SCN-007 genes, compositions, methods, and markers for SCN resistance

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

SCN-007 GENES, COMPOSITIONS, METHODS, AND MARKERS FOR SCN RESISTANCEREFERENCE TO A SEQUENCE LISTING SUBMITTED VIA EFS-WEB

[0001] The official copy of the sequence listing is submitted electronically via Patent Center as an XML formatted sequence listing as a file named 210814-US-PRV-1 ST.26, created October 3, 2023 and having a size of 156,067 bytes. The sequence listing comprised in this XML formatted document is part of the specification and is herein incorporated by reference in its entirety.TECHNICAL FIELD

[0002] The present disclosure relates to plant pest resistance and provides plants, plant breeding methods, methods of identifying, methods of selecting and method of generating plants with soybean cyst nematode (SCN) resistance in soybean. Also provided are compositions (e.g., nucleic acids, constructs, and the like) and methods for introducing or improving SCN resistance in a host plant such as by transgenic modification and / or genome editing.BACKGROUND

[0003] The soybean, Glycine max, is one of the major economic crops grown worldwide as a primary source of vegetable oil and protein. Growing demand for low cholesterol and high fiber diets has increased soybean’s importance as a food. Over 10,000 soybean varieties have now been introduced into the United States, of which a limited number form the genetic base of cultivars developed from hybridization and selection programs. Johnson and Bernard, The Soybean, Norman Ed., Academic Press, N.Y., pp. 1-73, 1963.

[0004] Soybean cyst nematode, (SCN, Heterodera glycines (HG) Ichinohe) is the single most damaging pest affecting soybean in the U.S. and in many other top soybean-producing countries of the world. 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. See Meinhardt et al. 2021, Plant Disease 105(10): 3238-43 and Wrather and Koenning 2006, JNematoL 38(2): 173- 80. The SCN resistance phenotype is a complex trait, which is controlled by multiple genes, both recessive and dominant. Concibido et al. 2004, Crop Sci. 44(4): 1121-31. SCN phenotyping is time consuming, cost and labor intensive.

[0005] SCN infection causes various symptoms that may include chlorosis of the leaves and stems, root necrosis, loss in seed yield, and suppression of root and shoot growth. The aboveground symptoms of SCN infection are not unique to SCN infection, and could be confused with nutrient deficiency, particularly iron deficiency, stress from drought, herbicide injury or another disease. The first signs of infection are groups of plants with yellowing leaves that have stunted growth. The pathogen may also be difficult to detect on the roots, as stunted roots are also a common symptom of stress or plant disease. Adult females and cysts of SCN are about 1 / 32 inch long and, thus, visible without magnification. Observation of adult females and cysts on the roots is the only accurate way to detect and diagnose SCN infection in the field.

[0006] The presence of SCN is usually not obvious at the time of initial soil infestation. The SCN population density must increase in the soil until it is sufficient to cause above-ground symptoms on plants or a decrease in yield. Population densities may take years to reach significant numbers. Thus, current SCN damage is the result of infestations that have been growing for years. Although soybean is the primary host of SCN, other legumes can serve as hosts, for example: green beans, snap beans, dry beans, red beans, lima beans, mung beans, bush beans, Adzuki beans, garden peas, and cowpeas. There are thirty days in the SCN life cycle. Thus, a single growing season encompasses multiple generations of the parasite. Moreover, SCN eggs may remain intact in soil for several years before hatching.

[0007] In the past, an SCN population was given a “race” designation by comparing its reproduction on a set of four soybean germplasm lines with that on a standard SCN-susceptible soybean cultivar. The most commonly used race scheme identified 16 races of SCN. The race designation allowed nematologists and soybean breeders to share information about the ability of certain SCN populations to reproduce on soybean varieties that contain certain genes for resistance to SCN.

[0008] In 2003, the HG Type Test was developed to replace the race test. This new test includes seven sources of resistance (germplasm lines) and the results are shown as a percentage, indicating how much the nematode population from a soil sample increased on each of the seven lines. This test indicates which sources of resistance would be good for a particular field being tested, and which would be poor. Since the genetic sources of resistance are currently limited in commercially available soybean varieties, it is important to rotate these “sources of resistance” to delay the build-up of a virulent SCN population.

[0009] Shortly after the discovery of SCN in the United States, sources of SCN resistance were identified. Ross and Brim 1957, Plant Dis. Rep. 41:923-4. Some lines, such as Peking and PI 88788, were quickly incorporated into breeding programs. Peking became widely used as a source of resistance due to its lack of agronomically undesirable traits, with Pickett as the first SCN resistant cultivar released. The recognition that certain SCN resistant populations could overcome resistant cultivars led to an extensive screen for additional sources of SCN resistance. PI 88788 emerged as a popular source of race 3 and 4 resistance, even though it had a cyst index greater than 10% (but less than 20%) against race 4, and Peking and its derivatives emerged as a popular source for races 1 and 3. PI 437654 was subsequently identified as having resistance to all known races and its SCN resistance was backcrossed into Forrest. Currently, there are more than 130 Pls known to have SCN resistance. PI 209332 and PI 90763 are other exemplary SCN resistant soybean breeding lines. Not all varieties with the same source of resistance have comparable yields, nor do they respond identically to SCN.

[0010] Resistant soybean varieties are available for management of SCN. SCN densities usually decrease when resistant soybeans are grown because most SCN juveniles are unable to feed and develop on the roots of the resistant varieties. However, in any naturally infested field, a few SCN juveniles (< 1%) will be able to reproduce on the resistant varieties currently available. The number of SCN juveniles that can reproduce on resistant soybean varieties can increase when resistant varieties are grown repeatedly. Eventually, the SCN population may be able to reproduce as well on a resistant variety as a susceptible variety if SCN-resistant soybeans are grown every time soybeans are produced in an infested field. Fortunately, the number of SCN juveniles that can reproduce on resistant varieties declines when susceptible soybean varieties are grown because these nematodes do not compete well for food with the other SCN juveniles in the soil that cannot feed on the resistant varieties.

[0011] SCN race 3 has been the most prominent race in Midwestern soybean producing states. Considerable effort has been devoted to the genetics and breeding for resistance to race 3. While both Peking and PI 88788 are resistant to SCN race 3, classical genetics studies suggest that they harbor different genes for race 3 resistance. Rao-Arelli and Anand 1988, Crop Sci. 28:650-2. Race 3 resistance is probably under the control of three or four different genes. Id see also Mansur et al. 1993, Crop Sci. 33: 1249-53. One major SCN resistance QTL that maps to linkage group G is rhgl. Concibido et al. 1996, Theor. Appl. Genet. 93:234-41. Other SCN resistance QTLs map tolinkage groups A2, Cl , M, D, J, L25, L26, and K. Id.,- U.S. Patent 5,491,081 . SCN resistance QTLs behave in a race-specific manner, at least by accounting for different proportions of the total phenotypic variation with respect to different SCN races. Concibido et al. 1997, Crop Sci. 37:258-64. However, the rhgl locus on linkage group G may be involved in resistance to a number of the identified SCN races. But see Qui et al. 1999, Theor. Appl. Genet. 98:356-64.

[0012] More recently, QTLs cqSCN-006 and cqSCN-007 for soybean cyst nematode resistance were identified in the glycine soja line PI 468916 and fine-mapped to an approximately 212.1 kb interval on chromosome 15 and an approximately 103.2 kb interval on chromosome 18, respectively, using the Williams 82 version 2 reference genome. See Yu and Diers 2017, Euphytica, 213: 1-11. However, the candidate genes responsible for these traits were not successfully identified. This may be due to natural genomic variation, including large scale structural variation, that prevents single reference assemblies from accurately identifying the specific genes or genomic differences that underlie such major phenotypic variations.

[0013] There remains 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 OF THE DISCLOSURE

[0014] Described herein are plants and plant materials comprising a heterologous nucleic acid associated with increased resistance to soybean cyst nematode (SCN). Also described herein are compositions and methods useful in producing, identifying and / or selecting plants and plant materials comprising the heterologous nucleic acid 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 to select SCN resistant plants, breeding for SCN resistant plants, creating transgenic SCN resistant plants, and / or using genome editing to introduce SCN resistance in plants. Also provided herein are plants and methods for making plants having the markers and / or heterologous nucleic acid associated with enhanced SCN resistance as compared to control plants. The compositions and methods can also be used to introgress SCN resistance into plants.

[0015] Provided herein is a heterologous nucleic acid comprising a sequence that (i) encodes a polypeptide having at least 90% amino acid sequence identity to any one of SEQ ID NOs: l, 4, 7,10, 13, 16, 19, 22, 25, 28, or 31 , (ii) comprises a sequence having at least 90% nucleotide sequence identity to any one of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, 29, or 32, or (iii) comprises a sequence having at least 90% nucleotide sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33. As used herein the term heterologous refers to 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. Thus, the disclosed heterologous nucleic acid can be a vector insert, in a transgenic construct, or it can be integrated at a non-native locus that differs from its natural or native location. Additionally, a heterologous nucleic acid can include any of the foregoing sequences (i), (ii), or (iii) which is operably linked to a heterologous promoter or enhancer.

[0016] In another aspect, provided is a method of modifying plant material, the method comprising introducing into the plant material’s genome a heterologous nucleic acid sequence or expressing the heterologous nucleic acid sequence in the plant material, wherein the heterologous nucleic acid (i) encodes a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs: l, 4, 7, 10, 13, 16, 19, 22, 25, 28, or 31, (ii) comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to any one of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, 29, or 32, or (iii) comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33. In particular examples, the heterologous nucleic acid is associated with increased resistance to soybean cyst nematode. In particular examples, the method comprises introducing into the plant material’s genome two or more heterologous nucleic acid sequences or expressing the two or more heterologous nucleic acid sequences in the plant material, wherein the two or more heterologous nucleic acid sequences are selected from the group consisting of a heterologous nucleic acid (i) encoding a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, or 31, (ii) comprising a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, 29, or 32, or (iii) comprising a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33. In particular examples, the two or more heterologousnucleic acid sequences comprise (a) a heterologous nucleic acid encoding SEQ ID NO: 22 and a heterologous nucleic acid encoding SEQ ID NO: 25; (b) a heterologous nucleic acid encoding SEQ ID NO: 22, a heterologous nucleic acid encoding SEQ ID NO: 25, and a heterologous nucleic acid encoding SEQ ID NO: 19; or (c) a heterologous nucleic acid encoding SEQ ID NO: 7 and a heterologous nucleic acid encoding SEQ ID NO: 10.

[0017] For example, provided is a method of modifying plant material, the method comprising introducing into the plant material’s genome a heterologous nucleic acid sequence or expressing the heterologous nucleic acid sequence in the plant material, wherein the heterologous nucleic acid encodes a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:7 or SEQ ID NO: 10 or SEQ ID NO: 19 or SEQ ID NO:22 or SEQ ID NO:25, or SEQ ID NO:31. Provided is a method of modifying plant material, the method comprising introducing into the plant material’s genome a heterologous nucleic acid sequence or expressing the heterologous nucleic acid sequence in the plant material, wherein the heterologous nucleic acid comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to SEQ ID NO:8 or SEQ ID NO: 11 or SEQ ID NO:20 or SEQ ID NO:23 or SEQ ID NO:26 or SEQ ID NO:32. Provided is a method of modifying plant material, the method comprising introducing into the plant material’s genome a heterologous nucleic acid sequence or expressing the heterologous nucleic acid sequence in the plant material, wherein the heterologous nucleic acid comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% 100% nucleotide sequence identity to SEQ ID NO: 9 or SEQ ID NO: 12 or SEQ ID NO:21 or SEQ ID NO: 24 or SEQ ID NO:27 or SEQ ID NO:33. Provided is a method of modifying plant material, the method comprising introducing into the plant material’s genome two or more heterologous nucleic acid sequences or expressing the two or more heterologous nucleic acid sequences in the plant material, wherein the two or more heterologous nucleic acid sequences encodes a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to two or more of SEQ ID NO:7 or SEQ ID NO: 10 or SEQ ID NO: 19 or SEQ ID NO:22 or SEQ ID NO 25, or SEQ ID NO:31.

[0018] In each of the foregoing instances of the disclosed method of modifying plant material, the plant material is preferably soybean and the heterologous nucleic acid can be inserted into a genomic locus on soybean chromosome Chr 1, Chr 2, Chr 3, Chr 4, Chr 5, Chr 6, Chr 7, Chr 8,Chr 9, Chr 10, Chr 1 1, Chr 12, Chr 13, Chr 14, Chr 15, Chr 16, Chr 17, Chr 19, or Chr 20. Alternatively, in each of the foregoing instances, the plant material is preferably soybean and the heterologous nucleic acid can be inserted into a genomic locus on soybean chromosome 18 at location other than its native locus, which corresponds to the locus spanning physical positions 53,484,561 to 53,614,866 of chromosome 18 in the publicly available soybean genome for Williams82, version 4 (Wm82, v4) (Valliyodan et al. 2019, Plant J. 100: 1066-82).

[0019] In each of the foregoing instances of the disclosed method of modifying plant material, the plant material is preferably soybean and the heterologous nucleic acid can be operably linked to its native promoter and enhancer. Alternatively, the heterologous nucleic acid can be the native gene sequence which is operably linked to a modified (e.g., by genome editing) gene regulatory sequence (e.g., promoter, enhancer, terminator) to alter native gene expression. The modified promoter or other regulatory sequence may be (i) modified to include additional copies of the native regulatory sequence, (ii) modified to include an altered, genome edited version of the native regulatory sequence, (iii) modified to include a different plant gene regulatory sequence from the same species, or (iv) a foreign synthetic regulatory sequence which as used herein indicates that the regulatory sequence is synthetic or from a different species and is not found in the native organism into which the regulatory sequence is introduced. 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

[0020] 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 heterologous nucleic acid by creating a double-stranded break in the plant’s genome. Thus, the heterologous nucleic acid can be introduced introducing the heterologous nucleic acid using TALEN, meganuclease, zinc finger nuclease, or CRISPR-Cas technology. In some examples, the method comprises introducing the heterologous nucleic acid using a Cas endonuclease.

[0021] 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 (i) encodes a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs: l, 4, 7, 10, 13, 16, 19, 22, 25, 28, or 31, (ii) comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to any one of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, 29, or 32, or (iii) comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33; 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.

[0022] For example, the plant having the crossing method can comprise screening the sample for and selecting a one or more progeny plants that comprise a nucleic acid that (i) encodes a polypeptide having at least 90% amino acid sequence identity to SEQ ID NO: 7, 10, 19, 22, 25 or 31, (ii) comprises a sequence having at least 90% nucleotide sequence identity to SEQ ID NO:8, 11, 20, 23, 26, or 32, or (iii) comprises a sequence having at least 90% nucleotide sequence identity to SEQ ID NO: 9, 12, 21, 24, 27, or 33.

[0023] In some examples of each instance of the foregoing crossing method, the screened-for nucleic acid is a heterologous nucleic acid on soybean chromosome Chr 1, Chr 2, Chr 3, Chr 4, Chr 5, Chr 6, Chr 7, Chr 8, Chr 9, Chr 10, Chr 11, Chr 12, Chr 13, Chr 14, Chr 15, Chr 16, Chr 17, Chr 19, or Chr 20. In other examples of each instance of the foregoing crossing method, the screened-for nucleic acid is a heterologous nucleic acid located on soybean chromosome 18 at location other than its native locus, which corresponds to physical positions 53,484,561 to 53,614,866 of chromosome 18 in the publicly available soybean genome Wm82, v4.

[0024] In certain examples of each instance of the foregoing crossing method, the screened for nucleic acid can be a heterologous nucleic acid 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.

[0025] Each of the foregoing crossing method 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.

[0026] 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.

[0027] In yet another aspect, provided herein is a modified (e.g., genome edited or transgenic) plant comprising a heterologous nucleic acid sequence, wherein the heterologous nucleic acid (i) encodes a polypeptide having at least 90% amino acid sequence identity to any one of SEQ ID NOs: l, 4, 7, 10, 13, 16, 19, 22, 25, 28, or 31, (ii) comprises a sequence having at least 90% nucleotide sequence identity to any one of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, 29, or 32, or (iii) comprises a sequence having at least 90% nucleotide sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33; and wherein the heterologous nucleic acid is associated with increased resistance to soybean cyst nematode. In particular examples, the modified (e.g., genome edited or transgenic) plant comprises two or more heterologous nucleic acid sequences, wherein the two or more heterologous nucleic acid sequences are selected from the group consisting of a heterologous nucleic acid (i) encoding a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NOs: l, 4, 7, 10, 13, 16, 19, 22, 25, 28, or 31, (ii) comprising a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, 29, or 32, or (iii) comprising a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33. In particular examples, the two or more heterologous nucleic acid sequences sequence comprise (a) a heterologous nucleic acid encoding SEQ ID NO:22 and a heterologous nucleic acid encoding SEQ ID NO: 25; (b) a heterologous nucleic acid encoding SEQ ID NO: 22, a heterologous nucleic acid encoding SEQ ID NO: 25, and a heterologous nucleic acid encoding SEQ ID NO: 19; or (c) a heterologous nucleic acid encoding SEQ ID NO: 7 and a heterologous nucleic acid encoding SEQ ID NO: 10.

[0028] For example, the modified (e.g., genome edited or transgenic) plant can comprise a heterologous nucleic acid sequence that encodes a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO: 7 or SEQ ID NO: 10 or SEQ ID NO: 19 or SEQ ID NO:22 or SEQ ID NO:25, or SEQ ID NO:31. The modified (e.g., genome edited or transgenic) plant can comprise a heterologous nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to SEQ ID NO:8 or SEQ ID NO: 11 or SEQ ID NO:20 or SEQ ID NO:23 or SEQ ID NO:26 or SEQ ID NO:32. The modified (e.g., genome edited or transgenic) plant can comprise a heterologous nucleic acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% 100% nucleotide sequence identity to SEQ ID NO: 9 or SEQ ID NO: 12 or SEQ ID NO:21 or SEQ ID NO: 24 or SEQ ID NO:27 or SEQ ID NO:33. The modified (e g., genome edited or transgenic) plant can comprise two or more heterologous nucleic acid sequences that encode two or more polypeptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:7 or SEQ ID NOTO or SEQ ID NO: 19 or SEQ ID NO:22 or SEQ ID NO:25, or SEQ ID NOT E

[0029] In some examples of each instance of the foregoing modified plant, the plant is a transgenic soybean plant and the heterologous nucleic acid comprises coding sequence that is operably linked to a heterologous promoter.

[0030] In other examples of each instance of the foregoing modified plant, the plant is a genome edited soybean plant and the heterologous nucleic acid is located on soybean chromosome Chr 1, Chr 2, Chr 3, Chr 4, Chr 5, Chr 6, Chr 7, Chr 8, Chr 9, Chr 10, Chr 11, Chr 12, Chr 13, Chr 14, Chr 15, Chr 16, Chr 17, Chr 19, or Chr 20. In still other examples of each instance of the foregoing crossing method, the plant the plant is a genome edited soybean plant and the heterologous nucleic acid is located on soybean chromosome 18 at location other than its native locus, which corresponds to physical positions 53,484,561 to 53,614,866 of chromosome 18 in the publicly available soybean genome Wm82, v4. In each of the foregoing examples of genome edited soybeanplants, the heterologous nucleic acid can further comprise and be operatively linked to its native promoter (though at a non-native locus).

[0031] Each of the modified plants disclosed herein can be used to control soybean cyst nematode (SCN) inasmuch as the modified plant expresses an effective amount a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs: l, 4, 7, 10, 13, 16, 19, 22, 25, 28, or 31. Thus, disclosed herein is the use of the modified plant disclosed herein to control SCN by expressing heterologous nucleic acid sequence the encodes a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to SEQ ID NO:7 or SEQ ID NO: 10 or SEQ ID NO: 19 or SEQ ID NO: 22 or SEQ ID NO: 25 or SEQ ID NO: 31.

[0032] 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 both of SEQ ID NO: 34 or SEQ ID NO: 39 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 NO: 34 or SEQ ID NO: 39 to thereby generate an amplicon. The amplicon is then contacted with one or more probes comprising one or more of SEQ ID NOs: 37, 38, 42, 43. 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.

[0033] In one example, the detection method comprises detecting the zygosity of the SNP allele at position 201 of one or both of SEQ ID NO: 34 or SEQ ID NO: 39 in a plant genome. Zygosity can be detected by (i) amplifying genomic DNA containing the SNP allele at position 201 of one or both of SEQ ID NO: 34 or SEQ ID NO: 39; to thereby generate an amplicon; (ii) contacting the amplicon with one or both of the following sets of probes: (1) a first set comprises a first probe comprising SEQ ID NO:37 and a second probe comprising SEQ ID NO:38 or (2) a second set comprises a first probe comprising SEQ ID NO:42 and a second probe comprising SEQ ID NO:43; and (iii) 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.

[0034] In each of the foregoing detection methods, the amplicon can be amplified using a (1) a first set of two primers: one primer comprising SEQ ID NO:35 and the other primer comprising SEQ ID NO:36 and / or (2) a second set of primers: one primer comprising SEQ ID NO:40 the other primer comprising SEQ ID NO:41.SEQUENCE LISTING

[0035] The nucleic acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, as defined in 37 C.F.R. § 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the displayed strand. In the accompanying sequence listing:

[0036] SEQ ID NO: 1 shows polypeptide sequence encoded by MSTRG.28982.1 gene.

[0037] SEQ ID NO:2 shows MSTRG.28982.1 gene coding sequence.

[0038] SEQ ID NO:3 shows MSTRG.28982.1 gene genomic sequence.

[0039] SEQ ID N0:4 shows polypeptide sequence encoded by 5199. dpgml8g580830.855.1 gene.

[0040] SEQ ID NO:5 shows 5199. dpgml8g580830.855.1 gene coding sequence.

[0041] SEQ ID NO:6 shows 5199. dpgml8g580830.855.1 gene genomic sequence.

[0042] SEQ ID NO:7 shows polypeptide sequence encoded by 5199.dpgml8g580840.855.1 gene.

[0043] SEQ ID NO:8 shows 5199.dpgml8g580840.855.1 gene coding sequence.

[0044] SEQ ID NO:9 shows 5199. dpgml8g580840.855.1 gene genomic sequence.

[0045] SEQ ID NO: 10 shows polypeptide sequence encoded by 5199. dpgml8g580850.855.1 gene.

[0046] SEQ ID NO: 11 shows 5199. dpgml8g580850.855.1 gene coding sequence.

[0047] SEQ ID NO: 12 shows 5199. dpgml8g580850.855.1 gene genomic sequence.

[0048] SEQ ID NO: 13 shows polypeptide sequence encoded by 5199. dpgml8g580860.855.1 gene.

[0049] SEQ ID NO: 14 shows 5199. dpgml8g580860.855.1 gene coding sequence.

[0050] SEQ ID NO: 15 shows 5199. dpgml8g580860.855.1 gene genomic sequence.

[0051] SEQ ID NO: 16 shows polypeptide sequence encoded by 5199. dpgml8g580870.855.1 gene.

[0052] SEQ ID NO: 17 shows 5199. dpgml8g580870.855.1 gene coding sequence.

[0053] SEQ ID NO: 18 shows 5199. dpgml8g580870.855.1 gene genomic sequence.

[0054] SEQ ID NO: 19 shows polypeptide sequence encoded by MSTRG.28988.1 gene.

[0055] SEQ ID NO:20 shows MSTRG.28988.1 gene coding sequence.

[0056] SEQ ID NO:21 shows MSTRG.28988.1 gene genomic sequence.

[0057] SEQ ID NO:22 shows polypeptide sequence encoded by MSTRG.28989.5 gene.

[0058] SEQ ID NO:23 shows MSTRG.28989.5 gene coding sequence.

[0059] SEQ ID NO:24 shows MSTRG.28989.5 gene genomic sequence.

[0060] SEQ ID NO:25 shows polypeptide sequence encoded by 5199. dpgm!8g580900.855.1 gene.

[0061] SEQ ID NO:26 shows 5199. dpgm!8g580900.855.1 gene coding sequence.

[0062] SEQ ID NO:27 shows 5199. dpgml8g580900.855.1 gene genomic sequence.

[0063] SEQ ID NO:28 shows polypeptide sequence encoded by 5199.dpgml8g580910.855.2 gene.

[0064] SEQ ID NO:29 shows 5199.dpgml8g580910.855.2 gene coding sequence.

[0065] SEQ ID NO:30 shows 5199.dpgml8g580910.855.2 gene genomic sequence.

[0066] SEQ ID NO:31 shows polypeptide sequence encoded by 5199. dpgml8g580920.855.1 gene.

[0067] SEQ ID NO:32 shows 5199. dpgm!8g580920.855.1 gene coding sequence.

[0068] SEQ ID NO:33 shows 5199. dpgm!8g580920.855.1 gene genomic sequence.

[0069] SEQ ID NO:34 shows S7-001 SNP and context flanking sequence.

[0070] SEQ ID NO:35 shows forward primer for amplifying S7-001.

[0071] SEQ ID NO:36 shows reverse primer for amplifying S7-001.

[0072] SEQ ID NO:37 shows probe sequence that hybridizes to one allele of S7-001.

[0073] SEQ ID NO:38 shows probe sequence that hybridizes to other allele of S7-001.

[0074] SEQ ID NO:39 shows S8-001 SNP and context flanking sequence.

[0075] SEQ ID NO:40 shows forward primer for amplifying S8-001.

[0076] SEQ ID NO:41 shows reverse primer for amplifying S8-001.

[0077] SEQ ID NO:42 shows probe sequence that hybridizes to one allele of S8-001.

[0078] SEQ ID NO:43 shows probe sequence that hybridizes to other allele of S8-001.DETAILED DESCRIPTIONTerms

[0079] 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 the desired allele in its genome. The desired allele can be, e.g., detected by a marker that is associated with a phenotype, at a QTL, a transgene, 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.

[0080] 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.

[0081] A “locus” is a position on a chromosome, e.g. where a trait nucleotide, gene, sequence, or marker is located.

[0082] 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 witha 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.

[0083] “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.

[0084] A “marker allele”, can refer to one of a plurality of polymorphic nucleotide sequences found at a marker locus in a population.

[0085] “Marker assisted selection” (of MAS) is a process by which individual plants are selected based on marker genotypes.

[0086] “Marker assisted counter- sei ection” is a process by which marker genotypes are used to identify plants that will not be selected, allowing them to be removed from a breeding program or planting.

[0087] A “marker haplotype” refers to a combination of alleles at two or more marker loci.

[0088] 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.

[0089] 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 fromexpressed 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 marker locus. 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.

[0090] 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.

[0091] 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.

[0092] 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) Proc. Natl. Acad. Sci. USA 80:4045-9.

[0093] 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.

[0094] “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.

[0095] 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 an 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 betweenmarker 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.

[0096] 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 gene and 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.

[0097] 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.

[0098] 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.

[0099] 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. Thus, the term heterologous can refer to a native nucleic acid sequence which is cisgenic (originating from the same speciesor a closely related species capable of interbreeding with the same species) that is introduced at a different, non-native genomic 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.

[0100] A “modified” plant is a plant comprising a mutated gene or a heterologous nucleic acid.

[0101] 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.

[0102] 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.

[0103] Nucleic acid molecules may be modified chemically or biochemically, or may contain nonnatural 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, internucleotide 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 IND gene present within the genome of a Brassica plant cell).

[0104] 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 genotypingtechnologies such as GoldenGate® and INFTNIUM® 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.

[0105] 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.

[0106] 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.

[0107] “Sequence identity” or “Percent (%) sequence identity”, as used herein in the context of two nucleic acid or polypeptide sequences, may refer to the residues in the two sequences 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 x 100).

[0108] 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 (19 ) Proc. Natl. Acad. Sei. 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 et 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 sequencealignment methods and homology calculations can be found in, e.g., Altschul et al. (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 acid sequences with even greater similarity to the reference sequences will show increasing percentage identity when assessed by this method.

[0109] 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

[0110] 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: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33. 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.

[0111] 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 toSanger 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 SNPs exemplified 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.

[0112] Primer design and linkage screening.

[0113] 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: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33. 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.

[0114] 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.

[0115] 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 singlenucleotide polymorphism (S\P) 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.

[0116] Additional markers may be identified as equivalent to any of the exemplary markers named herein (e.g., markers listed in Table 4 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 (z.e., 10%), then the additional marker is considered equivalent to the particular exemplary marker for the purposes of use in the presently disclosed methods.

[0117] Markers that are linked to any and all SCN resistance genes 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 4.

[0118] 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

[0119] 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. Since DNA marker assays are less laborious and take up less physical space than field phenotyping, much larger populations can be assayed, increasing the chances of finding a recombinant with the target segment from the donor line moved to the recipient line. The closer the linkage, the more useful the marker, as recombination is less likely to occur between the marker and the gene causing the trait, which can result in false positives. 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.

[0120] When a gene is introgressed by MAS, it is not only the gene that is introduced but also the flanking regions (Gepts. (2002). Crop Sei, 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 acrossover within 1 cM single meiosis map distance of the other side of the gene, generating a segment around the target gene of less than 2 cM based on a single meiosis map distance. This can be accomplished in two generations with markers, while it would have required on average 100 generations without markers (See Tanksley et al., supra). When the exact location of a gene is known, flanking markers surrounding the gene can be utilized to select for recombinations in different population sizes. For example, in smaller population sizes, recombinations may be expected further away from the gene, so more distal flanking markers would be required to detect the recombination.

[0121] 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 markerbased 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.

[0122] SSRs can be defined as relatively short runs of tandemly repeated DNA with lengths of 6 bp or less (Tautz (1989) Nucleic Acid Research VT. 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).

[0123] 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.

[0124] 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 wayssimilar 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 variability among germplasm, often due to insertions or deletions, such that the fragments generated by the amplification primers can be distinguished among polymorphic individuals, and such indels are known to occur frequently in maize (Bhattramakki et al. (2002). Plant Mol Biol 48, 539-547; Rafalski (2002b), supra).

[0125] 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 straight-forward. SNPs also have the promise of being relatively low-cost systems. These three factors together make SNPs highly attractive for use in MAS. Several methods are available for SNP genotyping, including but not limited to, hybridization, primer extension, oligonucleotide ligation, nuclease cleavage, mini sequencing, and coded spheres. Such methods have been reviewed in: Gut (2001) Hum Mutat 17 pp. 475-492; Shi (2001) Clin Chem 47, pp. 164-172; Kwok (2000) Pharmacogenomics 1, pp. 95-100; and Bhattramakki and Rafalski (2001) Discovery and application of single nucleotide polymorphism markers in plants. In: R. J. Henry, Ed, Plant Genotyping: The DNA Fingerprinting of Plants, CABI Publishing, Wallingford. A wide range of commercially available technologies utilize these and other methods to interrogate SNPs including Masscode. TM. (Qiagen), INVADER®. (Third Wave Technologies) and Invader PLUS®, SNAPSHOT®. (Applied Biosystems), TAQMAN®. (Applied Biosystems) and BEAD ARRAYS®. (Illumina).

[0126] 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 thatpopulation 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.

[0127] Single nucleotide polymorphic (SNP) markers can be used to select for the nucleic acid disclosed herein that is associated with SCN resistance, e.g., nucleic acid that (i) encodes a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs: l, 4, 7, 10, 13, 16, 19, 22, 25, 28, or 31, (ii) comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to any one of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, 29, or 32, or (iii) comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33. 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.

[0128] 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.

[0129] 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).

[0130] 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.

[0131] 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.

[0132] 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 of markers for the nucleic acid disclosed herein which (i) encodes a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, or 31, (ii) comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to any one of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, 29, or 32, or (iii) comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33.

[0133] 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 inlinkage disequilibrium (LD) with an allele at one or more of the polymorphic sites in the haplotype and thus could be used in a marker assisted selection program to introgress a gene allele or genomic fragment of interest. Two particular alleles at different polymorphic sites are said to be in LD if the presence of the allele at one of the sites tends to predict the presence of the allele at the other site on the same chromosome (Stevens, Mol. Diag. 4:309-17 (1999)). The marker loci can be located within 5 cM, 2 cM, or 1 cM (on a single meiosis based genetic map) of the SCN resistance trait QTL.

[0134] 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.

[0135] Recombinant Nucleic Acid Molecules, Variants and Fragments Thereof

[0136] Provided herein are isolated or recombinant nucleic acid molecules comprising nucleic acid that (i) encodes a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs:l, 4, 7, 10, 13, 16, 19, 22, 25, 28, or 31, (ii) comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to any one of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, 29, or 32, or (iii) comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33. Also provided are nucleic acids encoding biologically active portions of a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs: l, 4, 7, 10, 13, 16, 19, 22, 25, 28, or 31. 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.

[0137] 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 invitro. 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.

[0138] In some embodiments, an isolated nucleic acid molecule encodes the polypeptide of SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, or 31 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: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, or 31 is a non-genomic sequence.

[0139] A variety of polynucleotides that encode SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, or 31 or related proteins are contemplated. Such polynucleotides are useful for production of this polypeptide in host cells when operably linked to a suitable promoter, transcription termination and / or polyadenylation sequences. Such polynucleotides are also useful as probes for isolating homologous or substantially homologous polynucleotides that encode SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, or 31 or related proteins.

[0140] Provided herein are nucleic acid molecules associated with SCN resistance. Such a nucleic acids can have the sequence set forth in SEQ ID NO:2, 5, 8, 11, 14, 17, 20, 23, 26, 29, or 32, or in SEQ ID NO: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33, and variants, fragments and complements thereof. “Complement” is used herein to refer to a nucleic acid sequence that is sufficientlycomplementary 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.

[0141] In some examples, the nucleic acid molecule encoding SEQ ID NO: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, or 31 polypeptide 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 of the 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 polyadenylation site. In some examples, the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence.

[0142] Examples of a recombinant nucleic acid molecule provided herein include those (i) encoding a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs:l, 4, 7, 10, 13, 16, 19, 22, 25, 28, or 31, (ii) comprising a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to any one of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, 29, or 32, or (iii) comprising a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33. Wherein the recombinant nucleic acid molecule is associated with and capable of conferring SCN resistance when expressed in a plant, e.g., improved SCNresistance relative to an isogenic or near-isogenic plant lacking the recombinant nucleic acid molecule.

[0143] 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 encoding any one of SEQ ID NOs: l, 4, 7, 10, 13, 16, 19, 22, 25, 28, or 31. “Contiguous nucleotides” is used herein to refer to nucleotide residues that are immediately adjacent to one another. Fragments of the nucleic acid sequences will encode protein fragments that retain the biological activity of the full-length polypeptide and, hence, retain SCN resistance. “Retains SCN resistance” is used herein to refer to a polypeptide having at least about 10%, at least about 30%, at least about 50%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or more of the SCN resistance of the full-length polypeptide.

[0144] 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:l, 4, 7, 10, 13, 16, 19, 22, 25, 28, or 31, without altering the biological activity of the proteins. 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-mediated mutagenesis. Such variant nucleic acid sequences are also encompassed by the present disclosure.

[0145] 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.

[0146] The polynucleotides of the disclosure and fragments thereof are optionally used as substrates for a variety of recombination and recursive recombination reactions, in addition tostandard 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] 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 thelike 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.

[0151] 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. ( 9901) 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.

[0152] 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 encodingpolypeptides 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.

[0153] Nucleotide Constructs, Expression Cassettes and Vectors

[0154] The use of the term “construct” in connection with isolated and / or heterologous polynucleotides 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.

[0155] 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 nucleotide sequence disclosed herein which are associated with SCN resistance.

[0156] 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 nucleic acid that encodes a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs:l, 4, 7, 10, 13, 16, 19, 22, 25, 28, or 31. 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 leastone additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple DNA constructs.

[0157] 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.

[0158] 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 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.

[0159] In some embodiments the DNA construct comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to (i) any one of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, 29, or 32, or (iii) any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33. In some embodiments the DNA construct comprises a polynucleotide encoding a fusion protein, one part of the fusion protein comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to any one of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, or 31.

[0160] 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 of RNA ed. Cech (Liss, New York) 237-256 and Gallie et al. (1987) Gene 60:217-25), the CaMV 35S enhancer (see, e.g., Benfey et al. (1990) EMBO J. 9: 1685-96) and the enhancers of US Patent Number 7,803,992 may also be used. The above list of transcriptional enhancers is not meant to be limiting. Any appropriate transcriptional enhancer can be used in the embodiments.

[0161] 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).

[0162] 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) A o / . 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) Gene 91 : 151-158; Ballas et al. C ) Nucleic Acids Res. 17:7891-7903 and Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.

[0163] 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.

[0164] Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron 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 averagefor 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. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.

[0165] 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.

[0166] 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.

[0167] Plant Transformation

[0168] The methods of the embodiments involve introducing a polypeptide or polynucleotide 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.

[0169] “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).

[0170] 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 di cot, targeted for transformation. 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-mediated 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.(soybean); McCabe et al. (1988) Bio / Technology 6:923-926 (soybean); Finer and McMullen (1991) 7 / 7 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. (1988) 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-V n 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. (\ 990) 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) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens).

[0171] Methods to Introduce Genome Editing Technologies into Plants

[0172] In some examples disclosed herein, a nucleic acid that (i) encodes a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs: l, 4, 7, 10, 13, 16, 19, 22, 25, 28, or 31, (ii) comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to any one of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, 29, or 32, or (iii) comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33 is introduced into the genome of a plant (e.g. soybean) using genome editing technologies. For example, the foregoing nucleic acid 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 genomic sequence (having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33) or a coding sequence (having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to any one of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, 29, or 32) can be introduced into a desired location in a genome using a CRISPR-Cas system, for the purpose of site-specific insertion. The desired location in a plant genome can be any desired target site for insertion, such as a genomic region amenable for breeding or may be a target site located in a genomic window with an existing trait of interest. Existing traits of interest could be either an endogenous trait or a previously introduced trait. Thus, for example, a native gene (SCN susceptible allele) can be altered though genome editing to the SCN resistant allele disclosed herein. Alternatively or additionally, the disclosed SCN resistant allele (that (i) encodes a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs: l, 4, 7, 10, 13, 16, 19, 22, 25, 28, or 31, (ii) comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to any one of SEQ ID NOs:2, 5, 8, 11, 14, 17, 20, 23, 26, 29, or 32, or (iii) comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33) can be introduced by genome editing at a different genomic location (e.g., in soybean). The different genome location can be soybean chromosome Chr 1, Chr 2, Chr 3, Chr4, Chr 5, Chr 6, Chr 7, Chr 8, Chr 9, Chr 10, Chr 11 , Chr 12, Chr 13, Chr 14, Chr 15, Chr 16, Chr 17, Chr 19, or Chr 20. Alternatively, the different genome location can be on soybean chromosome 18 at location other than its native locus, which native locus corresponds to physical positions 53,484,561 to 53,614,866 of chromosome 18 in the publicly available soybean genome Wm82, v4.

[0173] 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).

[0174] In some examples, an SCN susceptible gene allele can be modified by genome editing technology to include the SCN resistance allele. 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 doublestranded break technologies such as TALENs, meganucleases, zinc finger nucleases, CRISPR- 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 addedinclude, 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.

[0175] 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).

[0176] In some aspects of the present disclosure, the polynucleotide sequences can be stacked with any combination of polynucleotide sequences of interest to create plants with a desired phenotype. This stacking can be accomplished by a combination of genes within a DNA construct, or by crossing one or more plants having transgenes with another plant line that comprises a desired combination. For example, the polynucleotides of the present disclosure or variants thereof can be stacked with any other polynucleotides of the disclosure (e.g., stack of genes encoding SEQ ID NOs: 22 and 25; SEQ ID NOs: 22, 25, and 19; or SEQ ID NOs: 7 and 10), or with other genes. The combinations generated can also include multiple copies of any one of the polynucleotides of interest. The polynucleotides of the present disclosure can also be stacked with any other gene or combination of genes to produce plants with a variety of desired trait combinations including and not limited to traits desirable for animal feed such as high oil genes, balanced amino acids, increased digestibility, insect, disease or herbicide resistance, avirulence and disease resistance genes, agronomic traits (e.g, male sterility, flowering time) and / or transformation technology traits (e.g., cell cycle regulation or gene targeting).

[0177] These stacked combinations can be created by any method including but not limited to cross breeding plants by any conventional or known methodology, or genetic transformation. If the traits are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. For example, a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation. The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. Forexample, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that can suppress the expression of the polynucleotide of interest. This can be combined with any combination of other suppression cassettes or overexpression cassettes to generate the desired combination of traits in the plant.

[0178] The following examples are offered to illustrate, but not to limit, the claimed subject matter. It is understood that the examples and embodiments described herein are for illustrative purposes only, and persons skilled in the art will recognize various reagents or parameters that can be altered without departing from the spirit of the disclosure or the scope of the appended claims.Example 1

[0179] To avoid the limitations of the previously reported single-reference assembly that failed to identify the specific genomic differences responsible for the soybean cyst nematode resistance of QTL cqSCN-007, a reference genome was generated for an SCN resistant line that carries the cqSCN-007 QTL. This public variety was developed following a cross to PI 468916. See Meinhardt et al. 2021, Plant Disease 105(10): 3238-43.

[0180] The reference genome was generated by high molecular weight genomic DNA sequencing on the PacBio Sequel2e platform (Pacific Biosciences of California, Inc, Menlo Park, CA USA), which generated HiFi long reads with greater than 99.9% predicted accuracy. HiFi reads were assembled with using a de novo assembler hifiasm. Cheng et al. 2021, Nat Methods 18: 170-175 and Cheng et al. 2022, Nature Biotechnology 40: 1332-1335. The resulting contigs were filtered for a minimum length of 70kb and minimum coverage of 1 Ox. Contigs were then placed into hybrid scaffolds using BioNano optical maps from Bionano Genomics, Inc. (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.

[0181] Paired-end RNA-seq and ISO-SEQ libraries (Pacific Biosciences of California) were then constructed from RNA extracted from root and shoot samples taken from the SCN resistant line that carries the cqSCN-007 QTL. The libraries were used to further gene annotation. RNAseq and ISO-SEQ reads were aligned to the SCN resistant line’s reference genome using HISAT2 and MiniMap2, respectively (Kim et al. 2019, Nat Biotechnol 37: 907-15 and Li et al. 2021, Bioinformatics 37: 4572-74) StringTie2 was then employed to assemble gene models based onaligned reads (Pertea et al. 2015, Nat Biotechnol 33:290-5). Augustus was also used to predict gene models and its results were merged with alignment-based assemblies using StringTie2 to create the final gene annotation set (Stanke and Waack 2003, Bioinformatics 19(Suppl 2): ii215— ii225). Nine susceptible genomes were used in the alignment assemblies

[0182] Following creation of the final gene annotation set, marker sequences correlated with cqSCN-007 were compared to the SCN resistant line’s reference genome using BLAST software to define the causal interval. This interval on chromosome 18 was found to contain a total of 11 genes shown in Table 1. “RLP” refers to the class of “receptor like proteins” that have an extracellular domain but lack an intracellular kinase domain.Table 1Example 2

[0183] A strategy of comparative genomics and functional annotation was used to identify and select candidate genes for further study from this interval. Genes listed in Table 1 were compared against the proteomes of genomes of nine soybean lines known to lack the cqSCN-007 resistanceQTL to assess their level of conservation in susceptible lines. None of the genes were present at 100% identity in any susceptible lines. Therefore, comparative genomics analysis did not clearly identify or exclude any of the candidate genes as being associated with SCN resistance.

[0184] The genes in Table 1 were further annotated by searching for functional domains using HMMER (Eddy SR, PLoS Comput Biol 7(10): el002195) and transmembrane domains with TMHMM (Krogh et al. 2021, J Mol. Biol. 305(3): 567-580. Four proteins having transmembrane domains (SEQ ID NO:s 7, 19, 22, and 25) were considered potential candidates due to their potential involvement in interaction between SCN and soy. One protein (SEQ ID NO: 10) containing a TolA domain, which is associated with membranes, and another protein (SEQ ID NO:31) with no known function were also selected for testing.Example 3

[0185] This example describes the development of transient and stable transgenic assays to confirm the SCN resistance phenotype conferred by a candidate gene identified in Example 2.

[0186] Two transient hairy root assays were developed and used to test the candidate genes of interest identified in Example 2. Agrobacterium rhizogenes lines were created with a vector comprising an expression construct encoding one of SEQ ID NOs: 7, 22, and 25, respectively. An Agrobacterium rhizogenes line carrying a control construct that does not carry any candidate gene was also created. In an in planta assay, primary roots of healthy soybean seedlings were removed by cutting. Each seedling’s exposed hypocotyl tissue was transfected with one of the A. rhizogenes lines and the seedling was then grown under conditions that allow hairy roots to develop. In an ex planta assay, explants (e.g., cotyledons) were harvested from healthy soybean seedlings. Explants were transfected with one of the A. rhizogenes lines and the explants were cultured under conditions that allowed hairy roots to develop.

[0187] Successfully transformed hairy roots were identified using a visual marker. Transformed roots were challenged by exposure to 2000 eggs / ml of race 2 (HG 1, 2, 5, 6, 7) SCN. The number of cysts that develop for each dose of SCN in hairy roots carrying each of the candidate genes encoding SEQ ID NOs: 7, 22, and 25, respectively, was then compared to the number of cysts in corresponding hairy roots carrying the control construct. Table 2 provides the number of cysts in the hairy roots and the relative expression level of the candidate genes.Table 2: Cyst counts and relative gene expression level in hairy roots* significant reduction compared to control, p value <0.05

[0188] Stable transgenic plants carrying each of the candidate genes encoding each of SEQ ID NOs: 7, 10, 22, 25, and 31, were created. Stable transgenic plants carrying a candidate gene encoding SEQ ID NO: 7, 22, or 25 and control plants that do not carry the candidate gene (but are otherwise isogenic to each of the transgenic plants) are compared by exposure to SCN in SCN bioassays and by exposure to SCN in field trials. Stable transgenic plants carrying a candidate gene encoding SEQ ID NO: 10 or 31 and control plants that do not carry the candidate gene (but are otherwise isogenic to each of the transgenic plants) were compared by exposure to SCN in SCN bioassays. The efficacy and expression data for three events encoding SEQ ID NO: 31 and three events encoding SEQ ID NO: 10 are provided in Tables 3A and 3B, respectively.Table 3A: Cyst counts and relative gene expression level in soybean plants expressing SEQ IDNO: 31Table 3B: Cyst counts and relative gene expression level in soybean plants expressing SEQ IDNO: 10* significant reduction compared to segregating plants that do not carry the candidate gene

[0189] The foregoing confirms that one or more of the candidate genes identified in Example 2 confers an SCN resistance phenotype.Example 4

[0190] This example provides molecular markers for selecting and developing soybean products having the cqSCN-007 QTL alleles associated with improved SCN resistance. Table 4 provides single nucleotide polymorphism (SNP) alleles on that can be used individually or in combination (i.e., a haplotype) to select the SCN resistant QTL on chromosome 18 described herein. Physical positions shown in Table 4 are based on the W82.a2.vl assembly (Brown et al. 2021 supra , chromosome 18; “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.Table 4

[0191] Table 5 provides primer and probe sequences that can be used to detect and select for one or more of the resistance alleles disclosed in Table 4.Table 5

[0192] A breeding population is genotyped for marker S7-001 and / or S8-001, individually or together. Plants having the favorable allele(s) of each shown in Table 2 are selected and used for further breeding. Markers S7-001 and / or S8-001 are also used individually or together to select for resistant plants having improved resistance to SCN.

Claims

WHAT IS CLAIMED:

1. A method of modifying plant material, the method comprising introducing into the plant material’s genome a heterologous nucleic acid sequence or expressing the heterologous nucleic acid sequence in the plant material, wherein the heterologous nucleic acid (i) encodes a polypeptide having at least 90% amino acid sequence identity to any one of SEQ ID NOs: 22, 25, 1, 4, 7, 10, 13, 16, 19, 28, or 31, (ii) comprises a sequence having at least 90% nucleotide sequence identity to any one of SEQ ID NOs: 23, 26, 2, 5, 8, 11, 14, 17, 20, 29, or 32, or (iii) comprises a sequence having at least 90% nucleotide sequence identity to any one of SEQ ID NOs: 24, 27, 3, 6, 9, 12, 15, 18, 21, 30, or 33; and wherein the heterologous nucleic acid is associated with increased resistance to soybean cyst nematode.

2. The method of claim 1, wherein the heterologous nucleic acid (i) encodes a polypeptide having at least 90% amino acid sequence identity to SEQ ID NO: 22, 25, 7, 10, 19, or 31, (ii) comprises a sequence having at least 90% nucleotide sequence identity to SEQ ID NO: 23, 26, 8,11, 20, or 32, or (iii) comprises a sequence having at least 90% nucleotide sequence identity to SEQ ID NO: 24, 27, 9, 12, 21, or 33.

3. The method of claim 1 or 2, wherein the plant material is soybean plant material and the heterologous nucleic acid is inserted into a location on soybean chromosome 1-17, 19, or 20.

4. The method of any one of claims 1-3, wherein the plant material is soybean plant material and the heterologous nucleic acid is inserted into a location other than its native locus corresponding to physical positions 53,484,561 to 53,614,866 of chromosome 18 in soybean genome Wm, 82v4.

5. The method of any one of claims 1-4, wherein the heterologous nucleic acid further comprises a heterologous promoter.

6. The method of any one of claims 1-4, wherein the heterologous nucleic acid further comprises its native promoter.

7. The method of any one of claims 1-6, wherein the method comprises introducing the heterologous nucleic acid using a double-stranded break.

8. The method of claim 7, wherein the method comprises introducing the heterologous nucleic acid using TALEN, meganuclease, zinc finger nuclease, or CRISPR-Cas technology.

9. The method of claim 7, wherein the method comprises introducing the heterologous nucleic acid using Cas endonuclease.

10. A method of introducing a gene associated with increased resistance to soybean cyst nematode (SCN) into a plant, said method comprising: a. crossing a plant having SCN resistance 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 (i) encodes a polypeptide having at least 90% amino acid sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, or 31, (ii) comprises a sequence having at least 90% nucleotide sequence identity to any one of SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, 32, or (iii) comprises a sequence having at least 90% nucleotide sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33; and d. selecting one or more progeny plants that have the screened-for nucleic acid.

11. The method of claim 10, wherein the plant having SCN resistance comprises the screened- for nucleic acid.

12. The method of claim 10 or 11, wherein the screened-for nucleic acid comprises a nucleic acid that (i) encodes a polypeptide having at least 90% amino acid sequence identity to SEQ ID NO: 7, 10, 19, 22, 25 or 31, (ii) comprises a sequence having at least 90% nucleotide sequence identity toSEQ ID NO: 8, 1 1, 20, 23, 26, or 32, or (iii) comprises a sequence having at least 90% nucleotide sequence identity to SEQ ID NO: 9, 12, 21, 24, 27, or 33.

13. The method of any one of claims 10-13, wherein the screened-for nucleic acid is a heterologous nucleic acid on soybean chromosome 1-17, 19, or 20.

14. The method of any one of claims 10-13, wherein the screened-for nucleic acid is a heterologous nucleic acid on soybean chromosome 18 at a locus other than its native locus corresponding to physical positions 53,484,561 to 53,614,866 of chromosome 18 in soybean genome Wm, 82v4.

15. The method of claim 14, wherein the heterologous nucleic acid has been 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.

16. The method of any one of claims 10-15, further comprising crossing the selected one or more progeny plants with the second plant to produce backcross progeny plants.

17. The method of claim 16, 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.

18. The method of claim 17, 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; andg. selecting one or more additional backcross progeny plants that have the screened for nucleic acid.

19. The method of claim 18, 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. 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.

20. A genome edited or transgenic plant comprising a heterologous nucleic acid sequence or expressing the heterologous nucleic acid sequence, wherein the heterologous nucleic acid (i) encodes a polypeptide having at least 90% amino acid sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, or 31, (ii) comprises a sequence having at least 90% nucleotide sequence identity to any one of SEQ ID NOs: 2, 5, 8, 11, 14, 17, 20, 23, 26, 29, or 32, or (iii) comprises a sequence having at least 90% nucleotide sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, or 33; and wherein the heterologous nucleic acid is associated with increased resistance to soybean cyst nematode.

21. The genome edited or transgenic plant of claim 20, wherein the heterologous nucleic acid (i) encodes a polypeptide having at least 90% amino acid sequence identity to SEQ ID NO: 7, 10, 19,22. 25 or 31, (ii) comprises a sequence having at least 90% nucleotide sequence identity to SEQ ID NO: 8, 11, 20, 23, 26, or 32, or (iii) comprises a sequence having at least 90% nucleotide sequence identity to SEQ ID NO: 9, 12, 21, 24, 27, or 33.

22. The genome edited or transgenic plant of claim 20 or 21 , wherein the plant is a soybean plant and the heterologous nucleic acid is inserted into a location on soybean chromosome 1-17, 19, or 20.

23. The genome edited or transgenic plant of any one of claims 20-22, wherein the plant is a soybean plant and the heterologous nucleic acid is inserted into a location other than its native locus corresponding to physical positions 53,484,561 to 53,614,866 of chromosome 18 in soybean genome Wm, 82v4.

24. The genome edited or transgenic plant of any one of claims 20-23, wherein the heterologous nucleic acid further comprises a heterologous promoter.

25. The genome edited or transgenic plant of any one of claims 20-24, wherein the heterologous nucleic acid further comprises its native promoter.

26. Use of the genome edited or transgenic plant of any one of claims 20-25 for control of soybean cyst nematode (SCN) comprising providing to the SCN an effective amount of a polypeptide having at least 90% amino acid sequence identity to any one of SEQ ID NOs: 1, 4, 7, 10, 13, 16, 19, 22, 25, 28, or 31.

27. The use of claim 26, comprising providing to the SCN an effective amount of a polypeptide having at least 90% amino acid sequence identity to SEQ ID NO: 7, 10, 19, 22, 25 or 31.

28. A method of detecting the presence or absence of a SNP allele at position 201 of one or both of SEQ ID NO: 34 or SEQ ID NO: 39 in a plant genome; the method comprising: amplifying genomic DNA containing the SNP allele at position 201 of one or more of SEQ ID NO: 34 or SEQ ID NO: 39; to thereby generate an amplicon; contacting the amplicon with one or more probes comprising one or more of SEQ ID NOs: 37, 38, 42, 43; 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.

29. The method of claim 28, wherein the method comprises detecting the zygosity of the SNP allele at position 201 of one or both of SEQ ID NO: 34 or SEQ ID NO: 39 in a plant genome; the method comprising: amplifying genomic DNA containing the SNP allele at position 201 of one or both of SEQ ID NO: 34 or SEQ ID NO: 39; to thereby generate an amplicon; contacting the amplicon with one or two of the following sets of probes: a first set comprising a first probe comprising SEQ ID NO:37 and a second probe comprising SEQ ID NO:38 and / or a second set comprising a first probe comprising SEQ ID NO:42 and a second probe comprising SEQ ID NO: 43; 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.

30. The method of claim 28 or 29, wherein the amplicon is amplified using a first set of two primers: one primer comprising SEQ ID NO:35 the other primer comprising SEQ ID NO:36, and / or a second set of two primers: one primer comprising SEQ ID NO:40 and the other primer comprising SEQ ID NO:41.

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