus of interest. The homologous (e.g., native or wild-type) copy of the first chromosome, which now contains a second chromosome telomeric region, can optionally be removed via breeding techniques. Figure 3B shows an example of this strategy consistent with a genome in which the events of Figure 1 were performed first (or performed in a parental genome).

[0050] Additional Details Regarding the Methods of Locus Relocation Comprising Reciprocal Chromosomal Translocations

[0051] The methods disclosed herein can be performed used to translocate genomic sequence between any two non-homologous chromosomes. Any locus of interest located on a chromosome can be relocated to any non-homologous chromosome. In some examples, a locus of interest (e.g., a QTL) can be relocated from chromosome 18 to chromosome 15. In some examples, the locus of interest comprises a soybean cyst nematode (SCN) QTL or a charcoal rot (CR) QTL.

[0052] In some examples, an SCN QTL located on chromosome 18 is relocated to chromosome 15. In some examples, the SCN QTL is relocated to be adjacent to a CR QTL.

[0053] In some examples, the second reciprocal translocation is effected by the third DSB and fourth DSB. The second reciprocal translocation can be between a modified chromosome 15 and a modified chromosome 18.

[0054] In some examples, the reciprocal translocation effected by the third DSB and fourth DSB is between a modified chromosome 15 and a naturally occurring chromosome 15.

[0055] In some examples, the targeted endonuclease utilizes a guide RNA targeting one or more of the following genomic sequences: SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

[0056] In some examples, the regenerated plant, comprising modified chromosome(s) as described herein may be used in breeding methods. In such examples, the method can comprise selecting a soybean plant cell that has undergone the first reciprocal translocation and, optionally, the second translocation and regenerating a soybean plant from the at least one selected soybean plant cell. In some examples, the regenerated plant is self-fertilized to produce a segregating population.

[0057] Selection can be performed using molecular markers that are specific to sequences that are unique to the construct generated by the relocation. In some examples, one or more molecular markers targeting a junction sequence is used for the selecting. For example, referring to the Figure 1 , 3A, and 3B the junction sequence can be the intervening sequence between the CR and SCN QTLs, which are located adjacent to each other in the construct shown. Alternatively or additionally, a junction sequence spanning from the telomeric regionof chromosome 15 into the SCN QTL could also be used in examples translocation is employed. Use of such molecular markers for selection of progeny plants is described in more detail in Section VI.

[0058] In some examples, the method can comprise relocating a locus of interest in order to break linkage between the locus of interest and an undesirable trait. For example, the locus of interest, when located on the second chromosome, can be linked to a trait for which elimination of the trait via breeding is desired (e.g., a trait which increased disease susceptibility or decreases yield). By relocating the locus of interest, the locus of interest may be retained while elimination the genomic sequence encoding the undesirable trait.

[0059] In some examples, the method comprises breeding the regenerated plant with another soybean plant. This can be performed for introgression of locus of interest into a new plant variety. Additionally, breeding can be used to eliminate various genomic modifications introduced during the method. For example, a progeny plant comprising the modified genomic structure (e.g., the relocated locus of interest) and lacking one or more: gene(s) encoding the targeted endonuclease; gene(s) encoding guide RNAs; or modified chromosomes that have not been restored can be generative via breeding.

[0060] In examples where the locus of interest comprises an SCN QTL allele, breeding can be performed with a plant comprising a different SCN QTL allele located on chromosome 18, thereby producing a progeny plant with SCN resistance traits provided by both QTL alleles. In these cases, there can be a SCN QTL allele that has been relocated to chromosome 15 and a different native SCN QTL allele present on a chromosome 18 derived from a breeding partner. Such a plant is expected to show resistance to increased varieties of SCN.

[0061] II. Methods of Locus Relocation Comprising Chromosomal Inversions between Chromosomal Arms

[0062] Provided herein is a method for modifying the genomic structure of a soybean plant cell. The method comprises introducing a targeted endonuclease into a soybean plant cell. The method comprises introducing a first DSB in a first arm of a chromosome of the soybean plant cell and a second DSB in a second arm of the chromosome of the soybean plant cell. The first DSB and the second DSB can be introduced into their respective chromosomal arms via the targeted endonuclease. The first DSB and the second DSB can separate each of the first and second arms into a distal portion and centromeric portion.

[0063] The first DSB can optionally be located distal to a locus of interest present in the first arm. In Figure 4 QTL1 is provided as an example of an optional locus of interest, and the scissors above QTL1 represent the site of the first DSB.

[0064] There need not be any particular locus interest in the first arm. For example, the locus of interest initially present in the second arm may be relocated to the first arm to gain any other benefit of relocation, without necessarily stacking multiple loci of interest. Nonetheless, Figure4 shows an example including two loci of interest (QTL1 and QTL2), with < first and second chromosomal arms.

[0065] The second DSB can be located proximal to a locus of interest present in the second arm. In Figure 4, arm 2 is provided as an example of a second arm, QTL2 is provided as an example of a locus of interest, and the scissors proximal to QTL2 represent the site of the second DSB.

[0066] The first DSB and the second DSB can effect a first chromosomal inversion. The distal portion of the first arm can be joined to the centromeric portion of the second arm, thereby creating a modified second arm. A distal portion of the second arm can be joined to the centromeric portion of the first arm, thereby creating a modified first arm. The translocation distal portion of the second arm comprises a locus of interest. Referring to Figure 4, the locus of interest in the second arm is shown as QTL2.

[0067] As shown in Figure 4, the first chromosomal inversion results in both a first arm and a second arm that comprise telomeric regions which are derived from the other respective arm. Depending on the desired outcome of downstream processes, this chromosomal configuration may or may not be desirable. For example, if a plant is crossed (e.g., self-crossed) such that a progeny plant comprises only pairs of modified chromosomes (e.g., the product chromosome of Figure 4), the progeny plant may perform well, agronomically. However, an optional, second inversion can be performed to restore the telomeric regions of the first and second chromosomal arms. With the naturally occurring telomeric regions restored, the modified first and second chromosomes can more easily participate in crossing over and / or recombination between homologous chromosomes provided by other plant varieties. Additionally, the optional second inversion results in the locus of interest (and any additional loci stacked therewith) being translocated to the second arm, from the first arm. Accordingly, a strategy for performing the second inversion is provided (Figure 5).

[0068] In this optional strategy, a third DSB is introduced (e.g., via the targeted endonuclease) in the modified first arm that is proximal to the locus of interest and a fourth DSB in the modified second arm to effect a second chromosomal inversion such that the first arm telomeric region and second arm telomeric region are restored to generate a modified, restored first arm and a modified, restored second arm. The modified, restored first arm comprises the locus of interest.

[0069] In some examples, a soybean plant cell that has undergone the first chromosomal inversion and, optionally, the second chromosomal inversion can be selected. A soybean plant can be regenerated from the at least one selected soybean plant cell.

[0070] III. Methods of Locus Relocation Comprising Chromosomal Inversions within a Chromosomal Arm

[0071] Provided herein is a method for modifying the genomic structure < cell. The method comprises introducing a targeted endonuclease into a soybean plant cell. The method comprises introducing a first DSB in an arm of a chromosome and a second DSB in the arm of the chromosome, wherein the first DSB is distal to a locus of interest and the second DSB is proximal to the locus of interest.

[0072] The first DSB and the second DSB allow for an inversion of a first genomic sequence that is located between the two DSBs. The first genomic sequence comprises the locus of interest. Figure 6 shows QTL1 as the locus of interest, the location of which is changed via the inversion. Optionally the arm may contain an additional locus of interest (e.g., QTL2 in Figure 6). Thus, the inversion can produce a modified arm and / or a stack of loci of interest as shown in Figure 6.

[0073] As shown in Figure 6, the first chromosomal inversion results in a first arm that comprises an inverted genomic sequence. Depending on the desired outcome of downstream processes, this chromosomal configuration may or may not be desirable. For example, if a plant is crossed (e.g., self-crossed) such that a progeny plant comprises only pairs of modified chromosomes (e.g., the product chromosome of Figure 6), the progeny plant may perform well, agronomically. However, an optional, second inversion can be performed to reduce the disruption to native chromosomal sequence of the chromosomal arm. With the naturally occurring sequence restored (except for loci of interest), the modified arm can more easily participate in crossing over and / or recombination between homologous chromosomes provided by other plant varieties. Accordingly, a strategy for performing the second inversion is provided (Figure 7).

[0074] In the optional procedure a third DSB distal to the locus of interest and a fourth DSB distal to the locus of interest are introduced into the modified arm. Thus a second chromosomal inversion is effected. The inversion is of a second genomic sequence located between the third DSB and fourth DSB, thereby restoring the chromosomal structure of the second genomic sequence in the modified arm of the chromosome.

[0075] In some examples, a soybean plant cell that has undergone the first chromosomal inversion and, optionally, the second chromosomal inversion can be selected. A soybean plant can be regenerated from the at least one selected soybean plant cell.

[0076] IV. Targeted Endonuclease

[0077] The targeted endonuclease used in any of the methods provided herein can comprise a CRISPR / Cas system, a zinc finger nuclease, a TALEN, or a meganuclease.

[0078] Targeted endonucleases cleave the phosphodiester bond within a polynucleotide chain, which can result in the induction of DNA repair mechanisms, including the non- homologous end-joining pathway and homologous recombination. Targeted endonucleases include a range of different enzymes. These include: TAL effector nucleases or TALENs (seee.g., LIS20110145940, Christian, M., T. Cermak, et al. 2010. Targeting C breaks with TAL effector nucleases. Genetics 186(2): 757-61 and Boch et al., (2009), Science 326(5959): 1509-12); zinc finger nucleases (see e.g. Kim, Y. G., J. Cha, et al. (1996). "Hybrid restriction enzymes: zinc finger fusions to Fokl cleavage”); and CRISPR-Cas endonucleases (see e.g. W02007 / 025097 application published March 1 , 2007). Any double-strand-break or -nick or -modification inducing agent may be used for the methods described herein such as, for example, Cas endonucleases, TALENs, zinc finger nucleases, and meganucleases.

[0079] Meganucleases, also known as homing endonucleases (HEases), which like restriction endonucleases, bind and cut at a specific recognition site, however the recognition sites for meganucleases are typically longer, about 18 bp or more (patent application PCT / LIS 12 / 30061 , filed on March 22, 2012). Meganucleases have been classified into four families based on conserved sequence motifs, the families are the LAGLIDADG, GIY-YIG, H- N-H, and His-Cys box families. These motifs participate in the coordination of metal ions and hydrolysis of phosphodiester bonds. HEases are notable for their long recognition sites, and for tolerating some sequence polymorphisms in their DNA substrates. The naming convention for meganuclease is similar to the convention for other restriction endonuclease. Meganucleases are also characterized by prefix F-, I-, or PI- for enzymes encoded by freestanding ORFs, introns, and inteins, respectively. One step in the recombination process involves polynucleotide cleavage at or near the recognition site. The cleaving activity can be used to produce a double-strand break. For reviews of site-specific recombinases and their recognition sites, see, Sauer (1994) Curr Op Biotechnol 5:521-7; and Sadowski (1993) FASEB 7:760-7.

[0080] Zinc finger nucleases (ZFNs) are engineered targeted endonucleases comprising a zinc finger DNA binding domain and a double-strand-break-inducing agent domain. Recognition site specificity is conferred by the zinc finger domain, which typically comprising two, three, or four zinc fingers, for example comprising a C2H2 structure, however other zinc finger structures are known and have been engineered. Zinc finger domains are amenable for designing polypeptides which specifically bind a selected polynucleotide recognition sequence. ZFNs include an engineered DNA-binding zinc finger domain linked to a nonspecific endonuclease domain, for example nuclease domain from a Type Ils endonuclease such as Fokl. Additional functionalities can be fused to the zinc-finger binding domain, including transcriptional activator domains, transcription repressor domains, and methylases. In some examples, dimerization of nuclease domain is required for cleavage activity. Each zinc finger recognizes three consecutive base pairs in the target DNA. For example, a 3 finger domain recognized a sequence of 9 contiguous nucleotides, with a dimerization requirement of the nuclease, two sets of zinc finger triplets are used to bind an 18 nucleotide recognition sequence.

[0081] “CRISPR” (Clustered Regularly Interspaced Short Palindromic Rep certain genetic loci encoding components of DNA cleavage systems, for example, used by bacterial and archaeal cells to destroy foreign DNA (Horvath and Barrangou, 2010, Science 327:167-170; W02007025097, published 01 March 2007). A CRISPR locus can consist of a CRISPR array, comprising short direct repeats (CRISPR repeats) separated by short variable DNA sequences (called spacers), which can be flanked by diverse Cas (CRISPR-associated) genes.

[0082] The term “Cas protein” refers to a polypeptide encoded by a Cas (CRISPR-associated) gene. A Cas protein includes but is not limited to: a Cas9 protein, a Cpf1 (Cas12a) protein, a C2c1 protein, a C2c2 protein, a C2c3 protein, Cas3, Cas3-HD, Cas 5, Cas7, Cas8, Casio, or combinations or complexes of these. A Cas protein may be a “Cas endonuclease” or “Cas effector protein”, that when in complex with a suitable polynucleotide component, is capable of recognizing, binding to, and optionally nicking or cleaving all or part of a specific polynucleotide target sequence. A Cas endonuclease described herein comprises one or more nuclease domains. The endonucleases of the disclosure may include those having one or more RuvC nuclease domains. A Cas protein is further defined as a functional fragment or functional variant of a native Cas protein, or a protein that shares at least 50%, between 50% and 55%, at least 55%, between 55% and 60%, at least 60%, between 60% and 65%, at least 65%, between 65% and 70%, at least 70%, between 70% and 75%, at least 75%, between 75% and 80%, at least 80%, between 80% and 85%, at least 85%, between 85% and 90%, at least 90%, between 90% and 95%, at least 95%, between 95% and 96%, at least 96%, between 96% and 97%, at least 97%, between 97% and 98%, at least 98%, between 98% and 99%, at least 99%, between 99% and 100%, or 100% sequence identity with at least 50, between 50 and 100, at least 100, between 100 and 150, at least 150, between 150 and 200, at least 200, between 200 and 250, at least 250, between 250 and 300, at least 300, between 300 and 350, at least 350, between 350 and 400, at least 400, between 400 and 450, at least 500, or greater than 500 contiguous amino acids of a native Cas protein, and retains at least partial activity.

[0083] A “Cas endonuclease” may comprise domains that enable it to function as a double- strand-break-inducing agent. A “Cas endonuclease” may also comprise one or more modifications or mutations that abolish or reduce its ability to cleave a double-strand polynucleotide (dCas). In some aspects, the Cas endonuclease molecule may retain the ability to nick a single-strand polynucleotide (for example, a D10A mutation in a Cas9 endonuclease molecule) (nCas9).

[0084] A “functional fragment “, “fragment that is functionally equivalent” and “functionally equivalent fragment” of a Cas endonuclease are used interchangeably herein, and refer to a portion or subsequence of the Cas endonuclease of the present disclosure in which the abilityto recognize, bind to, and optionally unwind, nick or cleave (introduce a sing break in) the target site is retained. The portion or subsequence of the Cas endonuclease can comprise a complete or partial (functional) peptide of any one of its domains such as for example, but not limiting to a complete of functional part of a Cas3 HD domain, a complete of functional part of a Cas3 Helicase domain, complete of functional part of a Cascade protein (such as but not limiting to a Cas5, Cas5d, Cas7 and Cas8b1).

[0085] The terms “functional variant”, “variant that is functionally equivalent” and “functionally equivalent variant” of a Cas endonuclease or Cas effector protein are used interchangeably herein and refer to a variant of the Cas effector protein disclosed herein in which the ability to recognize, bind to, and optionally unwind, nick or cleave all or part of a target sequence is retained.

[0086] A Cas endonuclease may also include a multifunctional Cas endonuclease. The term “multifunctional Cas endonuclease” and “multifunctional Cas endonuclease polypeptide” are used interchangeably herein and includes reference to a single polypeptide that has Cas endonuclease functionality (comprising at least one protein domain that can act as a Cas endonuclease) and at least one other functionality, such as but not limited to, the functionality to form a cascade (comprises at least a second protein domain that can form a cascade with other proteins). In one aspect, the multifunctional Cas endonuclease comprises at least one additional protein domain relative (either internally, upstream (5’), downstream (3’), or both internally 5’ and 3’, or any combination thereof) to those domains typical of a Cas endonuclease.

[0087] The terms “target site”, “target sequence”, “target site sequence, ’’target DNA”, “target locus”, “genomic target site”, “genomic target sequence”, “genomic target locus”, “target polynucleotide”, and “protospacer”, are used interchangeably herein and refer to a polynucleotide sequence such as, but not limited to, a nucleotide sequence on a chromosome, episome, a locus, or any other DNA molecule in the genome (including chromosomal, chloroplastic, mitochondrial DNA, plasmid DNA) of a cell, at which targeted endonuclease complex can recognize, bind to, and optionally nick or cleave. The target site can be an endogenous site in the genome of a cell, or alternatively, the target site can be heterologous to the cell and thereby not be naturally occurring in the genome of the cell, or the target site can be found in a heterologous genomic location compared to where it occurs in nature.

[0088] A “protospacer adjacent motif” (PAM) herein refers to a short nucleotide sequence adjacent to a target sequence (protospacer) that is recognized (targeted) by a guide polynucleotide / Cas endonuclease system described herein. The Cas endonuclease may not successfully recognize a target DNA sequence if the target DNA sequence is not followed by a PAM sequence. The sequence and length of a PAM herein can differ depending on the Cas protein or Cas protein complex used. The PAM sequence can be of any length but is typically1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotk“plant-optimized Cas endonuclease” herein refers to a Cas protein, including a multifunctional Cas protein, encoded by a nucleotide sequence that has been optimized for expression in a plant cell or plant.

[0089] A “plant-optimized nucleotide sequence encoding a Cas endonuclease”, “plant- optimized construct encoding a Cas endonuclease” and a “plant-optimized polynucleotide encoding a Cas endonuclease” are used interchangeably herein and refer to a nucleotide sequence encoding a Cas protein, or a variant or functional fragment thereof, that has been optimized for expression in a plant cell or plant. A plant comprising a plant-optimized Cas endonuclease includes a plant comprising the nucleotide sequence encoding for the Cas sequence and / or a plant comprising the Cas endonuclease protein. In one aspect, the plant- optimized Cas endonuclease nucleotide sequence is a maize-optimized, rice-optimized, wheat-optimized, soybean-optimized, cotton-optimized, or canola-optimized Cas endonuclease.

[0090] The terms “single guide RNA" and “sgRNA” are used interchangeably herein and relate to a synthetic fusion of two RNA molecules, a crRNA (CRISPR RNA) comprising a variable targeting domain (linked to a tracr mate sequence that hybridizes to a tracrRNA), fused to a tracrRNA (trans-activating CRISPR RNA). The single guide RNA can comprise a crRNA or crRNA fragment and a tracrRNA or tracrRNA fragment of the type II CRISPR / Cas system that can form a complex with a type II Cas endonuclease, wherein said guide RNA / Cas endonuclease complex can direct the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, optionally bind to, and optionally nick or cleave (introduce a single or double-strand break) the DNA target site.

[0091] The terms “Guide RNA” or “gRNA” are used interchangeably herein and encompass both sgRNAs and guide RNA species comprising un-fused crRNA and tracrRNA components.

[0092] A “CRISPR / Cas system” as used herein includes a CRISPR Associated (Cas) endonuclease and at least one guide RNA or genes encoding the endonuclease and guide RNA. Class I Cas endonucleases comprise multisubunit effector complexes (Types I, III, and IV), while Class 2 systems comprise single protein effectors (Types II, V, and VI) (Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15; Zetsche et al., 2015, Cell 163, 1-13; Shmakov et al., 2015, Molecular Cell 60, 1-13; Haft et a / ., 2005, Computational Biology, PLoS Comput Biol 1(6): e60; and Koonin et al. 2017, Curr Opinion Microbiology 37 Q7-78). In Class 2 Type II systems, the Cas endonuclease acts in complex with a guide RNA (gRNA) that directs the Cas endonuclease to cleave the DNA target to enable target recognition, binding, and cleavage by the Cas endonuclease. The gRNA comprises a Cas endonuclease recognition (CER) domain that interacts with the Cas endonuclease, and a Variable Targeting (VT) domain that hybridizes to a nucleotide sequence in a target DNA. In some aspects, thegRNA comprises a CRISPR RNA (crRNA) and a trans-activating CRISPR guide the Cas endonuclease to its DNA target. The crRNA comprises a spacer region complementary to one strand of the double strand DNA target and a region that base pairs with the tracrRNA, forming an RNA duplex. In some aspects, the gRNA is a “single guide RNA” (sgRNA) that comprises a synthetic fusion of crRNA and tracrRNA. In many systems, the Cas endonuclease-guide polynucleotide complex recognizes a short nucleotide sequence adjacent to the target sequence (protospacer), called a “protospacer adjacent motif’ (PAM).

[0093] Examples of a Cas endonuclease include but are not limited to Cas9 and Cpf1 . Cas9 (formerly referred to as Cas5, Csn1 , or Csx12) is a Class 2 Type II Cas endonuclease (Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15). A Cas9-gRNA complex recognizes a 3’ PAM sequence (NGG for the S. pyogenes Cas9) at the target site, permitting the spacer of the guide RNA to invade the double-stranded DNA target, and, if sufficient homology between the spacer and protospacer exists, generate a double-strand break cleavage. Cas9 endonucleases comprise RuvC and HNH domains that together produce double strand breaks, and separately can produce single strand breaks. For the S. pyogenes Cas9 endonuclease, the double-strand break leaves a blunt end. Cpf1 is a Class 2 Type V Cas endonuclease and comprises nuclease RuvC domain but lacks an HNH domain (Yamane et al., 2016, Cell 165:949-962). Cpf1 endonucleases create “sticky” overhang ends.

[0094] Some uses for Cas9-gRNA systems at a genomic target site include but are not limited to insertions, deletions, substitutions, or modifications of one or more nucleotides at the target site; modifying or replacing nucleotide sequences of interest (such as a regulatory elements); insertion of polynucleotides of interest; gene knock-out; gene-knock in; modification of splicing sites and / or introducing alternate splicing sites; modifications of nucleotide sequences encoding a protein of interest; amino acid and / or protein fusions; gene silencing by expressing an inverted repeat into a gene of interest; and induction of chromosomal translocations and / or inversions.

[0095] In some aspects, a “polynucleotide modification template” is provided that comprises at least one nucleotide modification when compared to the nucleotide sequence to be edited. A nucleotide modification can be at least one nucleotide substitution, addition, deletion, or chemical alteration. Optionally, the polynucleotide modification template can further comprise homologous nucleotide sequences flanking the at least one nucleotide modification, wherein the flanking homologous nucleotide sequences provide sufficient homology to the desired nucleotide sequence to be edited.

[0096] In some aspects, a polynucleotide of interest is inserted at a target site and provided as part of a “donor DNA” molecule. As used herein, “donor DNA” is a DNA construct that comprises a polynucleotide of interest to be inserted into the target site of a Cas endonuclease. The donor DNA construct further comprises a first and a second region ofhomology that flank the polynucleotide of interest. The first and second re of the donor DNA share homology to a first and a second genomic region, respectively, present in or flanking the target site of the cell or organism genome. The donor DNA can be tethered to the guide polynucleotide. Tethered donor DNAs can allow for co-localizing target and donor DNA, useful in genome editing, gene insertion, and targeted genome regulation, and can also be useful in targeting post-mitotic cells where function of endogenous HR machinery is expected to be highly diminished (Mali et al., 2013, Nature Methods Vol. 10: 957- 963). The amount of homology or sequence identity shared by a target and a donor polynucleotide can vary and includes total lengths and / or regions.

[0097] The process for editing a genomic sequence at a Cas9-gRNA double-strand-break site with a modification template generally comprises: providing a host cell with a Cas9-gRNA complex that recognizes a target sequence in the genome of the host cell and is able to induce a single- or double-strand-break in the genomic sequence, and optionally at least one polynucleotide modification template comprising at least one nucleotide alteration when compared to the nucleotide sequence to be edited. The polynucleotide modification template can further comprise nucleotide sequences flanking the at least one nucleotide alteration, in which the flanking sequences are substantially homologous to the chromosomal region flanking the double-strand break. Genome editing using double-strand-break-inducing agents, such as Cas9-gRNA complexes, has been described, for example in LIS20150082478 published on 19 March 2015, WO2015026886 published on 26 February 2015, W02016007347 published 14 January 2016, and W02016025131 published on 18 February 2016.

[0098] To facilitate optimal expression and nuclear localization for eukaryotic cells, the gene comprising the Cas endonuclease may be optimized as described in WO2016186953 published 24 November 2016, and then delivered into cells as DNA expression cassettes by methods known in the art. In some aspects, the Cas endonuclease is provided as a polypeptide. In some aspects, the Cas endonuclease is provided as a polynucleotide encoding a polypeptide. In some aspects, the guide RNA is provided as a DNA molecule encoding one or more RNA molecules. In some aspects, the guide RNA is provided as RNA or chemically modified RNA. In some aspects, the Cas endonuclease protein and guide RNA are provided as a ribo nucleoprotein complex (RNP).

[0099] Once a double-strand break is induced in the genome, cellular DNA repair mechanisms are activated to repair the break.

[0100] In some examples, the targeted endonuclease can comprise a Cas endonuclease, which can be a Cas9, Cas12f, Cas12a, Cpf1 or variants thereof. See e.g., US Patent No. 10,934,536 and International Application Publication WO 2022 / 082179.

[0101] V. Modified soybean plants

[0102] Provided herein are modified soybean plants generated by the r herein. A modified soybean plant can comprise a modified chromosome 15. The modified chromosome 15 can comprise both a SCN QTL and a CR QTL. The SCN QTL and the CR QTL can be located immediately adjacent to each other.

[0103] In the modified soybean plant, the SCN QTL can be distal to the CR QTL. The SCN QTL can be proximal to the CR QTL.

[0104] Chromosome 18 of the modified soybean plant can or can not comprise an SCN QTL. The SCN QTL allele on chromosome 18 can be a different allele than the SCN QTL allele on chromosome 15. Such a configuration can provide enhanced resistance to SCN.

[0105] VI. Marker Assisted Selection for Relocated Loci of Interest

[0106] A relocated QTL can be selected simultaneously with a flanking QTL using molecular assays. To enable efficient development of new material, plant breeders use genotypic data from molecular assays to select plants with favorable genetic alleles, including those alleles that condition disease resistance, improve agronomic performance, and impart higher yield. Marker assisted selection of plants obviates the need for phenotypic observation, which saves costs and time during the breeding process.

[0107] As described in Example 3, Marker assisted selection (MAS) can be used to identify plants that comprise a newly formed translocation or inversion junction. For example, a junction between to newly joined QTLs.

[0108] “Genetic markers”, “markers”, or “molecular markers” are nucleic acids that are polymorphic in a population and where the alleles of which can be detected and distinguished by one or more analytic methods, described below. The terms also refer to nucleic acid sequences complementary to the genomic sequences, such as nucleic acids used as probes. Markers corresponding to genetic polymorphisms between members of a population can be detected by analytic methods such as, for example, PCR-based sequence specific amplification methods, detection of restriction fragment length polymorphisms (RFLP), detection of isozyme markers, detection of polynucleotide polymorphisms by allele specific hybridization (ASH), detection of amplified variable sequences of the plant genome, detection of self-sustained sequence replication, detection of simple sequence repeats (SSRs), detection of single nucleotide polymorphisms (SNPs), detection of amplified fragment length polymorphisms (AFLPs), or next generation sequencing techniques. Other appropriate methods include detection of expressed sequence tags (EST s) and SSR markers derived from EST sequences and randomly amplified polymorphic DNA (RAPD). In addition to nucleic acids, “Genetic markers”, “markers”, or “molecular markers” may also refer to proteins or inherited phenotypes whose causal genes are known or expected to be linked to a favorable allele.

[0109] A common measure of linkage is the frequency with which traits < can be expressed as a percentage of cosegregation (recombination frequency) or in centiMorgans (cM). The cM is a unit of measure of genetic recombination frequency. One cM is equal to a 1 % chance that a trait at one genetic locus will be separated from a trait at another locus due to crossing over in a single generation (meaning the traits segregate together 99% of the time). Because chromosomal distance is approximately proportional to the frequency of crossing over events between traits, there is an approximate physical distance that correlates with recombination frequency.

[0110] Marker loci are themselves traits and can be assessed according to standard linkage analysis by tracking the marker loci during segregation. Thus, one cM is equal to a 1% chance that a marker locus will be separated from another locus, due to crossing over in a single generation.

[0111] The closer a marker is to a gene controlling a trait of interest, the more effective and advantageous that marker is as an indicator for the desired trait. Closely linked loci display an inter-locus cross-over frequency of about 10% or less such as, for example, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, or about 2% or less. In some examples, the relevant loci (e.g., a marker locus and a target locus) display a recombination frequency of about 1% or less, e.g., about 0.75% or less, about 0.5% or less, or about 0.25% or less. Thus, the loci are about 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.75 cM, 0.5 cM or 0.25 cM or less apart. Put another way, two loci that are localized to the same chromosome, and at such a distance that recombination between the two loci occurs at a frequency of less than 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are said to be “proximal to” each other.

[0112] Although particular marker alleles can co-segregate with a disease resistance trait, it is important to note that the marker locus is not necessarily responsible for the expression of the disease resistance phenotype. For example, it is not a requirement that the marker polynucleotide sequence be part of a gene that is responsible for the disease resistant phenotype (for example, is part of the gene open reading frame). The association between a specific marker allele and the disease resistance trait is due to the original “coupling” linkage phase between the marker allele and the allele in the ancestral line from which the allele originated. Eventually, with repeated recombination, crossing over events between the marker and genetic locus can change this orientation. For this reason, the favorable marker allele may change depending on the linkage phase that exists within the parent comprising resistance to the disease that is used to create segregating populations. This does not change the fact that the marker can be used to monitor segregation of the phenotype. It only changes which marker allele is considered favorable in a given segregating population.

[0113] Methods presented herein include detecting the presence of on alleles associated with disease resistance in a plant and then selecting plants that have favorable alleles at those marker loci. Markers have been identified herein as being associated with the disease resistance trait and hence can be used to predict disease resistance in a plant. Any marker within 50 cM, 40 cM, 30 cM, 20 cM, 15 cM, 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.75 cM, 0.5 cM or 0.25 cM (based on a single meiosis based genetic map) could also be used to predict disease resistance in a plant.

[0114] Molecular markers can be used in a variety of plant breeding applications (e.g., see Staub et al. (1996) Hortscience 31 : 729-741 ; Tanksley (1983) Plant Molecular Biology Reporter. 1 : 3-8). One area of interest is to increase the efficiency of backcrossing and introgressing genes using MAS. A molecular marker that demonstrates linkage with a locus affecting a desired phenotypic trait provides a useful tool for the selection of the trait in a plant population. This is particularly true where the phenotype is hard to assay. Since DNA marker assays are less laborious and take up less physical space than field phenotyping, much larger populations can be assayed, increasing the chances of finding a recombinant with the target segment from the donor line moved to the recipient line. The closer the linkage, the more useful the marker, as recombination is less likely to occur between the marker and the gene causing the trait, which can result in false positives. Use of flanking markers decreases the chances that false positive selection will occur as a double recombination event would be needed. In some cases, a marker is located within the gene itself, so that recombination cannot occur between the marker and the gene. In some examples, the methods disclosed herein produce a marker in a disease resistance gene, wherein the gene was identified by inferring genomic location from clustering of conserved domains or a clustering analysis.

[0115] When a gene is introgressed by MAS, it is not only the gene that is introduced but also the flanking regions (Gepts. (2002). Crop Sci; 42: 1780-1790). This is referred to as “linkage drag.” In the case where the donor plant is highly unrelated to the recipient plant, these flanking regions carry additional genes that may code for agronomically undesirable traits. Linkage drag may also result in reduced yield or other negative agronomic characteristics even after multiple cycles of backcrossing into the elite line. This is also sometimes referred to as “yield drag.” The size of the flanking region can be decreased by additional backcrossing, although this is not always successful, as breeders do not have control over the size of the region or the recombination breakpoints (Young et al. (1998) Genetics 120:579-585). In classical breeding it is usually only by chance that recombinations are selected that contribute to a reduction in the size of the donor segment (Tanksley et al. (1989). Biotechnology 7: 257- 264). Even after 20 backcrosses in backcrosses of this type, one may expect to find a sizeable piece of the donor chromosome still linked to the gene being selected. With markers however, it is possible to select those rare individuals that have experienced recombination near thegene of interest. In 150 backcross plants, there is a 95% chance that at I have experienced a crossover within 1 cM of the gene, based on a single meiosis map distance. Markers will allow unequivocal identification of those individuals. With one additional backcross of 300 plants, there would be a 95% chance of a crossover within 1 cM single meiosis map distance of the other side of the gene, generating a segment around the target gene of less than 2 cM based on a single meiosis map distance. This can be accomplished in two generations with markers, while it would have required on average 100 generations without markers (See Tanksley et al., supra). When the exact location of a gene is known, flanking markers surrounding the gene can be utilized to select for recombinations in different population sizes. For example, in smaller population sizes, recombinations may be expected further away from the gene, so more distal flanking markers would be required to detect the recombination.

[0116] Implementation of MAS can include: (i) Defining the population within which the marker-trait association will be determined, which can be a segregating population, or a random or structured population; (ii) monitoring the segregation or association of polymorphic markers relative to the trait, and determining linkage or association using statistical methods; (iii) defining a set of desirable markers based on the results of the statistical analysis, and (iv) the use and / or extrapolation of this information to the current set of breeding germplasm to enable marker-based selection decisions to be made. The markers described in this disclosure, as well as other marker types such as SSRs and FLPs, can be used in marker assisted selection protocols.

[0117] SSRs can be defined as relatively short runs of tandemly repeated DNA with lengths of 6 bp or less (Tautz (1989) Nucleic Acid Research 17: 6463-6471 ; Wang et al. (1994) Theoretical and Applied Genetics, 88:1-6). Polymorphisms arise due to variation in the number of repeat units, probably caused by slippage during DNA replication (Levinson and Gutman (1987) Mol Biol Evol 4: 203-221). The variation in repeat length may be detected by designing PCR primers to the conserved non-repetitive flanking regions (Weber and May (1989) Am J Hum Genet. 44:388-396). SSRs are highly suited to mapping and MAS as they are multi-allelic, codominant, reproducible and amenable to high throughput automation (Rafalski et al. (1996) Generating and using DNA markers in plants. In: Non-mammalian genomic analysis: a practical guide. Academic press, pp 75-135).

[0118] Various types of SSR markers can be generated, and SSR profiles can be obtained by gel electrophoresis of the amplification products. Scoring of marker genotype is based on the size of the amplified fragment.

[0119] Various types of FLP markers can also be generated. Most commonly, amplification primers are used to generate fragment length polymorphisms. Such FLP markers are in many ways similar to SSR markers, except that the region amplified by the primers is not typically ahighly repetitive region. Still, the amplified region, or amplicon, will have ; among germplasm, often due to insertions or deletions, such that the fragments generated by the amplification primers can be distinguished among polymorphic individuals, and such indels are known to occur frequently in maize (Bhattramakki et al. (2002). Plant Mol Biol 48, 539- 547; Rafalski (2002b), supra).

[0120] SNP markers detect single base pair nucleotide substitutions. Of all the molecular marker types, SNPs are the most abundant, thus potentially providing the highest genetic map resolution (Bhattramakki et al. 2002 Plant Molecular Biology 48:539-547). SNPs can be assayed at an even higher level of throughput than SSRs, in a so-called 'ultra-high- throughput' fashion, as SNPs do not require large amounts of DNA and automation of the assay may be straight-forward. SNPs also have the promise of being relatively low-cost systems. These three factors together make SNPs highly attractive for use in MAS. Several methods are available for SNP genotyping and / or detection, including but not limited to, hybridization, primer extension, oligonucleotide ligation, nuclease cleavage, minisequencing, and coded spheres. Such methods have been reviewed in: Gut (2001) Hum Mutat 17 pp. 475- 492; Shi (2001) Clin Chem 47, pp. 164-172; Kwok (2000) Pharmacogenomics 1 , pp. 95-100; and Bhattramakki and Rafalski (2001) Discovery and application of single nucleotide polymorphism markers in plants. In: R. J. Henry, Ed, Plant Genotyping: The DNA Fingerprinting of Plants, CABI Publishing, Wallingford. A wide range of commercially available technologies utilize these and other methods to interrogate SNPs including Masscode. TM. (Qiagen), INVADER®. (Third Wave Technologies) and Invader PLUS®, SNAPSHOT®. (Applied Biosystems), TAQMAN®. (Applied Biosystems) and BEADARRAYS®. (Illumina).

[0121] A number of SNPs together within a sequence, or across linked sequences, can be used to describe a haplotype for any particular genotype (Ching et al. (2002), BMC Genet. 3:19 pp Gupta et al. 2001 , Rafalski (2002b), Plant Science 162:329-333). Haplotypes can be more informative than single SNPs and can be more descriptive of any particular genotype. For example, a single SNP may be allele “T' for a specific line or variety with disease resistance, but the allele 'T' might also occur in the breeding population being utilized for recurrent parents. In this case, a haplotype, e.g. a combination of alleles at linked SNP markers, may be more informative. Once a unique haplotype has been assigned to a donor chromosomal region, that haplotype can be used in that population or any subset thereof to determine whether an individual has a particular gene. Using automated high throughput marker detection platforms makes this process highly efficient and effective.

[0122] Many of the markers presented herein can readily be used as single nucleotide polymorphic (SNP) markers to select for the P450 gene. Using PCR, the primers are used to amplify DNA segments from individuals (e.g., an inbred) that represent the diversity in the population of interest. The PCR products are sequenced directly in one or both directions.The resulting sequences are aligned, and polymorphisms are identified. T are not limited to single nucleotide polymorphisms (SNPs), but also include indels, CAPS, SSRs, and VNTRs (variable number of tandem repeats). Specifically, with respect to the fine map information described herein, one can readily use the information provided herein to obtain additional polymorphic SNPs (and other markers) within the region amplified by the primers disclosed herein. Markers within the described map region can be hybridized to BACs or other genomic libraries, or electronically aligned with genome sequences, to find new sequences in the same approximate location as the described markers.

[0123] In addition to SSR's, FLPs and SNPs, as described above, other types of molecular markers are also widely used, including but not limited to expressed sequence tags (ESTs), SSR markers derived from EST sequences, randomly amplified polymorphic DNA (RAPD), and other nucleic acid based markers.

[0124] Isozyme profiles and linked morphological characteristics can, in some cases, also be indirectly used as markers. Even though they do not directly detect DNA differences, they are often influenced by specific genetic differences. However, markers that detect DNA variation are far more numerous and polymorphic than isozyme or morphological markers (Tanksley (1983) Plant Molecular Biology Reporter 1 :3-8).

[0125] Sequence alignments or contigs may also be used to find sequences upstream or downstream of the specific markers listed herein. These new sequences, close to the markers described herein, are then used to discover and develop functionally equivalent markers. For example, different physical and / or genetic maps are aligned to locate equivalent markers not described within this disclosure but that are within similar regions. These maps may be within the species, or even across other species that have been genetically or physically aligned.

[0126] In general, MAS uses polymorphic markers that have been identified as displaying a significant likelihood of co-segregation with a trait such as the SLB disease resistance trait and / or genes disclosed herein. Such markers are presumed to map near a gene or genes that give the plant its disease resistant phenotype, and are considered indicators for the desired trait, or markers. Plants are tested for the presence of a desired allele in the marker, and plants containing a desired genotype at one or more loci are expected to transfer the desired genotype, along with a desired phenotype, to their progeny. Thus, plants with SLB disease resistance may be selected for by detecting one or more marker alleles, and in addition, progeny plants derived from those plants can also be selected. Hence, a plant containing a desired genotype in a given chromosomal region (i.e. a genotype associated with disease resistance) is obtained and then crossed to another plant. The progeny of such a cross would then be evaluated genotypically using one or more markers and the progeny plants with the same genotype in a given chromosomal region would then be selected as comprising disease resistance. 1

[0127] The SNPs could be used alone or in combination (i.e. a SNP haplot favorable resistant gene allele associated with GLS disease resistance. For example, a SNP haplotype can include a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , or 12 of the markers for SLB resistance in Table 1 of this disclosure. The 2-12 markers can be (1) the K22 markers or (2) the CIMBL83 / CML304 markers.

[0128] The skilled artisan would expect that there might be additional polymorphic sites at marker loci in and around a chromosome marker identified by the methods disclosed herein, wherein one or more polymorphic sites is in linkage disequilibrium (LD) with an allele at one or more of the polymorphic sites in the haplotype and thus could be used in a marker assisted selection program to introgress a gene allele or genomic fragment of interest. Two particular alleles at different polymorphic sites are said to be in LD if the presence of the allele at one of the sites tends to predict the presence of the allele at the other site on the same chromosome (Stevens, Mol. Diag. 4:309-17 (1999)). The marker loci can be located within 5 cM, 2 cM, or 1 cM (on a single meiosis based genetic map) of the disease resistance trait QTL.

[0129] Allelic frequency (and hence, haplotype frequency) can differ from one germplasm pool to another. Germplasm pools vary due to maturity differences, heterotic groupings, geographical distribution, etc. As a result, SNPs and other polymorphisms may not be informative in some germplasm pools.

[0130] Clauses of the disclosure

[0131] The present disclosure will be more completely understood with reference to the following numbered clauses.1. A method for modifying the genomic structure of a soybean plant cell, the method comprising: introducing a targeted endonuclease into a soybean plant cell; introducing a first double-strand break (DSB) in a first chromosome of the soybean plant cell and a second DSB in a second chromosome of the soybean plant cell; effecting a first reciprocal translocation of two sequences, a first sequence that is distal to the first DSB and a second sequence which is distal to the second DSB, such that a portion of the first chromosome comprising the first sequence is translocated to the second chromosome thereby creating a modified second chromosome, and a portion of the second chromosome comprising the second sequence is translocated to the first chromosome thereby creating a modified first chromosome, wherein the translocated second sequence comprises a locus of interest; and optionally introducing a third DSB in the modified first chromosome and a fourth DSB in the modified second chromosome to effect a second reciprocal translocation of a third sequence that is distal to the third DSB and fourth sequence that is distal to the fourth DSB,wherein the second reciprocal translocation restores the modified first chrc telomeric region, thereby generating a modified, restored first chromosome, wherein the third DSB is distal to the translocated locus of interest and the modified, restored first chromosome retains the locus of interest.2. A method for modifying the genomic structure of a soybean plant cell, the method comprising: introducing a targeted endonuclease into a diploid soybean plant cell comprising two copies of a first chromosome and two copies of a second chromosome; introducing a first double-strand break (DSB) into a first copy of the first chromosome and a second DSB into a first copy of the second chromosome; effecting a first reciprocal translocation of two sequences, a first sequence that is distal to the first DSB and a second sequence which is distal to the second DSB, such that a portion of the first copy of the first chromosome is translocated to the first copy of the second chromosome thereby creating a modified second chromosome, and a portion of the first copy of the second chromosome comprising the second sequence is translocated to the first copy of the first chromosome, thereby creating a modified first chromosome, wherein the second sequence comprises a locus of interest; and optionally introducing a third DSB in the modified first chromosome and a fourth DSB in a second homologous copy of the first chromosome to effect a second reciprocal translocation of a third sequence that is distal to the third DSB and a fourth sequence that is distal to the fourth DSB, thereby restoring the modified first chromosome’s telomere to generate a modified, restored first chromosome; wherein the third DSB is distal to the translocated locus of interest and the modified, restored first chromosome retains the locus of interest.3. A method for modifying the genomic structure of a soybean plant cell, the method comprising: introducing a targeted endonuclease into a soybean plant cell; introducing a first double-strand break (DSB) in a first arm of a chromosome and a second DSB in a second arm of the chromosome in the soybean plant cell, thereby separating each the first and second arms into a distal portion and a centromeric portion; effecting a first chromosomal inversion, such that the distal portion of the first arm is joined to the centromeric portion of the second arm thereby creating a modified second arm, and the distal portion of the second arm is joined to the centromeric portion of first arm thereby creating a modified first arm, wherein the translocated distal portion of the second arm comprises a locus of interest; and optionally introducing a third DSB in the modified first arm that is distal to the translocated locus of interest and a fourth DSB in the modified second arm to effect asecond chromosomal inversion such that the first arm telomeric region anc telomeric region are restored to generate a modified, restored first arm and a modified, restored second arm; wherein the modified, restored first arm comprises the translocated locus of interest.4. A method for modifying the genomic structure of a soybean plant cell, the method comprising: introducing a targeted endonuclease into a soybean plant cell; introducing a first double-strand break (DSB) in a first arm of a chromosome and a second DSB in the first arm of the chromosome, wherein the first DSB is distal to a locus of interest, the second DSB is between the centromere and the locus of interest, and a first genomic sequence is a portion of genomic sequence between the first DSB and second DSB; effecting a first chromosomal inversion of the first genomic sequence, thereby generating a modified chromosomal arm comprising an inversion of the first genomic sequence, which comprises the locus of interest; and optionally introducing a third DSB distal to the inverted locus of interest and a fourth DSB distal to the third DSB, effecting a second chromosomal inversion of a second genomic sequence located between the third DSB and fourth DSB, thereby restoring the chromosomal structure of the second genomic sequence on the modified chromosomal arm of the chromosome.5. The method of clause 1 or 2 wherein the reciprocal translocation effected by the first DSB and second DSB is between chromosome 15 and chromosome 18.6. The method of any one of clauses 1 , 2, or 5, wherein the locus of interest comprises a soybean cyst nematode (SCN) QTL or a charcoal rot (CR) QTL.7. The method of any one of clauses 1 , 5, or 6 wherein the second reciprocal translocation effected by the third DSB and fourth DSB is between a modified chromosome 15 and a modified chromosome 18.8. The method of any one of clauses 2, 5, or 6 wherein the second reciprocal translocation effected by the third DSB and fourth DSB is between a modified chromosome 15 and a naturally occurring chromosome 15.9. The method of any one of clauses 1 , 2, or 5-8, wherein the targeted endonuclease utilizes a guide RNA targeting one or more of the following sequences: SEQ ID NOs: 1-4.10. The method of any one of clauses 3 or 4, further comprising: selecting a soybean plant cell that has undergone the first chromosomal inversion and, optionally, the second chromosomal inversion and regenerating a soybean plant from the at least one selected soybean plant cell.11 . The method of any one of clauses 1 , 2, or 5-10, further comprising:selecting a soybean plant cell that has undergone the first reciprocal optionally, the second translocation and regenerating a soybean plant from the at least one selected soybean plant cell.12. The method of clause 10 or 11 , wherein the regenerated plant is self-fertilized to produce a segregating population.13. The method of any one of clauses 10-12, wherein one or more molecular markers targeting a junction sequence is used for the selecting.14. The method of any one of clauses 11-13, wherein the locus of interest, when located on the second chromosome, is linked to a trait for which elimination of the trait via breeding is desired.15. The method of any one of clauses 10-14, further comprising breeding the regenerated plant with another soybean plant.16. The method of clause 15, wherein breeding results in a progeny plant comprising the modified genomic structure and lacking one or more of: gene(s) encoding the targeted endonuclease; gene(s) encoding guide RNAs; or modified chromosomes that have not been restored.17. The method of clause 15 or 16, wherein the locus of interest comprises an SCN QTL allele, and the breeding is performed with a plant comprising a different SCN QTL allele located on chromosome 18, thereby producing a progeny plant with SCN resistance traits provided by both QTL alleles.18. The method of any one of clauses 1-17, wherein the targeted endonuclease comprises a CRISPR / Cas system, a zinc finger nuclease, a TALEN, or a meganuclease.19. The method of any one of clauses 1-18, wherein the locus of interest is at least 100 kbp in size, at least 300 kbp in size, at least 500 kbp in size, at least 1,000 kbp in size, or at least 2,000 kbp in size.20. The method of any one of clauses 1 , 2, 5-9, or 11-18 wherein the targeted endonuclease utilizes a guide RNA comprising one or more of the following sequences: SEQ ID NOs: 5-12.21. A modified soybean plant comprising a modified chromosome 15, the modified chromosome 15 comprising both a SCN QTL and a CR QTL.22. The modified soybean plant of clause 19, wherein the SCN QTL is distal to the CR QTL.23. A soybean plant (or a seed, plant part, or cell thereof) comprising a genome edited translocation of the Rhg1 gene locus to a chromosome other than chromosome 18.24. The soybean plant (or seed, plant part, or cell thereof) of claus translocated Rhg1 gene locus is translocated to a locus that is genetically linked and / or distal to a QTL for a trait of interest.25. The soybean plant (or seed, plant part, or cell thereof) of clause 23, wherein the translocated Rhg1 gene locus is translocated to a locus that is distal to a QTL that provides resistance to charcoal rot disease.26. The soybean plant (or seed, plant part, or cell thereof) of clause 25, wherein the translocated Rhg1 gene locus is distal to the QTL on chromosome 15 disclosed herein which is associated with resistance to charcoal rot disease.27. The soybean plant (or seed, plant part, or cell thereof) of any one of clauses 23-26, wherein the translocated Rhg1 gene locus comprises an Rhgla allele or Rhgl b allele.28. A soybean plant (or a seed, plant part, or cell thereof) that comprises homozygous copies of (i) a Rhgla allele associated with SCN resistance and (ii) a Rhglb allele associated with SCN resistance.29. The soybean plant (or a seed, plant part, or cell thereof) of clause 28, wherein the homozygous Rhgla alleles are located on chromosome 18 and the homozygous Rhgl b alleles are located on chromosome 15.30. The soybean plant (or a seed, plant part, or cell thereof) of clause 28, wherein the homozygous Rhgla alleles are located on chromosome 15 and the homozygous Rhgl b alleles are located on chromosome 18.

[0132] EXAMPLES

[0133] The present disclosure will be more completely understood with reference to the following examples. The examples are presented to aid in understanding of the present disclosure and are not intended to limit the scope of the present disclosure.

[0134] Example 1 - Creating translocations between soybean chromosome 18 and chromosome 15 to link SCN and a CR QTLs

[0135] This example demonstrates how the disclosed methods can be used to perform translocations of chromosomal fragments. This example also provides a reciprocal translocation that was induced to link a complex repeat region on chromosome 18 associated with soybean cyst nematode (SCN) resistance (SCN QTL) with a charcoal rot (CR) QTL on chromosome 15. The telomeric region containing the SCN QTL (~300kb Rhgl b locus) was relocated to chromosome 15, distal to the ~1.2 Mb CR QTL, as shown in Fig. 1. Based on the SCN QTL location (delimited by flanking markers) and the locations of nearby genes as well as the locations of repeat annotations, candidate regions were chosen and prioritized for guide RNA design based on the requirement of a minimum of 2kb from gene annotations and 1kbfrom repeat annotations. To move the end of chromosome 18, including the end of chromosome 15, the target regions were considered off the 3’ end of the QTL (proximal to centromere).

[0136] The first translocation experiment was conducted using Agrobacterium-mediated delivery of Cas9, two gRNAs, and selectable marker gene expression cassettes (Figure 2) into soybean embryo cells. Two different T-DNAs were used in the experiment (T-DNA-1 and T-DNA-2). Each T-DNA contained a gRNA targeting a site on chromosome 15 and a gRNA targeting a site on chromosome 18 (Table 1). Table 2 shows the spacers sequences used in the sgRNAs (SEQ ID NOs: 5-8) and the full length sgRNAs (SEQ ID NOs: 9-12). Each spacer sequence and sgRNA sequence corresponds to the indicated target site. Spacer sequences were modified to comprise a 5’ G nucleotide residue, compared to the initial residue indicated in the target site sequence.

[0137] Table 1. Summary of the target sites used in the soybean chromosome 15 / 18 reciprocal translocation experiment. Cas9 PAM (protospacer adjacent motif) sites are underlined. TS means “Target Site”.

[0138]

[0139] Table 2. Guide RNA sequences.

[0140] The first translocation was performed between chromosomes 18 and 15 to move SCN QTL from chromosome 18 to the desired location on chromosome 15 (Figure 1). Results of this experiment are summarized in Table 3.

[0141] Table 3. Summary of the first soybean chromosome 15 / 18 reciprocal translocation experiment.

[0142] A total of 6016 (3008 for each of the two T-DNAs used) TO plants were regenerated and analyzed for the presence of a new junctions between chromosome 15 and chromosome 18. Expected junctions indicating relocation of chromosome 18 telomeric fragment to chromosome 15 and chromosome 15 telomeric fragment to chromosome 18 (reciprocal translocation) was observed in 41 TO plants when analyzed by qPCR using primers and probes listed in Table 4. Notably, very similar frequencies of events with reciprocal translocations were observed for the two T-DNA vectors used in the experiment.

[0143] Table 4. List of primers and probes used in qPCR analysis of novel junctions

[0144] Sequence analysis of new junctions was performed by NGS using primer pairs listed in Table 4 and demonstrated the expected sequences resulted from ligation of two chromosomal fragments either with no insertions or deletions (INDELs) or with small INDELs, which are typical for products of the double-stranded break repair pathway in plants.

[0145] Example 2 - A second translocation to restore telomeric ends and recombination potential

[0146] During meiosis, reciprocal translocations may decrease gamete viability due to unbalanced segregation of chromosomes during meiosis. This could limit the genetic potential of lines with these translocations as their progeny may have lower reproductive success. Additionally, recombination may be suppressed in translocated segments of the genome, which limits genetic gain over time. To mitigate such limitations, a second translocation is performed to restore the original telomeric regions of both chromosome 15 and chromosome 18. This second translocation exchanges the chromosome 18 telomere segment upstream of the SCN QTL that was relocated to chromosome 15, with the respective telomeric fragment of the chromosome 15 relocated to chromosome 18 (Figure 3A). The resulting plant has an otherwise normal genotype with only a small desired genomic region of -300 kb containing SCN resistant QTL relocated to chromosome 15.

[0147] In a different experiment of the second translocation, chromosor restored by translocating the telomeric regions between modified and wild type chromosome 15 (Figure 3B). In this case, the second translocation is performed in embryo cells heterozygous for the translocation.

[0148] Plants homozygous for the QTL stack can then be obtained in consecutive generation(s) via breeding techniques. Based on the second translocation plant material used, chromosome 18 may be either wild type (containing a naturally occurring SCN QTL) or modified - without an SCN QTL.

[0149] Example 3 - Optimizing breeding efficiency by selecting multiple genetic loci using a single molecular assay

[0150] Molecular assays for selecting the SCN resistant QTL (Rgh1 locus on chromosome 18) and a CR QTL on chromosome 15 have been described. See, for example, US Patent Publications 20180084745 and 20130337442 and US Patent 9,957,577, each of which is incorporated by reference herein. Identification of individuals that contain homozygous resistant alleles at both loci requires genotyping plants with separate molecular makers that are tightly linked to each locus. Given that, prior to the present disclosure, the SCN and the CR resistance QTLs have resided on different chromosomes and segregate independently, only small portion of individuals in a segregating population will contain the favorable allelic state at both loci.

[0151] In contrast, the translocation of the SCN QTL to chromosome 15 places the SCN QTL in c / s linkage with the CR QTL. Accordingly, a plant comprising a QTL stack in a single locus as described in Example 1 and 2 is screened for with increased efficiency. The two previously independent loci are now inherited together in segregating populations, which in turn, increases the number of progeny that are homozygous at both resistance loci. Since a larger number of individuals in a population will contain a desired genetic state at these two loci, breeders are more efficient during population development and plant selection.

[0152] Furthermore, marker-assisted selection strategies are optimized by using a single assay that genotypes a novel translocation junction rather than multiple assays that target each locus independently. In this QTL relocation example, a breeder selects for the SCN and CR resistance simultaneously by identifying plants that contain the translocation junction. For example, an assay for detecting (and measuring the copy number of) the translocation can use the “translocation junction qPCR” primer pairs shown in Table 4. A quantitative PCR method is used to determine the zygosity of the translocated locus. Following genotyping, plants are selected that are homozygous (or heterozygous) for the translocation, and therefore, contain the resistant alleles for both SCN and CR resistance.

[0153] Example 4 - Creating homozygous Rhgla and Rhglb soybean germplasm using a translocated QTL

[0154] This example describes how to create novel homozygous ste breeding alleles in a single plant using chromosome engineering. Multiple allelic variants of the Rhg1 locus have been described, including Rhgla a 3-copy 31.2 KB repeat on chr18 derived in modern breeding material from the SCN resistant cultivar Peking, and Rhglb, a 9- copy 31.2 Kb repeat derived from the PI088788 resistance source (Lee, Tong Geon, et al. "Evolution and selection of Rhg1 , a copy-number variant nematode-resistance locus." Molecular ecology 24.8 (2015): 1774-1791). Rhgla, Rhglb and other low- and high- copy variants have been demonstrated to condition resistance to different SCN race types either independently or in combination with additional resistance genes. Given that these genetic loci occupy a similar region on Chr18, homozygous lines carrying two different alleles of the Rhg1 locus have not been created or uncovered.

[0155] A novel allelic combination is created by crossing and then self-fertilizing the progeny of a line containing a translocated Rhg1 QTL (on a chromosome other than Chr. 18) with another line containing a different Rhg1 allele either in its native or translocated position. Recovered progeny, for example, harbor homozygous Rhgla alleles on chromosome 18 and homozygous Rhglb alleles on a modified chromosome 15 segment, as described in Examples 1-3. This new combination provides a differential soybean cyst nematode response that is not achieved via traditional crossing and inbred line development.

[0156] Example 5 - Stacking of QTLs located on the same chromosome

[0157] This example describes an alternative approach QTL stacking when the desired QTLs are located on the same chromosome. In this case, chromosomal fragments containing QTLs can be accomplished using inversions instead of chromosomal translocations described above. Thus, if two QTLs located on different arms of a chromosome, double stranded breaks are generated on the telomere side of one QTL and on the centromere side of the second QTL as shown in Figure 4. Pericentric chromosomal inversion (inversion #1) results in bringing both QTLs next to each other. Optionally, to restore chromosomal structure, the second inversion (inversion #2) is performed (Figure 5). If stacking QTLs are located on the same chromosomal arm, the double stranded breaks are generated on the telomere side of both QTLs as shown on Figure 4B. In this case, inversion #1 would result in bringing both QTLs next to each other, while inversion #2 will restore the chromosomal structure (Figures 6 and 7).

[0158] Example 6 - Breeding using lines with QTL(s) that have been relocated

[0159] Breeding lines containing a translocated locus of interest, such as those outlined in examples 1-5, can be used to develop high-yielding commercial lines directly, or they can be used as breeding parents within a breeding program. A breeding scheme using such soybean lines is summarized as follows:

[0160] 1. Outcross a line containing a homozygous or hemi-zygous trai interest to a desirable breeding line to produce F1 seed. The desirable breeding line may contain favorable traits, such as high-yield, disease resistance, high-diversity, herbicide resistance, optimal maturity, drought resistance, etc. The desirable breeding line may contain favorable disease resistance alleles or haplotypes for increased diversity, as mentioned in example 5.

[0161] 2. Next, F1 individuals are self-fertilized and then genotypic and / or phenotypic selection methods are used within the F2 generation and during subsequent rounds of inbreeding to develop progeny containing the translocated QTL and additional traits of interest. Phenotypic selection is based on yield measurements in replicated multi-environment and / or multi-year field trials. Genotypic selection is based on marker-assisted selection for specific favorable alleles or following genomic selection using a genomic prediction.

[0162] 3. Derived inbred progeny lines with favorable phenotypes (e.g. high yield) then become a genetic resource for future breeding crosses or can be sold as commercial products.

[0163] 4. Alternatively, derived progeny containing the translocated QTL of examples 1-3 can be used as a resource for additional rounds of genome engineering. For example, the derived progeny may now contain additional favorable QTL that can be translocated to select genomic regions or linked to the previously translocated QTL to create a larger QTL stack. Following genome engineering, the newly developed material can re-enter the breeding pool following steps 1-3.

[0164] Example 7 - Experimental testing of soybean plants comprising translocated chromosomal sections linking the SCN and CR QTLs

[0165] This example demonstrates how plants with the translocation of Example 1 are used to maintain, relocate, and / or improve resistance to charcoal rot and soybean cyst nematodes. The genetic variety used to create the translocation of Example 1 is resistant to soybean cyst nematode, race 3, and charcoal rot. Experiments were conducted to demonstrate that resistance to both diseases is maintained following a targeted translocation between chr15 and Chr18. The “WT RES control” in the descriptions below refers to the variety used to develop material with a translocation, that is plants with the wild-type locations of the SCN and CR QTLs.

[0166] To phenotype plants for charcoal rot resistance, the method of US20170027121 was followed. In short, T2 seeds from four T1 plants that were each homozygous for the translocation of Example 1 were germinated in plastic pots. Both resistant and susceptible controls were also germinated in plastic pots. On the 8thday after planting, plants were inoculated with Macrophomina phaseolina by inserting a microsclerotia-infested toothpick into the plant. The inoculated plants were placed in a dew room for 48 hours and then moved to a growth chamber. Plants were assessed for level of charcoal rot tolerance 14 to 21 days post-inoculation and given a score from 1-9. A score of 1 indicates that the infected, completely wilting, dying, and / or dead. A score of 3 indicates that the plant had a long external and / or internal lesion and was partially wilted. A 5 score indicates the plant had an internal lesion greater than 1.3 cm long with no wilting observed. A score of 7 indicates the presence of an internal lesion of less than 1.3 cm with no wilting. A 9 score indicates that no charcoal rot symptoms were visible. Charcoal rot resistance ratings were compared between plants harboring the translocation and control resistant and susceptible plants that do not carry the translocation.

[0167] Plants carrying homozygous, balanced translocations (i.e., translocations inherited without loss of DNA) between chromosome 15 and chromosome 18 (Lines 1-4) had statistically equivalent resistance scores to plants of the same genetic background that do not have the translocation (“WT RES control”) and to plants of a resistant elite control from a different genetic background (“RES control”) (Table 5). Both the resistant control plants and plants with the translocation were significantly more resistant to charcoal rot than a susceptible control (“SUS control”).

[0168] Table 5. Average charcoal rot scores across T2 plants carrying homozygous balanced translocations between chromosome 15 and chromosome 18 (Lines 1-4) and control plants

[0169] To phenotype plants for soybean cyst nematode resistance, standard methods were followed. T2 seeds descended from four T 1 plants that were homozygous for the translocation of example 1 , and SCN resistant and susceptible controls were placed in a sand media within containers. The sand media was then treated with an SCN solution containing 2,000 J2 individuals designated as Race 3. Seven days after inoculation, germinated plants were removed from the sand and cyst counts were made. The SCN female cyst counts were compared between plants carrying the reciprocal translocations between chromosomes 15 and 18 and control plants that do not carry the translocation.

[0170] Plants carrying homozygous, balanced translocations between chromosome 15 and chromosome 18 had statistically equivalent cyst counts to plants of the same genetic background that do not have the translocation (“WT RES control”), see tablebelow. Interestingly, plants with the translocation had lower cysts count: control plants from the same genetic background; this suggests that moving the Rhg1 region to another genomic context could be used to improve overall SCN resistance to race 3. Both the resistant control plants and plants with the translocation were significantly more resistant than a susceptible control (“SUS control”).

[0171] Table 6. Average SCN cyst counts across T2 plants carrying homozygous balanced translocations between chromosome 15 and chromosome 18 (Lines 1-4) and control plants

[0172] These data show that an intentional translocation that places the Rhg1 region on chromosome 18 adjacent to a charcoal rot QTL on chr15 can be used to create new plants with genetically linked QTL that maintain resistance to charcoal rot and SCN. Additionally, they present an opportunity to create plants with improved resistance to SCN by modifying the genomic location of the Rhg1 region.

Claims

Claims1. A method for modifying the genomic structure of a soybean plant cell, the method comprising: introducing a targeted endonuclease into a soybean plant cell; introducing a first double-strand break (DSB) in a first chromosome of the soybean plant cell and a second DSB in a second chromosome of the soybean plant cell; effecting a first reciprocal translocation of two sequences, a first sequence that is distal to the first DSB and a second sequence which is distal to the second DSB, such that a portion of the first chromosome comprising the first sequence is translocated to the second chromosome thereby creating a modified second chromosome, and a portion of the second chromosome comprising the second sequence is translocated to the first chromosome thereby creating a modified first chromosome, wherein the translocated second sequence comprises a locus of interest; and optionally introducing a third DSB in the modified first chromosome and a fourth DSB in the modified second chromosome to effect a second reciprocal translocation of a third sequence that is distal to the third DSB and fourth sequence that is distal to the fourth DSB, wherein the second reciprocal translocation restores the modified first chromosome’s telomeric region, thereby generating a modified, restored first chromosome, wherein the third DSB is distal to the translocated locus of interest and the modified, restored first chromosome retains the locus of interest.

2. A method for modifying the genomic structure of a soybean plant cell, the method comprising: introducing a targeted endonuclease into a diploid soybean plant cell comprising two copies of a first chromosome and two copies of a second chromosome; introducing a first double-strand break (DSB) into a first copy of the first chromosome and a second DSB into a first copy of the second chromosome; effecting a first reciprocal translocation of two sequences, a first sequence that is distal to the first DSB and a second sequence which is distal to the second DSB, such that a portion of the first copy of the first chromosome is translocated to the first copy of the second chromosome thereby creating a modified second chromosome, and a portion of the first copy of the second chromosome comprising the second sequence is translocated to the first copy of the first chromosome, thereby creating a modified first chromosome, wherein the second sequence comprises a locus of interest; andoptionally introducing a third DSB in the modified first chromosome in a second homologous copy of the first chromosome to effect a second reciprocal translocation of a third sequence that is distal to the third DSB and a fourth sequence that is distal to the fourth DSB, thereby restoring the modified first chromosome’s telomere to generate a modified, restored first chromosome; wherein the third DSB is distal to the translocated locus of interest and the modified, restored first chromosome retains the locus of interest.

3. A method for modifying the genomic structure of a soybean plant cell, the method comprising: introducing a targeted endonuclease into a soybean plant cell; introducing a first double-strand break (DSB) in a first arm of a chromosome and a second DSB in a second arm of the chromosome in the soybean plant cell, thereby separating each the first and second arms into a distal portion and a centromeric portion; effecting a first chromosomal inversion, such that the distal portion of the first arm is joined to the centromeric portion of the second arm thereby creating a modified second arm, and the distal portion of the second arm is joined to the centromeric portion of first arm thereby creating a modified first arm, wherein the translocated distal portion of the second arm comprises a locus of interest; and optionally introducing a third DSB in the modified first arm that is distal to the translocated locus of interest and a fourth DSB in the modified second arm to effect a second chromosomal inversion such that the first arm telomeric region and second arm telomeric region are restored to generate a modified, restored first arm and a modified, restored second arm; wherein the modified, restored first arm comprises the translocated locus of interest.

4. A method for modifying the genomic structure of a soybean plant cell, the method comprising: introducing a targeted endonuclease into a soybean plant cell; introducing a first double-strand break (DSB) in a first arm of a chromosome and a second DSB in the first arm of the chromosome, wherein the first DSB is distal to a locus of interest, the second DSB is between the centromere and the locus of interest, and a first genomic sequence is a portion of genomic sequence between the first DSB and second DSB;effecting a first chromosomal inversion of the first genomic sequent generating a modified chromosomal arm comprising an inversion of the first genomic sequence, which comprises the locus of interest; and optionally introducing a third DSB distal to the inverted locus of interest and a fourth DSB distal to the third DSB, effecting a second chromosomal inversion of a second genomic sequence located between the third DSB and fourth DSB, thereby restoring the chromosomal structure of the second genomic sequence on the modified chromosomal arm of the chromosome.

5. The method of claim 1 or 2 wherein the reciprocal translocation effected by the first DSB and second DSB is between chromosome 15 and chromosome 18.

6. The method of any one of claims 1, 2, or 5, wherein the locus of interest comprises a soybean cyst nematode (SCN) QTL or a charcoal rot (CR) QTL.

7. The method of any one of claims 1 , 5, or 6 wherein the second reciprocal translocation effected by the third DSB and fourth DSB is between a modified chromosome 15 and a modified chromosome 18.

8. The method of any one of claims 2, 5, or 6 wherein the second reciprocal translocation effected by the third DSB and fourth DSB is between a modified chromosome 15 and a naturally occurring chromosome 15.

9. The method of any one of claims 1 , 2, or 5-8, wherein the targeted endonuclease utilizes a guide RNA targeting one or more of the following sequences: SEQ ID NOs: 1-4.

10. The method of any one of claims 3 or 4, further comprising: selecting a soybean plant cell that has undergone the first chromosomal inversion and, optionally, the second chromosomal inversion and regenerating a soybean plant from the at least one selected soybean plant cell.

11. The method of any one of claims 1 , 2, or 5-10, further comprising: selecting a soybean plant cell that has undergone the first reciprocal translocation and, optionally, the second translocation and regenerating a soybean plant from the at least one selected soybean plant cell.

12. The method of claim 10 or 11, wherein the regenerated plant is self-fertilized to produce a segregating population.

13. The method of any one of claims 10-12, wherein one or more mole targeting a junction sequence is used for the selecting.

14. The method of any one of claims 11-13, wherein the locus of interest, when located on the second chromosome, is linked to a trait for which elimination of the trait via breeding is desired.

15. The method of any one of claims 10-14, further comprising breeding the regenerated plant with another soybean plant.

16. The method of claim 15, wherein breeding results in a progeny plant comprising the modified genomic structure and lacking one or more of: gene(s) encoding the targeted endonuclease; gene(s) encoding guide RNAs; or modified chromosomes that have not been restored.

17. The method of claim 15 or 16, wherein the locus of interest comprises an SCN QTL allele, and the breeding is performed with a plant comprising a different SCN QTL allele located on chromosome 18, thereby producing a progeny plant with SCN resistance traits provided by both QTL alleles.

18. The method of any one of claims 1-17, wherein the targeted endonuclease comprises a CRISPR / Cas system, a zinc finger nuclease, a TALEN, or a meganuclease.

19. The method of any one of claims 1-18, wherein the locus of interest is at least 100 kbp in size.

20. The method of any one of claims 1 , 2, 5-9, or 11-18 wherein the targeted endonuclease utilizes a guide RNA comprising one or more of the following sequences: SEQ ID NOs: 5-12.

21. A modified soybean plant comprising a modified chromosome 15, the modified chromosome 15 comprising both a SCN QTL and a CR QTL.

22. The modified soybean plant of claim 19, wherein the SCN QTL is distal to the CR QTL.

23. A soybean plant (or a seed, plant part, or cell thereof) comprising a genome edited translocation of the Rhg1 gene locus to a chromosome other than chromosome 18.

24. The soybean plant (or seed, plant part, or cell thereof) of claim 23, wherein the translocated Rhg1 gene locus is translocated to a locus that is genetically linked and / or distal to a QTL for a trait of interest.

25. The soybean plant (or seed, plant part, or cell thereof) of claim translocated Rhg1 gene locus is translocated to a locus that is distal to a QTL that provides resistance to charcoal rot disease.

26. The soybean plant (or seed, plant part, or cell thereof) of claim 25, wherein the translocated Rhg1 gene locus is distal to the QTL on chromosome 15 disclosed herein which is associated with resistance to charcoal rot disease.

27. The soybean plant (or seed, plant part, or cell thereof) of any one of claims 23-26, wherein the translocated Rhg1 gene locus comprises an Rhgla allele or Rhglb allele.

28. A soybean plant (or a seed, plant part, or cell thereof) that comprises homozygous copies of (i) a Rhgla allele associated with SCN resistance and (ii) a Rhglb allele associated with SCN resistance.

29. The soybean plant (or a seed, plant part, or cell thereof) of claim 28, wherein the homozygous Rhgla alleles are located on chromosome 18 and the homozygous Rhglb alleles are located on chromosome 15.

30. The soybean plant (or a seed, plant part, or cell thereof) of claim 28, wherein the homozygous Rhgla alleles are located on chromosome 15 and the homozygous Rhglb alleles are located on chromosome 18.

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