Methods for identifying, selecting and producing crops resistant to anthracnose stem rot

Abstract

The present invention relates to plant breeding and methods for identifying and selecting plants resistant to anthracnose stem rot. Methods for identifying novel genes encoding proteins that provide plant resistance to anthracnose stem rot and uses thereof are provided. These disease resistance genes can be used to generate resistant plants through breeding, transgenic modification or genome editing.

Description

Technical Field

[0001] The present invention relates to plant breeding and methods for identifying and selecting plants resistant to anthracnose stalk rot. Methods for identifying novel genes encoding proteins that provide plant resistance to anthracnose stalk rot and uses thereof are provided. These disease resistance genes can be used to generate resistant plants through breeding, transgenic modification or genome editing.

[0002] References to Sequence Listings Submitted as Text Files via EFS-Web

[0003] An official copy of the sequence listing is submitted as a text file via EFS-Web in accordance with the American Standard Code for Information Interchange (ASCII) with the present specification, wherein the file name is 8054_Seq_List.txt, the creation date is August 22, 2019, and the size is 63Kb. The sequence listing submitted via EFS-Web is part of the specification and is incorporated herein by reference in its entirety. Background Art

[0004] Anthracnose stalk rot, caused by the fungal pathogen Colletotrichum graminicola (Ces.) Wils (Cg), is one of the major stalk rot diseases in maize (Zea mays L.). Anthracnose stalk rot is a major concern due to significant reductions in yield, grain weight and quality. Yield losses result from immature plant mortality (interruption of grain filling) as well as stalk breakage and lodging (resulting in loss of ears in the field). Anthracnose stalk rot occurs in all corn-growing regions and can cause losses of 10% to 20%.

[0005] While farmers can combat fungal infections such as anthracnose by using fungicides, these fungicides have side effects on the environment and require field monitoring and diagnostic techniques to determine which fungus is causing the infection so that the correct fungicide can be used. Using corn lines of genetic or transgenic origin that carry resistance would be more practical if the genes responsible for resistance could be incorporated into elite, high-yielding germplasm without reducing yield. The genetic sources of Cg resistance have been described (White, et al. (1979) Annu. Corn Sorghum Res. Conf. Proc. 34: 1-15; Carson. 1981. Sources of inheritance of resistance to anthracnose stalk rot of corn. Ph. D. Thesis, University of Illinois, Urbana-Champaign; Badu-Apraku et al. (1987) Phytopathology 77: 957-959; Toman et al. 1993. Phytopathology, 83: 981-986; Cowen, N et al. (1991) Maize Genetics Conference Abstracts 33; Jung et al. 1994. Theoretical and Applied Genetics, 89: 413-418). However, the introgression of resistance can be very complex.

[0006] Select by using the molecular marker that is associated with the anthracnose stem rot resistance trait, allow the selection based only on the gene composition of progeny. As a result, plant breeding can occur more rapidly, produce commercially acceptable maize plants with higher levels of anthracnose stem rot thus. There are multiple QTLs (for example rcg1 and rcg1b genes on chromosome 4 (WO 2008157432 and WO 2006107931)) that control anthracnose stem rot resistance, and each QTL has different effects on traits. Therefore, it is desirable to provide a composition and method for identifying and selecting a maize plant with newly conferred or enhanced anthracnose stem rot resistance. There is a continuous need for disease resistant plants and methods for finding disease resistance genes. Summary of the invention

[0007] Provided herein are compositions and methods useful in identifying and selecting plant disease resistance genes, or "R genes". The compositions and methods can be used to select disease resistant plants, produce transgenic resistant plants, and / or produce plants with resistance genome editing. Also provided herein are plants with newly conferred or enhanced resistance to various plant diseases compared to control plants. In some embodiments, the compositions and methods can be used to select anthracnose stem rot (ANTROT) disease resistant plants, produce transgenic anthracnose stem rot resistant plants, and / or produce plants with anthracnose stem rot resistance genome editing.

[0008] Anthracnose stem rot resistant plants can be crossed with a second plant to obtain progeny plants having the resistance gene allele. The disease resistance can be newly conferred or enhanced relative to a control plant that does not have the favorable allele. The anthracnose stem rot R gene allele can be further refined to a chromosome interval defined by and including a defined marker. In some embodiments, methods for identifying and / or selecting plants with anthracnose stem rot resistance are proposed. In some embodiments, the method for identifying and / or selecting plants with anthracnose stem rot resistance comprises detecting or selecting a genomic region comprising SEQ ID NO: 3 (from maize line TZI8) or SEQ ID NO: 5 (from maize line MS14), the genomic region having a promoter and terminator of SEQ ID NO: 4. The anthracnose stem rot resistance can be newly conferred or enhanced relative to a control plant that does not have the favorable allele. In another embodiment, the anthracnose stalk rot resistance region comprises a gene encoding an NLR02 polypeptide that confers or enhances anthracnose stalk rot resistance ("NLR02 gene"). In some embodiments, the NLR02 polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.

[0009] In another embodiment, a method for identifying and / or selecting plants with ANTROT resistance is provided, wherein one or more marker alleles linked and associated with any one of the following are detected in the plant: "C" at the left flank (conserved)_marker 104 (position 51 of the reference sequence SEQ ID NO: 6); "A" at the right flank (conserved)_marker 015 (position 51 of the reference sequence SEQ ID NO: 7); "T" at the left flank (optimized)_marker 115 (position 35 of the reference sequence SEQ ID NO: 8); and "C" at the right flank (optimized)_marker 157 (position 30 of the reference sequence SEQ ID NO: 9), and plants having the one or more marker alleles are selected. The one or more marker alleles may be linked at 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.9 cM, 0.8 cM, 0.7 cM, 0.6 cM, 0.5 cM, 0.4 cM, 0.3 cM, 0.2 cM or 0.1 cM or less on a genetic map based on a single meiosis. The selected plant can be crossed with a second plant to obtain a progeny plant having one or more marker alleles linked to and associated with any of the following: "C" at the left flank (conserved)_marker 104 (position 51 of the reference sequence SEQ ID NO: 6); "A" at the right flank (conserved)_marker 015 (position 51 of the reference sequence SEQ ID NO: 7); "T" at the left flank (optimized)_marker 115 (position 35 of the reference sequence SEQ ID NO: 8); and "C" at the right flank (optimized)_marker 157 (position 30 of the reference sequence SEQ ID NO: 9).

[0010] In another embodiment, methods for introgressing a QTL associated with anthracnose stem rot resistance are presented herein. In these methods, a population of plants is screened with one or more markers to determine whether any of the plants have a QTL associated with anthracnose stem rot resistance, and at least one plant having a QTL associated with anthracnose stem rot resistance is selected from the population. The QTL comprises a "C" at left flank (conserved)_marker 104 (position 51 of reference sequence SEQ ID NO: 6); an "A" at right flank (conserved)_marker 015 (position 51 of reference sequence SEQ ID NO: 7); a "T" at left flank (optimized)_marker 115 (position 35 of reference sequence SEQ ID NO: 8); and a "C" at right flank (optimized)_marker 157 (position 30 of reference sequence SEQ ID NO: 9).

[0011] In some embodiments, introgression of anthracnose stalk rot resistance genes from resistant lines into susceptible lines can be achieved through marker-assisted trait introgression, transgenics, or genome editing methods.

[0012] Embodiments include isolated polynucleotides comprising a nucleotide sequence encoding an NLR02 polypeptide capable of conferring resistance to anthracnose stalk rot, wherein the NLR02 polypeptide has an amino acid sequence that is at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, or at least 99% identical to SEQ ID NO: 2. In another embodiment, an isolated polynucleotide comprises a nucleotide sequence encoding an NLR02 polypeptide capable of conferring resistance to ANTROT, wherein the NLR02 polypeptide has an amino acid sequence that is at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% sequence identity to SEQ ID NO: 2. In some embodiments, the polynucleotide encoding the NLR02 polypeptide comprises a nucleic acid sequence that is at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% sequence identity to SEQ ID NO: 1.

[0013] Additional embodiments of the present disclosure include vectors comprising polynucleotides of the present disclosure, such as SEQ ID NO: 1, or recombinant DNA constructs comprising polynucleotides disclosed herein operably linked to at least one regulatory sequence. Plant cells and plants, each comprising a recombinant DNA construct of the embodiments disclosed herein, and seeds comprising the recombinant DNA constructs are also presented.

[0014] In some embodiments, the compositions and methods relate to modified plants having increased resistance to disease, wherein the allele causing the increased disease resistance comprises a nucleotide sequence encoding an NLR02 resistance gene, wherein the NLR02 resistance gene has at least 50%, at least 75%, at least 80%, at least 85%, at least 90%, and at least 95% identity, at least 96% identity, at least 97% identity, at least 98% identity, or at least 99% identity to the sequence shown in SEQ ID NO:2.

[0015] The methods embodied in the present disclosure relate to methods for transforming host cells, including plant cells, comprising transforming host cells with a polynucleotide of an embodiment of the present disclosure; for manufacturing comprising transforming plant cells with a recombinant DNA construct of an embodiment of the present disclosure and regenerating plants from the transformed plant cells, and conferring or enhancing disease resistance, the method comprising transforming plants with the recombinant DNA construct disclosed herein.

[0016] Also presented are methods for altering the expression level of a protein capable of conferring disease resistance on a plant or plant cell, the method comprising (a) transforming a plant cell with a recombinant DNA construct disclosed herein, and (b) culturing the transformed plant cell under conditions suitable for expressing the recombinant DNA construct, wherein expression of the recombinant DNA construct results in the production of altered levels of the protein capable of conferring disease resistance in the transformed host.

[0017] Also provided are plants identified and / or selected using any of the above methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 PH12P9 / TZI8 NLS in PH1V69 background is shown. NIL negative (left) and positive (right) for the TZI8 Chr06 region. DETAILED DESCRIPTION

[0019] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "a protein" includes reference to one or more proteins and their equivalents, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs unless expressly stated otherwise.

[0020] The NBS-LRR ("NLR") group of R genes is the largest class of R genes discovered to date. In Arabidopsis thaliana, more than 150 NLR genes are predicted to be present in the genome (Meyers, et al., (2003), Plant Cell, 15:809-834; Monosi, et al., (2004), Theoretical and Applied Genetics, 109:1434-1447), while in rice, approximately 500 NLR genes have been predicted (Monosi, (2004) supra). The NBS-LRR class of R genes consists of two subclasses. Class 1 NLR genes contain a TIR-Toll / interleukin-1-like domain at their N' terminus; they have been found only in dicots to date (Meyers, (2003) supra; Monosi, (2004) supra). The second type of NBS-LRR contains a coiled-coil domain or (nt) domain at its N-terminus (Bai, et al. (2002) Genome Research, 12: 1871-1884; Monosi, (2004) supra; Pan, et al. (2000) Journal of Molecular Evolution, 50: 203-213). Type 2 NBS-LRRs are found in both dicot and monocot species (Bai, (2002) supra; Meyers, (2003) supra; Monosi, (2004) supra; Pan, (2000) supra).

[0021] The NBS domain of the gene appears to play a role in signaling of plant defense mechanisms (van der Biezen, et al., (1998), Current Biology: CB, 8: R226-R227). The LRR region appears to be a region that interacts with pathogen AVR products (Michelmore, et al., (1998), Genome Res., 8: 1113-1130; Meyers, (2003) supra). Compared to the NB-ARC (NBS) domain, this LRR region is under greater selective pressure to diversify (Michelmore, (1998) supra; Meyers, (2003) supra; Palomino, et al., (2002), Genome Research, 12: 1305-1315). LRR domains can also be found in other contexts; these 20-29 residue motifs are arranged in tandem in many proteins that have diverse functions such as hormone-receptor interactions, enzyme inhibition, cell adhesion, and cell trafficking. Many recent studies have shown that LRR proteins are involved in early mammalian development, neural development, cell polarization, regulation of gene expression, and apoptosis signaling.

[0022] An allele is "associated with a trait" when the allele is part of or linked to a DNA sequence or allele that affects the expression of the trait. The presence of the allele is an indicator of how the trait will be expressed.

[0023] As used herein, "disease resistance" or "resistant to disease" refers to a plant that exhibits increased resistance to a disease compared to a control plant. Disease resistance can be manifested as fewer and / or smaller lesions, increased plant health, increased yield, increased root mass, increased plant vigor, less or no discoloration, increased growth, reduced necrotic area, or reduced wilting. In some embodiments, the allele can show resistance to one or more diseases.

[0024] Diseases affecting corn plants include, but are not limited to, bacterial leaf blight and stalk rot; bacterial leaf spot; bacterial stripe; chocolate spot; goss's bacterial wilt and blight; holcus spot; purple leaf sheath; seed rot-seedling blight; bacterial wilt; corn stunt; anthracnose leaf blight; anthracnose stalk rot; aspergillus ear and kernel rot; banded leaf and sheath spot; leaf and sheath spot; black bundle disease; black kernel rot; borde blanco; brown spot; blackspot; stalk rot; cephalosporium kernel rot; charcoal rot; corticium ear rot; curvularia leaf spot; didymella leaf spot; diplodia ear rot and stalk rot; diplodia ear rot; seed rot; corn seedling blight; diplodia leaf spot or leafstreak; downy mildew; mildews); brown stripe downy mildew; crazy top downy mildew; green ear downy mildew; graminicola downy mildew;Java downy mildew; Philippine downy mildew; Sorghum downy mildew; Spontaneum downy mildew; Sugarcane downy mildew; Dry earrot; Ergot; Horse's tooth; Corn eyespot; Fusarium ear and stalk rot; Fusarium blight; Seedling root rot; Gibberella ear and stalk rot; Gray ear rot; Gray leaf spot; Cercospora leaf spot; Helminthosporium root rot; hormodendrum ear rot; cladosporium rot; hyalothyridium leaf spot; late wilt; northern leaf blight; whiteblast; crown stalk rot; corn stripe; northern leaf spot; helminthosporium ear rot; penicillium ear rot; corn blue eye; blue mold; phaeocytostromastalk rot and root rot; phaeosphaeria leaf spot; physalospora ear rot. rot); botryosphaeria ear rot; pyrenochaeta stalk rot and root rot; pythium root rot; pythium stalk rot;red kernel disease; rhizoctonia ear rot; sclerotial rot; rhizoctonia root rot and stalk rot; rostratum leaf spot; common corn rust; southern corn rust; tropical corn rust; sclerotium ear rot; southern blight; selenophoma leaf spot; sheath rot; shuck rot; silage mold; common smut; false smut; head smut; southern corn leaf blight and stalk rot rot; southern leaf spot; tar spot; trichoderma ear rot and root rot; white ear rot, root and stalk rot; yellow leaf blight; zonate leaf spot; wheat striate mosaic; barley stripe mosaic; barley yellow dwarf; brome mosaic; cereal chlorotic mottle; maize lethal necrosis disease; cucumber mosaic; johnsongrass mosaic; maize bushy dwarf stunt); maize chlorotic dwarf; maize chlorotic mottle; maize dwarf mosaic;maize leaf fleck; maize pellucid ringspot; maize rayado fino; maize red leaf and red stripe; maize red stripe; maize ring mottle; maize rough dwarf; maize sterile stunt; maize streak; maize stripe; maize tassel abortion; maize vein enation; maize wallaby ear; maize white leaf; maize white line mosaic; millet red leaf; and northern cereal mosaic.

[0025] Diseases affecting plants include, but are not limited to, bacterial blight; bacterial leaf streak; foot rot; grain rot; sheath brown rot; blast; brown spot; crown sheath rot; downy mildew; eyespot; false smut; kernelsmut; leaf smut; leaf scald; narrow brown leaf spot; root rot; seedling blight; sheath blight; sheath rot; sheath spot; alternaria leaf spot; leafspot; and stem rot.

[0026] Diseases affecting soybean plants include, but are not limited to, alternaria leaf spot; anthracnose; black leaf blight; black root rot; brown spot; brown stem rot; charcoal rot; choanephora leaf blight; downy mildew; drench slerablight; frogeye leaf spot; leptosphaerulina leaf spot; mycoleptodiscus root rot; neocosmospora stem rot; phomopsis seed decay; phytophthora root and stem rot; and phytophthora root and stem rot. rot; phyllosticta leaf spot; phymatotrichum root rot; pod and stem blight; powdery mildew; purple seed stain; pyrenochaeta leaf spot; pythium rot; red crown rot; dactuliophora leaf spot; rhizoctonia aerial blight; rhizoctonia root and stem rot; rust; scab; sclerotinia stem rot; sclerotium blight; stem canker; stemphylium leaf blight blight; sudden death syndrome; target spot; yeast spot; lance nematode; lesion nematode; pin nematode;reniform nematode; ring nematode; root-knot nematode; sheath nematode; cyst nematode; spiral nematode; sting nematode; stubby root nematode; stunt nematode; alfalfa mosaic; bean podmottle; bean yellow mosaic; brazilian bud blight; chlorotic mottle; yellow mosaic; peanutmottle; peanut stripe; peanut stunt; chlorotic mottle; yellow mosaic; peanutmottle; peanut stripe; peanut stunt; chlorotic mottle; crinkle leaf; dwarf; severe stunt; and tobacco ringspot or bud blight.

[0027] Diseases affecting canola plants include, but are not limited to, bacterial blackrot; bacterial leaf spot; bacterial pod rot; bacterial soft rot; scab; crown gall; alternaria black spot; anthracnose; black leg; black mold rot; black root; brown girdling root rot; cercospora leaf spot; clubroot; downy mildew; fusarium wilt; gray mold; head rot; leaf spot; light leaf spot; and spot; pod rot; powdery mildew; ringspot; root rot; sclerotinia stem rot; seedrot (damping-off); root gall smut; southern blight; verticillium wilt; white blight; white leaf spot; staghead; yellows; crinkle virus; mosaic virus; yellows virus;

[0028] Diseases that affect sunflower plants include, but are not limited to, apical chlorosis; bacterial leaf spot; bacterial wilt; crown gall; erwinia stalk rot and head rot; alternaria leaf blight, stem spot and head rot; botrytis head rot; charcoal rot; downy mildew; fusarium stalk rot; fusarium wilt; myrothecium leaf and stem spot; phialophora yellows; phoma blackstem; phomopsis brown stem canker; canker; phymatotrichum root rot; phytophthora stem rot; powdery mildew; pythium seedling blight and root rot; rhizoctonia seedling blight; rhizopus head rot; sunflower rust; sclerotium basal stalk and root rot; septorial eaf spot; verticillium wilt; white rust; yellow rust; dagger-shaped; needle-shaped; lesion; reniform; knots; and chlorotic mottle;

[0029] Diseases affecting sorghum plants include, but are not limited to, bacterial leaf spot; bacterial leaf streak; bacterial leaf stripe; acremonium wilt; anthracnose; charcoal rot; crazy top downy mildew; damping-off and seed rot; ergot; fusarium head blight, root and stalk rot; grain storage mold; gray leaf spot; latter leaf spot; leaf blight; milo disease; oval leaf spot; pokkah boeng; pythium root rot; rough leaf spot; rust; pythium seedling blight and root rot; smut (covered kernel); smut (head); smut (loose kernel); sooty stripe; downy mildew; tar spot; target leaf spot; and zonate leafspot and sheath blight.

[0030] Disease resistant plants can have 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95 or 100% increased resistance compared to control plants. In some embodiments, plants can have 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95 or 100% increased plant health in the presence of disease compared to control plants.

[0031] As used herein, the term "chromosome interval" refers to a continuous linear span of genomic DNA present on a single chromosome of a plant. Genetic elements or genes located on a single chromosome interval are physically linked. The size of a chromosome interval is not particularly limited. In some aspects, the genetic elements located within a single chromosome interval are genetically linked, typically having, for example, a genetic recombination distance of less than or equal to 20 cM, or alternatively, less than or equal to 10 cM. ​​That is, two genetic elements within a single chromosome interval recombine at a frequency of less than or equal to 20% or 10%.

[0032] In the present application, the phrase "tightly linked" means that recombination between two linked loci occurs at a frequency equal to or less than about 10% (i.e., separated by no more than 10 cM on a genetic map). In other words, the tightly linked loci have at least a 90% chance of co-segregation. When the marker locus shows a significant probability of co-segregation (linkage) with the desired trait (e.g., resistance to ANTROT), the marker locus is particularly useful for the subject matter of the present disclosure. Tightly linked loci (e.g., a marker locus and a second locus) can show a recombination frequency between loci of 10% or less, preferably about 9% or less, still more preferably about 8% or less, yet more preferably about 7% or less, yet more preferably about 6% or less, yet more preferably about 5% or less, yet more preferably about 4% or less, yet more preferably about 3% or less, and yet more preferably about 2% or less. In a very preferred embodiment, the related locus shows about 1% or lower, for example about 0.75% or lower, more preferably about 0.5% or lower or more preferably about 0.25% or lower recombination frequency. Positioned on the same chromosome and having a distance that makes the reorganization between the two loci occur with a frequency less than 10% (for example, about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25% or less) is also considered to be "adjacent" to each other. In some cases, two different markers can have identical genetic map coordinates. In this case, the two markers are very adjacent to each other, so that the reorganization between the two occurs with this low frequency that cannot be detected.

[0033] The term "hybridized" or "crossing" refers to sexual crossing and involves the fusion of two haploid gametes by pollination to produce diploid progeny (e.g., cells, seeds, or plants). The term encompasses both pollination of one plant by another and selfing (or self-pollination, e.g., when the pollen and ovules are from the same plant).

[0034] An "elite line" is any line produced by breeding for the performance of superior agronomic traits.

[0035] "Exotic varieties," "tropical lines," or "exotic germplasm" are varieties derived from plants that are not part of an available elite line or germplasm variety. In the case of a cross between two plant or germplasm varieties, the offspring of the exotic germplasm are not closely related to the elite germplasm with which it was crossed. Most commonly, the exotic germplasm is not derived from any known elite line, but is selected to introduce new genetic elements (usually new alleles) into a breeding program.

[0036] "Favorable allele" is an allele of a specific locus (marker, QTL, gene, etc.) that confers or contributes to an agronomically desirable phenotype (e.g., disease resistance) and allows identification of plants with the agronomically desirable phenotype. A favorable allele of a marker is a marker allele that segregates with the favorable phenotype.

[0037] "Genetic marker" is a nucleic acid that is polymorphic in a population, and the alleles of the genetic marker can be detected and distinguished by one or more analytical methods (e.g., RFLP, AFLP, isozymes, SNPs, SSRs, etc.). The term also refers to a nucleic acid sequence that is complementary to a genomic sequence (e.g., nucleic acid) used as a probe. The marker corresponding to the genetic polymorphism between the population members can be detected by methods generally recognized in the art. These methods include, for example, sequence-specific amplification methods based on PCR, restriction fragment length polymorphism detection (RFLP), isozyme marker detection, polynucleotide polymorphism detection by allele-specific hybridization (ASH), amplification variable sequence detection of plant genomes, autonomous sequence replication detection, simple repeat sequence detection (SSR), single nucleotide polymorphism detection (SNP), or amplified fragment length polymorphism detection (AFLP). Known generally recognized methods are also used to detect expressed sequence tags (ESTs) and SSR markers derived from EST sequences, as well as randomly amplified polymorphic DNA (RAPD).

[0038] " Germplasm " refers to hereditary material, it belongs to or comes from individual (for example, plant), individual group (for example, plant strain, variety or family) or derived from strain, variety, species or culture clone, or more generally, all individuals of a certain species or multiple species (for example, maize germplasm collection (maize germplasm collection) or Andean germplasm collection (Andean germplasmcollection)). The germplasm can be a part of an organism, a cell, or can be separated from the organism or cell. Generally speaking, germplasm provides hereditary material with specific molecular composition, and the specific molecular composition provides physical basis for some or all of the genetic qualities of an organism or cell culture. As used herein, germplasm includes cells, seeds or tissues that can grow new plants thus, or can be cultured into plant parts of complete plants, such as leaves, stems, pollen or cells.

[0039] A "haplotype" is an individual's genotype at multiple genetic loci, ie, a combination of alleles. Typically, the genetic loci described by a haplotype are physically and genetically linked, ie, on the same chromosome segment.

[0040] The term "heterogeneity" is used to indicate that individuals within the group differ in their genotype at one or more specific loci.

[0041] The heterotic response or "hybrid vigor" of a material can be defined by its performance above the average of a parent (or high parent) when crossed to other dissimilar or unrelated groups.

[0042] A "heterotic group" comprises a group of genotypes that perform well when crossed with genotypes from a different heterotic group (Hallauer et al. (1998) Corn breeding, pp. 463-564. In GF Sprague and JW Dudley (eds.) Corn and corn improvement). Inbred lines are divided into heterotic groups and further subdivided into families within heterotic groups based on several criteria, such as pedigree, molecular marker-based association, and performance in hybrid combinations (Smith et al. (1990) Theor. Appl. Gen. 80: 833-840). In the United States, the two most widely used heterotic groups are called the "Iowa Stiff Stalk Synthetic" (also referred to herein as "rigid stalk") and "Lancaster" or "Lancaster Sure Crop" (sometimes referred to as NSS or non-rigid stalk).

[0043] Some heterotic groups have the traits required to be female, and others have the traits required to be male. For example, in maize, the yield results of the public inbreds released from a group called BSSS (Iowa rigid stalk synthesis group) have led to these inbreds and their derivatives becoming female pools in the central corn belt. BSSS inbreds have been hybridized with other inbreds (e.g., SD 105 and Maiz Amargo), and the general group of this material has been known for rigid stalk synthesis (Stiff Stalk Synthetics, SSS), even though not all inbreds are derived from the original BSSS group (Mikel and Dudley (2006) Crop Sci [Crop Science]: 46: 1193-1205). By default, all other inbreds that combine well with the SSS inbreds are assigned to the male pool, which is named NSS for lack of a better name, i.e. non-rigid stalks. This group includes several major heterotic groups such as Lancaster Surecrop, Iodent and Leaming Corn.

[0044] The term "homogeneity" means that members of a group have the same genotype at one or more specific loci.

[0045] The term "hybrid" refers to the progeny obtained between a cross of at least two genetically distinct parents.

[0046] The term "inbred line" refers to a line that has been bred to achieve genetic homogeneity.

[0047] The term "indel" refers to an insertion or deletion, wherein one strain can be referred to as having an inserted nucleotide or DNA fragment relative to a second strain, or the second strain can be referred to as having a deleted nucleotide or DNA fragment relative to the first strain.

[0048] The term "infiltration" refers to the phenomenon that the desired allele of the genetic locus is transmitted to another genetic background from a kind of genetic background.For example, the infiltration of the required allele at the designated locus can be passed to at least one filial generation via the sexual hybridization between two parents of the same species, wherein at least one of the parents has the required allele in its genome.Alternately, for example, allelic transmission can occur by the reorganization between two donor genomes, for example in fusion protoplasts, wherein at least one of the donor protoplasts has the desired allele in its genome.Required allele can, for example, detect at QTL, transgenics, etc. by the mark associated with phenotype.The offspring comprising the required allele can be repeatedly backcrossed with the strain with the required genetic background and select the required allele to produce the allele fixed in the genetic background of selection.

[0049] When "introgression" is repeated two or more times, the process is often referred to as "backcrossing."

[0050] A "line" or "breed" is a group of individuals with the same parents, which are usually inbred to some extent, and are usually homozygous and homogeneous (isogenic or nearly isogenic) at most loci. A "subline" refers to a subpopulation of inbred lines that is genetically different from other similar subpopulations of inbred lines that originated from the same ancestor.

[0051] As used herein, the term "linkage" is used to describe the degree of association of a marker locus with another marker locus or some other loci. The linkage relationship between a molecular marker and the locus that affects the phenotype is represented by "probability" or "adjusted probability". Linkage can be expressed as required restriction or scope. For example, in some embodiments, when any marker is separated from any other marker by less than 50, 40, 30, 25, 20 or 15 map distance units (or cM) on a single meiosis map (based on the genetic map of a population (such as, F2) that has carried out a round of meiosis; IBM2 map consists of multiple meiosis), the marker is linked (genetically or physically). In some respects, it is advantageous to limit the linkage range of brackets, for example, between 10cM and 20cM, between 10cM and 30cM or between 10cM and 40cM. The tighter the linkage of the marker and the second locus, the better the indication effect of the marker to the second locus. Thus, "closely linked loci", such as a marker locus and a second locus, exhibit an inter-locus recombination frequency of 10% or less, preferably about 9% or less, still more preferably about 8% or less, yet more preferably about 7% or less, yet more preferably about 6% or less, yet more preferably about 5% or less, yet more preferably about 4% or less, yet more preferably about 3% or less, and yet more preferably about 2% or less. In a very preferred embodiment, the associated loci exhibit a recombination frequency of about 1% or less, such as about 0.75% or less, more preferably about 0.5% or less, or yet more preferably about 0.25% or less. The two loci that are located on the same chromosome and have a distance such that recombination between the two loci occurs at a frequency of less than 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25% or less) are also considered to be "adjacent" to each other. Because one cM is the distance between two markers that show a recombination frequency of 1%, any marker is tightly linked (genetically and physically) to any other marker that is closely adjacent (e.g., at a distance equal to or less than 10 cM). Two tightly linked markers on the same chromosome may be located 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5, or 0.25 cM or closer to each other.

[0052] The term "linkage disequilibrium" refers to the non-random separation of genetic loci or proteus (or both). In either case, linkage disequilibrium means that the related loci are physically close enough along a section of chromosome so that they separate together with a frequency higher than random (i.e., non-random). The markers showing linkage disequilibrium are considered to be linked. The linked loci have a chance of more than 50% (e.g., about 51% to about 100%) to separate together. In other words, two markers of cosegregation have a recombination frequency less than 50% (and, by definition, separated less than 50cM on the same linkage group). As used herein, linkage can be present between two markers, or alternatively, between a marker and a locus that affects a phenotype. The marker locus can be "associated" (linkage) with proteus. The linkage degree of the marker locus and the locus that affects a phenotypic proteus is, for example, measured by the statistical probability (e.g., F statistics or LOD score) of the molecular marker and the cosegregation of the phenotype.

[0053] Linkage disequilibrium is most commonly measured using the measure r 2 Evaluation, the measure r 2 The calculation was performed using the formula from Hill, WG and Robertson, A, Theor. Appl. Genet. 38: 226-231 (1968). 2 =1, there is complete LD between the two marker loci, meaning that the markers have not yet undergone recombination segregation and have the same allele frequency. 2 The value depends on the population used. 2 Values ​​greater than 1 / 3 indicate sufficiently strong LD for mapping (Ardlie et al., Nature Reviews Genetics 3:299-309 (2002)). 2 When the value is greater than or equal to 0.33, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0, the alleles are in linkage disequilibrium.

[0054] As used herein, "linkage equilibrium" describes a situation in which two markers segregate independently, ie, randomly distribute among progeny. Markers that exhibit linkage equilibrium are considered unlinked (regardless of whether they are located on the same chromosome).

[0055] A "locus" is a position on a chromosome, eg, where a nucleotide, gene, sequence, or marker is located.

[0056] "Odds logarithm (LOD) value" or "LOD score" (Risch, Science [Science] 255: 803-804 (1992)) is used for genetic interval mapping to describe the degree of linkage between two marker loci. A LOD score of three between two markers indicates that the probability of linkage is 1000 times higher than the probability of no linkage, while a LOD score of two indicates that the probability of linkage is 100 times higher than the probability of no linkage. A LOD score greater than or equal to two can be used to detect linkage. The LOD score can also be used to display the strength of association between the marker locus and the quantitative trait in the "quantitative trait locus" mapping. In this case, the LOD score size depends on the compactness between the marker locus and the locus that affects the quantitative trait, as well as the size of the quantitative trait effect.

[0057] The term "plant" includes whole plants, plant cells, plant protoplasts, plant cells or tissue cultures from which plants can be regenerated, plant callus, plant clumps, and intact plant cells or plant parts in plants, such as seeds, flowers, cotyledons, leaves, stems, buds, roots, root tips, etc. As used herein, "modified plants" means any plant with genetic changes due to human intervention. The modified plants may have genetic changes introduced by plant transformation, genome editing, or conventional plant breeding.

[0058] "Marker" is to find the position on the hereditary or physical map or to find the linkage between the marker and the trait locus (the locus that affects the trait). The position detected by the marker can be known by detecting polymorphic alleles and their genetic location, or by hybridizing, sequence matching or amplifying the sequence that has been physically mapped. The marker can be a DNA marker (detecting DNA polymorphism), a protein (detecting the variation of the encoded polypeptide) or a simple inherited phenotype (such as a "waxy" phenotype). DNA markers can be developed from genomic nucleotide sequences or from expressed nucleotide sequences (for example, from RNA or cDNA spliced). According to DNA marker technology, the marker can be composed of complementary primers flanking the locus and / or complementary probes hybridized with the polymorphic alleles at the locus. DNA markers or genetic markers can also be used to describe genes, DNA sequences or nucleotides (rather than components for detecting the genes or DNA sequences) on the chromosome itself, and they are usually used when the DNA marker is associated with a specific trait in human genetics (for example breast cancer markers). The term marker locus is the locus (gene, sequence or nucleotide) detected by the marker.

[0059] Marker can be defined by the type of polymorphism detected by it and the marking technique for detecting the polymorphism. Marker types include but are not limited to: restriction fragment length polymorphism detection (RFLP), isozyme marker detection, randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphism detection (AFLP), simple repeat sequence detection (SSR), amplified variable sequence detection of plant genome, autonomous sequence replication detection, or single nucleotide polymorphism detection (SNP). SNP can be detected by, for example, by DNA sequencing, PCR-based sequence-specific amplification method, multiple nucleotide polymorphism detection by allele-specific hybridization (ASH), dynamic allele-specific hybridization (DASH), molecular beacons, microarray hybridization, oligonucleotide ligase analysis, Flap endonuclease, 5' endonuclease, primer extension, single-stranded conformation polymorphism (SSCP) or temperature gradient gel electrophoresis (TGGE). DNA sequencing (such as pyrophosphate sequencing technology) has the advantage of being able to detect a series of linked SNP alleles that constitute haplotype. Haplotype tends to be more informative (detecting higher levels of polymorphism) than SNP.

[0060] A "marker allele", alternatively an "allele of a marker locus" can refer to one of a plurality of polymorphic nucleotide sequences found at a marker locus in a population.

[0061] "Marker assisted selection" (MAS) is a method of selecting individual plants based on their marker genotype.

[0062] "Marker-assisted counter-selection" is a method whereby marker genotypes are used to identify plants that are not to be selected, allowing them to be removed from a breeding program or planting.

[0063] A "marker haplotype" refers to the combination of alleles at a marker locus.

[0064] "Marker locus" is a specific chromosome location in a species genome where specific markers can be found. The marker locus can be used to track the presence of a second linked locus (e.g., a linked locus that affects phenotypic trait expression). For example, the marker locus can be used to monitor the separation of alleles at genetically or physically linked loci.

[0065] As mentioned above, when identifying linked loci, the term "molecular marker" can be used to refer to genetic markers, or its coded product (e.g., protein) used as a reference point. The marker can be derived from a genomic nucleotide sequence or derived from an expressed nucleotide sequence (e.g., derived from RNA, cDNA, etc. of splicing), or derived from a coded polypeptide. The term also refers to a nucleic acid sequence complementary to a marker sequence or flanked by it, such as a probe or a nucleic acid of a primer pair capable of amplifying the marker sequence. "Molecular marker probe" is a nucleic acid sequence or molecule that can be used to identify the presence or absence of a marker locus, such as a nucleic acid probe complementary to a marker locus sequence. Alternatively, in some aspects, a marker probe refers to a probe that can distinguish any type (i.e., genotype) of a specific allele present at a marker locus. When nucleic acids are specifically hybridized in a solution, they are "complementary". When located in an insertion-deletion region, such as a non-collinear region as described herein, some of the markers described herein are also referred to as hybridization markers. This is because, by definition, the insertion region is a polymorphism about a plant without the insertion. Therefore, the marker only needs to indicate whether the insertion-deletion region exists. Any suitable marker detection technology can be used to identify such hybridization markers, such as the use of SNP technology in the examples provided herein.

[0066] An allele is "negatively" associated with a trait when the allele is linked to the trait and when the presence of the allele is an indication that the desired trait or form of the trait will not appear in a plant containing the allele.

[0067] The term "phenotype", "phenotypic trait" or "trait" may refer to the observable expression of a gene or gene series. The phenotype may be observable to the naked eye, or by any other evaluation method (e.g., weighing, counting, measuring (length, width, angle, etc.), microscopy, biochemical analysis, or electromechanical determination). In some cases, the phenotype is directly controlled by a single gene or genetic locus, i.e., a "monogenic trait" or "simple genetic trait". In the absence of large-scale environmental changes, monogenic traits may be separated in a population to give a "quality" or "discrete" distribution, i.e., the phenotype is attributed to discrete categories. In other cases, the phenotype is the result of multiple genes and may be considered to be a "polygenic trait" or "complex trait". Polygenic traits are separated in a population to give a "quantity" or "continuous" distribution, i.e., the phenotype cannot be separated into discrete categories. Both monogenic traits and polygenic traits may be affected by the environment in which they are expressed, but polygenic traits tend to have larger environmental components.

[0068] A "physical map" of a genome is a map showing the linear order of identifiable markers (including genes, markers, etc.) on chromosomal DNA. However, in contrast to a genetic map, the distances between markers are absolute (e.g., measured in base pairs or isolated and overlapping contiguous gene fragments) and are not based on genetic recombination (which may vary in different populations).

[0069] A "polymorphism" is a variation in the DNA between two or more individuals within a population. A polymorphism preferably has a frequency of at least 1% in a population. Useful polymorphisms can include single nucleotide polymorphisms (SNPs), simple sequence repeats (SSRs), or insertion / deletion polymorphisms (also referred to herein as "indels").

[0070] A "production marker" or "production SNP marker" is a marker that has been developed for high throughput purposes. Production SNP markers are developed to detect specific polymorphisms and are designed for use with a variety of chemistries and platforms.

[0071] The term "quantitative trait locus" or "QTL" refers to a region of DNA associated with differential expression of a quantitative phenotypic trait in at least one genetic background (e.g., in at least one breeding population). The region of a QTL encompasses or is tightly linked to one or more genes that affect the trait under consideration.

[0072] A "reference sequence" or "consensus sequence" is a defined sequence used as the basis for sequence alignment. A labeled reference sequence is obtained by sequencing multiple strains at the locus, aligning these nucleotide sequences in a sequence alignment program (e.g., Sequencher), and then obtaining the most common nucleotide sequence of the alignment. Polymorphisms found in these individual sequences are annotated in the consensus sequence. A reference sequence is usually not an exact copy of any individual DNA sequence, but rather represents a mix of available sequences and is used to design primers and probes for polymorphisms within the sequence.

[0073] An "unfavorable allele" of a marker is a marker allele that segregates with the unfavorable plant phenotype, thus providing the benefit of identifying plants that can be removed from a breeding program or planting.

[0074] The term "yield" refers to the productivity per unit area of ​​a particular plant product with commercial value. Yield is affected by both heredity and environmental factors. "Agronomy", "agronomic traits" and "agronomic trait performance" refer to the traits (and potential genetic elements) of a given plant variety, which contribute to yield during the growth period. Individual agronomic traits include seedling vigor, nutritional potential, stress tolerance, disease resistance or tolerance, herbicide resistance, branching, flowering, seed formation, seed size, seed density, lodging resistance, threshing, etc. Therefore, yield is the final apex of all agronomic traits.

[0075] Provided herein are marker loci that show statistically significant cosegregation with disease resistance traits that confer broad resistance to one or more specific diseases. These loci or additional linked loci and detection of resistance genes can be used in marker-assisted selection as part of a breeding program to produce plants that are resistant to one or more diseases.

[0076] Genetic mapping

[0077] It has been recognized that in quite some cases a specific genetic locus associated with a specific phenotype (e.g., disease resistance) can be located in the genome of an organism. Plant breeders can advantageously use molecular markers to identify desired individuals by detecting marker alleles that show a statistically significant probability of co-segregation with the desired phenotype, manifested as linkage disequilibrium. By identifying molecular markers or clusters of molecular markers that co-segregate with the desired trait, the plant breeder can rapidly select for the desired phenotype by selecting the appropriate molecular marker alleles (a method known as marker-assisted selection or MAS).

[0078] Various methods can be used to detect molecular markers or molecular marker clusters that are co-separated with the target trait (e.g., disease resistance trait). The basic concept of these methods is to detect markers of alternative genotypes (or allelotrope) with significantly different average phenotypes. Therefore, the difference size between the alternative genotypes (or allelotrope) between the marker loci or the significance level of the difference is compared. Infer that the trait gene is located at the position of one or more markers of the genotype difference with the maximum correlation closest to it. Two methods for detecting the target trait loci like this are: 1) association analysis (i.e., association positioning) based on colony and 2) traditional linkage analysis.

[0079] Relationship positioning

[0080] Understanding the degree and pattern of linkage disequilibrium (LD) in the genome is a prerequisite for developing effective, in order to identify and draw the association method of quantitative trait locus (QTL).Linkage disequilibrium (LD) refers to the non-random association of alleles in an individual collection.When LD is observed in the alleles at the linked locus, LD is measured as the LD decay of a specific region across chromosome.The scope of LD reflects the recombination history of this region.The average rate of LD decay in the genome can help predict the quantity and density of the markers required for whole genome association studies, and provides an estimated value of the resolution that can be expected.

[0081] Association or LD mapping aims to identify significant genotype-phenotype associations. It has been developed as a powerful tool for fine mapping in outcrossing species such as humans (Corder et al. (1994) "Protective effect of apolipoprotein-E type-2 allele for late-onset Alzheimer-disease," Nat Genet 7:180-184; Hastbacka et al. (1992) "Linkage disequilibrium mapping in isolated founder populations: diastrophic dysplasia in Finland," Nat Genet 2:204-211; Kerem et al. (1989) "Identification of the cystic fibrosis gene: genetic analysis [Identification of cystic fibrosis genes: genetic analysis], Science 245:1073-1080) and maize (Remington et al. (2001) "Structure of linkage disequilibrium and phenotype associations in the maize genome," Proc Natl Acad Sci USA 98:11479-11484; Thornsberry et al. (2001) "Dwarf8 polymorphisms associate with variation in flowering time," Nat Genet 28:286-289; reviewed by Flint-Garcia et al. (2003) "Structure of linkage disequilibrium in plants," Annu Rev Plant Biol. [Annual Review of Plant Biology] 54: 357-374), in which recombination between heterozygotes is frequent and leads to rapid decay of LD.In inbred species, where recombination between homozygous genotypes is not genetically detectable, the extent of LD is greater (i.e., larger blocks of linked markers are inherited together) and this greatly increases the power to detect association mapping (Wall and Pritchard, (2003) "Haplotype blocks and linkage disequilibrium in the human genome," Nat Rev Genet 4:587-597).

[0082] The recombination and mutation history of a population is a function of mating habits and the effective size and age of the population. Larger population sizes provide an enhanced probability of detecting recombination, while older populations are generally associated with higher levels of polymorphism, both of which promote a significant increase in the observed LD decay rate. On the other hand, smaller effective population sizes, such as those that have experienced a recent genetic bottleneck, tend to exhibit slower LD decay rates, leading to more extensive haplotype conservation (Flint-Garcia et al., (2003) "Structure of linkage disequilibrium in plants," Annu Rev Plant Biol. [Annual Review of Plant Biology] 54: 357-374).

[0083] Elite breeding lines provide a valuable starting point for association analyses. Association analyses use quantitative phenotypic scores (e.g., disease tolerance ratings from one to nine for each line) in the analysis (rather than considering only tolerance vs. resistance allele frequency distributions in the type of allele distributions among the groups analyzed). The availability of detailed phenotypic performance data collected over many years through breeding programs and the environment of a large number of elite lines provide valuable data sets for genetic marker association mapping analyses. This paves the way for seamless integration between research and application, taking advantage of historically accumulated data sets. However, understanding the relationship between polymorphism and recombination is useful for developing appropriate strategies for efficiently extracting maximum information from these resources.

[0084] This type of association analysis neither generates nor requires any map data, but is independent of map position. This analysis compares the phenotypic scores of plants with the genotypes at different loci. Subsequently, using the previously determined map locations of these markers, any suitable map (e.g., a composite map) can optionally be used to help visualize the distribution of identified QTL markers and / or QTL marker clusters.

[0085] Traditional linkage analysis is based on the same principle; however, LD is generated by creating a population from a small number of founders. Founders are selected to maximize the level of polymorphism within a structured population, and the level of co-segregation of polymorphic sites with a given phenotype is assessed. A large number of statistical methods have been used to identify significant marker-trait associations. One such method is the interval positioning method (Lander and Botstein, Genetics [Genetics] 121: 185-199 (1989), in which each of many positions along the genetic map (say, in intervals of 1 cM) is tested for the probability that the gene controlling the trait of interest is located at that position. Genotype / phenotype data are used to calculate the LOD score (logarithm of the probability ratio) for each tested position. When the LOD score is greater than a threshold, there is significant evidence that the gene controlling the trait of interest is located at that location on the genetic map (to be located between two specific marker loci).

[0086] Provided herein are marker loci that show statistically significant cosegregation with disease resistance traits as determined by traditional linkage analysis and genome-wide association analysis. Detection of these loci or additional linked loci can be used in marker-assisted breeding programs to produce plants with disease resistance.

[0087] Activities in a marker-assisted breeding program may include, but are not limited to, selecting among new breeding populations based on historical genotype and agronomic trait associations to identify which population has the highest frequency of favorable nucleic acid sequences, selecting for favorable nucleic acid sequences in progeny within a breeding population, selecting among parental lines based on predictions of progeny performance, and advancing lines in germplasm improvement activities based on the presence of favorable nucleic acid sequences.

[0088] Chromosome interval

[0089] Chromosome intervals associated with disease resistance traits are provided. Various methods can be used to identify chromosomal intervals. The boundaries of such chromosomal intervals are extended to encompass markers that will be linked to one or more genes controlling the target trait. In other words, the chromosomal interval is extended so that any marker (including the end markers that define the boundaries of the interval) within the interval can be used as a marker for disease resistance traits.

[0090] Conversely, if, for example, two markers that are very close show co-segregation with a desired phenotypic trait, it is sometimes unclear whether each of those markers identifies the same gene or two different genes or multiple genes. In any case, knowledge of how many genes are within a particular physical / genomic interval is not necessary for formulating or practicing which is presented in this disclosure.

[0091] Chromosome 6 interval may encompass any marker identified herein as being associated with the ANTROT resistance trait, including "C" at left flank (conserved)_marker 104 (position 51 of reference sequence SEQ ID NO: 6); "A" at right flank (conserved)_marker 015 (position 51 of reference sequence SEQ ID NO: 7); "T" at left flank (optimized)_marker 115 (position 35 of reference sequence SEQ ID NO: 8); and "C" at right flank (optimized)_marker 157 (position 30 of reference sequence SEQ ID NO: 9). Any marker located within these intervals can be used as a marker for ANTROT resistance and can be used in the context of the methods presented herein to identify and / or select plants with ANTROT resistance, regardless of whether the resistance is newly conferred or enhanced compared to control plants. In certain embodiments, markers located upstream and downstream of the NLR02 gene location are very closely linked genetically and physically and can therefore be used to select the NLR02 gene for trait gene introgression and product development.

[0092] Chromosomal intervals may also be defined by markers that are linked to (and in linkage disequilibrium with) disease resistance genes, and r 2 is a common measure of linkage disequilibrium (LD) in the context of association studies. If the LD between a chromosome 7 marker locus in the target interval and another closely adjacent chromosome 7 marker locus is r 2 If the value is greater than 1 / 3 (Ardlie et al., Nature Reviews Genetics 3:299-309 (2002)), the two loci are in linkage disequilibrium with each other.

[0093] Tags and linkage

[0094] A common measure of linkage is the frequency of co-segregation of traits. This can be expressed as a percent co-segregation (recombination frequency), or in centimorgans (cM). cM is a unit of measure for genetic recombination frequency. One cM is equal to a 1% chance that a trait at one genetic locus will separate from a trait at another locus due to hybridization in a single generation (meaning there is a 99% chance that these traits will separate in total). Since chromosome distance is roughly proportional to the frequency of hybridization events between traits, there is an approximate physical distance associated with recombination frequency.

[0095] The marker loci themselves are traits and can be assessed according to standard linkage analysis by tracking the marker loci during segregation. Thus, one cM equals a 1% chance that a marker locus will segregate from another locus due to hybridization in a single generation.

[0096] The closer the marker is to the gene controlling the desired trait, the more effective and advantageous the marker is as an indicator of the desired trait. Tightly linked loci show a hybridization frequency between loci of about 10% or less, preferably about 9% or less, still more preferably about 8% or less, yet more preferably about 7% or less, yet more preferably about 6% or less, yet more preferably about 5% or less, yet more preferably about 4% or less, yet more preferably about 3% or less, and yet more preferably about 2% or less. In a highly preferred embodiment, the related loci (e.g., marker loci and target loci) show a recombination frequency of about 1% or less, such as about 0.75% or less, more preferably about 0.5% or less, or yet more preferably about 0.25% or less. Therefore, the loci are separated by a distance of about 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.75 cM, 0.5 cM or 0.25 cM or less. In other words, two loci that are located on the same chromosome and have a distance such that recombination between the two loci occurs with a frequency of less than 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25% or less) are considered to be "adjacent" to each other.

[0097] Although specific marker alleles can be co-segregated with disease resistance traits, it is important to note that the marker locus does not necessarily cause the expression of the disease resistance phenotype. For example, it is not a requirement that the marker polynucleotide sequence is part of a gene that produces a disease resistance phenotype (e.g., part of a gene open reading frame). The association between a specific marker allele and a disease resistance trait is due to the initial "coupling" phase linkage between the marker allele and the allele in the ancestral strain from which the allele originated. Finally, hybridization events between the marker and the genetic locus can change this orientation through repeated recombination. For this reason, the favorable marker allele can change according to the linkage phase used to create a segregating population that is present in a parent with disease resistance. This does not change the fact that the marker can be used to monitor phenotypic segregation. It only changes which marker allele is considered favorable in a given segregating population.

[0098] The method proposed herein comprises the existence of one or more marker allelotrope associated with disease resistance in detection plant, and then identifies and / or selects to have the plant of favorable allelotrope at those marker loci.Mark has been identified as being associated with disease resistance proterties in this article, and therefore can be used for predicting the disease resistance in plant.Any mark (based on the genetic map of single meiosis) in 50cM, 40cM, 30cM, 20cM, 15cM, 10cM, 9cM, 8cM, 7cM, 6cM, 5cM, 4cM, 3cM, 2cM, 1cM, 0.75cM, 0.5cM or 0.25cM also can be used for predicting the disease resistance of plant.

[0099] Marker-Assisted Selection

[0100] Molecular markers can be used in a variety of plant breeding applications (e.g., see Staub et al. (1996) Hortscience 31:729-741; Tanksley (1983) Plant Molecular Biology Reporter. 1:3-8). One of the main areas of interest is the use of marker-assisted selection (MAS) to increase the efficiency of backcrossing and introgression. Molecular markers that exhibit linkage to loci that affect desired phenotypic traits provide a useful tool for selecting traits in plant populations. This is especially true in cases where phenotypes are difficult to measure. Because DNA marker assays are less labor-intensive and take up less physical space than field phenotyping, larger populations can be assayed, increasing the probability of finding recombinants with the target segment moved from the donor line to the recipient line. The tighter the linkage, the more useful the marker, because recombination is less likely to occur between the marker and the gene causing the trait, which can result in false positives. Flanking markers reduce the probability of false positive selections occurring because double recombination events are required. Ideally the gene itself has a marker so that recombination between the marker and the gene cannot occur. In some embodiments, the methods disclosed herein generate markers in disease resistance genes, wherein the genes are identified by inferring genomic locations from clustering or cluster analysis of conserved domains.

[0101] When a gene is introgressed by MAS, not only the gene but also the flanking regions are introduced (Gepts. (2002). Crop Sci [Crop Science]; 42: 1780-1790). This is called "linkage drag". In cases where the donor plant is very unrelated to the recipient plant, these flanking regions carry additional genes that can encode agronomically undesirable traits. Even after multiple cycles of backcrossing with elite lines, linkage drag may lead to reduced yield or other negative agronomic characteristics. This is sometimes also called "yield drag". The size of the flanking regions can be reduced by additional backcrossing, although this is not always successful because breeders cannot control the size of the region or the recombination breakpoints (Young et al., (1998) Genetics [Genetics] 120: 579-585). In classical breeding, recombination that helps reduce the size of the donor segment is usually selected only by chance (Tanksley et al. (1989). Biotechnology [Biotechnology] 7: 257-264). Even after 20 backcrosses of this type, it is expected that quite large donor chromosome fragments still linked to the gene will be selected. However, if markers are used, it is possible to select those rare individuals that have experienced recombination near the target gene. In 150 backcross plants, there is a 95% chance that at least one plant will experience hybridization within the 1cM (based on a single meiotic map distance) of the gene. Markers make it possible to clearly identify these individuals. Using another backcross of 300 plants, there is a 95% probability of hybridization within the 1cM single meiotic map distance on the other side of the gene, thereby producing a section near the target gene less than 2cM based on a single meiotic map distance. This can be achieved in two generations with markers, while an average of 100 generations (see Tanksley et al., the same) are required without markers. When the exact location of the gene is known, the flanking markers around the gene can be used to select recombination in different population sizes. For example, in smaller populations, it is expected that recombination may be further away from the gene, thus requiring more distal flanking markers to detect the recombination.

[0102] The main components of implementing MAS are: (i) defining a population in which the marker-trait association is to be determined, which can be a segregating population, or a random or structured population; (ii) monitoring the segregation or association of polymorphic markers with respect to the trait, and using statistical methods to determine linkage or association; (iii) defining a set of desired markers based on the results of the statistical analysis, and (iv) using and / or extrapolating this information to the current breeding germplasm set to enable marker-based selection decisions to be made. The markers described in this disclosure, as well as other marker types, such as SSR and FLP, can be used in marker-assisted selection schemes.

[0103] SSRs can be defined as relatively short sequences of tandemly repeated DNA of 6 bp or less in length (Tautz (1989) Nucleic Acid Research 17:6463-6471; Wang et al. (1994) Theoretical and Applied Genetics, 88:1-6). Polymorphisms arise from variations in the number of repeat units, which may be caused by slippage during DNA replication (Levinson and Gutman (1987) Mol Biol Evol 4:203-221). Variations in repeat length can be detected by designing PCR primers to conserved non-repeat flanking regions (Weber and May (1989) Am J Hum Genet. 44:388-396). Because SSRs are multiallelic, codominant, reproducible, and amenable to high-throughput automation, they are well suited for mapping and MAS (Rafalski et al. (1996) Generating and using DNA markers in plants. In: Non-mammalian genomic analysis: a practical guide. Academic press. pp. 75-135).

[0104] Various types of SSR markers can be generated, and the SSR profile can be obtained by gel electrophoresis of the amplified products. The scoring of marker genotypes is based on the size of the amplified fragments.

[0105] Various types of FLP markers can also be generated. Most commonly, fragment length polymorphisms are generated using amplification primers. Such FLP markers are similar to SSR markers in many ways, except that the regions amplified by the primers are usually not highly repetitive regions. Usually due to insertions or deletions, the amplified regions or amplicons still have enough variability between germplasms so that the fragments produced by the amplification primers can be distinguished in polymorphic individuals, and it is known that such insertions and deletions often occur in maize (Bhattramakki et al. (2002). Plant Mol Biol [Plant Molecular Biology] 48, 539-547; Rafalski (2002b), supra).

[0106] SNP markers detect single base pair nucleotide substitutions. Of all molecular marker types, SNPs are the most abundant and therefore have the potential to provide the highest genetic map resolution (Bhattramakki et al., 2002 Plant Molecular Biology 48: 539-547). Since SNPs do not require large amounts of DNA and automation of the assay can be direct, SNPs can be assayed in a so-called "ultra-high throughput" manner at even higher throughput levels than SSRs. SNPs also have the potential to be a relatively low-cost system. These three factors together make the use of SNPs in MAS highly attractive. Several methods are available for SNP genotyping, including but not limited to: hybridization, primer extension, oligonucleotide ligation, nuclease cleavage, microsequencing, and coded spheres. These methods have been reviewed in Gut (2001) Hum Mutat [Human Gene Mutation] 17, pp. 475-492; Shi (2001) Clin Chem [Clinical Chemistry] 47, pp. 164-172; Kwok (2000) Pharmacogenomics [Pharmacogenomics] 1, pp. 95-100; and Bhattramakki and Rafalski (2001) Discovery and application of single nucleotide polymorphism markers in plants. In: RJ 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 detect SNPs, including: Masscode.TM. (Qiagen), (Third Wave Technologies) and Invader (Applied Biosystems), (Applied Biosystems, Inc.) and (Illumina).

[0107] Many SNPs within a sequence or across linked sequences can be used to describe the haplotype of any particular genotype (Ching et al. (2002), BMC Genet. [BMC Genetics] 3: 19 pp Gupta et al. 2001, Rafalski (2002b), Plant Science [Plant Science] 162: 329-333). Haplotypes can be more informative than single SNPs and can describe any particular genotype in more detail. For example, a single SNP may be the allele "T" for a particular line or variety with disease resistance, but the allele "T" may also appear in the breeding population used for the recurrent parent. In this case, a haplotype (e.g., a combination of alleles at linked SNP markers) may be more informative. Once a unique haplotype is assigned to a donor chromosome region, the haplotype can be used in the population or any subpopulation thereof to determine whether an individual has a particular gene. The use of an automated high-throughput marker detection platform makes this method efficient and effective.

[0108] Many markers proposed herein can be easily used as single nucleotide polymorphism (SNP) markers to select NLR02 gene. Utilize PCR, primers are used to amplify DNA segments of individuals (preferably inbred lines) representing the diversity of the target population. The PCR product is directly sequenced in one or two directions. The obtained sequence is compared and polymorphism is identified. The polymorphism is not limited to single nucleotide polymorphism (SNP), and includes insertion and deletion, CAPS, SSR and VNTR (variable number of tandem repeats). Specifically, for the fine map information described herein, people can easily use the information provided herein to obtain other polymorphic SNPs (and other markers) in the region amplified by primers disclosed herein. The markers in the described map region can be hybridized with BAC or other genomic libraries, or electronically compared with the genomic sequence, to find new sequences in the same approximate location as the marker.

[0109] In addition to the above-mentioned SSR, FLP and SNP, 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.

[0110] Isozyme profiles and linked morphological traits can also be used indirectly as markers in some cases. Although they do not directly detect DNA differences, they are often affected by specific genetic differences. However, markers for detecting DNA variation are much more numerous and more polymorphic than isozyme or morphological markers (Tanksley (1983) Plant Molecular Biology Reporter: [Plant Molecular Biology Guide] 1: 3-8).

[0111] Sequence alignment or overlapping group can also be used to find the sequence of the upstream or downstream of the specific marker listed herein.Then use these new sequences close to the mark as herein described to find and develop functionally equivalent marks.For example, different physical and / or genetic maps are compared to locate the equivalent markers that are not described in the present disclosure but are located in the similar region.These maps may be in species, or even span other species that are compared genetically or physically.

[0112] In general, MAS uses polymorphic markers, which have been identified as having a significant possibility of co-segregation with traits such as ANTROT disease resistance traits. It is inferred that such markers are located near one or more genes that give plant disease resistance phenotypes on the map, and are considered to be the indication of desired traits or markers. The presence of the allele required in the marker in the test plant, and it is expected that the plant containing the desired genotype at one or more loci will transfer the desired genotype to its progeny together with the desired phenotype. Therefore, plants with ANTROT disease resistance can be selected by detecting one or more marker alleles, and in addition, progeny plants derived from these plants can also be selected. Therefore, plants containing the desired genotype (i.e., the genotype associated with disease resistance) in a given chromosome region are obtained, and then hybridized with another plant. The progeny of this hybridization is then evaluated for genotype using one or more markers, and then the progeny plants with the same genotype in a given chromosome region are selected as having disease resistance.

[0113] SNPs (i.e., SNP haplotypes) can be used alone or in combination to select favorable resistance gene alleles associated with ANTROT disease resistance. For example, the following SNP haplotypes: "C" at left flank (conservative)_marker 104 (reference sequence SEQ ID NO: 6 position 51); "A" at right flank (conservative)_marker 015 (reference sequence SEQ ID NO: 7 position 51); "T" at left flank (optimized)_marker 115 (reference sequence SEQ ID NO: 8 position 35); and "C" at right flank (optimized)_marker 157 (reference sequence SEQ ID NO: 9 position 30), or a combination thereof.

[0114] Those skilled in the art will expect that there may be additional polymorphic sites at the marker loci in and near the chromosomal markers identified by the methods disclosed herein, wherein one or more polymorphic sites are in linkage disequilibrium (LD) with the alleles at one or more of the polymorphic sites in the haplotype, and therefore can be used in a marker-assisted selection program to introgress a target gene allele or a target genomic fragment. If the presence of an allele at one of the sites tends to predict the presence of alleles at other sites on the same chromosome, then two specific alleles at different polymorphic sites are considered to be in LD (Stevens, Mol. Diag. [Molecular Diagnosis] 4: 309-17 (1999)). The marker locus can be located within 5cM, 2cM, or 1cM of the disease resistance trait QTL (on a genetic map based on a single meiosis).

[0115] The skilled artisan will appreciate that allele frequencies (and thus haplotype frequencies) may vary from one germplasm pool to another. Germplasm pools vary due to differences in maturity, heterosis grouping, geographic distribution, etc. Therefore, SNPs and other polymorphisms in certain germplasm pools may not be informative.

[0116] Plant composition

[0117] Plants identified, modified and / or selected by any of the above methods are also of interest.

[0118] Proteins and their variants and fragments

[0119] The present disclosure encompasses NLR02 polypeptides. As used herein, "NLR02 polypeptide" and "NLR02 protein" are used interchangeably to refer to one or more polypeptides having ANTROT resistance activity and are substantially identical to the NLR02 polypeptide of SEQ ID NO: 2. A variety of NLR02 polypeptides are contemplated.

[0120] As used herein, "substantially identical" refers to an amino acid sequence having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity. In some embodiments, the sequence identity is for the full length sequence of a polypeptide. When used in conjunction with a percentage of sequence identity in this article, the term "about" means + / - 1.0%.

[0121] As used herein, "recombinant protein" refers to a protein that is no longer in its native environment (e.g., in vitro or in a recombinant bacterial or plant host cell); a protein expressed from a polynucleotide that has been edited from its native version; or a protein expressed from a polynucleotide at a different genomic location relative to the native sequence.

[0122] As used herein, "substantially free of cellular material" refers to polypeptides that include protein preparations having less than about 30%, 20%, 10% or 5% (by dry weight) of non-target proteins (also referred to herein as "contaminating proteins").

[0123] "Fragments" or "biologically active portions" include polypeptide fragments or polynucleotide fragments that comprise a sequence sufficiently identical to an NLR02 polypeptide or polynucleotide, respectively, and exhibit disease resistance when expressed in a plant.

[0124] As used herein, "variant" refers to a protein or polypeptide having an amino acid sequence that is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the parent amino acid sequence.

[0125] In some embodiments, the NLR02 polypeptide comprises at least about 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157 , 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical amino acid sequence, wherein the NLR02 polypeptide has ANTROT resistance when expressed in a plant.

[0126] Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of NLR02 polypeptides can be prepared by mutations in DNA. This can also be accomplished by one of several forms of mutagenesis, such as site-specific double-strand break technology, and / or directed evolution. In some aspects, the changes encoded in the amino acid sequence will not substantially affect the function of the protein. Such variants will have the desired activity. However, it should be understood that the ability of NLR02 polypeptides to confer disease resistance can be improved by using these techniques on the compositions of the present disclosure.

[0127] Nucleic acid molecules and variants and fragments thereof

[0128] Provided are isolated or recombinant nucleic acid molecules comprising nucleic acid sequences encoding NLR02 polypeptides or biologically active portions thereof, and nucleic acid molecules sufficient to be used as hybridization probes to identify nucleic acid molecules encoding proteins having regions of sequence homology. As used herein, the term "nucleic acid molecule" refers to DNA molecules (e.g., recombinant DNA, cDNA, genomic DNA, plasmid DNA, mitochondrial DNA) and RNA molecules (e.g., mRNA) and analogs of DNA or RNA produced using nucleotide analogs. Nucleic acid molecules can be single-stranded or double-stranded, but are preferably double-stranded DNA.

[0129] As used herein, "isolated" nucleic acid molecules (or DNA) refer to nucleic acid sequences (or DNA) that are no longer in their natural environment (e.g., in vitro). "Recombinant" nucleic acid molecules (or DNA) used herein refer to nucleic acid sequences (or DNA) in recombinant bacteria or plant host cells; the nucleic acid sequence has been edited from its native sequence; or the nucleic acid sequence is located at a different position from the native sequence. In some embodiments, "isolated" or "recombinant" nucleic acids do not contain sequences that are naturally located on the sides of the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (i.e., sequences located at the 5' and 3' ends of the nucleic acid) (preferably protein-encoding sequences). For the purposes of this disclosure, "isolated" or "recombinant" excludes isolated chromosomes when used to refer to nucleic acid molecules. For example, in various embodiments, a recombinant nucleic acid molecule encoding an NLR02 polypeptide may contain less than about 5kb, 4kb, 3kb, 2kb, 1kb, 0.5kb, or 0.1kb of nucleic acid sequences that are naturally located on the sides of the nucleic acid molecule in the genomic DNA of the cell from which the nucleic acid is derived.

[0130] In some embodiments, the isolated nucleic acid molecule encoding the NLR02 polypeptide has one or more changes in the nucleic acid sequence compared to the native or genomic nucleic acid sequence. In some embodiments, changes in the native or genomic nucleic acid sequence include, but are 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 amino acid substitutions, insertions, deletions and / or additions compared to the native or genomic sequence; removal of one or more introns; deletion of one or more upstream or downstream regulatory regions; and deletion of 5' and / or 3' untranslated regions associated with the genomic nucleic acid sequence. In some embodiments, the nucleic acid molecule encoding the NLR02 polypeptide is a non-genomic sequence.

[0131] A variety of polynucleotides encoding NLR02 polypeptides or related proteins are contemplated. Such polynucleotides can be used to produce NLR02 polypeptides in host cells when operably linked to appropriate promoters, transcription termination and / or polyadenylation sequences. Such polynucleotides can also be used as probes for isolating homologous or substantially homologous polynucleotides encoding NLR02 polypeptides or related proteins.

[0132] In some embodiments, the nucleic acid molecule encoding the NLR02 polypeptide is a polynucleotide having a sequence shown in SEQ ID NO: 2, and variants, fragments, and complementary sequences thereof. As used herein, "complementary sequence" refers to a nucleic acid sequence that is sufficiently complementary to a given nucleic acid sequence so that it can hybridize with the given nucleic acid sequence to form a stable duplex. As used herein, "polynucleotide sequence variant" refers to a nucleic acid sequence that encodes the same polypeptide except for the degeneracy of the genetic code.

[0133] In some embodiments, the nucleic acid molecule encoding the NLR02 polypeptide is a non-genomic nucleic acid sequence. As used herein, "non-genomic nucleic acid sequence" or "non-genomic nucleic acid molecule" or "non-genomic polynucleotide" refers to a nucleic acid molecule having one or more changes in the nucleic acid sequence compared to a natural or genomic nucleic acid sequence. In some embodiments, changes in natural or genomic nucleic acid molecules include, but are not limited to: changes in nucleic acid sequences due to the degeneracy of the genetic code; optimization of nucleic acid sequences for expression in plants; changes in nucleic acid sequences introducing at least one amino acid substitution, insertion, deletion and / or addition compared to a natural or genomic sequence; removal of one or more introns 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 5' and / or 3' untranslated regions associated with the genomic nucleic acid sequence; insertion of heterologous 5' and / or 3' untranslated regions; and modification of polyadenylation sites. In some embodiments, the non-genomic nucleic acid molecule is a synthetic nucleic acid sequence.

[0134] In some embodiments, the nucleic acid molecule encoding the NLR02 polypeptide disclosed herein is a non-genomic polynucleotide having a nucleotide sequence similar to SEQ ID 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the nucleic acid sequence of NO:2, wherein the NLR02 polypeptide has ANTROT resistance activity when expressed in a plant.

[0135] In some embodiments, the nucleic acid molecule encodes an NLR02 polypeptide variant comprising one or more amino acid substitutions to the amino acid sequence of SEQ ID NO:2.

[0136] Nucleic acid molecules as fragments of these nucleic acid sequences encoding NLR02 polypeptides are also encompassed in the embodiments. "Fragment" as used herein refers to a portion of a nucleic acid sequence encoding an NLR02 polypeptide. A fragment of a nucleic acid sequence can encode a biologically active portion of an NLR02 polypeptide, or it can be a fragment that can be used as a hybridization probe or PCR primer using the methods disclosed below. A nucleic acid molecule as a fragment of a nucleic acid sequence encoding an NLR02 polypeptide comprises at least about 150, 180, 210, 240, 270, 300, 330, 360, 400, 450 or 500 consecutive nucleotides or as high as the number of nucleotides present in the full-length nucleic acid sequence encoding an NLR02 polypeptide identified by the methods disclosed herein, depending on the intended use. "Continuous nucleotides" as used herein refers to nucleotide residues that are immediately adjacent to each other. The fragments of the nucleic acid sequences of the embodiments will encode protein fragments that retain the biological activity of the NLR02 polypeptide and therefore retain disease resistance. As used herein, "retaining disease resistance" refers to a polypeptide having at least about 10%, at least about 30%, at least about 50%, at least about 70%, 80%, 90%, 95% or more disease resistance of the full-length NLR02 polypeptide set forth in SEQ ID NO:2.

[0137] "Percentage (%) of sequence identity" is determined relative to a reference sequence (subject sequence) as the percentage of amino acid residues or nucleotides in a candidate sequence (query sequence) that are identical to the corresponding amino acid residues or nucleotides in the reference sequence after aligning the sequences and introducing gaps (if necessary) to achieve maximum percentage sequence identity, and without considering any conservative substitutions of amino acids as part of sequence identity. Alignment for the purpose of determining percentage of sequence identity can be achieved in various ways, for example, using publicly available computer software, such as BLAST, BLAST-2. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the full length of the sequence being compared. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences (e.g., percentage of identity of the query sequence = number of identical positions between the query sequence and the subject sequence / total number of positions of the query sequence × 100).

[0138] In some embodiments, the NLR02 polynucleotide encodes an NLR02 polypeptide comprising an amino acid sequence that is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 2 throughout the entire length. In some embodiments, the NLR02 polynucleotide comprises a genomic sequence that includes introns, regulatory elements, and untranslated regions.

[0139] Embodiments also encompass nucleic acid molecules encoding NLR02 polypeptide variants. "Variants" of NLR02 polypeptides encoding nucleic acid sequences include those sequences that encode NLR02 polypeptides identified by the methods disclosed herein but that have conservative differences due to the degeneracy of the genetic code and those sequences that are fully identical as described above. Naturally occurring allelic variants can be identified using well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant nucleic acid sequences also include synthetically derived nucleic acid sequences that have been generated, for example, using site-directed mutagenesis, but still encode NLR02 polypeptides disclosed herein.

[0140] The skilled person will further appreciate that changes can be introduced by mutations in the nucleic acid sequence, thereby resulting in changes in the amino acid sequence of the encoded NLR02 polypeptide without changing the biological activity of the protein. Therefore, variant nucleic acid molecules can be produced in the following manner: one or more nucleotide substitutions, additions and / or deletions are introduced into the corresponding nucleic acid sequence disclosed herein, so 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 directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleic acid sequences are also encompassed by the present disclosure.

[0141] Alternatively, variant nucleic acid sequences can be prepared by randomly introducing mutations along all or part of the coding sequence (e.g., by saturation mutagenesis), and the resulting mutants can be screened for the ability to confer activity to identify mutants that retain activity. After mutagenesis, the encoded protein can be recombinantly expressed, and the activity of the protein can be determined using standard assay techniques.

[0142] The polynucleotides and fragments thereof disclosed herein are optionally used as substrates for various recombination and recursive recombination reactions, in addition to standard cloning methods such as those described by Ausubel, Berger and Sambrook, i.e., to produce other polypeptide homologues and fragments thereof having desired properties. Various such reactions are known. Methods for producing variants of any nucleic acid listed herein (these methods include recursive recombination of such polynucleotides with a second (or more) polynucleotides to form a variant polynucleotide library) are also embodiments of the present disclosure, as are the libraries produced, the cells comprising the libraries, and any recombinant polynucleotides produced by such methods. In addition, such methods optionally include selecting variant polynucleotides from such libraries based on activity, as where such recursive recombination is performed in vitro or in vivo.

[0143] Various diversity generation schemes (including nucleic acid recursive recombination schemes) are available. The programs can be used alone and / or in combination to produce one or more variants of nucleic acids or nucleic acid sets, and variants of encoded proteins. Individually or as a whole, these programs provide a robust and widely applicable way to produce diversified nucleic acids and nucleic acid sets (including, for example, nucleic acid libraries), which can be used, for example, for engineering or rapid evolution of nucleic acids, proteins, pathways, cells and / or organisms with new and / or improved characteristics.

[0144] Although for the sake of clarity, distinctions and classifications are made during the subsequent discussion, it should be understood that the techniques are generally not mutually exclusive. In fact, various methods can be used alone or in combination, in parallel or in series, so as to obtain different sequence variants.

[0145] The result of any diversity generation procedure described herein can be the generation of one or more nucleic acids, which can select or screen nucleic acids having or conferring desired properties or nucleic acids encoding proteins having or conferring desired properties. After diversification by one or more methods otherwise available herein or to the skilled person, any nucleic acid 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 form, by any assay in the art. Various related (or even unrelated) properties can be evaluated in series or in parallel as appropriate by the practitioner.

[0146] The nucleotide sequence of embodiment can also be used to separate corresponding sequence from different sources.In this way, such sequence (based on the sequence homology of the sequence identified by the method disclosed herein) can be identified using methods such as PCR, hybridization.The embodiment encompasses the sequence selected based on the sequence identity with the whole sequence shown herein or its fragment.Such sequence includes the sequence of the straight homologue as described sequence.Term "straight homologue" refers to the gene derived from the common ancestor gene and found in different species due to species formation.When its nucleotide sequence and / or its encoded protein sequence have the basic identity as defined elsewhere herein, the gene found in different species is considered to be straight homologue.

[0147] In the PCR method, oligonucleotide primers can be designed for use in the PCR reaction to amplify the corresponding DNA sequence 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 (2nd ed., Cold Spring Harbor Laboratory Press, Plainview, New York), hereinafter "Sambrook". See also, Innis, et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Known PCR methods 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.

[0148] In hybridization methods, all or part of the nucleic acid sequence can be used to screen cDNA or genomic libraries. Methods for constructing such cDNA and genomic libraries are disclosed in Sambrook and Russell, (2001), supra. The so-called hybridization probe can be a genomic DNA fragment, a cDNA fragment, an RNA fragment or other oligonucleotide, and can be labeled with a detectable group (such as 32P or any other detectable label, such as other radioisotopes, fluorescent compounds, enzymes or enzyme cofactors). The probe for hybridization can be prepared by labeling a synthetic oligonucleotide based on a nucleic acid sequence encoding a known polypeptide disclosed herein. Degenerate primers can be used in addition, and the degenerate primers are designed based on conserved nucleotides or amino acid residues in the nucleic acid sequence or the encoded amino acid sequence. Such probes typically include a region of the following nucleic acid sequence, and the nucleic acid sequence region hybridizes under stringent conditions with at least about 12, at least about 25, at least about 50, 75, 100, 125, 150, 175 or 200 continuous nucleic acids of the nucleic acid sequence encoding the polypeptide or its fragment or variant. Methods and stringency conditions for preparing probes for hybridization are disclosed in Sambrook and Russell, (2001), supra.

[0149] Nucleotide constructs, expression cassettes and vectors

[0150] The term "nucleotide construct" is used herein and is not intended to limit the embodiment to a nucleotide construct comprising DNA. It will be appreciated by those of ordinary skill in the art that nucleotide constructs, particularly polynucleotides and oligonucleotides consisting of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides, can also be used in the method disclosed herein. The nucleotide constructs, nucleic acids and nucleotide sequences of the embodiments additionally encompass all complementary forms of such constructs, molecules and sequences. In addition, the nucleotide constructs, nucleotide molecules and nucleotide sequences of the embodiments encompass all nucleotide constructs, molecules and sequences that can be used for the plant transformation method of the embodiments, including but not limited to those consisting of deoxyribonucleotides, ribonucleotides and combinations thereof. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. The nucleotide constructs, nucleic acids and nucleotide sequences of the embodiments also encompass all forms of nucleotide constructs, including but not limited to single-stranded forms, double-stranded forms, hairpins, stem-loop structures, etc.

[0151] Additional embodiments relate to transformed organisms, such as organisms selected from the group consisting of plant cells, bacteria, yeast, baculovirus, protozoa, nematodes, and algae. The transformed organism comprises: a DNA molecule of the embodiments, an expression cassette comprising the DNA molecule, or a vector comprising the expression cassette, which can be stably incorporated into the genome of the transformed organism.

[0152] The sequence of the embodiment is provided in a DNA construct for expression in a target organism. The construct will include 5' and 3' regulatory sequences that are operably connected to the sequence of the embodiment. As used herein, the term "operably connected" refers to the functional linkage between a promoter and / or regulatory sequence and a second sequence, wherein the promoter and / or regulatory sequence initiates, mediates and / or affects the transcription of a DNA sequence corresponding to the second sequence. Typically, being operably connected means that the connected nucleotide sequence is continuous, and two protein coding regions are connected in the same reading frame when necessary. The construct may contain at least one other gene to be co-transformed into an organism in addition. Alternatively, one or more other genes may be provided on a plurality of DNA constructs.

[0153] This DNA construct is provided with a plurality of restriction sites for inserting the polypeptide gene sequence of the present disclosure, which will be placed under the transcriptional regulation of the regulatory region.The DNA construct may additionally comprise a selectable marker gene.

[0154] In general, the DNA construct will include, in the 5' to 3' transcription direction: a transcription and translation initiation region (i.e., a promoter), the DNA sequence of the embodiment, and a transcription and translation termination region (i.e., a termination region) that functions in an organism as a host. For the host organism and / or sequence of the embodiment, the transcription initiation region (i.e., a promoter) may be natural, similar, exogenous, or heterologous. In addition, the promoter or regulatory sequence may be a natural sequence, or alternatively, a synthetic sequence. As used herein, the term "exogenous" means that the promoter is not found in the natural organism into which the promoter is introduced. As used herein, the term "heterologous" with respect to a sequence means that the sequence originates from an alien species, or, if from the same species, is a sequence that is substantially modified from its natural form in a composition and / or a genomic locus by deliberate human intervention. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region, and the transcription initiation region is heterologous to the coding sequence. When the promoter is a native or natural sequence, expression of the operably linked sequence is altered from wild-type expression, which results in a change in phenotype.

[0155] In some embodiments, the DNA construct comprises a polynucleotide encoding the NLR02 polypeptide of the embodiments. In some embodiments, the DNA construct comprises a polynucleotide encoding a fusion protein comprising the NLR02 polypeptide of the embodiments.

[0156] In some embodiments, the DNA construct may further include a transcription enhancer sequence. As used herein, the term "enhancer" refers to a DNA sequence that can stimulate promoter activity and may be an innate element or a heterologous element of a promoter inserted to enhance the level or tissue specificity of a promoter. Various enhancers may also be used, including, for example, introns with gene expression enhancing properties in plants (U.S. Patent Application Publication No. 2009 / 0144863), ubiquitin introns (i.e., maize ubiquitin intron 1 (see, e.g., NCBI sequence S94464)), ω enhancer or ω major 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), CaMV 35S enhancer (see, e.g., Benfey, et al., (1990) EMBO J. 9:1685-96), and enhancers of U.S. Pat. No. 7,803,992. The above list of transcription enhancers is not meant to be limiting. Any suitable transcription enhancer may be used in the embodiments.

[0157] The termination region may be native to the transcriptional initiation region, may be native to the operably linked DNA sequence of interest, may be native to the plant host, or may be derived from another source (i.e., foreign or heterologous to the promoter, sequence of interest, plant host, or any combination thereof).

[0158] 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) Mol. Gen. Cenet. [Molecular and general genetics] 262: 141-144; Proudfoot, (1991) Cell [Cell] 64: 671-674; Sanfacon et al., (1991) Genes Dev. [Genes and Development] 5: 141-149; Mogen et al., (1990) Plant Cell [Plant Cell] 2: 1261-1272; Munroe et al., (1990) Gene [Gene] 91: 151-158; Ballas et al., (1989) Nucleic Acids Res. [Nucleic Acids Research] 17: 7891-7903 and Joshi et al., (1987) Nucleic Acid Res. [Nucleic Acids Research] 15: 9627-9639.

[0159] When appropriate, the nucleic acid can be optimized to increase expression in the host organism. Thus, where the host organism is a plant, the synthetic nucleic acid can be synthesized using plant-preferred codons to improve expression. For a discussion of host preference usage, see, e.g., Campbell and Gowri, (1990) Plant Physiol. [Plant Physiology] 92: 1-11. For example, although the nucleic acid sequences of the embodiments can be expressed in both monocotyledonous and dicotyledonous plant species, the sequences can be modified to take into account the specific preferences and GC content preferences of monocotyledonous or dicotyledonous plants, as these preferences have shown differences (Murray et al. (1989) Nucleic Acids Res. [Nucleic Acids Research] 17: 477-498). Thus, plant preferences for specific amino acids can be derived from known gene sequences of plants.

[0160] It is known that there are other sequence modifications that can enhance gene expression in a cellular host. These include eliminating the following sequences: sequences encoding false polyadenylation signals, sequences encoding exon-intron splice site signals, sequences encoding transposon-like repetitive sequences, and other sequences that are fully characterized and may be detrimental to gene expression. The GC content of the sequence can be adjusted to the average level of a given cellular host, as calculated by reference to known genes expressed in the host cell. As used herein, the term "host cell" refers to a cell that contains a vector and supports replication and / or expression of an expression vector. The host cell can be a prokaryotic cell such as Escherichia coli, or a eukaryotic cell such as yeast, insect, amphibian or mammalian cell, or a monocot or dicot plant cell. The example of a monocot host cell is a maize host cell. When possible, the modified sequence is to avoid the occurrence of predictable hairpin secondary mRNA structures.

[0161] In preparing the expression cassette, the various DNA fragments may be manipulated to provide a DNA sequence in the proper orientation and, where appropriate, in the proper reading frame. To this end, adapters or linkers may be employed to connect the DNA fragments, or other manipulations may be involved to provide convenient restriction sites, remove excess DNA, remove restriction sites, etc. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitution (e.g., conversion and transversion) may be involved.

[0162] Many promoters can be used to implement the embodiments. A promoter can be selected based on the desired results. The nucleic acid can be combined with a constitutive, tissue-preferred, inducible or other promoter for expression in a host organism.

[0163] Plant transformation

[0164] The method of the embodiment involves introducing a polypeptide or polynucleotide into a plant. As used herein, "introducing" means presenting the polynucleotide or polypeptide to the plant in such a way that the sequence enters the interior of the plant cell. The method of the embodiment does not depend on a specific method for introducing one or more polynucleotides or one or more polypeptides into a plant, as long as the polynucleotide or polypeptide enters the interior of at least one cell of the plant. Methods for introducing one or more polynucleotides or one or more polypeptides into a plant include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0165] As used herein, "stable transformation" means that the nucleotide construct introduced into a plant is integrated into the genome of the plant and can be inherited by its progeny. As used herein, "transient transformation" means that a polynucleotide is introduced into the plant and is not integrated into the genome of the plant, or a polypeptide is introduced into the plant. As used herein, "plant" refers to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, and embryos and progeny thereof. Plant cells can be differentiated or undifferentiated (e.g., callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, and pollen).

[0166] Transformation protocols and protocols for introducing nucleotide sequences into plants can vary depending on the type of plant or plant cell (ie, monocot or dicot) being targeted for transformation. Suitable methods for introducing nucleotide sequences into plant cells and subsequently inserting them 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 (U.S. Pat. Nos. 5,563,055 and 5,981,840), direct gene transfer (Paszkowski et al., (1984) EMBO J 3:2717-2722), and ballistic particle acceleration (see, e.g., U.S. Pat. Nos. 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, Gamborg and Phillips, eds. (Springer-Verlag, Berlin); and McCabe et al., (1988) Biotechnology 6:923-926); and Lecl transformation method (WO 00 / 28058). For potato transformation methods, see Tu et al., (1998) Plant Molecular Biology 37:829-838 and Chong et al., (2000) Transgenic Research 9:71-78. Additional transformation methods can be found in Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio / Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol.[In Vitro Cell Biology and Developmental Biology] 27P: 175-182 (soybean); Singh et al., (1998) Theor. Appl. Genet. [Theoretical and Applied Genetics] 96: 319-324 (soybean); Datta et al., (1990) Biotechnology [Biotechnology] 8: 736-740 (rice); Klein et al., (1988) Proc. Natl. Acad.. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 85: 4305-4309 (maize); Klein et al., (1988) Biotechnology [Biotechnology] 6: 559-563 (maize); U.S. Patent Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al., (1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) The Experimental Manipulation of Ovule Tissues [Experimental Manipulation of Ovule Tissue], Chapman et al., eds. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al., (1990) Plant Cell Reports 9:415-418 and Kaeppler et al., (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al., (1992) 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 [Nature Biotechnology] 14: 745-750 (maize via Agrobacterium tumefaciens).

[0167] Methods for introducing genome editing technology into plants

[0168] In some embodiments, the polynucleotide composition can be introduced into the genome of the plant using genome editing technology, or the polynucleotides previously introduced in the plant genome can be edited using genome editing technology. For example, the identified polynucleotides can be introduced into the desired position in the plant genome using double-strand break technology (such as TALEN, large-range nucleases, zinc finger nucleases, CRISPR-Cas, etc.). For example, for the purpose of site-specific insertion, the identified polynucleotides can be introduced into the desired position in the genome using the CRISPR-Cas system. The required position in the plant genome can be any target site required for insertion, such as a genomic region suitable for breeding, or it can be a target site located in a genomic window with an existing target trait. The existing target trait may be an endogenous trait or a previously introduced trait.

[0169] In some embodiments, in the case where the ANTROT resistance NLR02 gene allele has been identified in the genome, the polynucleotide sequence that can be changed or modified using genome editing technology. Site-specific modifications that can be introduced into the desired NLR02 gene allele polynucleotide include modifications produced using any method for introducing site-specific modifications, including but not limited to using gene repair oligonucleotides (e.g., U.S. Publication 2013 / 0019349), or by using double-strand break technology, such as TALEN, large-range nucleases, zinc finger nucleases, CRISPR-Cas, etc. Such technology can be used to modify the previously introduced polynucleotides by inserting, deleting or replacing nucleotides in the introduced polynucleotides. Alternatively, double-strand break technology can be used to add additional nucleotide sequences to the introduced polynucleotides. Additional sequences that can be added include additional expression elements (such as enhancer sequences and promoter sequences). In another embodiment, genome editing technology can be used to position additional disease resistance proteins near the NLR02 polynucleotide composition in the genome of the plant to produce molecular stacks of disease resistance proteins.

[0170] "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, which comprises at least one change when compared to an unaltered target sequence. Such "changes" include, for example: (i) substitution of at least one nucleotide, (ii) deletion of at least one nucleotide, (iii) insertion of at least one nucleotide, or (iv) any combination of (i)-(iii).

[0171] Examples

[0172] The following examples are provided to illustrate but not limit the claimed subject matter. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that those skilled in the art will recognize that various reagents or parameters may be changed without departing from the spirit of the disclosure or the scope of the appended claims.

[0173] Example 1. QTL mapping

[0174] Anthracnose stem rot (ANTROT), caused by the fungal pathogen Colletotrichum graminearum, is the most common stem disease of maize. As part of our efforts to identify natural maize genes that confer resistance to ANTROT, we created an F1 population by crossing the ANTROT-resistant line TZI8 with the (moderately) susceptible line PHI12P9. The material was backcrossed with PHI12P9 to produce the BC1 mapping population. Work on this mapping population was done in conjunction with efforts described in patent application WO / 2015 / 088970, which protects a similar locus identified from the inbred line PH14J. Approximately 10 days after flowering, the seeds were inoculated with a suspension of spores of Colletotrichum graminearum (approximately 5 x 10 5 spores / m1) were injected into the stems, and separated after about 4-6 weeks. Individual plants were genotyped with SNP markers, and visual scoring was used to phenotype the severity of ANTROT: ANTSUM. The severity was determined according to a visual score of 1-10, with 1 representing the highest resistance and 10 representing high susceptibility. Scoring of 1-3 was considered to be very resistant, 4-6 points were intermediate values, and 7-10 points were classified as susceptible. Our initial positioning placed the quantitative trait locus (QTL) of ANTROT resistance between 23.38 and 125.58 cM on Chr06, with a related peak at 82.67 cM (MZA4127-4).

[0175] Example 2. QTL fine mapping and candidate gene identification

[0176] To fine map the resistance gene, our mapping population was further backcrossed to the susceptible parent PH12P9 to generate the BC4 segregating population and these plants were characterized using previously described methods. Additional SNP markers were generated for fine mapping using a combination of 56K SNPs and exome capture data collected for TZI8 and PH12P9.

[0177] For a population of approximately 3,500 segregating BC4 individuals, the peak correlation between genotype and phenotype was 78.88 cM on Chr06 (p value 1.45E-118). Plants with the TZI8 allele at 78.88 cM had an ANTSUM score approximately 2 points lower than plants with the PH12P9 allele (Table 1). The flanking markers for our conservative and optimized intervals are shown below (Table 2). The Kruskal-Wallis method was used with The software package (version 7.11.0) analyzes positioning data to compare numerical and categorical variables.

[0178] Table 1. Chr06 TZI8 ANTROT QTL fine mapping interval.

[0179]

[0180] The p-values ​​presented represent the correlation between the genotype at a given genetic position and the ANTROT phenotype (ANTSUM). The genetic positions are based on the Corteva B73 genome (version 2). The flanking markers listed in Table 2 are marked with * (optimized right flanking markers were added after subsequent experiments). The peak correlation between genotype and phenotype occurs at 78.88 cM on Chr06 and is shown in bold.

[0181] Table 2. Positions of flanking markers of the Chr06 TZI8 ANTROT QTL.

[0182]

[0183]

[0184] The locations are based on the Corteva B73 genome (version 2), and the public B73 genome (version 4) also includes physical locations.

[0185] To obtain candidate genes within our mapping interval, we used a gene generated from a near isogenic line (NIL) in the Chr06 region. Whole genome sequence with TZI8 as donor and PH12P9 as recurrent parent. Based on FGENESH gene model predictions, there are two nucleotide binding sites - leucine-rich repeat (NLR) genes and a wall-associated kinase (WAK) in our designated conserved genetic interval. These are the only genes in our gene family interval with known roles in host defense against pathogens. RNASeq reads from TZI8NIL were aligned to the genome to confirm our predicted gene model.

[0186] One of the two NLR genes (NLR02) is a unique fusion of two NLRs that has unexpected evidence of expression in TZI8 but appears to be nonfunctional in B73. This is the only gene candidate in our optimized mapping interval. The optimized flanking markers we identified exclude other NLRs (NLR01) and WAK.

[0187] Due to the close proximity of the two NLRs, it was difficult to find recombination between these two genes. Only four individual recombinations indicated that NLR01 was outside our QTL interval, maintaining the possibility that this gene may be involved in resistance to the TZI8 locus. However, NLR01 has 100% identity at both the gene and protein levels with several susceptible lines, including B73 and PH1V69. Therefore, the NLR01 gene was not advanced in the test. Three recombinant families indicated that the QTL was to the left of marker 157, excluding WAK. As mentioned above, this marker appeared twice in a very small interval (Table 1). Both positions were to the left of WAK. However, our more conservative interval still includes this gene.

[0188] Example 3. QTL mapping in other c6 supply systems

[0189] In addition to TZI8, two other lines with very similar ANTROT resistance QTLs have been identified on Chr06. The QTL region is similar to the region identified from PH14J described in patent application WO / 2015 / 088970.

[0190] The other two strain donors were introgressed into susceptible strains and then backcrossed three times into PH1TW8 and / or PH2SNA. The segregating populations were then ANTROT scored as described in Example 1. The current fine mapping intervals (Table 3) presented below are our conservative intervals for these strains based on segregating individuals from multiple families.

[0191] Table 3. Positions of flanking markers of Chr06 QTL in other donors.

[0192] Donor Genetic interval (cM) Physical interval (bp) Inbred Line A Chr06-78.11-81.52 131752260-134517145 PH14J Chr06-78.11-79.52 131752260-134183792 Inbred Line B Chr06-78.11-81.52 131752260-134517145

[0193] The locations are based on the Corteva B73 genome (version 2), and the public B73 genome (V4) also includes the physical locations.

[0194] Example 4. Transgenic Validation of NLR02 Candidate Gene

[0195] Transformation of susceptible lines with constructs containing NLR02 to validate the gene.Two different NLR02 constructs were designed: 1) gene insertion with the native TZI8 promoter and 2) cDNA insertion with the constitutive H2B promoter.

[0196] T1 segregating seeds were tested in the greenhouse (as described below) and will be tested in the field and scored for ANTSUM (as described in Example 1) to identify resistant transformants of susceptible lines. The NLR02 gene is expected to improve the ANTSUM score in the susceptible background compared to the null value. When infiltrated into susceptible lines, (NIL) positives at the TZI8 Chr06 interval have reduced ANTROT symptoms compared to the susceptible lines.

[0197] The T1 seedlings of the TZI8 transgenic isolated in the PH1V69 background were inoculated with ANTROT in the greenhouse using the leaf sheath assay. For the assay, the seedlings were scored on a scale of 1-9, with 9 being the most resistant and 1 being the most susceptible. Plants expressing one or two copies of NLR02 had reduced ANTROT symptoms in a susceptible background compared to the null value (Table 4). Arrows indicate ANTROT infection sites. T1 seeds will also be tested in the field to confirm the validation results of the greenhouse assay.

[0198] Table 4. Efficacy of PHP94983 (H2B + TZI8 CDS) against anthracnose stem rot in the greenhouse.

[0199]

[0200] Plants with one or two copies of the TZI8 gene (H2B+TZI8 CDS) were more resistant to ANTROT infection in a greenhouse-based leaf sheath assay. For the assay, seedlings were scored on a scale of 1-9, with 9 being most resistant and 1 being most susceptible.

[0201] Reads from RNASeq experiments were aligned to determine if PH14J and PH1V69 have the same allele as TZI8 of NLR02 in our Chr06 interval. PH14J has a similar predicted gene model, but has unique alleles of several SNPs in the predicted coding region, which has been confirmed by long-range PCR and Sanger sequencing. In addition to the TZI8 allele described above, a vector will be constructed and tested in transgenic plants to test the efficacy of the PH14J allele of NLR02 (SEQ ID NO: 5).

Claims

1. A method for identifying a corn plant having increased resistance to anthracnose stalk rot, the method comprising: a. Detecting in the plant a resistance QTL associated with increased anthracnose stalk rot resistance and located on maize chromosome 6, wherein the flanking markers and corresponding positions of the resistance QTL are: "C" corresponding to position 51 of the reference sequence SEQ ID NO:6 and corresponding to the left flank of genomic position 133129280 at conserved marker 104; "A" corresponding to position 51 of the reference sequence SEQ ID NO:7 and corresponding to the right flank of genomic position 133685620 at conserved_marker 015; A "T" at position 35 of the reference sequence SEQ ID NO: 8 and corresponding to the left flank of genomic position 133369833; and A "C" at position 30 of the reference sequence SEQ ID NO:9 and corresponding to the right flank optimization_marker 157 of genomic position 133380242,133380887; and b. identifying said plant as having a QTL, wherein said plant has increased resistance to anthracnose stem rot, The genomic locations are provided based on version 4 of the B73 genome.

2. The method of claim 1, further comprising counter-selecting said plants from a breeding program.

3. A method of identifying a corn plant having increased resistance to anthracnose stalk rot, the method comprising: a. Detecting any of the following in the genome of a plant: i. a polynucleotide encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2; or ii. a polynucleotide consisting of a sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3; as well as b. If either i or ii is detected, the plant is identified as having increased resistance to anthracnose stem rot.

4. A method for increasing resistance to anthracnose stalk rot in corn plants, the method comprising expressing in the plant a heterologous polynucleotide encoding a heterologous polypeptide consisting of the amino acid sequence shown in SEQ ID NO:2; wherein when compared to a control plant not containing the heterologous polynucleotide, the corn plant expressing the heterologous polypeptide has increased resistance to anthracnose stalk rot.

5. The method of claim 4, wherein: The heterologous polynucleotide further comprises a heterologous promoter.

6. The method of claim 4, further comprising obtaining a progeny corn plant derived from a plant expressing the heterologous polynucleotide, wherein the progeny corn plant comprises the heterologous polynucleotide in its genome and exhibits increased resistance to anthracnose stalk rot when compared to a control plant not comprising the heterologous polynucleotide.

7. The method of claim 4, wherein: The heterologous polypeptide is encoded by the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:

3.

8. A method of introducing a heterologous NLR02 polynucleotide into a corn plant cell, wherein the heterologous NLR02 polynucleotide is associated with increased resistance to anthracnose stalk rot, the method comprising: A heterologous NLR02 polynucleotide is introduced into a corn plant cell, wherein the heterologous NLR02 polynucleotide is inserted into the corn plant cell genome and wherein the heterologous NLR02 polynucleotide encodes a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:

2.

9. The method of claim 8, wherein the polypeptide is encoded by the sequence shown in SEQ ID NO: 1 or SEQ ID NO:

3.

10. The method of claim 8 or 9, further comprising regenerating a corn plant from a corn plant cell comprising a heterologous NLR02 polynucleotide inserted into the genome of the corn plant cell, wherein the regenerated corn plant comprises resistance to anthracnose stalk rot compared to a control plant lacking the heterologous NLR02 gene.

Images