Compositions and methods for increasing resistance to soybean cyst nematode

Abstract

Provided are plants, cells, tissues, and germplasm thereof comprising gene and marker alleles associated with increased resistance to soybean cyst nematode (SCN). Also provided are breeding methods and methods of identifying and selecting plants having alleles associated with increased resistance to SCN. Transgenic and genome edited plants comprising the gene alleles associated with increased SCN resistance are provided.

Description

COMPOSITIONS AND METHODS FOR INCREASING RESISTANCE TO SOYBEANCYST NEMATODEREFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0001] The official copy of the sequence listing is submitted electronically via Patent Center as an XML formatted sequence listing with a file named 210934_SequenceLi sting created on October 29, 2024, and having a size of 93,355 bytes and is filed concurrently with the specification. The sequence listing comprised in this XML formatted document is part of the specification and is herein incorporated by reference in its entirety.BACKGROUND

[0002] Soybean diseases are major threat for soybean production, resulting in yield losses and decrease in grain quality. Soybean cyst nematode (SCN) is one of the most devastating pests to soybean across all major soybean growing regions. Estimated yearly yield reductions due to SCN infection have exceeded millions of tons and resulted in losses of more than $1 billion annually in the United States (Meinhardt et al. 2021, Plant Disease 105(10): 3238-43 and Wrather et al. 2006, J Nematol. 38(2): 173-80).

[0003] SCN management strategies rely on native resistance genes. Several of these resistance genes have been cloned, including Rhgl, Rhg2, and Rhg4. Rhgl and Rhg2 both encode modified a-SNAPs (Soluble N-ethylmaleimide-Sensitive Factor Attachment Proteins), which have been implicated in SCN resistance (Lakhssassi, Naoufal, et al., Scientific Reports 7.1 (2017): 45226).

[0004] Accordingly, there is a need to identify and use genes that provide improved resistance to SCN and methods for creating and selecting plants having genes that are associated with improved resistance to SCN.SUMMARY

[0005] Described herein are plants and plant materials comprising an introduced genetic modification decreasing expression or activity of SNAP14. Further described herein are plants and plant materials comprising an introduced silencing nucleic acid, the silencing nucleic acid decreasing expression of SNAP 14. Also described herein are compositions and methods useful in producing, identifying and / or selecting plants and plant materials comprising the SNAP14resistance gene associated with increased resistance to soybean cyst nematode (SCN). Additionally, the disclosure provides compositions and methods for the use of plant molecular markers that are linked to an SCN resistance phenotype. These compositions and methods can be used for selecting SCN resistant plants, breeding for SCN resistant plants, creating SCN resistant plants, and / or using genome editing to produce SCN resistance in plants. Also provided herein are plants and methods for making plants having the markers and / or the decreased SNAP14 expression associated with enhanced SCN resistance as compared to control plants. The compositions and methods can also be used to introgress SCN resistance into plants.

[0006] In another aspect, provided is a method of modifying plant material, comprising introducing into the plant material’s genome a genetic modification (i) decreasing expression of an endogenous polynucleotide comprising a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 2 or 5 as compared to a control plant material (e g., plant material not comprising the genetic modification) or (ii) decreasing expression and / or activity of an endogenous polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 3 or 6. In particular examples, the genetic modification is at a genomic locus associated with resistance to soybean cyst nematode. In certain embodiments, the genetic modification is at a genomic locus comprising a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1 or 3.

[0007] For example, provided is a method of modifying plant material, comprising introducing into the genome of the plant material a genetic modification at a genomic locus comprising a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1 or 3, wherein the genetic modification decreases (i) expression of an endogenous polynucleotide comprising a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 2 or 5 as compared to a control plant material (e.g., plant material not comprising the genetic modification) or (ii) expression and / or activity of an endogenous polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 3 or 6.

[0008] In another aspect, provided is a method of modifying plant material, comprising introducing into the plant material’s genome a silencing nucleic acid, the silencing nucleic acid (i) decreasing expression of an endogenous polynucleotide comprising a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 2 or 5 as compared to a control plant material (e.g., plant material not comprising the genetic modification) or (ii) decreasing expression and / or activity of an endogenous polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 3 or 6.

[0009] In each of the foregoing instances of the disclosed method of modifying plant material, the plant material is preferably soybean.

[0010] In each of the foregoing instances of the disclosed method of modifying plant material, the plant material is preferably soybean, and the method comprises introducing the genetic modification by creating a double-stranded break in the plant’s genome. Thus, the genetic modification can be introduced using TALEN, meganuclease, zinc finger nuclease, or CRISPR- Cas technology. In some examples, the method comprises introducing the genetic modification using a Cas endonuclease. In each of the foregoing instances of the disclosed method of modifying plant material, the plant material is preferably soybean, and the method comprises introducing the silencing nucleic acid by creating a double-stranded break in the plant’s genome. Thus, the silencing nucleic acid can be introduced using TALEN, meganuclease, zinc finger nuclease, or CRISPR-Cas technology. In some examples, the method comprises introducing the silencing nucleic acid using a Cas endonuclease.

[0011] In another aspect, provided herein is a method of introducing a gene associated with increased resistance to soybean cyst nematode (SCN) into a plant by crossing two plants. The crossing method can comprise (a) crossing a plant having SCN resistance trait with a plant from a second plant line (“second plant”) to obtain progeny plants; (b) obtaining a sample containing nucleic acid from each of one or more of the progeny plants; (c) screening the sample for a nucleic acid that comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to SEQ ID NO: 56 or 57; and selecting one or more progeny plants that have the screened-for nucleic acid. Typically, the plant having the SCN resistance trait comprises the screened-for nucleic acid. Incertain aspects, one or both of the two plants crossed in the methods described herein comprise at least one additional SCN resistance gene or QTL, such as, for example, the SCN resistance genes or SCN resistance QTLs described herein.

[0012] For example, the crossing method can comprise screening the sample for and selecting one or more progeny plants that comprise a nucleic acid that comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to SEQ ID NO: 56 or 57.

[0013] In certain examples of each instance of the foregoing crossing method, the screened for nucleic acid can be a modified SNAP 14 gene that was introduced to the plant having SCN resistance or was introduced to a progenitor of the plant having SCN resistance by genome editing or by transgenic modification.

[0014] Each of the foregoing crossing methods disclosed herein can further comprise crossing the selected one or more progeny plants with the second plant to produce backcross progeny plants. Samples containing nucleic acid from one or more backcross progeny plant can each be screened for the presence of the disclosed screened-for nucleic acid, and one or more backcross progeny plants having the screened-for nucleic acid can be selected.

[0015] This further process can be repeated such that the crossing method includes crossing the selected one or more backcross progeny plants with the second plant to produce additional backcross progeny plants; obtaining a sample containing nucleic acid from one or more of the additional backcross progeny plants; screening each sample for the screened-for nucleic acid; and selecting one or more additional backcross progeny plants that have the screened for nucleic acid. This method can include additional rounds of backcrossing such that the method further includes (a) crossing the selected one or more additional backcross progeny plants with the second plant to produce further additional backcross progeny plants, (b) obtaining samples containing nucleic acid from one or more further additional backcross progeny plants; (c) screening each sample for the screened-for nucleic acid; and (d) selecting one or more further additional backcross progeny plants that have the screened-for nucleic acid. Optionally, steps (a)- (d) can be repeated one or more times using the selected one or more further additional backcross progeny plants as the selected additional backcross progeny plants.

[0016] In yet another aspect, provided herein is a modified (e.g., genome edited or transgenic) plant comprising decreased expression of an endogenous polynucleotide comprising a nucleicacid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 2 or 5 and / or (ii) decreased expression and / or activity of an endogenous polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 3 or 6, the genome edited or transgenic plant having increased resistance to soybean cyst nematode as compared to a control plant.

[0017] For example, the modified (e.g., genome edited or transgenic) plant can comprise a genetic modification at a SNAP 14 genomic locus decreasing (i) expression of an endogenous polynucleotide comprising a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 2 or 5 and / or (ii) expression and / or activity of an endogenous polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 3 or 6. Alternatively, or in addition to, the modified (e.g., genome edited or transgenic) plant can comprise an introduced silencing nucleic acid, the silencing nucleic acid decreasing (i) expression of an endogenous polynucleotide comprising a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 2 or 5 and / or (ii) expression and / or activity of an endogenous polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 3 or 6

[0018] In some examples of each instance of the foregoing modified plant, the plant is a transgenic soybean plant. In some examples of each instance of the foregoing modified plant, the genome edited or transgenic plant further comprises at least one additional SCN resistance gene or SCN resistance QTL, such as, for example, the SCN resistance genes or SCN resistance QTLs described herein. In some examples, the at least one additional SCN resistance gene is introduced into the genome edited or transgenic plant by introgression or genome editing.

[0019] Each of the modified or transgenic plants disclosed herein can be used to control soybean cyst nematode (SCN). Thus, disclosed herein is the use of the modified plants disclosed herein to control SCN.

[0020] In an additional aspect, the disclosure provides a method of detecting the presence or absence of a SNP allele at position 201 of one or more of SEQ ID NOs: 9, 14, 19, 24, 29, 34, 39,or 44 in a plant genome. The method can comprise amplifying genomic DNA containing the SNP allele at position 201 of one or more of SEQ ID NOs: 9, 14, 19, 24, 29, 34, 39, or 44 to thereby generate an amplicon. The amplicon is then contacted with one or more probes comprising one or more of SEQ ID NOs: 17-18, 22-23, 27-28, 32-33, 37-38, 42-43, or 47-48. The method the includes detecting whether the one or more probes bind to the amplicon to thereby determine the presence or absence of the one or more SNP allele.

[0021] In one example, the detection method comprises detecting the zygosity of the SNP allele at position 201 of one or more of SEQ ID NOs: 9, 14, 19, 24, 29, 34, 39, or 44 in a plant genome. Zygosity can be detected by (a) amplifying genomic DNA containing the SNP allele at position 201 of one or more of SEQ ID NOs: 9, 14, 19, 24, 29, 34, 39, or 44; to thereby generate an amplicon; (b) contacting the amplicon with one or both of the following sets of probes: (i) a first probe comprising SEQ ID NO: 12 and a second probe comprising SEQ ID NO: 13; (ii) a first probe comprising SEQ ID NO: 17 and a second probe comprising SEQ ID NO: 18; (iii) a first probe comprising SEQ ID NO: 22 and a second probe comprising SEQ ID NO: 23; (iv) a first probe comprising SEQ ID NO: 27 and a second probe comprising SEQ ID NO: 28; (v) a first probe comprising SEQ ID NO: 32 and a second probe comprising SEQ ID NO: 33; (vi) a first probe comprising SEQ ID NO: 37 and a second probe comprising SEQ ID NO: 38; (vii) a first probe comprising SEQ ID NO: 42 and a second probe comprising SEQ ID NO: 43; and / or (viii) a first probe comprising SEQ ID NO: 47 and a second probe comprising SEQ ID NO: 48; and (c) detecting whether one or both probes from each set of the contacted probes bind to the amplicon to thereby determine the zygosity of the one or more SNP allele.

[0022] In each of the foregoing detection methods, the amplicon can be amplified using a (1) a first set of two primers: (i) a first primer comprising SEQ ID NO: 10 and a second primer comprising SEQ ID NO: 11; (ii) a first primer comprising SEQ ID NO: 15 and a second primer comprising SEQ ID NO: 16; (iii) a first primer comprising SEQ ID NO: 20 and a second primer comprising SEQ ID NO: 21; (iv) a first primer comprising SEQ ID NO: 25 and a second primer comprising SEQ ID NO: 26; (v) a first primer comprising SEQ ID NO: 30 and a second primer comprising SEQ ID NO: 31; (vi) a first primer comprising SEQ ID NO: 35 and a second primer comprising SEQ ID NO: 36; (vii) a first primer comprising SEQ ID NO: 40 and a second primer comprising SEQ ID NO: 41; and (viii) a first primer comprising SEQ ID NO: 45 and a second primer comprising SEQ ID NO: 46.

[0023] In an additional aspect, the disclosure provides method of introgressing a soybean cyst nematode (SCN) resistance gene into a soybean plant, the SCN resistance gene comprising a polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 56 or 57, comprising crossing an SCN resistant soybean plant comprising a polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 56 or 57 with a second soybean plant to produce progeny, screening the progeny with a nucleic acid marker to detect progeny comprising a polymorphism genetically linked to the SCN resistance gene, and selecting progeny that comprise the polymorphism to obtain soybean plants that contain the SCN resistance gene.

[0024] In one example of the method of introgressing a SCN resistance gene, the SCN resistant soybean plant, the second soybean plant or both comprise at least one additional SCN resistance gene or SCN resistance QTL, such as, for example, those described herein. In one example of the method of introgressing a SCN resistance gene, the selected for progeny comprise the at least one additional SCN resistance gene or QTL.

[0025] In one example of the method of introgressing a SCN resistance gene, the polymorphism is within 20 centimorgan (cM), 15 cM, 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, or 1 cM of the nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 57. In one example of the method of introgressing a SCN resistance gene, the polymorphism is within about 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb, 20 kb, 21 kb, 22 kb, 23 kb, 24 kb, 25 kb, 26 kb, 27 kb, 28 kb, 29 kb, 30 kb, 35 kb, 40 kb, 45 kb, 50 kb, 55 kb, 60 kb, 65 kb, 70 kb, 75 kb, 80 kb, 85 kb, 90 kb, 95 kb, 100 kb, 110 kb, 120 kb, 130 kb, 140 kb, 150 kb, 160 kb, 170 kb, 180 kb, 190 kb, or about 200 kb of the nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 57. In one example, the polymorphism is a single nucleotide polymorphism (SNP). In one example, the SNP comprises a T at S06754, a T at S02874, a G at SI 3434, an A at SI 3424, an A at SI 2351, a C at S00288, a T at S07921, a T at S03921, or any combination thereof. In one example, the polymorphism comprises the presence of a polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ IDNOs: 56 or 57. In one example, the polymorphism comprises the presence of a polynucleotide fragment comprising at least 10, 15, 20, 25, 30, 35, 40, 45, 50. 100, 200, 400, or 500 or more contiguous nucleic acids of SEQ ID NO: 56.

[0026] In an additional aspect, the disclosure provides methods for producing a soybean plant having increased resistance to SCN, comprising genotyping a soybean population comprising a plurality of soybean plants or soybean germplasm for the presence of at least one maker genetically linked to a locus comprising or corresponding to an SCN resistance gene having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 57, selecting from the soybean population one or more soybean plants or soybean germplasm comprising the at least one marker, and crossing the selected soybean plant or soybean germplasm with a second soybean plant or soybean germplasm to produce a progeny population, wherein at least one soybean plant or soybean germplasm of the progeny population comprises the at least one marker and has increased resistance to SCN as compared to a control plant.

[0027] In one example of the method for producing a soybean plant having increased resistance to SCN, selected soybean plant or soybean germplasm, the second soybean plant, or both the selected soybean plant or soybean germplasm and second soybean plant comprise at least one additional SCN resistance gene or QTL, such as, for example, those described herein. In one example of the method for producing a soybean plant having increased resistance to SCN, the selected for progeny comprise at least one soybean plant or soybean germplasm of the progeny population further comprises the additional SCN resistance gene or QTL.

[0028] In one example of the method for producing a soybean plant having increased resistance to SCN, the at least one marker genetically linked to the locus is within 20 cM, 15 cM, 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, or 1 cM of the locus comprising or corresponding to an SCN resistance gene having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 57. In one example of the for producing a soybean plant having increased resistance to SCN the at least one marker genetically linked to the locus is within about 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb, 17 kb, 18 kb, 19 kb, 20 kb, 21 kb, 22 kb, 23 kb, 24 kb, 25 kb, 26 kb, 27 kb, 28 kb, 29 kb, 30 kb, 35 kb, 40 kb, 45 kb, 50 kb, 55 kb, 60 kb, 65 kb, 70 kb, 75 kb, 80 kb, 85 kb, 90 kb, 95 kb, 100 kb, 110 kb, 120 kb, 130 kb, 140 kb, 150kb, 160 kb, 170 kb, 180 kb, 190 kb, or about 200 kb of the nucleic acid having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 57. In one example, the marker is selected from the group consisting of a T at S06754, a T at S02874, a G at SI 3434, an A at SI 3424, an A at SI 2351, a C at S00288, a T at S07921, a T at S03921, the presence of a polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 49, the presence of a polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 56 or a fragment thereof. In one example, the genotyping comprises amplifying a nucleic acid sequence comprising the at least one marker and detecting the resulting amplified nucleic acid comprising the marker. In one example, the amplification comprising amplification of at least a portion of one or more genomic regions of the soybean genome comprising SEQ ID NO: 9, 14, 19, 24, 29, 34, 39, or 44. In one example, the amplification comprises providing one or more nucleic acid primers, wherein the nucleic acid primers comprise the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 15, 16, 20, 21, 25, 26, 30, 31, 35, 36, 40, 41, 45, 46, 50, or 51. In one example, the detecting comprises hybridization with one or more nucleic acid probes, the one or more nucleic acid probes comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 12-13, 17-18, 22-23, 27-28, 32-33, 37-38, 42-43, 47-48, or 52.BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0029] The disclosure can be more fully understood from the following detailed description and the accompanying Sequence Listing, which form a part of this application. The sequence descriptions (Table 1) and sequence listing attached hereto comply with the rules governing nucleotide and amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §§1.831-1.835.Table 1: Sequence Listing Description - Polypeptides and Nucleotides Encoding the PolypeptideDETAILED DESCRIPTIONTerms

[0030] As used herein, “decrease in expression” “decreased expression” or the like refers to any detectable reduction in expression of a gene and / or the corresponding encoded polypeptide. Similarly, “decrease in activity” “decreased activity” or the like refers to any detectable reduction in the activity (e.g., enzymatic activity) of the encoded polypeptide. The method by which the expression or activity of a gene or polypeptide described herein is decreased is not particularly limited and can be done using methods known in the art such at RNAi, gene knockdown, gene knockout, or targeted amino acid modification.

[0031] As used herein a “gene knockout” is used to refer to gene in which there is no detectable expression of the mRNA or protein encoded by the gene, whereas “gene knockdown” is used to refer to a gene in which there is reduced expression, activity or amount of the mRNA or protein encoded by the gene. As used herein, “decreased expression”, “decreased activity” or “decreased stability” encompasses both gene knockout and gene knockdown.

[0032] 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 via a sexual cross between two parents of the same species, where at least one of the parents has the desired allele in 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 thedesired allele in its genome. The desired allele can be, e.g., detected by a marker that is associated with a phenotype, at a QTL, an edit (e.g., gene or nucleotide deletion or frame-shift mutation), or the like. Offspring comprising the desired allele may be repeatedly backcrossed to a line having a desired genetic background and selected for the desired allele, to result in the allele becoming fixed in a selected genetic background.

[0033] The process of “introgressing” is often referred to as “backcrossing” when the process is repeated two or more times. Backcrossing methods may be used to introduce a nucleic acid sequence into plants. The backcrossing technique has been widely used for decades to introduce new traits into plants. N. Jensen, Ed., Plant Breeding Methodology, John Wiley & Sons, Inc., 1988. In a typical backcross protocol, the original variety of interest (recurrent parent) is crossed to a second variety (non-recurrent parent) that carries a gene of interest to be transferred. The resulting progeny from this cross are then crossed again to the recurrent parent, and the process is repeated until a plant is obtained wherein essentially all of the desired morphological and physiological characteristics of the recurrent plant are recovered in the converted plant, in addition to the transferred gene from the non -recurrent parent.

[0034] A “locus” is a position on a chromosome, e.g. where a trait nucleotide, gene, sequence, or marker is located.

[0035] A “marker” is a means of finding a position on a genetic or physical map, or else linkages among markers and trait loci (loci affecting traits). The position that the marker detects may be known via detection of polymorphic alleles and their genetic mapping, or else by hybridization, sequence match or amplification of a sequence that has been physically mapped. A marker can be a DNA marker (detects DNA polymorphisms), a protein (detects variation at an encoded polypeptide), or a simply inherited phenotype (such as the ‘waxy’ phenotype). A DNA marker can be developed from genomic nucleotide sequence or from expressed nucleotide sequences (e.g., from a spliced RNA or a cDNA). Depending on the DNA marker technology, the marker may consist of primers complementary to sequence flanking the locus and / or probes that hybridize to polymorphic alleles at the locus. A DNA marker, or a genetic marker, may also be used to describe the gene, DNA sequence or nucleotide on the chromosome itself (rather than the components used to detect the gene or DNA sequence) and is often used when that DNA marker is associated with a particular trait in human genetics (e.g. a marker for breast cancer). The term marker locus is the locus (gene, sequence or nucleotide) that the marker detects.

[0036] “Marker(s)” can refer to the type of polymorphism that they detect and also the marker technology used to detect the polymorphism. Marker types include but are not limited to, e.g., detection of restriction fragment length polymorphisms (RFLP), detection of isozyme markers, randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLPs), detection of simple sequence repeats (SSRs), detection of amplified variable sequences of the plant genome, detection of self-sustained sequence replication, or detection of single nucleotide polymorphisms (SNPs). SNPs can be detected e.g. via DNA sequencing, PCR-based sequence specific amplification methods, detection of polynucleotide polymorphisms by allele specific hybridization (ASH), dynamic allele-specific hybridization (DASH), molecular beacons, microarray hybridization, oligonucleotide ligase assays, Flap endonucleases, 5’ endonucleases, primer extension, single strand conformation polymorphism (SSCP) or temperature gradient gel electrophoresis (TGGE). DNA sequencing, such as the pyrosequencing technology has the advantage of being able to detect a series of linked SNP alleles that constitute a haplotype. Haplotypes tend to be more informative (detect a higher level of polymorphism) than SNPs.

[0037] A “marker allele”, can refer to one of a plurality of polymorphic nucleotide sequences found at a marker locus in a population.

[0038] A “marker haplotype” refers to a combination of alleles at a marker locus.

[0039] A “marker locus” is a specific chromosome location in the genome of a species where a specific marker can be found. A marker locus can be used to track the presence of a second linked locus, e.g., one that affects the expression of a phenotypic trait. For example, a marker locus can be used to monitor segregation of alleles at a genetically or physically linked locus.

[0040] The term “molecular marker” may be used to refer to a genetic marker, as defined above, or an encoded product thereof (e.g., a protein) used as a point of reference when identifying a linked locus. A molecular marker can be derived from genomic nucleotide sequences or from expressed nucleotide sequences (e.g., from a spliced RNA, a cDNA, etc.), or from an encoded polypeptide. The term also refers to nucleic acid sequences complementary to or flanking the marker sequences, such as nucleic acids used as probes or primer pairs capable of amplifying the marker sequence. A “molecular marker probe” is a nucleic acid sequence or molecule that can be used to identify the presence of a marker locus, e.g., a nucleic acid probe that is complementary to a marker locus sequence. Alternatively, in some aspects, a marker probe refers to a probe of any type that is able to distinguish (i.e., genotype) the particular allele that is present at a markerlocus. Nucleic acids are “complementary” when they specifically hybridize in solution. Some of the markers described herein are also referred to as hybridization markers when located on an indel region, such as the non-collinear region described herein. This is because the insertion region is, by definition, a polymorphism vis a vis a plant without the insertion. Thus, the marker need only indicate whether the indel region is present or absent. Any suitable marker detection technology may be used to identify such a hybridization marker, e.g. SNP technology is used in the examples provided herein.

[0041] An allele “negatively” correlates with a trait when it is linked to it and when presence of the allele is an indicator that a desired trait or trait form will not occur in a plant comprising the allele.

[0042] In some examples disclosed herein, the presence of a gene allele or marker in a plant may be detected through the use of a nucleic acid probe. A probe may be a DNA molecule or an RNA molecule. RNA probes can be synthesized by means known in the art, for example, using a DNA molecule template. A probe may contain all or a portion of the nucleotide sequence of the marker and additional, contiguous nucleotide sequence from the plant genome. This is referred to herein as a “contiguous probe.” The additional, contiguous nucleotide sequence is referred to as “upstream” or “downstream” of the original marker, depending on whether the contiguous nucleotide sequence from the plant chromosome is on the 5' or the 3' side of the original marker, as conventionally understood. As is recognized by those of ordinary skill in the art, the process of obtaining additional, contiguous nucleotide sequence for inclusion in a marker may be repeated nearly indefinitely (limited only by the length of the chromosome), thereby identifying additional markers along the chromosome. All above-described markers may be used in some embodiments of the disclosed methods.

[0043] An oligonucleotide probe sequence may be prepared synthetically or by cloning. An oligonucleotide probe may be labeled or unlabeled. A variety of techniques exist for labeling nucleic acid molecules, including, for example and without limitation by adding a composition that is detectable which by spectroscopic, radioisotopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Thus, a particular allele may be detected using, for example, autoradiography, fluorography, or other similar detection techniques, depending on the particular label to be detected. Useful detectable labels include biotin (for staining with labeled streptavidin conjugate), magnetic beads, fluorescent dyes,radiolabels, enzymes, and colorimetric labels. Other detectable labels include ligands that bind to antibodies or specific binding targets labeled with fluorophores, chemiluminescent agents, and enzymes. Labeled probe detection techniques include the use of fluorescent dyes. Examples of fluorescent dyes include HEX fluorescent dye, VIC fluorescent dye, FAM fluorescent dye, JOE fluorescent dye, TET fluorescent dye, Cy 3 fluorescent dye, Cy 3.5 fluorescent dye, Cy 5 fluorescent dye, Cy 5.5 fluorescent dye, Cy 7 fluorescent dye, or ROX fluorescent dye. See generally, e.g., Leary et al. (1983) roc. Natl. Acad. Set. USA 80:4045-9.

[0044] A probe may be an exact copy of a marker to be detected. A probe may also be a nucleic acid molecule comprising, or consisting of, a nucleotide sequence which is substantially identical to a cloned segment of the subject organism’s (for example, soybean) chromosomal DNA. As used herein, the term “substantially identical” may refer to nucleotide sequences that are more than 85% identical. For example, a substantially identical nucleotide sequence may be 85.5%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% identical to the reference sequence.

[0045] “Marker-assisted selection” (MAS) is a process by which phenotypes are selected based on marker genotypes. Marker assisted selection includes the use of marker genotypes for identifying plants for inclusion in and / or removal from a breeding program or planting.

[0046] Molecular marker technologies generally increase the efficiency of plant breeding through MAS. A molecular marker allele that demonstrates linkage disequilibrium with a desired phenotypic trait (e.g., a SCN resistance described herein) provides a useful tool for the selection of the desired trait in a plant population. The key components to the implementation of a MAS approach are the creation of a dense (information rich) genetic map of molecular markers in the plant germplasm; the detection of at least one QTL based on statistical associations between marker and phenotypic variability; the definition of a set of particular useful marker alleles based on the results of the QTL analysis; and the use and / or extrapolation of this information to the current set of breeding germplasm to enable marker-based selection decisions to be made.

[0047] The closer a particular marker is to a gene that encodes a polypeptide that contributes to a particular phenotype (whether measured in terms of genetic or physical distance), the more tightly-linked is the particular marker to the phenotype. In view of the foregoing, it will be appreciated that the closer (whether measured in terms of genetic or physical distance) that a marker is linked to a particular gene, the more likely the marker is to segregate with that geneand its associated phenotype. Thus, the genetic markers disclosed herein can be used in MAS programs to identity plants (e g., soybean) that have or can generate progeny that have increased SCN resistance (when compared to parental varieties and / or otherwise isogenic plants lacking the SCN resistance genotype), to identify individual plants comprising this increased SCN trait, and to breed this trait into other plant varieties to improve their SCN resistance.

[0048] A “marker set” or a “set” of markers or probes refers to a specific collection of markers (or data derived therefrom) that may be used to identify individuals comprising a trait of interest. While each marker in a marker set may possess utility with respect to trait identification, individual markers selected from the set and subsets including some, but not all, of the markers may also be effective in identifying individuals comprising the trait of interest.

[0049] A “modified gene” is a gene that has been altered through human intervention. Such a “modified” gene has a nucleic acid sequence that differs from the sequence of the corresponding non-mutated gene by at least one nucleotide addition, deletion, or substitution. The modified gene can be altered by introducing one or more double strands break which are specifically targeted to a genomic sequence by a targeted nuclease using TALEN, meganuclease, zinc finger nuclease, or CRISPR-Cas technology.

[0050] As used herein, the term “heterologous” in reference to a nucleic acid means a nucleic acid sequence that originates from a foreign species or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. For example, as used herein, a “heterologous” gene can refer to a gene sequence (that is the same or only minimally altered) as the native gene, but which has been deliberately inserted at a genomic locus that differs from the native gene’s locus. Where the promoter is a native or natural sequence, the expression of the operably linked sequence can be altered from the wild-type expression, which results in an alteration in phenotype.

[0051] A “modified” plant is a plant comprising a mutated gene or a heterologous nucleic acid.

[0052] As used herein the term “native gene” refers to a gene as found in its natural endogenous location with its own regulatory sequences. In the context of this disclosure, a “transgenic”, “mutated”, “modified” gene is not a native gene.

[0053] As used herein, a “nucleic acid” or “nucleic acid molecule” is a polymeric form of nucleotides, which can include both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide,deoxynucleotide, or a modified form of either type of nucleotide. A “nucleic acid molecule” as used herein is synonymous with “nucleic acid”, “nucleotide sequence”, “nucleic acid sequence”, and “polynucleotide.” The term includes single- and double-stranded forms of DNA. A nucleic acid molecule can include either or both naturally occurring and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide linkages.

[0054] Nucleic acid molecules may be modified chemically or biochemically, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, intemucleotide modifications, such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., peptides), intercalators (e.g., acridine, psoralen, etc ), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.). The term “nucleic acid molecule” also includes any topological conformation, including single- stranded, double-stranded, partially duplexed, triplexed, hairpinned, circular, and padlocked conformations. An “endogenous nucleic acid sequence” refers to a nucleic acid sequence within a plant cell, (e.g. an endogenous allele of an SNAP 14 gene present within the genome of a soybean plant cell).

[0055] The term “single-nucleotide polymorphism” (SNP) refers to a DNA sequence variation occurring when a single nucleotide in the genome (or other shared sequence) differs between members of a species or paired chromosomes in an individual. High-throughput genotyping technologies such as GoldenGate® and INFINIUM® assays (Illumina, San Diego, CA) may be used in accurate and quick genotyping methods by multiplexing SNPs from 384-plex to >100,000-plex assays per sample.

[0056] As used herein, “phenotype” means the detectable characteristics (e.g. SCN susceptibility or SCN resistance) of a cell or organism which can be influenced by genotype.

[0057] As used herein, the term “plant material” refers to any processed or unprocessed material derived, in whole or in part, from a plant. For example, and without limitation, a plant material may be a plant part, a seed, a fruit, a leaf, a root, a plant tissue, a plant tissue culture, a plant explant, or a plant cell.

[0058] “Sequence identity” or “percent (%) sequence identity”, or the like as used herein in the context of two nucleic acid or polypeptide sequences, may refer to the residues in the twosequences that are the same when aligned for maximum correspondence over a specified comparison window. Sequence identity is determined as the percentage of amino acid residues or nucleotides in a candidate sequence (query) that are identical with the respective amino acid residues or nucleotides in the reference sequence (subject), after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any amino acid conservative substitutions as part of the sequence identity.Alignment for purposes of determining percent sequence identity can be achieved in various ways, for instance, using publicly available computer software such as BLAST, BLAST-2. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (e.g., percent identity of query sequence = number of identical positions between query and subject sequences / total number of positions of query sequence * 100).

[0059] Methods for aligning sequences for comparison are well-known in the art. Various programs and alignment algorithms are described in, for example: Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Set. U.S.A. 85:2444; Higgins and Sharp (1988) Gene 73:237-44; Higgins and Sharp (1989) CABIOS 5: 151-3; Corpet et al. (1988) Nucleic Acids Res. 16: 10881-90; Huang el al. (1992) Comp. Appl. Biosci. 8: 155-65; Pearson et al. (1994) Methods Mol. Biol. 24:307-31; Tatiana et al. (1999) FEMS Microbiol. Lett. 174:247-50. A detailed consideration of sequence alignment methods and homology calculations can be found in, e.g., Altschul etal. (1990) J. Mol. Biol. 215:403-10. The National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST™; Altschul et al. (1990)) is available from several sources, including the National Center for Biotechnology Information (Bethesda, MD), and on the internet, for use in connection with several sequence analysis programs. A description of how to determine sequence identity using this program is available on the internet under the “help” section for BLAST™. For comparisons of nucleic acid sequences, the “Blast 2 sequences” function of the BLAST™ (Blastn) program may be employed using the default BLOSUM62 matrix set to default parameters. Nucleic acidsequences with even greater similarity to the reference sequences will show increasing percentage identity when assessed by this method.

[0060] The terms “trait” and “phenotype” are used interchangeably herein. For the purposes of the present disclosure, a trait of particular interest is SCN resistance.Markers linked to SCN resistance trait

[0061] Markers may be selected that are physically located in, near, or between on or more of the genomic sequences corresponding to any one of SEQ ID NOs: 1, 4, or 57. For example, polymorphic markers among parental soybean lines are selected to screen SCN resistance mapping populations to determine which, if any, of the polymorphic markers are linked to the SCN resistance trait. Such markers may segregate so that one allele of the SNP marker appears exclusively in SCN resistant individuals, and the other allele of the SNP marker appears exclusively in SCN susceptible individuals. Mapping populations may be generated by crossing one variety that is SCN resistant with another variety that is SCN susceptible. In embodiments, a mapping population may comprise about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 95, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, or more individuals. In some embodiments, SCN resistant soybean plant may be crossed with one or more SCN susceptible plant(s) to create mapping populations.

[0062] In some embodiments, the polymorphic markers may be single nucleotide polymorphisms (SNPs) linked to or within the genomic sequence corresponding to the SCN resistance trait of interest. These SNP markers may be detected by sequencing through the region containing the gene or QTL using any DNA sequencing methods known in the art, including but not limited to Sanger sequencing or high throughput sequencing (“Next Generation”) methodologies that enable short or long sequence reads through the region of interest. In such embodiments, where genotyping by sequencing is used for the detection of SNP markers, primers corresponding to the flanking sequences of the region containing the SNPs in gene or QTL of interest may be used for the sequencing chemistries in order to sequence through the region of interest. In such embodiments, when different genotypes are used for sequencing through the region of interest for the detection of SNPs exemplified herein, other SNPs may be identified in addition to the SNPs exemplified herein. In such embodiments, the SNPsexemplified herein by themselves (individual SNPs) or in combination with other SNPs linked to exemplified sequences (haplotypes) may be utilized for differentiating genotypes towards marker assisted selection of plants for the SCN resistance trait of interest.Primer design and linkage screening

[0063] Oligonucleotide probes (e.g., primers) may be designed to specifically detect markers that are physically located in, near, genomic sequences corresponding to any one of SEQ ID NOs: 1, 4, or 57. In general, an oligonucleotide probe may be designed that specifically hybridizes to only one allele of a marker. In some cases, two oligonucleotide probes are designed to detect an SNP marker, such that each specifically hybridizes to the SNP allele to which the other probe does not specifically hybridize. As is understood by those of skill in the art, the length or composition of oligonucleotide probes for a particular marker may be varied according to established principles without rendering the probe non-specific for one allele of the marker.

[0064] In some examples, oligonucleotide probes may be primers. In specific examples, primers may be designed to detect markers in a KASPar™ genotyping assay. In particular, primers may be designed to detect markers linked to the SCN resistance phenotype in soybean using a KASPar™ genotyping assay. In these and further embodiments, the detection system may provide a high-throughput and convenient format for genotyping individuals in a mapping population, which may greatly facilitate the identification of individuals carrying a particular gene or trait and may also greatly facilitate the implementation or execution of a marker-assisted selection program.

[0065] In specific embodiments, the oligonucleotide probes may be primers designed to detect markers in a TAQMAN® genotyping assay. This method utilizes primers specific to the marker closely linked to the SCN resistance gene and fluorescent labeled probes containing a single nucleotide polymorphism (SNP). The SNP probe associated with resistance is labeled with a fluorescent dye such as FAM while the probe associated with susceptibility is labeled with a different fluorescent dye such as VIC. The data is analyzed as the presence or absence of a fluorescent dye signal. The detection system may provide a high-throughput and convenient format, such as multiplexing for genotyping individuals in a mapping population, which may greatly facilitate the identification of individuals carrying a particular gene or trait and may also greatly facilitate the implementation or execution of a marker-assisted selection program.

[0066] Additional markers may be identified as equivalent to any of the exemplary markers named herein (e.g., markers listed in Table 3 herein), for example, by determining the frequency of recombination between the exemplary marker and an additional marker. Such determinations may utilize a method of orthogonal contrasts based on the method of Mather (1931), The Measurement of Linkage in Heredity, Methuen & Co., London, followed by a test of maximum likelihood to determine a recombination frequency. Allard (1956) Hilgardia 24:235-78. If the value of the recombination frequency is less than or equal to 0.10 (i.e., 10%), then the additional marker is considered equivalent to the particular exemplary marker for the purposes of use in the presently disclosed methods.

[0067] Markers that are linked to any and all SCN resistance genes may be identified in embodiments of the invention. Additionally, markers that are linked to edited plants comprising a deletion, frameshift mutation or the link in the SCN resistance gene may be identified in embodiments of the invention. Further, markers that control any and all of resistance contributing loci for all SCN HG races may be identified in embodiments of the invention. For example, a means for providing SCN resistance in soybean is a marker selected from the group consisting of the markers listed in Table 3.

[0068] A means for identifying soybean plants having the SCN resistance phenotype may be a molecule that presents a detectable signal when added to a sample that includes the marker sequence, e.g., a probe with a detectable label that specifically hybridizes to an SNP marker allele that is linked to the SCN resistance phenotype may be a means for identifying soybean plants having the SCN resistance phenotype. In some examples, a means for identifying soybean plants having the SCN resistance phenotype is a probe that specifically hybridizes to a marker that is linked to the SCN resistance phenotype.Marker assisted selection

[0069] Molecular markers can be used in a variety of plant breeding applications (e.g. see Staub et al. (1996) Hortscience 31:729-41; Tanksley (1983) Plant Molecular Biology Reporter. 1 : 3-8). One of the main areas of interest is to increase the efficiency of backcrossing and introgressing genes using marker-assisted selection (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. SinceDNA 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. Having flanking markers decreases the chances that false positive selection will occur as a double recombination event would be needed. In the most preferred case, a marker is located within the gene itself, so that recombination cannot occur between the marker and the gene. In some embodiments, 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.

[0070] 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 the gene of interest. In 150 backcross plants, there is a 95% chance that at least one plant will 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 haverequired 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.

[0071] The key components to the implementation of MAS are: (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.

[0072] 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).

[0073] 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.

[0074] 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 a highly repetitive region. Still, the amplified region, or amplicon, will have sufficient variabilityamong 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).

[0075] SNP markers detect single base pair nucleotide substitutions. Of all the molecular marker types, SNPs are the most abundant, thus having the potential to provide 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 straightforward. 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, including but not limited to, hybridization, primer extension, oligonucleotide ligation, nuclease cleavage, mini sequencing, and coded spheres. Such methods have been reviewed in: Gut (200V) 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).

[0076] 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.

[0077] Single nucleotide polymorphic (SNP) markers can be used to select for the nucleic acid disclosed herein that is associated with SCN resistance, e.g., (i) nucleic acid having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity to SEQ ID NO: 57, (ii) a SNAP14 gene comprising an nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to SEQ ID NO: 56, or (iii) a SNAP14 gene having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to SEQ ID NOs: 1-2 or 4-5 comprising a gene modification decreasing expression and / or activity of the polynucleotide or encoded polypeptide. Using PCR, the primers are used to amplify DNA segments from individuals (preferably 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. The polymorphisms 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.

[0078] 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.

[0079] 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).

[0080] 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.

[0081] In general, MAS uses polymorphic markers that have been identified as having a significant likelihood of co-segregation with a trait such as the SCN resistance trait. Such markers are presumed to map near a gene or genes that give the plant its disease or pest 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 SCN 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 or pest 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 having disease or pest resistance.

[0082] The SNPs could be used alone or in combination (i.e. a SNP haplotype) to select for a favorable resistant gene allele associated with SCN resistance. For example, a SNP haplotype can include a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 markers to select for the nucleic acid disclosed herein which comprises a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to SEQ ID NO: 56 or 57.

[0083] There may 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 theallele at the other site on the same chromosome (Stevens, Mol. Diag. 4:309-17 (1999)). The marker loci can be located within 20 cM, 15 cM, 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, or 1 cM (on a single meiosis based genetic map) of the SCN resistance trait QTL.

[0084] 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.Genome Edited Plants

[0085] Provided are plants and plant material comprising an introduced genetic modification to generate a modified SNAP 14 gene having decreased expression as compared to expression of a SNAP 14 gene in a control plant not comprising the introduced genetic modification. In certain embodiments, the non-modified SNAP14 gene comprises (i) a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1 or 4 or (ii) a nucleotide sequence encoding a polypeptide comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 3 or 6.

[0086] A genetic modification in the genome of a plant to generate a modified gene can be made by introducing a double strand break (DSB) or nick. According to this approach, mutations, such as deletions, insertions, inversions and / or substitutions may be introduced at a target site via imperfect repair of the DSB or nick to produce a knock-out or knock-down of an endogenous gene (i.e., a SNAP14 gene). Such mutations may be generated by imperfect repair of the targeted locus even without the use of a donor template molecule. The DSB may be repaired via a Non- Homologous End Joining (NHEJ) pathway in the absence of any additional composition, via template-directed repair in the presence of a polynucleotide modification template, or via homologous recombination with a heterologous polynucleotide (donor DNA molecule). The HDR pathway repairs double-stranded DNA breaks and includes homologous recombination (HR) and single-strand annealing (SSA) (Lieber (2010) Annu. Rev. Biochem. 79: 181-211).

[0087] A “knockout” of a target gene, such as a SNAP 14 gene, may be achieved by inducing a DSB or nick at or near the endogenous locus of the target gene that results in non-expression of the protein or expression of a non-functional protein encoded by the target gene, whereas a“knock-down” of a gene may be achieved in a similar manner by inducing a DSB or nick at or near the endogenous locus of the target gene (e.g., a SNAP 14 gene) that is repaired imperfectly and reduces its expression but does not eliminate function of the encoded protein. For example, the site of the DSB or nick within the endogenous locus may be in the upstream or 5’ region of a targeted SNAP14 gene (e.g., a promoter and / or enhancer sequence) to affect or reduce its level of expression. Similarly, such targeted knockout or knockdown mutations of a SNAP 14 gene may be generated with a donor template molecule to direct a particular or desired mutation at or near the target site via repair of the DSB or nick. The donor template molecule may comprise a homologous sequence with or without an insertion sequence and comprising one or more mutations, such as one or more deletions, insertions, inversions and / or substitutions, relative to the targeted genomic sequence at or near the site of the DSB or nick. For example, targeted knockout mutations of a SNAP14 gene may be achieved by deleting or inverting at least a portion of the gene or by introducing a frame shift or premature stop codon into the coding sequence of the gene. A deletion of a portion or all of a SNAP14 gene may also be introduced by generating DSBs or nicks at two target sites and causing a deletion of the intervening target region flanked by the target sites.

[0088] In some aspects, the genome editing techniques described herein can combine the introduction of a DSB with the introduction of an “exogenous” donor DNA molecule to produce a “knock-in” at a target site. An “exogenous” donor molecule, donor template, or donor template molecule (collectively a donor template) is a molecule that is not native to a specified system (e.g., a germplasm, variety, and / or plant) with respect to a nucleotide sequence and / or genomic location (i.e., locus) for a polynucleotide. Exogenous or heterologous polynucleotides or polypeptides may be molecules that have been artificially supplied to a biological system (e.g., a plant cell, a plant gene, a particular plant species or variety, and / or a plant chromosome) and are not native to that particular biological system. Thus, the designation of a nucleic acid as “exogenous” may indicate that the nucleic acid originated from a source other than a naturally- occurring source. Site-specific integration of an exogenous nucleic acid at a SNAP 14 locus may be accomplished by any technique known to those of skill in the art.

[0089] The genetic modification in the genome of a plant to generate a modified gene as described herein can be an insertion, deletion, or substitution of one or more nucleotides at the plant genome target site. For example, a Cas endonuclease-induced double stranded break can beused to introduce a modification at the target site that results in one or more of the following: reduced expression of a polynucleotide encoding a polypeptide; reduced activity of a polypeptide; generation of one or more alternative spliced transcripts of a polynucleotide encoding a polypeptide; deletion of one or more active sites of a polypeptide; frameshift mutation in one or more exons of a polynucleotide encoding a polypeptide; deletion of a substantial portion or the full length of a polynucleotide encoding a polypeptide; repression of an enhancer motif present within a regulatory region operably linked to a coding sequence for a polypeptide; or modification of one or more nucleotides of a regulatory element in a promoter, intron, 3’UTR, or terminator that is operably linked to the expression of the polynucleotide encoding the polypeptide.Recombinant Nucleic Acid Molecules, Variants and Fragments Thereof

[0090] Provided herein are isolated or recombinant nucleic acid molecules comprising a nucleic acid sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to any one of SEQ ID NOs: 1-2, 4-5 or 57. Additionally provided are nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules encoding proteins with regions of sequence homology are provided. As used herein, the term “nucleic acid molecule” refers to DNA molecules (e.g., recombinant DNA, cDNA, genomic DNA, plastid DNA, mitochondrial DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. In some examples, the nucleic acid molecule can be single-stranded. In some examples, the nucleic acid molecule can be double-stranded.

[0091] An “isolated” nucleic acid molecule (e.g., RNA or DNA) is used herein to refer to a nucleic acid sequence (e.g., RNA or DNA) that is no longer in its natural environment, for example in vitro. A “recombinant” nucleic acid molecule (e.g., RNA or DNA) is used herein to refer to a nucleic acid sequence (e.g., RNA or DNA) that is in a recombinant bacterial or plant host cell; has been edited from its native sequence; or is located in a different location than the native sequence. In some embodiments, an “isolated” or “recombinant” nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For purposes of the disclosure, “isolated” or“recombinant” when used to refer to nucleic acid molecules excludes isolated chromosomes. For example, in various embodiments, the recombinant nucleic acid molecules encoding a SCN resistant gene allele can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleic acid sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.

[0092] In some embodiments, an isolated nucleic acid molecule encodes the polypeptide of SEQ ID NO: 3 or 6 that has one or more changes in the nucleic acid sequence compared to the native or genomic nucleic acid sequence. In some embodiments, the change in the native or genomic nucleic acid sequence includes but is not limited to: changes in the nucleic acid sequence due to the degeneracy of the genetic code; changes in the nucleic acid sequence due to the amino acid substitution, insertion, deletion and / or addition compared to the native or genomic sequence; removal of one or more intron; deletion of one or more upstream or downstream regulatory regions; and deletion of the 5’ and / or 3’ untranslated region associated with the genomic nucleic acid sequence. In some embodiments, the nucleic acid molecule encoding SEQ ID NO: 3 or 6 is a non-genomic sequence.

[0093] Provided herein are nucleic acid molecules associated with SCN resistance. Such a nucleic acids can have the sequence set forth in SEQ ID NOs: 57, and variants, fragments and complements thereof. “Complement” is used herein to refer to a nucleic acid sequence that is sufficiently complementary to a given nucleic acid sequence such that it can hybridize to the given nucleic acid sequence to thereby form a stable duplex. A reverse complement is a complement formed by exchanging each A with T, T with A, C with G, and G with C in a sequence and then reversing the 5’ to 3’ order of the exchanged sequence, such that the reverse complement of 5’-ACCTGAG-3’ is 5’-CTCAGGT-3’. “Polynucleotide sequence variants” is used herein to refer to a nucleic acid sequence that except for the degeneracy of the genetic code encodes the same polypeptide.

[0094] In some examples, the nucleic acid molecule is a non-genomic nucleic acid sequence. As used herein a “non-genomic nucleic acid sequence” or “non-genomic nucleic acid molecule” or “non-genomic polynucleotide” refers to a nucleic acid molecule that has one or more change in the nucleic acid sequence compared to a native or genomic nucleic acid sequence. In some examples, the change to a native or genomic nucleic acid molecule includes but is not limited to: changes in the nucleic acid sequence due to the degeneracy of the genetic code; optimization ofthe nucleic acid sequence for expression in plants; changes in the nucleic acid sequence to introduce at least one amino acid substitution, insertion, deletion and / or addition compared to the native or genomic sequence; removal of one or more intron associated with the genomic nucleic acid sequence; insertion of one or more heterologous introns; deletion of one or more upstream or downstream regulatory regions associated with the genomic nucleic acid sequence; insertion of one or more heterologous upstream or downstream regulatory regions; deletion of the 5’ and / or 3’ untranslated region associated with the genomic nucleic acid sequence; insertion of a heterologous 5’ and / or 3’ untranslated region; and modification of a poly adenylation site. In some examples, the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence.

[0095] Examples of a recombinant nucleic acid molecule provided herein include those associated with and capable of conferring SCN resistance when expressed in a plant, e.g., improved SCN resistance relative to an isogenic or near-isogenic plant lacking the recombinant nucleic acid molecule comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to SEQ ID NO: 57.

[0096] Nucleic acid molecules that are fragments of these nucleic acid sequences are also encompassed by the disclosure. A fragment of a nucleic acid sequence may encode a biologically active portion of the encoded polypeptide or it may be a fragment that can be used as a hybridization probe or PCR primer. Nucleic acid molecules that are fragments can comprise at least about 150, 180, 210, 240, 270, 300, 330, 360, 400, 450, or 500 contiguous nucleotides or up to the number of nucleotides present in a full-length nucleic acid sequence. “Contiguous nucleotides” is used herein to refer to nucleotide residues that are immediately adjacent to one another.

[0097] The skilled artisan will further appreciate that changes can be introduced by mutation of the nucleic acid sequences thereby leading to changes in the amino acid sequence of the encoded polypeptides of any of SEQ ID NOs: 3 or 6. Thus, variant nucleic acid molecules can be created by introducing one or more nucleotide substitutions, additions and / or deletions into the corresponding nucleic acid sequence disclosed herein, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Such variant nucleic acid sequences are also encompassed by the present disclosure.

[0098] Alternatively, variant nucleic acid sequences can be made by introducing mutations randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for ability to confer activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly, and the activity of the protein can be determined using standard assay techniques.

[0099] The polynucleotides of the disclosure and fragments thereof are optionally used as substrates for a variety of recombination and recursive recombination reactions, in addition to standard cloning methods as set forth in, e.g., Ausubel, Berger and Sambrook, i.e., to produce additional polypeptide homologues and fragments thereof with desired properties. A variety of such reactions are known. Methods for producing a variant of any nucleic acid listed herein comprising recursively recombining such polynucleotide with a second (or more) polynucleotide, thus forming a library of variant polynucleotides are also examples of the disclosure, as are the libraries produced, the cells comprising the libraries and any recombinant polynucleotide produced by such methods. Additionally, such methods optionally comprise selecting a variant polynucleotide from such libraries based on activity, as is wherein such recursive recombination is done in vitro or in vivo.

[0100] A variety of diversity generating protocols, including nucleic acid recursive recombination protocols are available. The procedures can be used separately, and / or in combination to produce one or more variants of a nucleic acid or set of nucleic acids, as well as variants of encoded proteins. Individually and collectively, these procedures provide robust, widely applicable ways of generating diversified nucleic acids and sets of nucleic acids (including, e.g., nucleic acid libraries) useful, e.g., for the engineering or rapid evolution of nucleic acids, proteins, pathways, cells and / or organisms with new and / or improved characteristics.

[0101] While distinctions and classifications are made in the course of the ensuing discussion for clarity, it will be appreciated that the techniques are often not mutually exclusive. Indeed, the various methods can be used singly or in combination, in parallel or in series, to access diverse sequence variants.

[0102] The result of any of the diversity generating procedures described herein can be the generation of one or more nucleic acids, which can be selected or screened for nucleic acids with or which confer desirable properties or that encode proteins with or which confer desirable properties. Following diversification by one or more of the methods herein or otherwise available to one of skill, any nucleic acids that are produced can be selected for a desired activity or property, e.g. such activity at a desired pH, etc. This can include identifying any activity that can be detected, for example, in an automated or automatable format, by any of the assays in the art. A variety of related (or even unrelated) properties can be evaluated, in serial or in parallel, at the discretion of the practitioner.

[0103] The nucleotide sequences disclosed herein can also be used to isolate corresponding sequences from a different source. In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences based on their sequence homology to the sequences identified by the methods disclosed herein. Sequences that are selected based on their sequence identity to the entire sequences set forth herein or to fragments thereof are encompassed by the disclosure. Such sequences include sequences that are orthologs of the sequences. The term “orthologs” refers to genes derived from a common ancestral gene and which are found in different species as a result of speciation. Genes found in different species are considered orthologs when their nucleotide sequences and / or their encoded protein sequences share substantial identity as defined elsewhere herein.

[0104] In a PCR approach, oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from cDNA or genomic DNA extracted from any organism of interest. Methods for designing PCR primers and PCR cloning are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York), hereinafter “Sambrook”. See also, Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known methods of PCR include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, vector-specific primers, partially- mismatched primers, and the like.

[0105] In hybridization methods, all or part of the nucleic acid sequence can be used to screen cDNA or genomic libraries. Methods for construction of such cDNA and genomic libraries are disclosed in Sambrook and Russell (2001), supra. The so-called hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments or other oligonucleotides and may be labeled with a detectable group such as32P or any other detectable marker, such as other radioisotopes, a fluorescent compound, an enzyme or an enzyme co-factor. Probes for hybridization can be made by labeling synthetic oligonucleotides based on the known polypeptide-encoding nucleic acid sequences disclosed herein. Degenerate primers designed on the basis of conserved nucleotides or amino acid residues in the nucleic acid sequence or encoded amino acid sequence can additionally be used. The probe typically comprises a region of nucleic acid sequence that hybridizes under stringent conditions to at least about 12, at least about 25, at least about 50, 75, 100, 125, 150, 175 or 200 consecutive nucleotides of nucleic acid sequences encoding polypeptides or a fragment or variant thereof. Methods for the preparation of probes for hybridization and stringency conditions are disclosed in Sambrook and Russell (2001 ), supra.Silencing Nucleic Acids, Constructs. Expression Cassettes and Vectors

[0106] Provided here are silencing nucleic acids targeting an endogenous polynucleotide comprising a nucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 1, 2, 4, or 5 along with constructs, expression cassettes and vectors comprising the silencing nucleic acids.

[0107] As used herein a “silencing nucleic acid” refers to a nucleic acid sequence which is capable of suppressing, reducing, or eliminating the level or expression of a target polynucleotide or the polypeptide encoded thereby. The silencing nucleic acid employed can reduce or eliminate the expression level of the target sequence by influencing the level of the target RNA transcript or, alternatively, by influencing translation and thereby affecting the level of the encoded polypeptide. Silencing nucleic acids of the methods described herein may include, but are not limited to, a sense suppression element, an antisense suppression element, a double stranded RNA, a siRNA, an amiRNA, a miRNA, or a hairpin suppression element.

[0108] In certain embodiments of the methods described herein, the silencing nucleic acid results in activity or amount of protein encoded by the target polynucleotide that is at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45% or 50% and less than 90%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% of the activity or amount of the protein in a wild-type control plant.

[0109] In certain embodiments of the compositions and methods described herein, the silencing nucleic acid results in expression or amount of mRNA encoded by the target polynucleotide that is at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45% or 50% and less than 90%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2% or 1% of the expression or amount of mRNA in a wild-type control plant or seed.

[0110] As used herein, a “sense suppression element” comprises a polynucleotide designed to express an RNA molecule corresponding to at least a part of a target messenger RNA in the "sense" orientation. Expression of the RNA molecule comprising the sense suppression element reduces or eliminates the level of the target polynucleotide or the polypeptide encoded thereby. The polynucleotide comprising the sense suppression element may correspond to all or part of the sequence of the target polynucleotide, all or part of the 5' and / or 3' untranslated region of the target polynucleotide, all or part of the coding sequence of the target polynucleotide, or all or part of both the coding sequence and the untranslated regions of the target polynucleotide. Typically, a sense suppression element has substantial sequence identity to the target polynucleotide, typically greater than about 65% sequence identity, greater than about 85% sequence identity, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. The sense suppression element of the methods described herein can be any length so long as it allows for the suppression of the SNAP 14 sequence. In certain embodiments, the sense suppression element is at least 15, 16, 17, 18, 19, 20, 22, 25, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 900, 1000, 1100, 1200, 1300 nucleotides or longer of the target polynucleotides set forth in any of SEQ ID NOs: 1, 2, 4, or 5. In other embodiments, the sense suppression element can be, for example, about 15-25, 19-35, 19-50, 25-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1050, 1050-1100, 1100-1200, 1200-1300,1300-1400, 1400-1500, 1500-1600, 1600-1700, 1700-1800 nucleotides or longer of the target polynucleotides set forth in any of SEQ ID NO: 1, 2, 4, or 5.

[0111] As used herein, an “antisense suppression element” comprises a polynucleotide which is designed to express an RNA molecule complementary to all or part of a target messenger RNA. Expression of the antisense RNA suppression element reduces or eliminates the level of the target polynucleotide. The polynucleotide for use in antisense suppression element may correspond to all or part of the complement of the sequence encoding the target polynucleotide, all or part of the complement of the 5' and / or 3' untranslated region of the target polynucleotide, all or part of the complement of the coding sequence of the target polynucleotide, or all or part of the complement of both the coding sequence and the untranslated regions of the target polynucleotide. In addition, the antisense suppression element may be fully complementary (i.e., 100% identical to the complement of the target sequence) or partially complementary (i.e., less than 100% identical to the complement of the target sequence) to the target polynucleotide. In certain embodiments, the antisense suppression element comprises at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence complementarity to the target polynucleotide. Antisense suppression may be used to inhibit the expression of multiple proteins in the same plant. Furthermore, the antisense suppression element can be complementary to a portion of the target polynucleotide. Generally, sequences of at least 15, 16, 17, 18, 19, 20, 22, 25, 50, 100, 200, 300, 400, 450 nucleotides or greater of the sequence set forth in any of SEQ ID NO: 1, 2, 4, or 5 may be used.

[0112] A “double stranded RNA” or “dsRNA” refers to a polyribonucleotide structure formed either by a single self-complementary RNA molecule or a polyribonucleotide structure formed by the expression of least two distinct RNA strands. The dsRNA molecule(s) employed in the methods and compositions of the invention mediate the reduction of expression of a target sequence, for example, by mediating RNA interference “RNAi” or gene silencing in a sequencespecific manner. In the context of the present disclosure, the dsRNA is capable of reducing or eliminating the level or expression of a target polynucleotide or the polypeptide encoded thereby in a plant.

[0113] The dsRNA can reduce or eliminate the expression level of the target sequence by influencing the level of the target RNA transcript, by influencing translation and thereby affecting the level of the encoded polypeptide, or by influencing expression at the pre-transcriptional level (i.e., via the modulation of chromatin structure, methylation pattern, etc., to alter gene expression). Accordingly, as used herein, the term “dsRNA” is meant to encompass other terms used to describe nucleic acid molecules that are capable of mediating RNA interference or gene silencing, including, for example, short-interfering RNA (siRNA), doublestranded RNA (dsRNA), micro-RNA (miRNA), hairpin RNA, short hairpin RNA (shRNA), post-transcriptional gene silencing RNA (ptgsRNA), and others.

[0114] In certain embodiments, at least one strand of the duplex or double-stranded region of the dsRNA shares sufficient sequence identity or sequence complementarity to the target polynucleotide to allow for the dsRNA to reduce the level of expression of the target sequence.

[0115] In certain embodiments, the dsRNA comprises a hairpin RNA. A hairpin RNA comprises an RNA molecule that is capable of folding back onto itself to form a double stranded structure. Multiple structures can be employed as hairpin elements. In certain embodiments, the dsRNA suppression element comprises a hairpin element which comprises in the following order, a first segment, a second segment, and a third segment, where the first and the third segment share sufficient complementarity to allow the transcribed RNA to form a double-stranded stem-loop structure. The “second segment” of the hairpin comprises a "loop" or a "loop region.” These terms are used synonymously herein and are to be construed broadly to comprise any nucleotide sequence that confers enough flexibility to allow self-pairing to occur between complementary regions of a polynucleotide (i.e., segments 1 and 3 which form the stem of the hairpin). For example, in certain embodiments, the loop region may be substantially single stranded and act as a spacer between the self-complementary regions of the hairpin stem-loop. In some embodiments, the loop region can comprise a random or nonsense nucleotide sequence and thus not share sequence identity to a target polynucleotide. In other embodiments, the loop region comprises a sense or an antisense RNA sequence or fragment thereof that shares identity to a target polynucleotide. In specific embodiments, the loop region can be optimized to be as short as possible while still providing enough intramolecular flexibility to allow the formation of the base-paired stem region. Accordingly, the loop sequence is generally less than 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 50, 25, 20, 19, 18, 17, 16, 15, 10 nucleotides or less.

[0116] Any region of the target polynucleotide can be used to design the domain of the silencing element that shares sufficient sequence identity to allow expression of the hairpin transcript to decrease the level of the target polynucleotide. For instance, the domain can be designed to sharesequence identity to the 5' untranslated region of the target polynucleotide(s), the 3' untranslated region of the target polynucleotide(s), exonic regions of the target polynucleotide(s), intronic regions of the target polynucleotide(s), and any combination thereof. In specific embodiments, a domain of the silencing element shares sufficient homology to at least about 15, 16, 17, 18, 19, 20, 22, 25 or 30 consecutive nucleotides from about nucleotides 1-50, 25-75, 75-125, 50-100,125-175, 175-225, 100-150, 150-200, 200-250, 225-275, 275-325, 250-300, 325-375, 375-425,300-350, 350-400, 425-475, 400-450, 475-525, 450-500, 525-575, 575-625, 550-600, 625-675,675-725, 600-650, 625-675, 675-725, 650-700, 725-825, 825-875, 750-800, 875-925, 925-975,850-900, 925-975, 975-1025, 950-1000, 1000-1050, 1025-1075, 1075-1125, 1050-1100, 1125-1175, 1100-1200, 1175-1225, 1225-1275, 1200-1300, 1325-1375, 1375-1425, 1300-1400, 1425- 1475, 1475-1525, 1400-1500, 1525-1575, 1575-1625, 1625-1675, 1675-1725, 1725-1775, 1775- 1825, 1825-1875, 1875-1925, 1925-1975, 1975-2025, 2025-2075, 2075-2125, 2125-2175, 2175- 2225, 1500-1600, 1600-1700, 1700-1800, 1800-1900, 1900-2000 of the target sequence, such as, for example, the SNAP 14 genes set forth in SEQ ID NOs: 1, 2, 4, or 5. In certain embodiments, to optimize the siRNA sequences employed in the hairpin, the synthetic oligodeoxyribonucleotide / RNAse H method can be used to determine sites on the target mRNA that are in a conformation that is susceptible to RNA silencing.

[0117] The hairpin silencing element may also be designed such that the sense sequence or the antisense sequence do not correspond to a target polynucleotide. In this embodiment, the sense and antisense sequence flank a loop sequence that comprises a nucleotide sequence corresponding to all or part of the target polynucleotide. Thus, it is the loop region that determines the specificity of the RNA interference.

[0118] In addition, transcriptional gene silencing (TGS) may be accomplished through use of a hairpin suppression element where the inverted repeat of the hairpin shares sequence identity with the promoter region of a target polynucleotide to be silenced.

[0119] In other embodiments, the dsRNA can comprise a small RNA (sRNA). sRNAs can comprise both microRNA (miRNA) and short-interfering RNA (siRNA). miRNAs are regulatory agents comprising about 19 ribonucleotides which are highly efficient at inhibiting the expression of target polynucleotides. For miRNA interference, the silencing element can be designed to express a dsRNA molecule that forms a hairpin structure containing a 19-nucleotide sequence that is complementary to the target polynucleotide of interest. The miRNA can besynthetically made, or transcribed as a longer RNA which is subsequently cleaved to produce the active miRNA. Specifically, the miRNA can comprise 19 nucleotides of the sequence having homology to a target polynucleotide in sense orientation and 19 nucleotides of a corresponding antisense sequence that is complementary to the sense sequence.

[0120] When expressing an miRNA, it is recognized that various forms of an miRNA can be transcribed including, for example, the primary transcript (termed the “pri-miRNA”) which is processed through various nucleolytic steps to a shorter precursor miRNA (termed the “pre- miRNA”); the pre-miRNA; or the final (mature) miRNA is present in a duplex, the two strands being referred to as the miRNA (the strand that will eventually basepair with the target) and miRNA*. The pre-miRNA is a substrate for a form of dicer that removes the miRNA / miRNA* duplex from the precursor, after which, similarly to siRNAs, the duplex can be taken into the RISC complex.

[0121] The methods and compositions of the disclosure employ silencing elements that when transcribed “form” a dsRNA molecule. Accordingly, the heterologous polynucleotide being expressed need not form the dsRNA by itself but can interact with other sequences in the plant cell to allow the formation of the dsRNA. For example, a chimeric polynucleotide that can selectively silence the target polynucleotide can be generated by expressing a chimeric construct comprising the target sequence for a miRNA or siRNA to a sequence corresponding to all or part of the gene or genes to be silenced. In this embodiment, the dsRNA is “formed” when the target for the miRNA or siRNA interacts with the miRNA present in the cell. The resulting dsRNA can then reduce the level of expression of the gene or genes to be silenced. The construct can be designed to have a target for an endogenous miRNA or alternatively, a target for a heterologous and / or synthetic miRNA can be employed in the construct. If a heterologous and / or synthetic miRNA is employed, it can be introduced into the cell on the same nucleotide construct as the chimeric polynucleotide or on a separate construct. As discussed elsewhere herein, any method can be used to introduce the construct comprising the heterologous miRNA.

[0122] In certain embodiments of the methods described herein, the silencing nucleic acid targets an endogenous SNAP14 gene whereby decreased expression results in increased resistance to SCN as compared to a control plant, such as, for example, a plant not comprising the silencing nucleic acid. In certain embodiments, the silencing nucleic acid targets at least one gene comprising a nucleic acid sequence that is at least, or at least about, 60%, 65%, 70%, 75%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of SEQ ID NOs: 1, 2, 4, or 5.

[0123] The use of the term “construct” in connection with silencing nucleic acids herein is not intended to limit the disclosure to constructs comprising DNA. Polynucleotide constructs, particularly polynucleotides and oligonucleotides composed of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides, may also be employed in the methods disclosed herein. The isolated polynucleotide constructs, nucleic acids, and nucleotide sequences disclosed herein additionally encompass all complementary forms (e.g., the reverse complement) of each sequence disclosed for such a construct. Further, polynucleotide constructs and nucleotide sequences disclosed herein can encompass any such constructs, molecules, and sequences suitable for use in a method for transforming plant material disclosed herein. Such constructs can include naturally occurring molecules and / or synthetic analogues. The disclosed nucleotide constructs, nucleic acids, and nucleotide sequences also encompass all forms of nucleotide constructs including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures and the like.

[0124] Transformed organisms disclosed herein include plant cells, bacteria, yeast, baculovirus, protozoa, nematodes and algae. The transformed organism comprises a disclosed sequence (e.g., as part of a construct, expression cassette, or vector comprising the silencing nucleic acid disclosed herein) which are associated with SCN resistance.

[0125] The disclosed sequences can be used in constructs for expression in the plant of interest, e.g. soybean. Constructs can include 5’ and 3’ regulatory sequences operably linked to a coding sequence for a silencing nucleic acid. The term “operably linked” as used herein refers to a functional linkage between a promoter and / or a regulatory sequence and a second sequence, wherein the promoter and / or regulatory sequence initiates, mediates, and / or affects transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary, to join two protein coding regions in the same reading frame. The construct may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple DNA constructs.

[0126] Such a DNA construct is provided with a plurality of restriction sites for insertion of the polypeptide gene sequence of the disclosure to be under the transcriptional regulation of the regulatory regions. The DNA construct may additionally contain selectable marker genes.

[0127] The DNA construct will generally include in the 5' to 3' direction of transcription: a transcriptional and translational initiation region (e.g., a promoter), a DNA sequence (e.g., silencing nucleic acid) of the embodiments, and a transcriptional and translational termination region (e.g., termination region) functional in the organism serving as a host. The transcriptional initiation region (e.g., the promoter) may be native, analogous, foreign or heterologous to the host organism and / or to the sequence of the embodiments. Additionally, the promoter or regulatory sequence may be the natural sequence or alternatively a synthetic sequence. The term “foreign” as used herein indicates that the promoter is not found in the native organism into which the promoter is introduced. As used herein, the term “heterologous” in reference to a sequence means a sequence that originates from a foreign species or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence. Where the promoter is a native or natural sequence, the expression of the operably linked sequence is altered from the wild-type expression, which results in an alteration in phenotype.

[0128] In some examples, the DNA construct may also include a transcriptional enhancer sequence. As used herein, the term an “enhancer” refers to a DNA sequence which can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Various enhancers include, for example, introns with gene expression enhancing properties in plants (US Patent Application Publication Number 2009 / 0144863, the ubiquitin intron (i.e., the maize ubiquitin intron 1 (see, for example, NCBI sequence S94464)), the omega enhancer or the omega prime enhancer (Gallie et al. (1989) Molecular Biology ofRNA ed. Cech (Liss, New York) 237-256 and Gallie et al. (1987) Gene 60:217-25), the CaMV 35S enhancer (see, e.g., Benfey et al. (V99QI) EMBO J . 9: 1685-96) and the enhancers of US Patent Number 7,803,992 may also be used. The above list of transcriptional enhancers is not meant to be limiting. Any appropriate transcriptional enhancer can be used in the embodiments.

[0129] The termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, may be native with the plant host or may be derived from another source (i.e., foreign or heterologous to the promoter, the sequence of interest, the plant host or any combination thereof).

[0130] Convenient termination regions are available from the Ti-plasmid of A. tumefaciens. such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau et al. (1991)Afo / . Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Ge / ze 91 : 151-158; Ballas et al. (\9 9) Nucleic Acids Res. 17:7891-7903 and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

[0131] Where appropriate, a nucleic acid may be optimized for increased expression in the host organism. Thus, where the host organism is a plant, the synthetic nucleic acids can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri (1990) Plant Physiol. 92: 1-11 for a discussion of host-preferred usage. For example, although nucleic acid sequences of the embodiments may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. (1989) Nucleic Acids Res. 17:477-498). Thus, the plant-preferred for a particular amino acid may be derived from known gene sequences from plants.

[0132] Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exonintron splice site signals, transposon-like repeats, and other well -characterized sequences that may be deleterious to gene expression. The GC content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. The term “host cell” as used herein refers to a cell which contains a vector and supports the replication and / or expression of the expression vector is intended. Host cells may be prokaryotic cells such as E. coli or eukaryotic cells such as yeast, insect, amphibian or mammalian cells or monocotyledonous or dicotyledonous plant cells. An example of a monocotyledonous host cell is a maize host cell.

[0133] In preparing the expression cassette, the various DNA fragments may be manipulated so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

[0134] A number of promoters can be used in the practice of the embodiments. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, tissue-preferred, inducible or other promoters for expression in the host organism.Plant Transformation

[0135] The methods of the embodiments involve introducing a polypeptide or polynucleotide (e.g., silencing nucleic acid) into a plant. “Introducing” is as used herein means presenting to the plant the polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a cell of the plant. The methods of the embodiments do not depend on a particular method for introducing a polynucleotide or polypeptide into a plant, only that the polynucleotide(s) or polypeptide(s) gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide(s) or polypeptide(s) into plants include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0136] “Stable transformation” as used herein means that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. “Transient transformation” as used herein means that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant or a polypeptide is introduced into a plant. “Plant” as used herein refers to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and progeny of the same. Plant cells can be differentiated or undifferentiated (e.g. callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells and pollen).

[0137] Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted fortransformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-modiisAeA transformation (US Patent Numbers 5,563,055 and 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722) and ballistic particle acceleration (see, for example, US Patent Numbers 4,945,050; 5,879,918; 5,886,244 and 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin) and McCabe et al. (1988) Biotechnology 6:923-926) and Led transformation (WO 00 / 28058). For potato transformation see, Tu et al. (1998) Plant Molecular Biology 37:829-838 and Chong et al. (2000) Transgenic Research 9:71- 78. Additional transformation procedures can be found in Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio / Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol.27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta et al. ( 1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. ( \ V) Biotechnology 6:559-563 (maize); US Patent Numbers 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988) Plant Physiol. 91 :440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311 :763-764; US Patent Number 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl.Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) / Vcm / Cell 4:1495- 1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford \995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745- 750 (maize via Agrobacterium tumefaciens).Methods to Introduce Genome Editing Technologies into Plants

[0138] In some examples disclosed herein, a genetic modification (i) decreasing expression of a nucleic acid comprising a sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to any one of SEQ ID NOs: 1-2, 4-5 or 57, and / or (ii) decreasing activity of a polypeptide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs: 3 or 6, is introduced into the genome of a plant (e.g. soybean) using genome editing technologies. For example, the foregoing genetic modification can be introduced into a desired location in the genome of a plant through the use of endonuclease or double-stranded break technologies such as TALENs, meganucleases, zinc finger nucleases, CRISPR-Cas, and the like. For example, a nucleotide deletion, nucleotide insertion, or nucleotide substitution can be introduced into a desired location in a SNAP14 gene using a CRISPR-Cas system. The desired location in the SNAP14 gene can be any desired target site in the gene.

[0139] The use of double-stranded break technologies such as Cas endonuclease-gRNA complexes, has been described, for example in U.S. Patent Application Publications 2015 / 0082478, and 2015 / 0059010, International Application Publications WO2015 / 026886, W02016 / 007347, and WO2016 / 25131, and US Patent No. 10,934,536. As used herein, a Cas endonuclease refers to a polypeptide encoded by a Cas (CRISPR-associated) gene. A Cas protein includes but is not limited to: a Cas9 protein, a Cpfl (Casl2) protein, a C2cl protein, a C2c2 protein, a C2c3 protein, Cas3, Cas3-HD, Cas 5, Cas7, Cas8, CaslO, or combinations or complexes of these. When complexed with a guide polynucleotide, the guide polynucleotide / Cas endonuclease complex”, (or “guide polynucleotide / Cas endonuclease system”, “ guide polynucleotide / Cas complex”, “guide polynucleotide / Cas system” and “guided Cas system” or “Polynucleotide-guided endonuclease”, “PGEN”” are capable of directing the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and nick or cleave (introduce a single or double-strand break) the DNA target site. A guided Cas system referred to herein can comprise Cas protein(s) and suitable polynucleotide component(s) of any known CRISPR systems (Horvath and Barrangou, 2010, Science 327:167-170; 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).

[0140] Site specific modifications can include those any method for introducing site specific modification, including, but not limited to, through the use of gene repair oligonucleotides (e.g. US Publication 2013 / 0019349), or through the use of double-stranded break technologies such asTALENs, meganucleases, zinc finger nucleases, CRTSPR-Cas, and the like. Such technologies can be used to modify the previously introduced polynucleotide through the insertion, deletion or substitution of nucleotides within the introduced polynucleotide. Alternatively, double-stranded break technologies can be used to add additional nucleotide sequences to the introduced polynucleotide. Additional sequences that may be added include, additional expression elements, such as enhancer and promoter sequences. In another embodiment, genome editing technologies may be used to position additional disease or pest resistant genes in close proximity to the SCN resistant gene within the genome of a plant, in order to generate molecular stacks with multiple disease / pest resistant proteins.

[0141] An “altered target site,” “altered target sequence.” “modified target site,” and “modified target sequence” are used interchangeably herein and refer to a target sequence as disclosed herein that comprises at least one alteration when compared to non-altered target sequence. Such “alterations” include, for example: (i) replacement of at least one nucleotide, (ii) a deletion of at least one nucleotide, (iii) an insertion of at least one nucleotide, or (iv) any combination of (i) - (iii).

[0142] The following are examples of specific embodiments of some aspects of the invention. The examples are offered for illustrative purposes only and are not intended to limit the scope of the invention in any way.EXAMPLE 1

[0143] This example demonstrates the identification of a candidate gene within a QTL for soybean cyst nematode resistance.

[0144] An SCN race 2 resistance QTL from the donor cultivars Hartwig and PI090763 has been previously reported on chromosome 14. The QTL interval contains the SNAP gene homolog, GmSNAP14 (Glyma.l4g054900). To investigate whether variation within SNAP14 is associated with the resistance phenotype, whole genome references for Hartwig and PI090763 we generated and sequences from the predicted SNAP14 gene models were compared to Glyma.l4g054900 from the w82.a2 reference genome.

[0145] To generate the references, genomic DNA was sequenced on the PacBio Sequel2e platform (Pacific Biosciences of California, Inc, Menlo Park, CA USA) to generate HiFi long reads with greater than 99.9% predicted accuracy. HiFi reads were assembled with the de novoassembler hifiasm (github.com / chhylpl23 / hifiasm) with default parameters. The resulting contigs were filtered for a minimum length of 70kb and minimum coverage of lOx. Contigs were placed into hybrid scaffolds using BioNano optical maps (San Diego, CA USA). Hybrid scaffolds were ordered and orientated relative to the W82.a5 assembly available from Soybase. Brown et al. 2021, NucL Acids Res. 49(D1): D1496-D15012. doi: 10.1093 / nar / gkaal 107.

[0146] Genomic analysis revealed that both Hartwig and PI090763 contain a 9,724 bp insertion between exons 3 and 4 of SNAP 14 relative to the Williams82 reference genome, which likely renders the gene non-functional.EXAMPLE 2

[0147] This example demonstrates that PI090763 and Hartwig carry a non-functional SNAP14 allele.

[0148] To determine the impact of the 9,724 bp insertion in SNAP14, identified in Example 1, on transcription, RNAseq analysis was performed on root samples that were infected with SCN race 3 (HG Type 7).

[0149] Twelve seeds from PI09763, Hartwig and the susceptible control Williams82 were placed in a media within containers. The media was then treated with an SCN solution. Seven days after inoculation, germinated plants were removed, briefly rinsed with tepid water, and severed at the stem / root junction; roots from three plants of each variety were pooled separately into single 50 mL tubes, frozen with liquid nitrogen, and stored at -80°C. Three replicates of pooled root tissue were collected per genotype. Total RNA was collected from ground frozen tissue using a Qiagen RNeasy plant kit (Qiagen, Germantown, MD, USA) and then spiked with the Ambion ERCC Mix 1 (ThermoFisher Scientific, Inc.) as an internal control; mRNA libraries were prepared from the isolated RNA using the Illumina® Stranded mRNA Prep, Ligation kit (Illumina.com) and sequencing was performed on an Illumina Novaseq 6000 instrument. On average, 31.4 million reads were generated per replicate.

[0150] Paired-end RNA-seq and PacBio Iso-seq libraries were then aligned to the Hartwig, PI090763 and Williams82 genomes using HISAT2 and MiniMap2, respectively. Analysis of aligned reads revealed that the SNAP 14 is not expressed in Hartwig or PI090763 in either of the treated or untreated root and shoot samples. However, aligned RNAseq reads were identified in all samples from Williams82, suggesting that the genomic insertion inhibits proper transcriptionof Hartwig and PI090763 SNAP14 alleles. The combination of genetic and genomic data suggests disruption of SNAP14 confers enhanced SCN resistance in soybean.EXAMPLE 3

[0151] This example demonstrates the loss of function allele of GmSNAP14 conditions resistance to SCN race 2.

[0152] To demonstrate that the insertion in SNAP 14 of Example 1 conditions resistance to SCN race 2, two experimental approaches are taken. First, a wild-type functional GmSNAP14 allele was introduced into PI090763 resistant line to test if the resistance to SCN race 2 in PI090763 was lost. Briefly, a sequence encoding the wild-type functional GmSNAPl 4 polypeptide of SEQ ID NO: 3 or SEQ ID NO: 6 or an empty vector control was introduced into the donor line PI090763 using 4^ / v> / / c7c / 7z / / 7?-iYiediated transformation. Transgene expression in the plants was evaluated using a quantitative RT-PCR approach. Roots of transgenic plants were then treated with SCN race 2 or a mock treatment as in Example 2. A comparison was made between the female indices from the control plants expressing the empty vector and the PI90763 transgenic plants expressing the GmSNAP14 polypeptide of SEQ ID NO: 3 or SEQ ID NO: 6. As shown in Table 2, plants expressing the wild-type functional GmSNAP14 polypeptide had an increase in the average cyst counts as compared to the control plants. These data indicate that SCN resistance is conditioned by a non-functional SNAP14 allele.Table 2: : Cyst counts in hairy roots a functional SNAP 14 gene*: significant increase compared to control, p value <0.05.

[0153] A second validation experiment where GmSNAP14 is rendered non-functional in a susceptible line is performed to demonstrate that loss-of-function of GmSNAP14 can condition resistance. The approach is as follows: a soybean cultivar, such as Peking or a derivative, isselected that has the Rhgl, Rhg4, and Rhg2 SCN resistant alleles and a functional GmSNAP14 allele; a CRISPR / Cas9 system using guide RNAs of SEQ ID NO: 7, 8, or 58 is used to generate GmSNAP14 frameshift mutations within the selected cultivar; plants with loss-of-function variants are selected and subjected to SCN bioassay with race 2. Plants with loss-of-function variants have a reduced female index and total cyst count when compared to wild-type controls.EXAMPLE 4

[0154] This example demonstrates how molecular markers are used to track and select for a favorable allele of GmSNAP14 for developing soybean products with improved SCN resistance.

[0155] 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.

[0156] Table 3 provides single nucleotide polymorphism (SNP) alleles and molecular assays that can be used individually or in combination (e.g., a haplotype) for selecting an SCN resistant QTL on Chrl4. Marker S2000BU is used to specifically genotype the insertion in GmSNAP14. The “M” allele is associated with resistance while the “W” allele is associated with susceptibility or decreased resistance. Physical positions shown in Table 3 are based on the W82.a2.vl assembly (Brown et al. 2021 supra , chromosome 14; “SNP alleles” refers to the expected SNP variation shown at nucleotide position 201 of the indicated context sequence; “Res” indicates the favorable, resistance allele to SCN and “Sus” indicates the less favorable allele, which is relatively more susceptible to SCN. Tables 4 and 5 provide primer and probe sequences that can be used to detect and select for one or more of the resistance alleles disclosed in Table 3.Table 3: Molecular Markers for Selecting and Developing Soybean Products Having the Chrl4 QTL Associated with Improved SCN ResistanceTable 4: Primer Sequences for Detecting the Markers on Chrl4Table 5: Probe Sequences for Detecting the Markers on Chrl4

[0157] A population or germplasm with improved resistance to SCN is generated by genotyping with marker S2000BU and breeding with plants that have the “M” allele. Similarly, the SNP markers of Table 3 are tightly linked to the insertion and are used either independently from or in addition to marker S2000BU to select for resistant plants.EXAMPLE 5

[0158] This example demonstrates how creating non-functional alleles of GmSNAP14 using a gene-editing approach will improve SCN resistance.

[0159] Introgression of native alleles from exotic material or older varieties, like PI090763 and Hartwig, into current elite cultivars through breeding is complicated by the unintended transfer of unfavorable linked DNA that decrease yield (e g., linkage drag). To eliminate the negative effects of linked loci, a CRISPR / Cas9 approach that abolishes or diminishes GmSNAP14 function in elite breeding material is performed. In one example, the guide RNA of Example 3 is used with Cas9 to generate novel frameshift null alleles of GmSNAP14. The plants with null GmSNAP14 mutations are used to create cultivars with improved SCN resistance. A marker assisted selection strategy as demonstrated in Example 4 can be used to select for plants harboring newly generated Gm SNAP 14 alleles. The new alleles can be further crossed into additional elite material through breeding.

[0160] Alternative methods that decrease or inhibit GmSNAP14 function, such as RNAi, random chemical mutagenesis, or targeted mutagenesis with meganucleases or different CAS enzymes, may also be used to target GmSNAP14 to increase SCN resistance.EXAMPLE 6

[0161] This example demonstrates how breeding for a loss-of-function allele of GmSNAP14 is used to create broader spectrum SCN resistance.

[0162] Breeding lines carrying Rhgla, Rhg4 and Rhg2 QTL from the SCN resistant line Peking were used as recurrent parents to introgress SCN resistant alleles at the SNAP02 and SNAP14 genes, derived from PI090763, via marker assisted backcrossing to create a novel stack of SCN resistant genes. BC3F3 individuals carrying the novel gene stack were screened with multiple races of SCN, including race 2 to which Peking is susceptible. Screening results showed soybean lines carrying the novel stack, i.e., Rhgla, Rhg4 and Rhg2 from Peking and SNAP02 and SNAP14 from PI090763, were resistant to SCN races 1, 2, 3 and 5.

[0163] The SCN resistant line Hartwig also carries a resistant allele at SNAP14 and has been used to create novel gene stacks that result in broader SCN resistance. Breeding lines carrying the Rhgla, Rhg4 and Rhg2 QTL from the SCN resistant line Peking were used as recurrent parents to introgress SCN resistant alleles at the SNAP14 gene and at a second SCN resistance QTL from Hartwig via marker assisted backcrossing to create a novel stack of SCN resistant genes. BC3F3 individuals carrying the novel gene stack were screened with multiple races of SCN, including race 2 to which Peking is susceptible. Screening results showed that soybean lines carrying the novel stack, i.e., Rhgla, Rhg4 and Rhg2 from Peking and SNAP14 and the second SCN resistance QTL from Hartwig were resistant to SCN races 1, 2, 3 and 5.

[0164] All publications and patent applications in this specification are indicative of the level of ordinary skill in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated by reference.

[0165] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative only and not limiting.

[0166] Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of theteachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0167] Units, prefixes and symbols may be denoted in their SI accepted form. Unless otherwise indicated, nucleic acids are written left to right in 5’ to 3’ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Numeric ranges are inclusive of the numbers defining the range. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

Claims

We claim:

1. A method of modifying plant material, the method comprising introducing into the genome of the plant material a genetic modification (i) decreasing expression of an endogenous polynucleotide comprising a nucleotide sequence having at least 95% identity to SEQ ID NO: 2 or 5 as compared to a control plant material or (ii) decreasing expression and / or activity of an endogenous polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 3 or 6.

2. The method of claim 1, wherein the plant material is soybean plant material.

3. The method of any one of claims 1-2, wherein the method comprises introducing the genetic modification using a double-stranded break.

4. The method of claim 3, wherein the method comprises introducing the genetic modification acid using TALEN, meganuclease, zinc finger nuclease, or CRISPR-Cas technology.

5. The method of claim 3, wherein the method comprises introducing the genetic modification using Cas endonuclease.

6. A method of modifying plant material, the method comprising introducing into the genome of the plant material a silencing nucleic acid, the silencing nucleic acid (i) decreasing expression of an endogenous polynucleotide comprising a nucleotide sequence having at least 95% identity to SEQ ID NO: 2 or 5 as compared to a control plant material or (ii) decreasing expression and / or activity of an endogenous polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 3 or 6.

7. A method of introducing a gene associated with increased resistance to soybean cyst nematode (SON) into a plant, the method comprising: a. crossing a first plant having SON resistance with a second plant to obtain progeny plants; b. obtaining a sample containing nucleic acid from each of one or more of the progeny plants;c. screening the sample for a nucleic acid that comprises a sequence having at least 90% nucleotide sequence identity to SEQ ID NO: 56 or 57; and d. selecting one or more progeny plants that have the screened-for nucleic acid.

8. The method of claim 9, wherein the first plant having SCN resistance comprises the screened-for nucleic acid.

9. The method of any one of claims 7-8, further comprising crossing the selected one or more progeny plants with the second plant to produce backcross progeny plants.

10. The method of claim 9, further comprising: a. obtaining a sample containing nucleic acid from one or more of the backcross progeny plants; b. screening each sample for the screened for screened-for nucleic acid; and c. selecting one or more backcross progeny plants that have the screened for nucleic acid.

11. The method of claim 10, further comprising d. crossing the selected one or more backcross progeny plants with the second plant to produce additional backcross progeny plants; e. obtaining a sample containing nucleic acid from one or more of the additional backcross progeny plants; f. screening each sample for the screened-for nucleic acid; and g. selecting one or more additional backcross progeny plants that have the screened for nucleic acid.

12. The method of claim 11, further comprising one or more repeated steps of h. crossing the selected one or more additional backcross progeny plants with the second plant to produce further additional backcross progeny plants,i. obtaining samples containing nucleic acid from one or more further additional backcross progeny plants; j. screening each sample for the screened-for nucleic acid; k. selecting one or more further additional backcross progeny plants that have the screened for nucleic acid; and l. optionally repeating steps h.-k. one or more times using the selected one or more further additional backcross progeny plants as the selected additional backcross progeny plants.

13. A genome edited or transgenic plant comprising decreased expression of an endogenous polynucleotide comprising a nucleotide sequence having at least 95% identity to SEQ ID NO: 2 or 5 and / or (ii) decreased expression and / or activity of an endogenous polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 3 or 6, the genome edited or transgenic plant having increased resistance to soybean cyst nematode as compared to a control plant.

14. The genome edited or transgenic plant of claim 13, wherein the genome edited or transgenic plant is soybean.

15. The genome edited or transgenic plant of claim 13 or 14, wherein the genome edited plant comprises a genetic modification introduced using a double-stranded break.

16. The genome edited or transgenic plant of claim 15, wherein the genome edited plant comprises a genetic modification introduced using TALEN, meganuclease, zinc finger nuclease, or CRISPR-Cas technology.

17. The genome edited or transgenic plant of claim 16, wherein the genome edited plant comprises a genetic modification introduced using Cas endonuclease.

18. The genome edited or transgenic plant of claim 13 or 14, wherein the transgenic plant comprises a silencing nucleic acid targeting the endogenous polynucleotide.

19. The genome edited or transgenic plant of any one of claims 13-18, wherein the genome edited or transgenic plant further comprises at least one additional SCN resistance gene or SCN resistance QTL.

20. The genome edited or transgenic plant of claim 19, wherein the at least one additional SCN resistance gene or SCN resistance QTL is selected from the group consisting of Rghla, Rhg4, Rhg2, or a second SCN resistance QTL from Hartwig.

21. The genome edited or transgenic plant of claim 19 or 20, wherein the at least one additional SCN resistance gene is introduced into the genome edited or transgenic plant by introgression or genome editing.

22. Use of the genome edited or transgenic plant of any one of claims 13-21 for control of soybean cyst nematode (SCN).

23. A method of detecting the presence or absence of a SNP allele at position 201 of one or more of SEQ ID NOs: 9, 14, 19, 24, 29, 34, 39, or 44 in a plant genome; the method comprising: a. amplifying genomic DNA containing the SNP allele at position 201 of one or more of SEQ ID NOs: 9, 14, 19, 24, 29, 34, 39, or 44; to thereby generate an amplicon; b. contacting the amplicon with one or more probes comprising one or more of SEQ ID NOs: 17-18, 22-23, 27-28, 32-33, 37-38, 42-43, or 47-48; and c. detecting whether the one or more probes bind to the amplicon to thereby determine the presence or absence of the one or more SNP allele.

24. The method of claim 23, wherein the method comprises detecting the zygosity of the SNP allele at position 201 of one or more of SEQ ID NOs: 9, 14, 19, 24, 29, 34, 39, or 44 in a plant genome; the method comprising: a. amplifying genomic DNA containing the SNP allele at position 201 of one or more of SEQ ID NOs: 9, 14, 19, 24, 29, 34, 39, or 44; to thereby generate an amplicon; b. contacting the amplicon with one or more of the following sets of probes:(i) a first probe comprising SEQ ID NO: 12 and a second probe comprising SEQ ID NO: 13;(ii) a first probe comprising SEQ ID NO: 17 and a second probe comprising SEQ ID NO: 18;(iii) a first probe comprising SEQ ID NO: 22 and a second probe comprising SEQ ID NO: 23;(iv) a first probe comprising SEQ ID NO: 27 and a second probe comprising SEQ ID NO: 28;(v) a first probe comprising SEQ ID NO: 32 and a second probe comprising SEQ ID NO: 33;(vi) a first probe comprising SEQ ID NO: 37 and a second probe comprising SEQ ID NO: 38;(vii) a first probe comprising SEQ ID NO: 42 and a second probe comprising SEQ ID NO: 43; and / or(viii) a first probe comprising SEQ ID NO: 47 and a second probe comprising SEQ ID NO: 48; and c. detecting whether one or both probes from each set of the contacted probes bind to the amplicon to thereby determine the zygosity of the one or more SNP allele.

25. The method of claim 23 or 24, wherein the amplicon is amplified using one or more of the following sets of primers:(i) a first primer comprising SEQ ID NO: 10 and a second primer comprising SEQ ID NO: 11;(ii) a first primer comprising SEQ ID NO: 15 and a second primer comprising SEQ ID NO: 16;(iii) a first primer comprising SEQ ID NO: 20 and a second primer comprising SEQ ID NO: 21;(iv) a first primer comprising SEQ ID NO: 25 and a second primer comprising SEQ ID NO: 26;(v) a first primer comprising SEQ ID NO: 30 and a second primer comprising SEQ ID NO : 31;(vi) a first primer comprising SEQ ID NO: 35 and a second primer comprising SEQ ID NO: 36;(vii) a first primer comprising SEQ ID NO: 40 and a second primer comprising SEQ ID NO: 41; and(viii) a first primer comprising SEQ ID NO: 45 and a second primer comprising SEQ ID NO: 46.

26. A method of introgressing a soybean cyst nematode (SCN) resistance gene into a soybean plant, the SCN resistance gene comprising a polynucleotide having at least 95% sequence identity to SEQ ID NO: 57, the method comprising: i) crossing an SCN resistant soybean plant comprising a polynucleotide encoding a polypeptide having at least 95% sequence identity to SEQ ID NO: 57 with a second soybean plant to produce progeny; ii) screening the progeny with a nucleic acid marker to detect progeny comprising a polymorphism genetically linked to the SCN resistance gene; and iii) selecting progeny that comprise the polymorphism to obtain soybean plants that contain the SCN resistance gene.

27. The method of claim 26, wherein the polymorphism is within 10 centimorgans of the nucleic acid having at least 95% sequence identity to SEQ ID NO: 57.

28. The method of claim 26 or 27, wherein the polymorphism comprises a single nucleotide polymorphism (SNP).

29. The method of claim 28, wherein the SNP comprises a T at S06754, a T at S02874, a G at S13434, an A at S13424, an A at S12351, a C at S00288, a T at S07921, a T at S03921, or any combination thereof.

30. The method of claim 26 or 27, wherein the polymorphism comprises the presence of a polynucleotide having at least 95% sequence identity to SEQ ID NO: 49 or 56.

31. The method of any one of claims 26-30, wherein the first plant, the second plant, or both the first plant and second plant comprise at least one additional SCN resistance gene or QTL.

32. The method of claim 31, wherein the at least one additional SCN resistance gene or SCN resistance QTL is selected from the group consisting of Rghla, Rhg4, Rhg2, or a second SCN resistance QTL from Hartwig.

33. The method of claim 31 or 32, wherein the selected for progeny comprise the at least one additional SCN resistance gene or QTL.

34. A method for producing a soybean plant having increased resistance to soybean cyst nematode (SCN), the method comprising: i) genotyping a soybean population comprising a plurality of soybean plants or soybean germplasm for the presence of at least one maker genetically linked to a locus comprising or corresponding to an SCN resistance gene comprising a polynucleotide having at least 95% sequence identity to SEQ ID NO: 57; ii) selecting from the soybean population one or more soybean plants or soybean germplasm comprising the at least one marker; and iii) crossing the selected soybean plant or soybean germplasm with a second soybean plant or soybean germplasm to produce a progeny population, wherein at least one soybean plant or soybean germplasm of the progeny population comprises the at least one marker and has increased resistance to SCN as compared to a control plant.

35. The method of claim 34, wherein the at least one marker genetically linked to the locus is within 20 centimorgans of the locus comprising or corresponding to an SCN resistance gene comprising a polynucleotide having at least 95% sequence identity to SEQ ID NO: 57.

36. The method of claim 34 or 35, wherein the marker is selected from the group consisting of aT at S06754, a T at S02874, a G at SI 3434, an A at SI 3424, an A at S 12351, a C at S00288,a T at S07921 , a T at S03921 , the presence of a polynucleotide having at least 95% sequence identity to any one of SEQ ID NO: 49, the presence of a polynucleotide having at least 95% sequence identity to any one of SEQ ID NO: 56 or a fragment thereof.

37. The method of any one of claims 34-36, wherein genotyping comprises amplifying a nucleic acid sequence comprising the at least one marker and detecting the resulting amplified nucleic acid comprising the marker.

38. The method of claim 37, wherein the amplification comprising amplification of at least a portion of one or more genomic regions of the soybean genome comprising SEQ ID NO: 9, 14, 19, 24, 29, 34, 39, or 44.

39. The method of claim 37 or 38, wherein the amplification comprises providing one or more nucleic acid primers, wherein the nucleic acid primers comprise the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 15, 16, 20, 21, 25, 26, 30, 31, 35, 36, 40, 41, 45, 46, 50, or 51.

40. The method of any one of claims 37-39, wherein the detecting comprises hybridization with one or more nucleic acid probes, the one or more nucleic acid probes comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 12-13, 17-18, 22-23, 27- 28, 32-33, 37-38, 42-43, 47-48, or 52.

41. The method of any one of claims 34-40, wherein the selected soybean plant or soybean germplasm, the second soybean plant, or both the selected soybean plant or soybean germplasm and second soybean plant comprise at least one additional SCN resistance gene or QTL.

42. The method of claim 41, wherein the at least one additional SCN resistance gene or SCN resistance QTL is selected from the group consisting of Rghla, Rhg4, Rhg2, or a second SCN resistance QTL from Hartwig.

43. The method of claim 41 or 42, wherein the selected for progeny comprise at least one soybean plant or soybean germplasm of the progeny population further comprises the additional SCN resistance gene or QTL.