Maize plants comprising resistance against southern corn rust and compositions and methods for selecting and producing same

Abstract

Provided herein are methods for selecting maize plants comprising a QTL associated with resistance to southern rust. Also provided are methods of producing a plant or plant cell comprising a QTL associated with resistance to southern rust. Also provided are resistant plants or plant cells themselves, as well as resistant plant seeds.

Description

Technical Field

[0001] This disclosure relates to maize plants containing resistance to southern maize rust, and compositions and methods for selecting and producing them. References to sequence lists

[0002] An official copy of this sequence list is submitted electronically as an XML file named "108486-WO-SEC-1Sequence Listing" created on September 17, 2024. The XML file is 74,908 bytes in size, and the sequence list is submitted along with this specification. The sequence list contained in this XML file document is part of this specification and is incorporated herein by reference in its entirety. Background Technology

[0003] Southern rust (“SR”) is a disease of maize plants caused by the fungal pathogen *Puccinia polysora*. In many maize-producing areas, *Puccinia polysora* cannot survive the winter, but under suitable climatic conditions, the pathogen can spread from warmer, wetter regions to colder winters. Because SR has the potential to reduce maize crop yield and / or quality, it is a concern for farmers and grain producers. SR can be particularly detrimental to maize due to its potential for rapid development and spread under favorable conditions. While technologies exist to mitigate the impact of SR on maize crops, further development of new technologies is needed to manage and minimize its potential effects. Summary of the Invention

[0004] This disclosure provides corn plants containing resistance to southern rust (“SR”). Resistance can be provided via the QTL and NLR01 gene disclosed herein. Breeding and / or genetic engineering techniques can be used to produce plants containing resistance provided via the QTL and NLR01 gene. When SR is a problem, the plants provided herein can increase agricultural yields and / or reduce or eliminate the need for fungicide application.

[0005] This document provides a method for selecting maize plants containing QTLs associated with resistance to SR as disclosed herein. The method includes obtaining nucleic acids from one or more maize plants, screening the nucleic acids for QTLs on maize chromosome 10 (including for one or more marker alleles disclosed in Table 1, e.g., haplotypes), and selecting maize plants containing one or more marker alleles (e.g., haplotypes) to select maize plants containing the QTLs.

[0006] This article also provides a method for selecting maize plants containing a QTL associated with resistance to southern rust. The method includes obtaining nucleic acids from one or more maize plants. The method includes screening the nucleic acids for a haplotype on maize chromosome 10 containing one or more marker alleles (e.g., haplotypes) disclosed in Table 1 and / or one or more of the following: (a) “A” at C01800-1, corresponding to position 201 of SEQ ID NO: 3; (b) “G” at C06790-1, corresponding to position 201 of SEQ ID NO: 4; or (c) “T” at C00429-801, corresponding to position 84 of SEQ ID NO: 8. The method includes selecting maize plants containing this haplotype, thereby selecting maize plants containing the QTL.

[0007] This article also provides a method for selecting maize plants containing a QTL associated with resistance to southern rust. The method includes obtaining nucleic acids from one or more maize plants and screening the nucleic acids for a haplotype on maize chromosome 10 containing one or more marker alleles disclosed in Table 1 and / or one or more of the following: (d) “T” at C00431-802, corresponding to position 210 of SEQ ID NO: 5; (e) “C” at C002TCN-001, corresponding to position 61 of SEQ ID NO: 6; (f) “T” at C002TCM-001, corresponding to position 61 of SEQ ID NO: 9; or (g) “A” at C06817-1, corresponding to position 201 of SEQ ID NO: 10. The method includes selecting maize plants containing the haplotype, thereby selecting maize plants containing the QTL.

[0008] This article also provides a method for selecting corn plants containing SR resistance. The method includes screening a population of corn plants for a gene or a protein encoded by that gene, wherein the protein contains an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 30. The method further includes selecting corn plants containing that gene or the protein encoded by it.

[0009] This document also provides a method for producing recombinant corn plants or cells thereof, the recombinant corn plants or cells thereof containing a heterologous gene associated with resistance to SR. The method includes introducing a heterologous NLR gene sequence encoding a protein having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 30 into one or more cells derived from a first corn plant, thereby producing at least one modified cell whose genome contains the heterologous NLR gene sequence. The method includes selecting at least one of the modified corn plant cells, and optionally regenerating the recombinant plant containing the heterologous NLR gene. This document also provides a recombinant plant or seed produced from a recombinant plant, wherein the plant or seed contains a heterologous gene encoding a protein having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 30.

[0010] This article also provides a method for producing recombinant corn plant cells containing a heterologous gene associated with resistance to SR. The method includes introducing an NLR gene sequence into one or more plant cells derived from a first plant; inserting the NLR gene sequence into the genome of at least one of the plant cells to produce one or more modified plant cells; and selecting the modified plant cells. The NLR gene sequence encodes a protein containing an amino acid sequence that is at least 95% identical to that of SEQ ID NO: 30.

[0011] This article also provides a recombinant plant comprising a heterologous protein having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 30. Furthermore, this article also provides a recombinant plant seed comprising a heterologous gene encoding a protein having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 30. Brief description of sequence lists

[0012] This disclosure can be more fully understood from the following detailed description and sequence table, which form part of this application.

[0013] These sequence descriptions and the accompanying sequence listings follow the rules governing the disclosure of nucleotide and / or amino acid sequences in patent applications as set forth in 37 CFR §§1.831-1.834 and WIPO ST.26. The notation and format used for nucleotide and amino acid sequence data follow the provisions set forth in 37 CFR §1.832. Unless otherwise stated, the use of a single letter to represent an amino acid or nucleotide residue is not meaningful (e.g., “atgc” is the same as “ATGC”, and “MRYG” is the same as “mryg”). Although only one strand is shown, each disclosed nucleotide sequence should be understood to encompass its complementary strand (i.e., the reverse complementary sequence).

[0014] SEQ ID NO: 1 is a C00422-801 labeled reference sequence, in which the SNP at position 201 is represented as "y", meaning "c or t". The SNP associated with anti-SR resistance contains "t" at position 51.

[0015] SEQ ID NO: 2 is a C01770-1 labeled reference sequence, in which the SNP at position 201 is represented as "y", meaning "c or t". The SNP associated with anti-SR resistance contains "c" at position 201.

[0016] SEQ ID NO: 3 is a C01800-1 labeled reference sequence, in which the SNP at position 201 is denoted as "w", meaning "a or t". The SNP associated with anti-SR resistance contains "a" at position 201.

[0017] SEQ ID NO: 4 is a C06790-1 labeled reference sequence, in which the SNP at position 201 is represented as "s", meaning "g or c". The SNP associated with anti-SR resistance contains "g" at position 201.

[0018] SEQ ID NO: 5 is a C00431-802 labeled reference sequence, in which the SNP at position 210 is denoted as "k", meaning "g or t". The SNP associated with anti-SR resistance contains "t" at position 210.

[0019] SEQ ID NO: 6 is a C002TCN-001 tagged reference sequence, in which the SNP at position 61 is denoted as "m", meaning "a or c". The SNP associated with anti-SR resistance contains "c" at position 61.

[0020] SEQ ID NO: 7 is a C06813-1 labeled reference sequence, in which the SNP at position 201 is represented as “w”, meaning “a or t”. The SNP associated with anti-SR resistance contains “t” at position 201.

[0021] SEQ ID NO: 8 is a C00429-801 labeled reference sequence, in which the SNP at position 84 is represented as “w”, meaning “a or t”. The SNP associated with anti-SR resistance contains “t” at position 84.

[0022] SEQ ID NO: 9 is a C002TCM-001 tagged reference sequence, where the SNP at position 61 is denoted as "k", meaning "g or t". The SNP associated with anti-SR resistance contains "t" at position 51.

[0023] SEQ ID NO: 10 is a C06817-1 labeled reference sequence, in which the SNP at position 201 is denoted as "r", meaning "a or g". The SNP associated with anti-SR resistance contains "a" at position 201.

[0024] SEQ ID NO: 11 is a C06824-1 labeled reference sequence, in which the SNP at position 201 is denoted as "r", meaning "a or g". The SNP associated with anti-SR resistance contains "g" at position 201.

[0025] SEQ ID NO: 12 is a C06834-1 labeled reference sequence, in which the SNP at position 201 is represented as "y", meaning "c or t". The SNP associated with anti-SR resistance contains "c" at position 201.

[0026] SEQ ID NO: 13 is a C01957-1 labeled reference sequence, in which the SNP at position 201 is represented as "y", meaning "t or c". The SNP associated with anti-SR resistance contains "t" at position 201.

[0027] SEQ ID NO: 14 is a C06838-1 labeled reference sequence, in which the SNP at position 201 is represented as "y", meaning "t or c". The SNP associated with anti-SR resistance contains "t" at position 201.

[0028] SEQ ID NO: 15 is a C06848-1 labeled reference sequence, in which the SNP at position 201 is denoted as "r", meaning "a or g". The SNP associated with anti-SR resistance contains "g" at position 201.

[0029] SEQ ID NO: 16 is a PZA9035-19 labeled reference sequence, in which the SNP at position 211 is represented as "w", meaning "a or t". The SNP associated with anti-SR resistance contains "a" at position 211.

[0030] SEQ ID NO: 17 is a C12422-001 labeled reference sequence, in which the SNP at position 201 is represented as "y", meaning "c or t". The SNP associated with anti-SR resistance contains "t" at position 201.

[0031] SEQ ID NO: 18 is a PZA10357-20 labeled reference sequence, in which the SNP at position 121 is denoted as "r", meaning "a or g". The SNP associated with anti-SR resistance contains "g" at position 121.

[0032] SEQ ID NO: 19 is a C06839-1 labeled reference sequence, in which the SNP at position 201 is denoted as "s", meaning "c or g". The SNP associated with anti-SR resistance contains "g" at position 201.

[0033] SEQ ID NO: 20 is a C002MY1-001 tagged reference sequence, in which the SNP at position 61 is denoted as "m", meaning "a or c". The SNP associated with anti-SR resistance contains "c" at position 61.

[0034] SEQ ID NO: 21 is the CSR_2373961 tagged reference sequence, where the SNP at position 51 is denoted as "r", meaning "a or g". The SNP associated with anti-SR resistance contains "g" at position 51.

[0035] SEQ ID NO: 22 is the CSR_2409943 tagged reference sequence, where the SNP at position 51 is denoted as "m", meaning "a or c". The SNP associated with anti-SR resistance contains "c" at position 51.

[0036] SEQ ID NO: 23 is the CSR_2643297 tagged reference sequence, where the SNP at position 51 is denoted as "k", meaning "g or t". The SNP associated with anti-SR resistance contains "g" at position 51.

[0037] SEQ ID NO: 24 is the CSR_2807867 tagged reference sequence, where the SNP at position 51 is represented as "y", meaning "c or t". The SNP associated with anti-SR resistance contains "c" at position 51.

[0038] SEQ ID NO: 25 is the CSR_2837481 tagged reference sequence, where the SNP at position 101 is denoted as "y", meaning "c or t". The SNP associated with anti-SR resistance contains "c" at position 101.

[0039] SEQ ID NO: 26 is the CSR_2839126 tagged reference sequence, where the SNP at position 101 is denoted as "m", meaning "a or c". The SNP associated with anti-SR resistance contains "a" at position 101.

[0040] SEQ ID NO: 27 is the promoter sequence driving the expression of the NLR01 gene in inbred line A. The promoter consists of 1,990 base pairs preceding the transcription start site.

[0041] SEQ ID NO: 28 is the genomic sequence corresponding to the precursor mRNA produced by the NLR01 gene of inbred line A.

[0042] SEQ ID NO: 29 is a terminator associated with the NLR01 gene of inbred line A. This terminator consists of 1,500 base pairs at the 3' end of the transcription termination site.

[0043] SEQ ID NO: 30 is the amino acid sequence encoded by the NLR01 gene of inbred line A.

[0044] SEQ ID NO: 31 is the promoter sequence driving the expression of the NLR02 gene in inbred line A. The promoter consists of 2,000 base pairs at the 5' end of the transcription start site.

[0045] SEQ ID NO: 32 is the genomic sequence corresponding to the precursor mRNA produced by the NLR02 gene from inbred line A.

[0046] SEQ ID NO: 33 is a terminator associated with the NLR02 gene of inbred line A. This terminator consists of 1,000 base pairs at the 3' end of the transcription termination site.

[0047] SEQ ID NO: 34 is the amino acid sequence encoded by the NLR02 gene of inbred line A.

[0048] SEQ ID NO: 35 shows the codon corresponding to the amino acid in SEQ ID NO: 30 (e.g., NLR01 cDNA sequence).

[0049] SEQ ID NO: 36 shows the codon corresponding to the amino acid in SEQ ID NO: 34 (e.g., NLR02 cDNA sequence). Detailed Implementation

[0050] This disclosure provides a gene that provides resistance to SR, and also provides a marker that has been identified as genetically linked to a locus (e.g., a QTL) that provides resistance to SR. This disclosure also provides methods for selecting resistant plants or counterselecting susceptible plants using a gene sequence, a protein encoded by that gene, and the disclosed marker. This document also provides maize plants and methods for preparing maize plants that contain increased resistance to SR relative to control plants (e.g., SR-susceptible parental lines). A causal gene that provides newly conferred or increased resistance when expressed in a plant is also provided, as well as plants that express and / or encode these causal genes.

[0051] definition

[0052] The following definitions are provided to aid in understanding this disclosure.

[0053] This disclosure is not limited to specific instances, which can naturally vary. The terms and examples used herein are for the purpose of describing specific aspects of this disclosure only and are not intended to be limiting. As used herein, singular and singular forms of terms such as “a” and “the” include plural referents unless the context clearly specifies otherwise. Thus, for example, the terms “plant,” “the plant,” or “a plant” also include multiple plants; and depending on the context, the term “plant” may also include genetically similar or identical offspring of that plant; the term “nucleic acid” optionally includes multiple copies of the nucleic acid molecule; similarly, the term “probe” optionally (and typically) covers many similar or identical probe molecules.

[0054] Unless otherwise indicated, nucleic acids are written from left to right in a 5' to 3' direction. Numerical ranges described in the specification include numbers within defined ranges and include every integer or any non-integer fraction within those ranges. Unless otherwise defined, 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 pertains. In describing and claiming protection for the subject matter of this current disclosure, the following terms will be used in accordance with the definitions set forth below and throughout the specification.

[0055] The term "allelic gene" refers to one of two or more different nucleotide sequences that appear at a specific locus.

[0056] "Allele frequency" refers to the frequency (proportion or percentage) of an allele present at a locus within an individual, a strain, or a population of strains. For example, diploid individuals with genotypes "AA," "Aa," or "aa" have allele frequencies of 1.0, 0.5, or 0.0, respectively, for the allele "A." Allele frequencies within a strain can be estimated by averaging the allele frequencies of a sample of individuals from that strain. Similarly, allele frequencies within a population of strains can be calculated by averaging the allele frequencies of the strains that make up the population. For a population of finite numbers of individuals or strains, allele frequency can be expressed as a count of the individuals or strains (or any other specified grouping) that contain that allele.

[0057] In the context of nucleic acid amplification, the term "amplification" refers to any method by which an additional copy of a selected nucleic acid (or its transcribed form) is produced. Typical amplification methods include a variety of polymerase-based replication methods, including polymerase chain reaction (PCR), ligase-mediated methods (such as ligase chain reaction (LCR)), and RNA polymerase-based amplification methods (e.g., via transcription).

[0058] When an allele is part of or linked to a DNA sequence that influences trait expression, that allele is “associated” with that trait. The presence of an allele is an indicator of how the trait will be expressed. For example, if an allele is part of or linked to a gene that provides resistance to SR, then that allele may be linked to resistance to SR. Similarly, haplotypes or QTLs can be “associated” with traits.

[0059] A centimolar (cM) is a unit of measurement for recombination frequency. One cM equals a 1% chance that a marker at one locus will separate from a marker at a second locus due to crossing over in a single generation.

[0060] As used herein, the term "chromosomal interval" refers to a continuous linear span of genomic DNA located on a single chromosome of a plant. Genetic elements or genes located on a single chromosomal interval are physically linked. There is no particular limitation on the size of a chromosomal interval. In some respects, genetic elements located within a single chromosomal interval are genetically linked, typically having a genetic recombination distance of, for example, less than or equal to 20 cM, or alternatively, less than or equal to 10 cM. ​​That is, two genetic elements within a single chromosomal interval recombine at a frequency of less than or equal to 20% or 10%.

[0061] A chromosome is a single helical DNA molecule containing many genes that function and move as a whole during cell division and are therefore considered linked. It can also be called a "linkage group".

[0062] The term "continuous DNA" refers to an uninterrupted extension of genomic DNA, represented by partially overlapping fragments or clusters.

[0063] The term “hybrid” or “hybrid” refers to sexual hybridization and involves the fusion of two haploid gametes through pollination to produce diploid offspring (e.g., cells, seeds, or plants). The term encompasses both pollination of one plant by another and self-pollination (or self-pollination, such as when pollen and ovules come from the same plant).

[0064] Plants referred to as "diploid" in this article have two sets of chromosomes.

[0065] Plants referred to in this paper as “double haploids” are developed by doubling the number of haploid chromosome sets (i.e., half the normal number of chromosomes). Double haploid plants have two identical sets of chromosomes, and all loci are considered homozygous.

[0066] A “superior strain” is any strain produced through breeding and selection that focuses on superior agronomic traits.

[0067] "Exotic corn varieties" or "exotic corn germplasm" are varieties of corn plants that are not derived from any available superior corn lines or germplasm varieties. In the case of hybridization between two corn plant or germplasm varieties, the offspring of the exotic germplasm are not closely related to the superior germplasm from which it is hybridized. Most commonly, the exotic germplasm is not derived from any known superior corn line, but is selected to introduce new genetic elements (usually new alleles) into the breeding program.

[0068] A “favorable allele” is an allele at a specific locus that confers or contributes to an agronomically desired phenotype (e.g., SR resistance). A marked favorable allele is a marked allele that is segregated (e.g., linked) to a favorable phenotype.

[0069] A “fragment” is intended to represent a portion of a nucleotide sequence. Fragments can be used as hybridization probes or PCR primers using the methods disclosed herein.

[0070] A genetic map is a description of the genetic linkages between loci on one or more chromosomes (or linkage groups) within a given species, typically presented as a graph or table. For each genetic map, the distance between loci is measured by the frequency with which alleles at those loci appear together in the population (recombination frequency of the loci). Alleles can be detected using DNA or protein markers or observable phenotypes. A genetic map is the product of the population used for mapping, the types of markers used, and the polymorphic potential of each marker between different populations. The genetic distance between loci can vary for different genetic maps. However, universal markers can be used to link information from one map to another. Those skilled in the art can use the locations of universal markers to identify the locations of markers and other target loci on each individual genetic map. Although small variations in marker order are common due to factors such as the detection of markers at alternating repeat loci in different populations, differences in statistical methods used to orient these markers, novel mutations, or laboratory errors, the locus order should not change between maps.

[0071] "Genetic mapping" is the localization of a genetic marker on a genetic map relative to surrounding genetic markers on the same linkage group, where a specified marker can be found within a given species.

[0072] "Genetic mapping" is a method of defining linkages at loci using standard genetic principles of genetic markers, population segregation and recombination frequencies targeting those markers.

[0073] “Genetic marker,” “marker,” or “molecular marker” refers to a polymorphic nucleic acid in a population, and the alleles of that genetic marker can be detected and distinguished by one or more analytical methods described below. These terms also refer to nucleic acid sequences complementary to genomic sequences (e.g., nucleic acids) used as probes. Markers corresponding to genetic polymorphism among population members can be detected by analytical methods such as PCR-based sequence-specific amplification methods, restriction fragment length polymorphism detection (RFLP), isoenzyme labeling detection, polynucleotide polymorphism detection via allele-specific hybridization (ASH), amplified variable sequence detection in plant genomes, autonomous sequence replication detection, simple repeat sequence detection (SSR), single nucleotide polymorphism detection (SNP), amplified fragment length polymorphism detection (AFLP), or next-generation sequencing technologies. Other suitable methods include detection of expressed sequence tags (ESTs) and SSR markers derived from EST sequences, and random amplified polymorphic DNA (RAPD). In addition to nucleic acids, “genetic marker,” “marker,” or “molecular marker” can also refer to proteins or genetic phenotypes whose causal genes are known or expected to be linked to favorable alleles.

[0074] For example, this article provides markers, such as the SNPs in Table 1, that are linked to favorable alleles that provide SR resistance. As used herein, “marker allele” refers to a specific sequence at a marker locus linked to a favorable allele.

[0075] "Genetic recombination frequency" is the frequency of crossing over (recombination) events between two genetic loci. Recombination frequency can be observed by following the segregation of markers and / or traits after meiosis.

[0076] "Genome" refers to the total DNA or the entire set of genes carried by an individual.

[0077] The term "genotype" is the genetic makeup of an individual (or a group of individuals) at one or more genetic loci. A genotype is defined by one or more alleles inherited by that individual from one or more known loci of its parents. The term genotype can be used to refer to the genetic makeup of an individual at a single locus, at multiple loci, or more generally, to the genetic makeup of an individual across all genes in its genome.

[0078] "Germium" refers to genetic material that belongs to or originates from an individual (e.g., a plant), a group of individuals (e.g., a plant strain, variety, or family), or a clone derived from a strain, variety, species, or culture; or more generally, all individuals of one or more species (e.g., a maize germplasm collection or an Andean germplasm collection). Germplasm can be a part of an organism or cell, or can be isolated from that organism or cell. Generally, germplasm provides genetic material with a specific molecular structure that provides the 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 from which new plants can grow, or plant parts, such as leaves, stems, pollen, or cells, that can be cultured into a whole plant.

[0079] Plants known as "haploids" have a single set of chromosomes (genome).

[0080] A "haplotype" is the genotype of an individual at multiple genetic loci, that is, a combination of alleles. Typically, the genetic loci described by haplotypes are physically and genetically linked, that is, located on the same segment of chromosome.

[0081] The term "heterogeneity" is used to indicate that individuals within a group have different genotypes at one or more specific loci.

[0082] The heterosis response, or "heterogeneity," of a material can be defined by its performance exceeding the average of its parents (or high parent) when crossed with other dissimilar or unrelated groups.

[0083] An individual is "heterozygous" if more than one allele type exists at a given locus (e.g., a diploid individual with one copy of each of two different alleles).

[0084] The term "homogeneity" means that members of a group have the same genotype at one or more specific loci.

[0085] An individual is "homozygous" if it has only one type of allele at a given locus (for example, a diploid individual has a copy of the same allele at each of the loci on two homologous chromosomes).

[0086] The term "hybrid" refers to offspring obtained from a cross between at least two genetically distinct parents.

[0087] "Hybridization" or "nucleic acid hybridization" refers to the pairing of complementary RNA and DNA strands, as well as the pairing of complementary DNA single strands.

[0088] The term "hybridization" refers to the formation of base pairs between complementary regions of nucleic acid chains.

[0089] The term "inbred line" refers to a line that has been bred to achieve genetic homogeneity.

[0090] The term "indel" refers to an insertion or deletion, in which one strain may be referred to as containing an inserted nucleotide or DNA fragment relative to a second strain, or the second strain may be referred to as containing a deleted nucleotide or DNA fragment relative to the first strain.

[0091] As used herein, “introduction” means presenting a polynucleotide or polypeptide to a plant or plant cell in such a manner that the sequence enters the interior of the cell. The methods disclosed herein are not dependent on any specific method for introducing a polynucleotide or polypeptide into a plant, as long as the polynucleotide or polypeptide enters the interior of at least one cell of the plant. Methods for introducing polynucleotides or polypeptides into plants include, but are not limited to, stable transformation, transient transformation, and virus-mediated transformation.

[0092] As used herein, "stable transformation" means that the nucleotide construct introduced into a plant integrates into the plant's genome and can be inherited by its offspring. As used herein, "transient transformation" means the introduction of a polynucleotide into a plant that does not integrate into the plant's genome, or the introduction of a polypeptide into a plant.

[0093] The transformation scheme and the scheme for introducing nucleotide sequences into plants can vary depending on the type of plant or plant cell to be targeted for transformation (i.e., monocots or dicots). Suitable methods for introducing nucleotide sequences into plant cells and subsequently inserting them into the plant genome include microinjection (Crossway et al. (1986) Biotechniques [Biotechnology] 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 83:5602-5606), Agrobacterium-mediated transformation (US Patent Nos. 5,563,055 and 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J [Journal of the European Society for Molecular Biology] 3:2717-2722), and ballistic (biolistic) particle acceleration (see, for example, US Patent Nos. 4,945,050; 5,879,918; 5,886,244 and 5,932,782; Tomes et al. (1995) in Plant Cell, In *Tissue, and Organ Culture: Fundamental Methods*, edited by Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al. (1988) *Biotechnology* 6:923-926; as well as the Lecl transformation method (WO 00 / 28058). For potato transformation, see Tu et al. (1998) *Plant Molecular Biology* 37:829-838 and Chong et al. (2000) *Transgenic Research* 9:71-78. Other transformation methods can be found in the following literature: 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.[Plant Physiology] 87:671-674 (Soybean); McCabe et al. (1988) Bio / Technology 6:923-926 (Soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182 (Soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (Soybean); Datta et al. (1990) Biotechnology 8:736-740 (Rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (Corn); Klein et al. (1988) Biotechnology 6:559-563 (Corn); US Patent Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988) Plant Physiol. 91:440-444 (Corn); Fromm et al. (1990) Biotechnology 8:833-839 (Corn); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; US Patent 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, edited by Chapman et al. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet.[Theoretical and Applied Genetics] 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens).

[0094] The term "introgression" refers to the phenomenon of a desired allele at a genetic locus being transferred from one genetic background to another. For example, introgression of a desired allele at a designated locus can be transferred to at least one offspring via sexual hybridization between two parents of the same species, where at least one of these parents carries the desired allele in its genome. Alternatively, allele transfer can occur, for example, via recombination between two donor genomes, such as in fusion protoplasts, where at least one of the donor protoplasts carries the desired allele in its genome. The desired allele can be detected, for example, by markers associated with the phenotype, at QTLs, transgenes, etc. In any case, offspring containing the desired allele can be repeatedly backcrossed with lines containing the desired genetic background and selected for the desired allele to produce alleles fixed in the selected genetic background.

[0095] When a sexual hybridization process is repeated two or more times, the "introgression" process is usually referred to as "backcrossing".

[0096] "Backcrossing" refers to the method of crossing the offspring of a hybrid with one of its parents. In a backcross scheme, the "donor" parent is the parent plant that is to be introgressed with one or more desired genes, loci, or specific phenotypes. The "recipient" parent (used once or multiple times) or "recurrent" parent (used twice or more times) is the parent plant with which the gene or locus is backcrossed. Repeated backcrossing can result in alleles being "introgressed" into the recipient or recurrent parent line. For example, see Ragot, M. et al. (1995), Marker-assisted backcrossing: a practical example, in Techniques et Utilisations des Marqueurs Moleculaires Les Colloques, Vol. 72, pp. 45-56, and Openshaw et al. (1994), Marker-assisted Selection in Backcross Breeding, Analysis of Molecular Marker Data, pp. 41-43. The initial cross produces the F1 generation; then the term “BC1” refers to the second use of the recurrent parent, “BC2” to the third use, and so on. Similarly, “BC5F2” refers to the second generation produced by the sixth use of the recurrent parent.

[0097] A "strain" or "variety" is a group of individuals that share the same parents, are usually inbred to some extent, and are typically homozygous and homogeneous (homogeneous or nearly homogeneous) at most loci. A "substrain" refers to a subgroup of inbred lines that are genetically distinct from other similar inbred subgroups that originated from the same ancestor.

[0098] As used herein, the term "linkage" describes the degree to which one marker locus is associated with another marker locus, a favorable allele, or some other locus. Linkage between a molecular marker and a locus influencing the phenotype (e.g., a favorable allele) is expressed as a "probability" or "adjusted probability." Linkage can be expressed as a desired limitation or range. For example, in some instances, markers are linked (genetically or physically) 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 meiotic map (a genetic map based on a population that has undergone one round of meiosis, such as F2). In some respects, limiting the range of linkage in brackets is advantageous, for example, between 10 cM and 20 cM, between 10 cM and 30 cM, or between 10 cM and 40 cM. ​​The stronger the linkage of a marker to a second locus, the better the marker indicates the second locus.

[0099] The term “linkage disequilibrium” refers to the non-random segregation of a genetic locus or trait (or both). In either case, linkage disequilibrium means that the related loci are physically close enough along a segment of chromosome that they segregate together at a frequency higher than random (i.e., non-random). Markers exhibiting linkage disequilibrium are considered linked. Linked loci have a greater than 50% chance (e.g., from about 51% to about 100%) of co-segregation. In other words, two co-segregating markers have a recombination frequency of less than 50% (and, by definition, less than 50 cM separated on the same linkage group). As used herein, linkage can exist between two markers, or alternatively, between a marker and a locus influencing the phenotype.

[0100] The most common measure of chain imbalance is r. 2 The metric was evaluated and calculated using the formula from the following literature: Hill, WG and Robertson, A, Theor. Appl. Genet. [Theoretical and Applied Genetics] 38:226-231 (1968). When r 2 When r = 1, there is a complete LD between the two marker loci, meaning that these markers have not yet undergone recombination segregation and have the same allele frequency. 2 The value will depend on the group used. 2 A value greater than 1 / 3 indicates a sufficiently strong LD for mapping (Ardlie et al., Nature Reviews Genetics 3:299-309 (2002)). Therefore, when r between paired marker loci... 2When 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 allele is in linkage disequilibrium.

[0101] As used in this article, “linkage equilibrium” describes a situation where two markers are independently separated, i.e., randomly assigned in offspring. Markers showing linkage equilibrium are considered unlinked (regardless of whether they are located on the same chromosome).

[0102] The "log dominance (LOD) value" or "LOD score" (Risch, Science 255:803-804 (1992)) is used in 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 that of no linkage, while a LOD score of two indicates that the probability of linkage is 100 times that of no linkage. LOD scores greater than or equal to two can be used to detect linkage. The LOD score can also be used in quantitative trait loci mapping to show the strength of the association between marker loci and quantitative traits. In this case, the size of the LOD score depends on the tightness of the association between the marker locus and the locus affecting the quantitative trait, as well as the magnitude of the quantitative trait effect.

[0103] "Maize" refers to plants of the genus and species Zea mays, and is also known as "corn".

[0104] The term "maize plant" or "plant" includes both the whole maize plant and parts of a maize plant. Such parts include, for example, maize plant cells, maize plant protoplasts, maize plant cell cultures or maize tissue cultures from which maize plants can regenerate, maize plant callus, maize plant clumps or maize plant cells as part of a larger plant structure, maize seeds, maize cores, maize flowers, maize cotyledons, maize leaves, maize stems, maize buds, maize roots, maize root tips, etc.

[0105] Marker-assisted selection (MAS) is a method for selecting individual plants based on marker genotypes.

[0106] "Marker-assisted reverse selection" is a method that uses marker genotypes to identify plants that will not be selected, thereby removing these plants from the breeding process or planting.

[0107] "Marker haplotype" refers to the combination of alleles at a marker locus.

[0108] A "marker locus" is a specific chromosomal location in a species' genome where a specific marker can be found. Marker loci can be used to track the presence of second-linked loci (e.g., linked loci that influence the expression of phenotypic traits). For example, marker loci can be used to monitor the segregation of alleles at genetically or physically linked loci.

[0109] A “labeled probe” is a nucleic acid sequence or molecule that can be used to identify the presence of a marker locus by nucleic acid hybridization, such as a nucleic acid probe complementary to a marker locus sequence. A labeled probe containing 30 or more consecutive nucleotides of the marker locus (“all or part” of the marker locus sequence) can be used for nucleic acid hybridization. Alternatively, in some respects, a labeled probe refers to a probe capable of distinguishing any type (i.e., genotype) of a specific allele present at a marker locus.

[0110] A nucleotide is a monomeric unit that makes up DNA or RNA polymers, consisting of a purine or pyrimidine base, a pentose sugar, and a phosphate group. Nucleotides (usually found as their 5'-monophosphate esters) are represented by their single-letter names as follows: "A" represents adenosine or deoxyadenosine (for RNA or DNA, respectively), "C" represents cytidine or deoxycytidine, "G" represents guanosine or deoxyguanosine, "U" represents uridine, "T" represents deoxythymidine, "R" represents purine (A or G), "Y" represents pyrimidine (C or T), "K" represents G or T, "H" represents A, C, or T, "I" represents inosine, and "N" represents any nucleotide.

[0111] "Polymorphism" is a variation in the DNA between two or more individuals within a population. Polymorphism preferably has a frequency of at least 1% in the population. Useful polymorphisms can include SNPs, simple repeat sequences (SSRs), or insertion / deletion polymorphisms (also referred to herein as "indels").

[0112] An allele is “associated” with a trait when it is linked to that trait, and when the presence of that allele is an indication that the trait or form of the trait will appear in a plant containing that allele.

[0113] The term "offspring" refers to the offspring produced by hybridization.

[0114] "Offspring plants" are plants produced through hybridization between two plants.

[0115] The term “quantitative trait locus” or “QTL” refers to a DNA region associated with differential expression of a quantitative phenotypic trait in at least one genetic context (e.g., in at least one breeding population). The region of a QTL encompasses or is closely linked to a nucleic acid sequence (e.g., one or more genes) that influences the trait under consideration.

[0116] As used in this article, a “recombinant” plant, plant cell, or nucleic acid is a plant, plant cell, or nucleic acid that contains a heterologous nucleic acid sequence (which may encode a heterologous protein / peptide or non-coding RNA).

[0117] As used herein, "heterologous" in relation to a nucleotide or amino acid sequence means a sequence originating from a foreign species, or, if from the same species, a sequence substantially modified relative to its natural form in terms of its composition and / or its natural genomic locus through deliberate human intervention. For example, a promoter operatively linked to a heterologous polynucleotide may originate from a species different from from which the polynucleotide is derived, or, if from the same species, one or both may be substantially modified relative to their original form or genetic locus, or the promoter may not be the natural promoter of the operatively linked polynucleotide. In another instance, a heterologous polynucleotide sequence of this disclosure may be "heterologous" because it is located at a different genomic locus than its natural or naturally occurring genomic locus. For example, a heterologous NLR01 gene sequence may be located on a different chromosome or chromosomal location than the natural NLR01 gene, or it may be located between different genes compared to the natural NLR01 gene. Heterologous genes can be inserted into the genome via, for example, transformation and / or site-specific nuclease-based methods. Such heterologous genes can then be transferred into the genome of plant lines through breeding methods (such as introgression). A nucleotide construct is "heterologous" to a plant or plant cell if it contains one or more nucleotide sequences that are heterologous to the plant / plant cell when incorporated into the genome.

[0118] A "reference sequence" or "shared sequence" is a defined sequence used as the basis for sequence alignment. A labeled reference sequence is obtained by sequencing multiple lines at a given locus, comparing these nucleotide sequences in a sequence alignment program (such as Sequencher), and then obtaining the most universal nucleotide sequence for that alignment. Polymorphisms found in these individual sequences are annotated in the shared sequence. The reference sequence is typically not an exact copy of any individual DNA sequence, but rather represents a mixture of available sequences and is used to design primers and probes targeting polymorphisms within that sequence.

[0119] As used herein, “southern rust (SR) resistance” refers to increased resistance or tolerance to the fungal pathogen that causes SR compared to a control plant containing less resistance. The effects of resistance can vary, ranging from a slight increase in tolerance to the fungal pathogen (e.g., partial suppression of pathogenesis) to complete resistance, rendering the plant unaffected by the presence of the fungal pathogen. The disclosures herein, including examples, provide materials and methods for providing increased resistance to the fungal pathogen that causes SR.

[0120] The term "yield" refers to the productivity of a specific plant product per unit area that has commercial value. For example, corn yield is generally measured in bushels of seed per acre or metric tons of seed per hectare per season. Yield is influenced by both genetic and environmental factors. "Agronomy," "agronomic traits," and "agronomic trait performance" refer to traits (and potential genetic elements) of a given plant variety that contribute to yield during its growth period. Individual agronomic traits include emergence vigor, nutrient potential, stress tolerance, disease resistance or tolerance, herbicide resistance, branching, flowering, seed formation, seed size, seed density, lodging resistance, and threshing ability. Therefore, yield is the ultimate culmination of all agronomic traits.

[0121] Sequence alignment and identity percentage calculations can be determined using a variety of comparison methods designed for detecting homologous sequences, including but not limited to the MEGALIGN® program of the LASERGENE® Bioinformatics Computing Package (DNASTAR®, Madison, Wisconsin). Unless otherwise stated, the multiple alignments of sequences presented herein were performed using the CLUSTAL V alignment method (Higgins and Sharp, CABIOS. [Computer Applications in Biology] 5:151153 (1989)) and default parameters (vacancy penalty = 10, vacancy length penalty = 10). The default parameters for sequential alignment and protein sequence identity percentage calculations using the CLUSTAL V method are KTUPLE = 1, vacancy penalty = 3, window (WINDOW) = 5, and stored diagonals (DIAGONALS SAVED) = 5. For nucleic acids, these parameters are KTUPLE = 2, vacancy penalty = 5, window = 4, and stored diagonals = 4. After aligning sequences using the CLUSTAL V procedure, it is possible to obtain the "Percentage of Identity" and "Divergence" values ​​by referring to the "Sequence Distance" table in the same procedure. Unless otherwise stated, the percentage of identity and divergence provided and requested herein are calculated in this manner.

[0122] The standard recombinant DNA and molecular cloning techniques used in this article are well known in the field and are described more fully in the following literature: Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter referred to as "Sambrook").

[0123] As used herein, a “heterologous NLR gene sequence” is a nucleotide sequence encoding a protein having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO: 30 or 34. Alternatively or additionally, a heterologous NLR gene sequence comprises a natural genomic sequence from inbred line A that encodes a protein comprising SEQ ID NO: 30, a protein comprising SEQ ID NO: 34, or both.

[0124] QTL location and disease-causing gene

[0125] This disclosure provides a QTL on the short arm of chromosome 10 of corn that has been identified as associated with SR resistance (see examples). Three different inbred lines were used in the work described herein—inbred line A (SR resistant), inbred line B (SR susceptible), and inbred line C (SR susceptible). The markers for the SR-resistant QTL present on chromosome 10 of inbred line A are located in lateral chromosomal regions including markers C00422-801 and C002MY1-001. These markers contain SNPs that vary between inbred lines A and at least one of inbred lines B and C. SEQ ID NO: 27-34 discloses sequences associated with two different genes, at least one of which is considered a pathogenic gene for the QTL in inbred line A.

[0126] Genetic mapping

[0127] Specific genetic loci associated with particular phenotypes (such as disease resistance) can be mapped in an organism's genome. 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, exhibiting linkage disequilibrium. By identifying molecular markers or clusters of molecular markers that co-segregate with the target trait, breeders can rapidly select for the desired phenotype by choosing appropriate molecular marker alleles (a method known as marker-assisted selection or MAS).

[0128] Several methods can be used to detect molecular markers or clusters of molecular markers that co-segregate with a target trait (e.g., disease resistance). The basic idea behind these methods is to detect markers of alternative genotypes (or alleles) with significantly different mean phenotypes. Therefore, the magnitude or significance level of the difference between alternative genotypes (or alleles) at marker loci is compared. The location of the trait gene closest to one or more markers with the greatest correlation in genotype differences is inferred. Two methods for detecting loci of a target trait are: 1) population-based association analysis (i.e., association mapping) and 2) traditional linkage analysis.

[0129] Related plotting

[0130] Understanding the extent and patterns of linkage disequilibrium (LD) in the genome is a prerequisite for developing effective association methods to identify and map quantitative trait loci (QTLs). LD refers to non-random association of alleles in a set of individuals. When LD is observed in alleles at linked loci, it is measured as LD decay across a specific region of the chromosome. The extent of LD reflects the recombination history of that region. The average rate of LD decay in the genome can help predict the number and density of markers needed for genome-wide association studies and provide estimates with predictable resolution.

[0131] Association or LD mapping aims to identify significant genotype-phenotype associations. It has been developed and utilized as a powerful tool for fine mapping in crossbred species such as humans (Corder et al. (1994) "Protective effect of apolipoprotein-E type-2 allele for late-onset Alzheimer-disease," Nat Genet [Nature Genetics] 7:180-184; Hastbacka et al. (1992) "Linkage disequilibrium mapping in isolated founder populations: diastrophic dysplasia in Finland," Nat Genet [Nature Genetics] 2:204-211; Kerem et al. (1989) "Identification of the cystic fibrosis gene: genetic analysis," Science [Science] 245:1073-1080) and maize (Remington et al. (2001) “Structure of linkage disequilibrium and phenotype associations in themaize genome,” Proc Natl Acad SciUSA [Proceedings of the National Academy of Sciences] 98:11479-11484; Thornsberry et al. (2001) “Dwarf8 polymorphisms associate with variation in flowering time,” Nat Genet [Nature Genetics] 28:286-289; Reviewed by Flint-Garcia et al. (2003) “Structure of linkage disequilibrium in plants [Structure of linkage disequilibrium in plants],” Annu Rev Plant Biol. [Annual Review of Plant Biology] 54:357-374), where recombination between heterozygotes is frequent and leads to rapid decay of LD.In inbred species, recombination between homozygous genotypes is not genetically detectable, and the degree of LD is greater (i.e., larger linkage marker blocks are inherited together), which greatly enhances the detection capability of association mapping (Wall and Pritchard, (2003) "Haplotype blocks and linkage disequilibrium in the human genome", Nat Rev Genet 4:587-597).

[0132] The recombination and mutation history of a population is a function of mating habits as well as the effective size and age of the population. Larger population sizes provide enhanced likelihood of recombination detection, while older populations are generally associated with higher levels of polymorphism, both of which lead to a significantly faster rate of LD decay. On the other hand, smaller effective population sizes, such as those that have recently experienced genetic bottlenecks, tend to exhibit slower rates of LD decay, resulting in broader haplotype conservation (Flint-Garcia et al., (2003) “Structure of linkage disequilibrium in plants”, Annu Rev Plant Biol. 54:357-374).

[0133] Superior breeding lines provide a valuable starting point for association analysis. This analysis uses quantitative phenotypic scores (e.g., disease tolerance grades from one to nine for each line) (rather than considering only the frequency distribution of tolerance versus resistance alleles in the inter-group allele distribution types analyzed). The availability of detailed phenotypic performance data collected over many years through breeding programs and the environment of a large number of superior lines provide valuable datasets for genetic marker association mapping analysis. This paves the way for seamless integration between research and application and leverages historically accumulated datasets. However, understanding the relationship between polymorphism and recombination is useful for developing appropriate strategies to effectively extract the maximum information from these resources.

[0134] This type of association analysis neither generates nor requires any atlas data, and is independent of atlas location. This analysis compares the plant's phenotypic score to the genotype at different loci. Subsequently, using the previously identified atlas locations of these markers, any suitable atlas (e.g., composite atlases) can optionally be used to aid in observing the distribution of identified QTL markers and / or QTL marker clusters.

[0135] 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 the structured population, and the level of co-segregation of polymorphic loci with a given phenotype is assessed. Numerous statistical methods have been used to identify significant marker-trait associations. One such method is the interval mapping approach (Lander and Botstein, Genetics 121:185-199 (1989), where each of many locations along a genetic map (say, in 1 cM intervals) is tested for the probability that a gene controlling the desired trait is located at that location. Genotype / phenotype data are used to calculate the LOD score (logarithm of the probability ratio) for each tested location. When the LOD score is greater than a threshold, there is significant evidence that the gene controlling the desired trait is located at that location on the genetic map (between two specific marker loci).

[0136] This article presents marker loci that show statistically significant cosegregation with disease resistance traits, as identified through conventional linkage analysis and genome-wide association analysis. Detection of these loci, or other linked loci, can be used in marker-assisted breeding programs to produce plants that contain disease resistance.

[0137] Activities in marker-assisted breeding programs may include, but are not limited to: selecting from 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 favorable nucleic acid sequences in the progeny of the breeding population; selecting from parental lines based on predictions of progeny performance; and advancing lines in germplasm improvement activities based on the presence of favorable nucleic acid sequences.

[0138] Chromosomal intervals

[0139] Chromosomal regions associated with disease resistance traits are provided. Various methods can be used to identify these chromosomal regions. The boundaries of such chromosomal regions can be extended to encompass markers linked to one or more genes controlling the desired trait. In other words, chromosomal regions can be extended such that any marker located within the region (including terminal markers defining the boundaries of the region) can be used as a marker for the disease resistance trait.

[0140] Conversely, for example, if two very close markers show co-segregation with the desired phenotypic trait, it is sometimes difficult to distinguish whether each of those markers identifies the same gene or two different genes or multiple genes. In any case, knowledge about how many genes are within a particular physical / genomic region is unnecessary for developing or practicing certain instances of this disclosure.

[0141] Therefore, this paper discloses regions on chromosome 10 of maize. The disclosed regions on chromosome 10 may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the SR resistance markers disclosed in Table 1. Optionally, a region may contain and be flanked by two of the markers disclosed in Table 1. Other markers in Table 1 may optionally be located between two flanking markers, depending on the genomic location shown.

[0142] In specific instances, the interval on chromosome 10 may cover 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the SR resistance markers disclosed in Table 1, and further cover C01800-1, C06790-1, and / or C00429-801. In specific instances, the interval on chromosome 10 may cover 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the SR resistance markers disclosed in Table 1, and further cover C00431-802, C002TCN-001, C002TCM-001, and / or C06817-1. C01800-1, C06790-1, C00429-801, C00431-802, C002TCN-001, C002TCM-001, and C06817-1 are described in Table 2. Any marker located within these intervals can be used as an SR resistance marker and can be used in the context of the methods presented herein to select plants that are SR resistant compared to control plants or plants lacking the SR resistance QTL. The markers disclosed herein are located upstream or downstream of the NLR01 and / or NLR02 gene locations and are genetically and physically tightly linked to these genes. Therefore, these markers can be used to select the NLR01 and / or NLR02 genes for trait introgression and product development.

[0143] Markers and Linkages

[0144] A common measure of linkage is the frequency of cosegregation of traits. This can be expressed as a percentage of cosegregation (recombination frequency) or in centimoles (cM). cM is a unit of measurement for the frequency of genetic recombination. One cM equals a 1% chance that a trait at one locus will segregate from a trait at another locus due to crossing over in a single generation (meaning there is a total 99% chance of these traits segregating). Since chromosome distance is roughly proportional to the frequency of crossing over events between traits, there exists an approximate physical distance associated with recombination frequency.

[0145] A marker locus is a trait in itself and can be evaluated during segregation by tracking the marker locus and performing standard linkage analysis. Therefore, a cM equals a 1% chance that a marker locus will segregate from another locus due to crossing over in a single generation.

[0146] The closer a marker is to the gene controlling the desired trait, the more effective and advantageous the marker is as an indicator of that trait. Closely linked loci show crossing-over frequencies of about 10% or less, such as about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, or about 2% or less. In some instances, related loci (e.g., marker loci and target loci) show recombination frequencies of about 1% or less, such as about 0.75% or less, about 0.5% or less, or about 0.25% or less. Therefore, these loci are approximately 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 located on the same chromosome and having a distance that allows recombination between the two loci to occur 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 considered to be “neighbors” to each other.

[0147] Although specific marker alleles can co-segregate with the disease resistance trait, it is important to note that the marker locus does not necessarily induce the expression of that disease resistance phenotype. For example, it is not necessary for the marker polynucleotide sequence to be part of the gene that produces the disease resistance phenotype (e.g., part of the gene's reading frame). The association between specific marker alleles and the disease resistance trait is due to the initial "coupling" between the marker allele and the allele in the ancestral line from which the allele originated. This orientation can be altered by repeated recombination events between the marker and the genetic locus. For this reason, favorable marker alleles can change based on the linkage present in the parent containing the disease resistance trait, which is used to create a segregating population. This does not change the fact that markers can be used to monitor phenotypic segregation. It only changes which marker allele is considered favorable in a given segregating population.

[0148] The method presented in this paper involves detecting the presence of one or more marker alleles associated with disease resistance in plants, and then selecting plants that possess favorable alleles at those marker loci. The markers have been identified in this paper as being associated with the disease resistance trait and can therefore be used to predict disease resistance in plants. Any marker within the range of 50 cM, 40 cM, 30 cM, 20 cM, 15 cM, 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.75 cM, 0.5 cM, or 0.25 cM (based on a genetic map from a single meiotic division) can also be used to predict plant disease resistance.

[0149] Marker-assisted selection

[0150] Molecular markers have applications in a variety of plant breeding fields (e.g., see Staub et al. (1996) Hortscience [Horticultural Science] 31: 729-741; Tanksley (1983) Plant Molecular Biology Reporter. [Journal of Plant Molecular Biology] 1: 3-8). One area of ​​interest is the use of marker-assisted selection (MAS) to increase the efficiency of backcrossing and gene introgression. Molecular markers demonstrating linkage to loci influencing desired phenotypic traits provide a useful tool for selecting traits in plant populations. This is especially true where phenotype determination is difficult. Because DNA marker determination is less labor-intensive and requires less physical space than field phenotypic analysis, it can be performed on larger populations, increasing the probability of finding recombinants with target segments that have moved from donor lines to recipient lines. The tighter the linkage, the more useful the marker, as recombination is less likely to occur between the marker and the gene causing the trait, which can lead to false positives. The use of flanking markers reduces the probability of false positive selection because double recombination events are required. In some cases, the marker is located within the gene itself, preventing recombination between the marker and the gene. In some instances, the methods disclosed herein generate markers in disease resistance genes, where the gene is identified by inferring its genomic location from clustering or cluster analysis of conserved domains.

[0151] When genes are introduced via MAS, not only the gene itself but also flanking regions are introduced (Gepts. (2002). Crop Sci; 42: 1780-1790). This is called "linkage cumbersomes." In cases where the donor and recipient plants are highly unrelated, these flanking regions carry additional genes that can encode traits that are agronomically unwanted. Linkage cumbersomes can lead to reduced yield or other negative agronomic traits even after multiple backcrosses with improved lines. This is sometimes also called "yield cumbersomes." The size of flanking regions can be reduced through further backcrosses, although this is not always successful because breeders cannot control the size of the region or the recombination breakpoint (Young et al., (1998) Genetics 120:579-585). In classical breeding, recombinations that help reduce the size of donor segments are often chosen only by chance (Tanksley et al. (1989). Biotechnology 7: 257-264). Even after 20 backcrosses in such backcrosses, a large region on the donor chromosome may still be found linked to the selected gene. However, with markers, it is possible to select rare individuals that have undergone recombination near the target gene. In 150 backcrossed plants, there is a 95% probability that at least one plant will undergo crossing over within 1 cM (based on the single meiotic map distance) of the gene. Marking allows for the definitive identification of these individuals. A further backcross using 300 plants yields a 95% probability of crossing over within 1 cM of the single meiotic map distance on the other side of the gene, resulting in a segment near the target gene within less than 2 cM based on the single meiotic map distance. This can be achieved in two generations with markers, compared to an average of 100 generations without markers (see Tanksley et al., ibid.). When the exact location of a gene is known, flanking markers around the gene can be used to select for recombination at different population sizes. For example, in smaller populations, recombination is expected to be further away from the gene, thus requiring more distant flanking markers to detect the recombination.

[0152] The implementation of MAS may include: (i) defining a population in which marker-trait associations will be determined, which may be a segregating population or a random or structured population; (ii) monitoring the segregation or association of polymorphic markers relative to the trait and using statistical methods to determine linkage or association; (iii) defining a set of desired markers based on the results of statistical analysis; and (iv) using and / or extrapolating this information to current breeding germplasms to enable marker-based selection decisions. 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.

[0153] SSRs can be defined as relatively short sequences of tandem repeat 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). Polymorphism arises from variations in the number of repeat units, which may be due to 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-repeatable flanking regions (Weber and May (1989) Am J Hum Genet. 44: 388-396). Because SSRs are multi-allelic, codominant, regenerable, and suitable for 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).

[0154] Various types of SSR markers can be generated, and SSR profiles can be obtained by gel electrophoresis of the amplification products. The marker genotype score is based on the size of the amplified fragment.

[0155] Various types of FLP markers can also be generated. Most commonly, amplification primers are used to generate fragment length polymorphisms. Except that the regions amplified by the primers are usually not highly repetitive, such FLP markers are similar to SSR markers in many ways. Usually, due to insertions or deletions, the amplified regions or amplicones still have enough variability across germplasm to allow the fragments generated by the amplification primers to be distinguished in polymorphic individuals, and such insertions and deletions are known to occur frequently in maize (Bhattramakki et al. (2002). Plant Mol Biol [Plant Molecular Biology] 48, 539-547; Rafalski (2002b), ibid.).

[0156] SNP markers detect single-base pair nucleotide substitutions. Among all molecular marker types, SNPs are the most abundant, thus potentially offering the highest genetic map resolution (Bhattramakki et al., 2002 Plant Molecular Biology 48:539-547). Because SNPs do not require large amounts of DNA and the automation of assays can be straightforward, they can be determined in a so-called “ultra-high throughput” manner, at throughput levels even higher 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. SNP genotyping and / or detection can be performed using several methods, including but not limited to: hybridization, primer extension, oligonucleotide ligation, nuclease digestion, microsequencing, and coded spheres. These methods have been reviewed in the following literature: Gut (2001) Hum Mutat [Human 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. [Single Nucleotide Polymorphism Markers in Plants] in: RJ Henry, ed., Plant Genotyping: The DNA Fingerprinting of Plants, CABI Publishing, Wallingford. [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. These commercially available technologies include: Masscode™ (Qiagen), INVADER® (Third Wave Technologies) and Invader PLUS®, SNAPSHOT® (Applied Biosystems), TAQMAN® (Applied Biosystems), and BEADARRAYS® (Illumina).

[0157] Haplotypes of any particular genotype can be described using numerous SNPs, either within a sequence or across linked sequences (Ching et al. (2002), BMC Genet. 3:19 pp; Gupta et al. 2001, Rafalski (2002b), Plant Science 162:329-333). Haplotypes can be more informative than a single SNP and can describe any particular genotype in more detail. For example, a single SNP might be the allele "T" for a specific line or variety with disease resistance, but it could also be present in a breeding population used for recurrent parents. In such cases, haplotypes (e.g., combinations of alleles at linked SNP markers) may be more informative. Once a unique haplotype is assigned to a donor chromosome region, that haplotype can be used in that population or any subpopulation to determine whether an individual possesses the specific gene. The use of automated, high-throughput marker detection platforms makes this method efficient and effective.

[0158] The markers presented herein can be readily used as single nucleotide polymorphism (SNP) markers to select target genes or traits. Using PCR, primers are used to amplify DNA segments representing the diversity of a target population (e.g., inbred lines). The PCR products are sequenced directly in one or both directions. The resulting sequences are aligned and polymorphisms are identified. Polymorphisms are not limited to single nucleotide polymorphisms (SNPs) but also include insertions / deletions, CAPS, SSRs, and VNTRs (variable number of tandem repeats). In particular, the fine mapping information described herein allows for the easy identification of additional polymorphic SNPs (and other markers) within regions amplified using the primers disclosed herein. Markers within the described mapping regions can be hybridized with BAC or other genomic libraries, or electronically aligned with genomic sequences, to find new sequences in the same approximate locations as the markers.

[0159] In addition to SSR, FLP, and SNP mentioned above, other types of molecular markers are also widely used, including but not limited to: expressed sequence tags (EST), SSR tags derived from EST sequences, randomly amplified polymorphic DNA (RAPD), and other nucleic acid-based markers.

[0160] Isozyme profiles and linkage morphological features can also be used indirectly as markers in certain situations. Although they do not directly detect DNA differences, they are often influenced by specific genetic differences. However, there are far more and more diverse markers for detecting DNA variation than isozymes or morphological markers (Tanksley (1983) Plant Molecular Biology Reporter 1:3-8).

[0161] Sequence alignment or contigs can also be used to discover upstream or downstream sequences of the specific markers listed herein. These new sequences, which are close to the markers described herein, are then used to discover and develop functionally equivalent markers. For example, alignments of different physical and / or genetic maps can be performed to locate equivalent markers not described in this disclosure but located in similar regions. These maps may be intra-species or even across other species for which genetic or physical alignments are performed.

[0162] Generally, MAS uses polymorphic markers that have been identified as having a significant probability of co-segregating with traits such as SR resistance and / or genes disclosed herein. Such markers are presumed to be located on the map near one or more genes that give the plant a resistance phenotype and are considered indicators of the desired trait or marker. The presence of the desired allele in the marker is tested in the plant, and plants containing the desired genotype at one or more loci are expected to transfer the desired genotype along with the desired phenotype to their progeny. Therefore, plants with SR resistance can be selected by detecting one or more marker alleles, and further, progeny plants derived from these plants can be selected. Thus, plants containing the desired genotype (i.e., the genotype associated with resistance) in a given chromosomal region are obtained and then crossed with another plant. The progeny of such crosses are then genotypically evaluated using one or more markers, and progeny plants with the same genotype in a given chromosomal region are then selected to contain resistance.

[0163] SNPs (i.e., SNP haplotypes) can be used alone or in combination to select favorable resistance gene alleles associated with SR resistance. For example, an SNP haplotype may contain a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the SR resistance markers in Table 1 of this disclosure.

[0164] Those skilled in the art will anticipate the presence of additional polymorphic loci at or near the marker loci identified by the methods disclosed herein, where one or more polymorphic loci are in linkage disequilibrium (LD) with alleles at one or more polymorphic loci in the haplotype, and thus can be used in marker-assisted selection procedures to infiltrate target gene alleles or target genomic fragments. Two specific alleles at different polymorphic loci are considered to be in LD if the presence of an allele at one of these loci tends to predict the presence of an allele at other loci on the same chromosome (Stevens, Mol. Diag. [Molecular Diagnostics] 4:309-17 (1999)). This marker locus may be located within 5 cM, 2 cM, or 1 cM of the disease resistance trait QTL (on a genetic map based on a single meiotic division).

[0165] Allele frequencies (and consequently haplotype frequencies) can vary between germplasm banks. Germplasm banks vary due to differences in maturity, heterosis grouping, geographical distribution, and other factors. Therefore, SNPs and other polymorphisms may not be available in some germplasm banks.

[0166] Methods for identifying and / or selecting plants containing SR resistance

[0167] This document provides a method for identifying and optionally selecting maize plants containing QTLs associated with resistance to SR. The method includes obtaining nucleic acid samples from each of one or more maize plants. The method includes screening each nucleic acid sample for a QTL on maize chromosome 10 (short arm). The method may include screening samples for the presence or absence of one or more marker alleles shown in Table 1. The QTL may be a QTL disclosed herein that is associated with resistance to SR, is located on maize chromosome 10 (short arm), and is present in inbred line A.

[0168] In some instances, the method includes screening each nucleic acid sample (e.g., from one or more members of a corn plant population) for at least one marker allele disclosed in Table 1. If a marker allele is present, the sample (and the plant from which it originates) can be identified as containing a marker allele associated with SR resistance.

[0169] In some instances, the method includes screening each nucleic acid for haplotypes containing at least two marker alleles disclosed in Table 1, thereby identifying samples as containing haplotypes associated with anti-SR resistance.

[0170] In some instances, the method involves screening nucleic acids against all marker alleles disclosed in Table 1 to identify samples containing haplotypes associated with anti-SR resistance.

[0171] Nucleic acid screening can include any procedure suitable for determining the genotype of one or more target plants. For example, screening can include PCR, Sanger sequencing, and / or next-generation sequencing.

[0172] Table 1 provides marker alleles (e.g., single nucleotide polymorphisms (SNPs)) linked to QTLs. The right column of Table 1 provides each SNP and flanking genomic sequence for context. In the sequences, residues associated with SR resistance are listed before the slash, and residues associated with SR susceptibility are listed after the slash. SNPs are shown in bold and in parentheses. Sequences of only one strand are provided, but the disclosed SNPs and methods disclosed herein cover all complementary forms (e.g., SNPs in the context of the opposite complementary strand).

[0173] Table 1

[0174]

[0175] Table 2

[0176]

[0177] In some instances, the method includes screening nucleic acids from corn plants for one or more additional markers. These additional markers are also located on chromosome 10 and are provided in Table 2. Table 2 is formatted in the same way as Table 1. Markers C01800-1, C06790-1, and / or C00429-801 can be used to distinguish haplotypes of inbred line B (susceptible to SR) from haplotypes of inbred line A (resistant to SR). Markers C00431-802, C002TCN-001, C002TCM-001, and / or C06817-1 can be used to distinguish haplotypes of inbred line B (susceptible to SR) from haplotypes of inbred line A (resistant to SR).

[0178] The markings C01800-1, C06790-1 and / or C00429-801 can be used in combination with any of the markings provided in Table 1.

[0179] The markings C00431-802, C002TCN-001, C002TCM-001 and / or C06817-1 may be used in combination with any of the markings provided in Table 1.

[0180] As a result of a fine-mapping experiment to locate markers at the positions of one or more causal genes closer to the QTL, the markers CSR_2373961, CSR_2409943, CSR_2643297, CSR_2807867, CSR_2837481, and CSR_2839126 were identified. In some instances, the method may include screening against one or more of these markers (alone or in combination with other markers in Table 1 or Table 2).

[0181] In some instances, the method includes selecting corn plants containing one or more markers (e.g., from a population). In some instances, selecting corn plants includes selecting plants for further use, for example, in breeding activities (e.g., hybridization, backcrossing, or gene introgression). Selecting plants containing at least one marker can provide suitable donors of the SR resistance trait during breeding activities.

[0182] In some instances, the method may further include isolating nucleic acids from maize plants or maize plant populations. Nucleic acid isolation can facilitate the detection of one or more markers. Nucleic acid isolation may include any method suitable for providing genomic material for PCR, sequencing procedures, and / or the identification of at least one marker.

[0183] In some instances, the selected and / or screened plants are members of a plant population. It can be known that the population contains a mixture of plants that contain or do not contain a resistance QTL on chromosome 10 (e.g., a population of progeny plants obtained by crossing plants containing the QTL with plants that do not contain the QTL). The method may include obtaining nucleic acid samples from each of multiple corn plants in the population, and screening each sample for one or more alleles associated with the QTL. The method may include selecting one or more corn plants that contain one or more screened marker alleles associated with the QTL.

[0184] In some instances, a method is provided for producing corn plants containing resistance to SR. The method includes selecting a plant containing a QTL on chromosome 10 and crossing the selected plant with a second corn plant. The second corn plant may be derived from a plant population used for backcrossing and / or introgression into a plant population lacking the SR resistance trait. In some instances, the second plant lacks the QTL. In some instances, the method includes obtaining progeny plants from the cross containing at least one marker allele listed in Tables 1 and / or 2 and / or containing SR resistance induced by the QTL. In some instances, the resistance to SR in both the parent and progeny is increased compared to the QTL-deficient second plant (e.g., the parent).

[0185] Maize plants may contain a portion of a QTL but still exhibit associated SR resistance (e.g., plants from which a portion of the QTL has been removed by recombination). Thus, in some instances, the selected marker allele is located in a chromosomal region containing the QTL or in a region that still contains the pathogenic gene (e.g., a portion reduced by recombination).

[0186] In some instances, the selected marker alleles are located in lateral chromosomal regions that include markers C06824-1 and C06834-1. These regions may also contain one or more of the other markers provided in Tables 1 and 2.

[0187] In some instances, the selected marker alleles are located in chromosomal regions that are flanked by markers CSR_2373961 and CSR_2839126. These regions may also include one or more of other markers CSR_2409943, CSR_2643297, CSR_2807867, or CSR_2837481.

[0188] In some instances, the selected marker alleles are located in chromosomal regions containing and flanking C00422-801 and C002MY1-001. C00422-801 is located at position 1,238,011 on chromosome 10 of the B73 version 4.0 genome. C002MY1-001 is located at position 9,161,724 on chromosome 10 of the B73 version 4.0 genome. In some instances, the regions flanking C00422-801 and C002MY1-001 contain all the interpolation markers in Table 1.

[0189] In some instances, the selected marker alleles are located in chromosomal regions containing and flanking C00422-801 and C06839-1. C00422-801 is located at position 1,238,011 on chromosome 10 of the B73 version 4.0 genome. C06839-1 is located at position 4,213,003 on chromosome 10 of the B73 version 4.0 genome. In some instances, the regions flanking C00422-801 and C06839-1 contain all the interpolation markers in Table 1.

[0190] In some instances, the selected marker alleles are located in chromosomal regions containing and flanking CSR_2373961 and CSR_2839126. CSR_2373961 is located at position 2,262,394 on chromosome 10 of the B73 version 4.0 genome. CSR_2839126 is located at position 2,756,463 on chromosome 10 of the B73 version 4.0 genome. In some instances, the regions flanking CSR_2373961 and CSR_2839126 contain all the interpolation markers in Table 1.

[0191] In some instances, methods for selecting corn plants containing QTLs associated with resistance to southern rust (SR) can be used during methods for producing corn plants resistant to southern rust. In such instances, the method for producing SR-resistant corn plants includes crossing a first parent corn plant with a second parent corn plant to produce one or more progeny plants. The first parent contains a QTL associated with SR resistance. The second parent does not contain the QTL. The method includes obtaining nucleic acid samples from one or more of the progeny plants. The method includes selecting one or more progeny plants containing a QTL associated with SR resistance according to the methods disclosed herein.

[0192] In some instances, methods for producing corn plants containing SR resistance include backcrossing. Backcrossing can provide progeny that contain agronomical traits more similar to a second parent variety (e.g., a recurrent parent) while also containing SR resistance QTLs from a first variety (e.g., a QTL donor). The method may include crossing one or more selected progeny plants with a second parent plant to produce one or more backcross progeny plants, and obtaining nucleic acid samples from the one or more backcross progeny plants. The method may also include selecting one or more backcross progeny plants containing QTLs associated with SR resistance.

[0193] In some instances, methods for producing corn plants containing SR resistance include one or more subsequent backcrosses. The method may include crossing one or more selected backcross progeny plants with a second parent plant to produce additional backcross progeny plants. The additional backcross progeny plants may more completely reproduce the agronomic characteristics of the second parent plant. The method may include obtaining nucleic acid samples from one or more additional backcross progeny plants, and selecting one or more additional backcross progeny plants containing QTLs associated with SR resistance. Optionally, the method includes repeating the backcross, nucleic acid acquisition, and selection activities to obtain additional backcross progeny plants containing QTLs associated with SR resistance. When grown under the same environmental conditions, the additional backcross progeny plants may contain the agronomic characteristics of the recurrent parent plant.

[0194] Methods for selecting corn plants encoding SR resistance genes

[0195] This document provides a method for selecting maize plants containing resistance to SR. The method includes screening a population of maize plants for a protein or a gene encoding the protein. The protein has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO: 30. The method also includes selecting maize plants containing the protein or a gene encoding the protein.

[0196] In some instances, screening a corn plant population for a protein or gene includes screening for phenotypes associated with the gene, screening for the gene or protein itself, or screening for genotypes linked to the gene. Screening can include any procedure suitable for determining the genotype of the target plant. For example, screening can include PCR, Sanger sequencing, and / or next-generation sequencing.

[0197] In some instances, the protein has 95% amino acid sequence identity with SEQ ID NO: 30 or 34.

[0198] In some instances, the protein has 96% amino acid sequence identity with SEQ ID NO: 30 or 34.

[0199] In some instances, the protein has 97% amino acid sequence identity with SEQ ID NO: 30 or 34.

[0200] In some instances, the protein has 98% amino acid sequence identity with SEQ ID NO: 30 or 34.

[0201] In some instances, the protein has 99% amino acid sequence identity with SEQ ID NO: 30 or 34.

[0202] In some instances, the protein has 100% amino acid sequence identity with SEQ ID NO: 30 or 34.

[0203] In some instances, the method involves crossing a selected corn plant with a second corn plant. In some instances, the method involves obtaining progeny plants containing a gene encoding the protein. In some instances, the second plant lacks the QTL. In some instances, the progeny plants derived from the cross can then be used for further breeding activities and / or as a seed source. In some instances, the progeny plants exhibit increased resistance to southern rust compared to the parent plant (e.g., a parent plant lacking the gene). In some instances, the gene can be introgressed into plant breeding lines (e.g., improved lines) using the progeny plants via repeated backcrosses.

[0204] In some instances, the protein is encoded by a gene containing a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequence of SEQ ID NO: 28, 32, 35, or 36.

[0205] In some instances, the protein is encoded by a gene containing a nucleotide sequence comprising SEQ ID NO: 28, 32, 35, or 36.

[0206] In some instances, the protein is encoded by a gene containing a nucleotide sequence that has at least 95% sequence identity with the sequence of SEQ ID NO: 28, 32, 35 or 36.

[0207] In some instances, the protein is encoded by a gene containing a nucleotide sequence that has at least 96% sequence identity with the sequence of SEQ ID NO: 28, 32, 35 or 36.

[0208] In some instances, the protein is encoded by a gene containing a nucleotide sequence that has at least 97% sequence identity with the sequence of SEQ ID NO: 28, 32, 35 or 36.

[0209] In some instances, the protein is encoded by a gene containing a nucleotide sequence that has at least 98% sequence identity with the sequence of SEQ ID NO: 28, 32, 35 or 36.

[0210] In some instances, the protein is encoded by a gene containing a nucleotide sequence that has at least 99% sequence identity with the sequence of SEQ ID NO: 28, 32, 35 or 36.

[0211] This document also provides a method for producing corn plants containing SR resistance. The method includes screening for a gene and / or protein as described herein, and selecting corn plants containing that gene and / or protein. The method includes crossing the selected corn plant with a second corn plant, and obtaining progeny plants containing that gene and / or protein. Compared to the second plant, the progeny plants exhibit increased SR resistance. The second plant may lack that gene and / or protein.

[0212] SR resistance gene

[0213] In various aspects of this disclosure, the NLR01 or NLR02 gene that confers SR resistance is used to generate recombinant plants. Alternatively, plants may be screened against said genes. For example, these genes can be detected (independently or in combination) to determine whether a given plant contains SR resistance, these genes can be transformed into plants, or these genes can be added to the plant genome using site-specific genome editing techniques (e.g., techniques based on CRISPR, TALEN, large-scale nucleases, or zinc finger nucleases). The NLR01 and NLR02 gene sequences conferring SR resistance were identified in inbred line A. Unless otherwise stated, any NLR01 or NLR02 gene sequence presented herein (e.g., any nucleic acid or protein sequence from SEQ ID NO: 27-34) may be used in any aspect of this disclosure where an NLR01 / NLR02 gene / protein sequence is required. Additionally, the gene sequences presented herein comprise protein sequences, DNA-coding sequences (e.g., cDNA sequences), or genomic DNA sequences including one or more of promoter, terminator, exon, and intron sequences.

[0214] Methods for producing recombinant corn plant cells and / or plants I

[0215] This document provides a method for producing recombinant maize plants or plant cells containing a heterologous NLR nucleic acid sequence (e.g., NLR01 disclosed herein) associated with resistance to SR. The method includes introducing the heterologous NLR gene sequence into one or more maize plant cells (or the plant itself) derived from an SR-susceptible maize plant, thereby producing at least one modified cell whose genome contains the heterologous NLR gene sequence. The method includes selecting the modified maize plant cells. Prior to performing the method of this invention, the susceptible maize plant may lack the heterologous NLR nucleic acid sequence. Alternatively, the method can be used to introduce additional copies of the heterologous NLR nucleic acid sequence.

[0216] In this method, genome editing techniques can be used to specifically modify at least one target site in the genome of one or more corn plant cells. For example, the SR susceptibility NLR gene allele can be altered through site-specific mutagenesis to encode the SR resistance NLR gene allele disclosed herein. Alternatively or additionally, the SR resistance NLR gene allele disclosed herein can be introduced at different locations through genome editing.

[0217] In some instances, the method involves introducing a polynucleotide-modified template containing a heterologous NLR nucleic acid sequence into one or more corn plant cells. This template can be integrated into the genome at a site-specific modification site via, for example, homologous recombination.

[0218] Heterogeneous NLR nucleic acid sequences, polynucleotide modification templates, and / or site-specific modifications can be successfully introduced into only a portion of a cell. Given this possibility, selection steps can be performed to ensure that a given cell has incorporated various modifications into its genome. Selection may include using molecular markers and / or sequencing or any other suitable technique to distinguish modified cells from unmodified cells.

[0219] In some instances, site-specific modifications are induced by CRISPR-associated endonucleases. Any CRISPR-associated endonuclease suitable for introducing site-specific modifications can be used. Site-specific modifications can include, for example, double-strand breaks or any other modifications that tend to lead to genomic modifications to include the NLR nucleic acid sequences disclosed herein that provide anti-SR resistance.

[0220] In some instances, the method involves growing modified corn plants from selected corn plant cells. The modified plants can exhibit increased resistance to SR compared to the first corn plant.

[0221] In some instances, the first corn plant contains an endogenous locus, and a heterologous NLR nucleic acid sequence replaces the endogenous locus. For example, the endogenous locus may contain an ortholog or paralog of the NLR01 gene or the NLR02 gene disclosed herein.

[0222] In some instances, a heterologous NLR nucleic acid sequence is inserted into a locus that does not naturally contain an NLR gene (e.g., the long arm of chromosome 10 of maize, or loci on chromosomes 1, 2, 3, 4, 5, 6, 7, 8, or 9). In some instances, the NLR nucleic acid sequence is inserted into a locus as part of a molecular stack. In some instances, the molecular stack contains one or more polynucleotides encoding additional maize disease resistance genes or other target genes.

[0223] In some instances, the heterologous NLR nucleic acid sequence contains a nucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 28, 32, 35, or 36.

[0224] In some instances, the heterologous NLR nucleic acid sequence contains a nucleotide sequence that is at least 95% identical to SEQ ID NO: 28, 32, 35 or 36.

[0225] In some instances, the heterologous NLR nucleic acid sequence contains a nucleotide sequence that has at least 96% identity with SEQ ID NO: 28, 32, 35 or 36.

[0226] In some instances, the heterologous NLR nucleic acid sequence contains a nucleotide sequence that is at least 97% identical to SEQ ID NO: 28, 32, 35 or 36.

[0227] In some instances, the heterologous NLR nucleic acid sequence contains a nucleotide sequence that is at least 98% identical to SEQ ID NO: 28, 32, 35 or 36.

[0228] In some instances, the heterologous NLR nucleic acid sequence contains a nucleotide sequence that is at least 99% identical to SEQ ID NO: 28, 32, 35 or 36.

[0229] In some instances, the heterologous NLR nucleic acid sequence contains the nucleotide sequence of SEQ ID NO: 28, 32, 35 or 36.

[0230] In some instances, the method includes site-specific modification of at least one target site in the genome of a corn plant cell, and the introduction of a polynucleotide modification template containing a heterologous NLR nucleotide sequence into the plant cell.

[0231] In some instances, site-specific modifications are achieved by CRISPR-associated endonucleases.

[0232] In some instances, the site-specific modification includes single-strand breaks or double-strand breaks (DSBs) produced by site-specific endonucleases, such as TALEN, broad-spectrum nucleases, zinc finger nucleases, and CRISPR-associated (Cas) protein / guide polynucleotide complexes. In some instances, the introduction of site-specific modifications can be combined with the introduction of polynucleotide modification templates.

[0233] In some instances, the template is introduced into the cell by any method suitable for delivering the polynucleotide-modified template into the cell nucleus, such as, but not limited to, transient introduction methods, transfection, electroporation, microinjection, particle-mediated delivery, topical application, whisker-mediated delivery, delivery via cell-penetrating peptides, or direct delivery mediated by mesoporous silica nanoparticles (MSN).

[0234] In some instances, polynucleotide modified templates can be introduced into cells as single-stranded polynucleotide molecules, double-stranded polynucleotide molecules, or as part of circular DNA (vector DNA). Polynucleotide modified templates can also be ligated with guide polynucleotides and / or Cas endonucleases. Ligated templates can allow colocalization of targets and template DNA, enabling genome editing and targeted genome regulation, and can also be used to target cells in late mitotic phases where the function of endogenous homologous recombination (HR) mechanisms is expected to be significantly reduced (Mali et al., 2013, Nature Methods, Vol. 10: 957-963). Polynucleotide modified templates can be transiently present in cells or introduced via viral replicons.

[0235] As used herein, a polynucleotide modification template refers to a polynucleotide that contains at least one nucleotide modification when compared to the target nucleotide sequence to be edited. A nucleotide modification is a substitution, addition, or deletion of at least one nucleotide. In some instances, the polynucleotide modification template may further comprise a homologous nucleotide sequence flanked by at least one nucleotide modification, wherein the flanked homologous nucleotide sequence provides sufficient homology to support the incorporation of the polynucleotide modification template into the genome of the recipient plant cell.

[0236] Methods for editing genomic sequences using both site-specific modifications and modification templates typically involve providing host cells with a site-specific endonuclease (or a nucleic acid encoding a site-specific endonuclease) that recognizes a target sequence in a chromosomal sequence, and inducing site-specific modifications (e.g., DSBs) in the genomic sequence via the site-specific endonuclease. The method also includes providing at least one polynucleotide modification template. The endonuclease can be provided to cells by any suitable method (e.g., transient introduction, transfection, microinjection, local application, and / or indirectly via a recombinant construct). The endonuclease can be provided directly to cells as a protein or as a guide polynucleotide complex, or indirectly via a recombinant construct. The endonuclease can be transiently introduced into cells or incorporated into the host cell's genome. In the case of a CRISPR-Cas system, as described in WO 2016073433, cell-penetrating peptides (CPPs) can be used to facilitate the uptake of endonucleases and / or guide polynucleotides into cells.

[0237] TAL effector nucleases (TALENs) are a class of sequence-specific nucleases that can be used to create double-strand breaks at specific target sequences in the genomes of plants or other organisms. (See Miller et al. (2011) Nature Biotechnology 29:143-148).

[0238] Endonucleases are enzymes that cleave phosphodiester bonds within a polynucleotide chain. Endonucleases include restriction endonucleases, which cleave DNA at specific sites without damaging bases; and broad-spectrum nucleases, also known as homing endonucleases (HE enzymes), which are similar to restriction endonucleases, binding and cleaving at specific recognition sites; however, for broad-spectrum nucleases, the recognition sites are typically longer, approximately 18 bp or longer (patent application PCT / US12 / 30061, filed March 22, 2012). Broad-spectrum nucleases are classified into four families based on conserved sequence motifs: LAGLIDADG, GIY-YIG, HNH, and the His-Cys box family. These motifs are involved in the coordination of metal ions and the hydrolysis of phosphodiester bonds. HE enzymes are notable for their long recognition sites and their tolerance to some sequence polymorphisms in their DNA substrates. The nomenclature conventions for broad-spectrum nucleases are similar to those for other restriction endonucleases. Large-scale nucleases are also characterized by prefixes F-, I-, or PI- for enzymes encoded by independent ORFs, introns, and intepids. One step in recombinant methods involves polynucleotide cleavage at or near the recognition site. The cleavage activity can be used to generate double-strand breaks. For a review of site-specific recombinases and their recognition sites, see Sauer (1994) Curr Op Biotechnol [Biotechnical Insights] 5:521-7; and Sadowski (1993) FASEB [Journal of the Federation of American Societies for Experimental Biology] 7:760-7. In some instances, recombinases belong to the integrase or resolvase family.

[0239] Zinc finger nucleases (ZFNs) are engineered double-strand break inducers consisting of a zinc finger DNA-binding domain and a double-strand break-inducer domain. Site-specific recognition is conferred by the zinc finger domain, which typically contains two, three, or four zinc fingers, such as a C2H2 structure; however, other zinc finger structures are known and have been engineered. The zinc finger domain is suitable for designing peptides that specifically bind to selected polynucleotide recognition sequences. ZFNs consist of an engineered DNA-binding zinc finger domain linked to a non-specific endonuclease domain (e.g., the nuclease domain from type IIs endonucleases such as FokI). Additional functionalities can be incorporated into the zinc finger binding domain; these include transcription activator domains, transcription repressor domains, and methyltransferases. In some instances, dimerization of the nuclease domain is required for cleavage activity. Each zinc finger recognizes three consecutive base pairs in the target DNA. For example, the 3-finger domain recognizes a sequence of 9 consecutive nucleotides, while two sets of zinc finger triplets are used to bind to a recognition sequence of 18 nucleotides due to the dimerization requirements of nucleases.

[0240] For example, genome editing using DSB inducers (such as the Cas9-gRNA complex) has been described in U.S. patent applications US 2015-0082478 A1, WO 2015 / 026886 A1, WO 2016007347 and WO 201625131 (all of which are incorporated herein by reference).

[0241] In this document, the terms “Cas gene” or “Cas protein” refer to genes that are typically coupled, associated with, or located near or adjacent to flanking CRISPR loci in bacterial systems. The terms “Cas” and “CRISPR-associated” are used interchangeably herein. The term “Cas endonuclease” refers to a protein or protein complex encoded by a Cas gene. When complexed with suitable polynucleotide components, Cas endonucleases as used herein are capable of recognizing, binding to all or part of a specific DNA target sequence, and optionally nicking or cleaving all or part of the specific DNA target sequence. Cas endonucleases as described herein comprise one or more nuclease domains. Cas endonucleases of this disclosure include those comprising HNH or HNH-like nuclease domains and / or RuvC or RuvC-like nuclease domains. Cas endonucleases of this disclosure may include Cas9 protein, Cpf1 protein, C2c1 protein, C2c2 protein, C2c3 protein, Cas3, Cas5, Cas7, Cas8, Cas10, or complexes thereof.

[0242] As used herein, the terms “guided polynucleotide / Cas endonuclease complex,” “guided polynucleotide / Cas endonuclease system,” “guided polynucleotide / Cas complex,” “guided polynucleotide / Cas system,” and “guided Cas system” are used interchangeably herein and refer to at least one guiding polynucleotide and at least one Cas endonuclease capable of forming a complex, wherein the guiding polynucleotide / Cas endonuclease complex guides the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave the DNA target site (introducing single-strand or double-strand breaks). The guiding polynucleotide / Cas endonuclease complex as used herein may comprise one or more Cas proteins and one or more suitable polynucleotide components from any of the four known CRISPR systems (Horvath and Barrangou, 2010, Science [Science] 327:167-170) (e.g., type I, type II, or type III CRISPR systems). Cas endonucleases unwind the DNA double helix at the target sequence and optionally cleave at least one DNA strand, as mediated by recognition of the target sequence by a polynucleotide (e.g., but not limited to crRNA or guide RNA) complexed with the Cas protein. Such recognition and cleavage of the target sequence by the Cas endonuclease typically occurs if the correct prespacer adjacent motif (PAM) is located at or adjacent to the 3' end of the DNA target sequence. Alternatively, the Cas protein described herein may lack DNA cleavage or cleavage activity, but may still specifically bind to the DNA target sequence when complexed with a suitable RNA component. (See also U.S. Patent Applications US 2015-0082478 A1 and US 2015-0059010 A1, both of which are incorporated herein by reference in their entirety.)

[0243] A guiding polynucleotide / Cas endonuclease complex can cleave one or both strands of a DNA target sequence. A guiding polynucleotide / Cas endonuclease complex capable of cleaving both strands of a DNA target sequence typically comprises a Cas protein having all of its endonuclease domains in a functional state (e.g., wild-type endonuclease domains or variants thereof retaining some or all activity in each endonuclease domain). Therefore, a wild-type Cas protein or a variant thereof (retaining some or all activity in each endonuclease domain of the Cas protein) is a suitable example of a Cas endonuclease capable of cleaving both strands of a DNA target sequence. The Cas9 protein, containing functional RuvC and HNH nuclease domains, is an example of a Cas protein capable of cleaving both strands of a DNA target sequence. A guiding polynucleotide / Cas endonuclease complex capable of cleaving one strand of a DNA target sequence can be characterized herein as containing cleavage enzyme activity (e.g., partial cleavage capability). Cas cleavage enzymes typically contain a functional endonuclease domain that allows Cas to cleave only one strand of the DNA target sequence (i.e., form a nick). For example, a Cas9 nickase may comprise (i) a mutated, dysfunctional RuvC domain and (ii) a functional HNH domain (e.g., a wild-type HNH domain). As another example, a Cas9 nickase may comprise (i) a functional RuvC domain (e.g., a wild-type RuvC domain) and (ii) a mutated, dysfunctional HNH domain. Non-limiting examples of Cas9 nickases suitable for use herein are disclosed in U.S. Patent Application Publication No. 2014 / 0189896, which is incorporated herein by reference.

[0244] In some instances, a pair of Cas9 nickases can be used to increase the specificity of DNA targeting. Generally, this is done by providing two Cas9 nickases that, by associating with RNA components having different guide sequences, target and nick the DNA sequence on the opposite strand of the region to be targeted. Such a nearby cleavage of each DNA strand produces a double-strand break (i.e., a DSB with a single-stranded overhang), which is then recognized as a substrate for non-homologous end joining (NHEJ) (which tends to produce imperfect repair leading to mutations) or homologous recombination (HR). Each nick in these instances can, for example, be at least about 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 (or any integer between 5 and 100) bases apart from each other. One or both Cas9 nickase proteins described herein can be used for Cas9 nickase pairs. For example, Cas9 nickases with a mutated RuvC domain but a functional HNH domain (i.e., Cas9 HNH+ / RuvC-) (e.g., Streptococcus pyogenes Cas9 HNH+ / RuvC-) can be used. Each Cas9 nickase (e.g., Cas9 HNH+ / RuvC-) is directed to a specific DNA site adjacent to each other (up to 100 base pairs apart) by using the appropriate RNA components described herein (with guide RNA sequences that target each nickase to each specific DNA site).

[0245] In some instances, the Cas protein may be part of a fusion protein that includes one or more heterologous protein domains (e.g., 1, 2, 3 or more domains other than the Cas protein). Such a fusion protein may include any additional protein sequence, as well as an optional linker sequence between any two domains (e.g., between the Cas protein and the first heterologous domain). Examples of protein domains that can fuse with the Cas protein described herein include, but are not limited to, epitope tags (e.g., histidine [His], V5, FLAG, influenza hemagglutinin [HA], myc, VSV-G, thioredoxin [Trx]); reporter molecules (e.g., glutathione-5-transferase [GST], horseradish peroxidase [HRP], chloramphenicol acetyltransferase [CAT], β-galactosidase, β-glucuronidase [GUS], luciferase, green fluorescent protein [GFP], HcRed, DsRed, cyan fluorescent protein [CFP], yellow fluorescent protein [YFP], blue fluorescent protein [BFP]); and domains containing one or more of the following activities: methyltransferase activity, demethyltransferase activity, transcriptional activation activity (e.g., VP16 or VP64), transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Cas proteins can also fuse with proteins that bind to DNA molecules or other molecules, such as maltose-binding protein (MBP), S-tags, Lex A DNA-binding domains (DBD), GAL4A DNA-binding domains, and herpes simplex virus (HSV) VP16. For further examples of Cas proteins, see PCT patent applications PCT / US16 / 32073, filed May 12, 2016, and PCT / US16 / 32028, filed May 12, 2016 (both applications are incorporated herein by reference).

[0246] In some instances, the guiding polynucleotide / Cas endonuclease complex can bind to a DNA target sequence but does not cleave any strand at the target sequence. Such a complex can contain a Cas protein in which all nuclease domains are mutated and dysfunctional. For example, the Cas9 protein of this article, which can bind to a DNA target sequence but does not cleave any strand at the target sequence, can contain a mutated, dysfunctional RuvC domain and a mutated, dysfunctional HNH domain. Cas proteins of this article that bind to but do not cleave target DNA sequences can be used to regulate gene expression, for example, in this case, the Cas protein can be fused with a transcription factor (or a portion thereof) (e.g., a repressor or activator, such as any of those disclosed herein). In other respects, an inactivated Cas protein can be fused with another protein containing endonuclease activity, such as the FokI endonuclease.

[0247] In this article, "Cas9" (formerly known as Cas5, Csn1, or Csx12) refers to the Cas endonuclease of a type II CRISPR system that forms a complex with cr and tracr nucleotides or with a single-guided polynucleotide, and is used to specifically recognize and cleave all or part of a DNA target sequence. The Cas9 protein contains a RuvC nuclease domain and an HNH (HNH) nuclease domain, each capable of cleaving a single strand of DNA at the target sequence (the synergistic effect of the two domains results in double-strand cleavage, while the activity of one domain results in a single cleavage). Typically, the RuvC domain contains subdomains I, II, and III, with domain I located near the N-terminus of Cas9, and subdomains II and III located in the middle of the protein, flanking the HNH domain (Hsu et al., Cell, 157:1262-1278). Type II CRISPR systems include DNA cleavage systems utilizing a Cas9 endonuclease complexed with at least one polynucleotide component. For example, Cas9 can complex with CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). In another instance, Cas9 can complex with a single guide RNA.

[0248] In some instances, Cas endonucleases may comprise modified forms of the Cas9 polypeptide. These modified forms of the Cas9 polypeptide may include amino acid changes (e.g., deletions, insertions, or substitutions) that reduce the nuclease activity of the naturally occurring Cas9 protein. For example, in some cases, the modified form of the Cas9 protein has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the corresponding wild-type Cas9 polypeptide (US Patent Application US 20140068797 A1) nuclease activity. In some cases, the modified form of the Cas9 polypeptide does not possess substantial nuclease activity and is referred to as catalytically inactivated "Cas9" or "inactivated Cas9 (dCas9)". Catalytically inactivated Cas9 variants include Cas9 variants containing mutations in the HNH and RuvC nuclease domains. These catalytically inactivated Cas9 variants are capable of interacting with sgRNA and binding to the target site in vivo but cannot cleave either strand of the target DNA.

[0249] In some instances, catalytically inactivated Cas9 can be fused to a heterologous sequence (US Patent Application US20140068797 A1). Suitable fusion couplers include, but are not limited to, peptides that provide activity by directly acting on target DNA or indirectly increasing transcription on peptides associated with that target DNA (e.g., histones or other DNA-binding proteins). Other suitable fusion couplers include, but are not limited to, peptides that provide methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinase activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristylation activity, or demyristylation activity. Further suitable fusion couplers include, but are not limited to, peptides that directly provide increased transcription of the target nucleic acid (e.g., transcription activators or fragments thereof, proteins or fragments thereof that recruit transcription activators, small molecule / drug-responsive transcription regulators, etc.). Catalytically inactivated Cas9 can also be fused to FokI nucleases to generate double-strand breaks (Guilinger et al., Nature Biotechnology, Vol. 32, No. 6, June 2014).

[0250] In some instances, the method involves growing plants from plant cells containing heterologous NLR gene sequences. In some instances, the plants exhibit increased resistance to SR. In some instances, plants derived from this method can then be used for further breeding activities and / or as a seed source. In some instances, plants produced by this method can be used to infiltrate NLR genes (e.g., NLR01 or NLR02) into plant breeding lines (e.g., improved lines) via repeated backcrosses.

[0251] Heterologous NLR loci can be inserted into any location in the plant cell genome suitable for heterologous NLR gene expression and optionally without interfering with endogenous gene expression. In some instances, the heterologous NLR nucleotide sequence replaces the endogenous NLR locus. In such instances, at least one allele of the endogenous NLR gene is eliminated or rendered nonfunctional and replaced by a heterologous NLR gene encoded on a polynucleotide modified template. In such instances, at least the coding region of the endogenous NLR gene is replaced or rendered nonfunctional. Introns, promoter sequences, and terminator sequences may also optionally be replaced or rendered nonfunctional.

[0252] In some instances, heterologous NLR nucleic acid sequences are inserted into loci different from endogenous NLR loci. In some instances, heterologous NLR nucleic acid sequences are inserted together with other genes also inserted into the same locus into loci different from endogenous NLR loci, forming molecular stacks. In some instances, the molecular stacks contain stacks of disease resistance genes.

[0253] Method II for producing recombinant corn plant cells and / or plants

[0254] This article also provides a method for producing recombinant corn plant cells containing a heterologous gene associated with resistance to SR. The method includes introducing an NLR nucleic acid sequence into one or more plant cells derived from a first corn plant. Prior to performing the method of the present invention, the first corn plant may lack the NLR nucleic acid sequence. The NLR nucleic acid sequence may be heterologous to the first corn plant.

[0255] This method involves inserting an NLR nucleic acid sequence into the genome of at least one plant cell to generate one or more modified plant cells. The method includes selecting the modified plant cells. The NLR nucleotide may comprise any NLR nucleotide sequence disclosed herein and may encode any NLR amino acid sequence disclosed herein. The NLR nucleic acid sequence may encode a protein comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the amino acid sequence identical to SEQ ID NO: 30 or 34. The NLR gene sequence may encode a protein comprising at least 95% of the amino acid sequence identical to SEQ ID NO: 30 or 34. The NLR nucleic acid sequence may encode a protein comprising the amino acid sequence of SEQ ID NO: 30 or 34.

[0256] In some instances, selection may include using molecular markers and / or sequencing or any other suitable technology to distinguish modified cells from unmodified cells.

[0257] In some instances, the method involves growing modified plants from selected plant cells. The modified plants may exhibit increased resistance to SR compared to the first plant. In some instances, selection may be based on any outcome indicating the presence of an NLR gene. For example, sequencing may be performed to determine if the gene has been inserted and / or to select plant cells for the use of a selection marker. In some instances, the method involves crossing newly grown modified corn plants with a second corn plant (e.g., a corn plant lacking the NLR gene) to obtain progeny plants containing the NLR gene. These progeny plants can then be used for further breeding activities (e.g., backcrossing and / or introgression) and / or as a seed source.

[0258] In some instances, the introduction of NLR nucleotide sequences has been involved in bacterial-mediated transformation.

[0259] In some instances, the introduction of NLR nucleotide sequences includes gene gun conversion.

[0260] In some instances, the NLR nucleic acid sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity with SEQ ID NO: 28, 32, 35, or 36. In some instances, the NLR nucleic acid sequence has at least 95% nucleotide sequence identity with SEQ ID NO: 28, 32, 35, or 36. In some instances, the NLR nucleic acid sequence comprises the sequence of SEQ ID NO: 28, 32, 35, or 36.

[0261] Recombinant plants and seeds

[0262] Recombinant maize plants comprising the NLR nucleic acid sequence described herein are provided. This document provides a recombinant maize plant comprising a heteronucleotide encoding a protein having at least 90% or at least 95% amino acid sequence identity with SEQ ID NO: 30 or 34. In some instances, the heteronucleotide encodes a protein having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO: 30, and the heteronucleotide is located at a chromosomal locus other than the native NLR01 locus on the short arm of chromosome 10 (e.g., the heteronucleotide is located on the long arm of maize chromosome 10 or on chromosomes 1, 2, 3, 4, 5, 6, 7, 8, or 9). In some instances, the heterologous protein has 100% amino acid sequence identity with SEQ ID NO: 30 or 34, and the heterologous nucleic acid is located at a chromosomal locus other than the native NLR01 locus on the short arm of chromosome 10 (e.g., the heterologous nucleic acid is located on the long arm of chromosome 10 or on chromosomes 1, 2, 3, 4, 5, 6, 7, 8, or 9). In some instances, the heterologous nucleic acid comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity with SEQ ID NO: 28 or 35, and the heterologous nucleic acid is located at a chromosomal locus other than the native NLR01 locus on the short arm of chromosome 10 (e.g., the heterologous nucleic acid is located on the long arm of chromosome 10 or on chromosomes 1, 2, 3, 4, 5, 6, 7, 8, or 9). In some instances, the heterologous protein is encoded by a gene containing the nucleotide sequence of SEQ ID NO: 28 or 35, and the heterologous nucleic acid is located at a chromosomal locus other than the native NLR01 locus on the short arm of chromosome 10 (e.g., the heterologous nucleic acid is located on the long arm of chromosome 10 of corn or on chromosomes 1, 2, 3, 4, 5, 6, 7, 8 or 9).

[0263] Also provided are recombinant maize plant seeds containing the NLR nucleic acid sequence described herein. Therefore, a recombinant plant seed is provided containing a heterologous nucleic acid encoding a protein having at least 90% or at least 95% amino acid sequence identity with SEQ ID NO: 30, wherein the heterologous nucleic acid is located at a chromosomal locus other than the natural NLR01 locus on the short arm of chromosome 10 (e.g., the heterologous nucleic acid is located on the long arm of maize chromosome 10 or on chromosomes 1, 2, 3, 4, 5, 6, 7, 8, or 9). In some instances, the protein has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO: 30, and the heteronucleotide is located at a chromosomal locus other than the native NLR01 locus on the short arm of chromosome 10 (e.g., the heteronucleotide is located on the long arm of chromosome 10 or on chromosomes 1, 2, 3, 4, 5, 6, 7, 8, or 9). In some instances, the protein has amino acid sequence identity with SEQ ID NO: 30 or 34, and the heteronucleotide is located at a chromosomal locus other than the native NLR01 locus on the short arm of chromosome 10 (e.g., the heteronucleotide is located on the long arm of chromosome 10 or on chromosomes 1, 2, 3, 4, 5, 6, 7, 8, or 9). In some instances, the gene contains a nucleotide sequence having at least 90% or at least 95% nucleotide sequence identity with SEQ ID NO: 28 or 35, and the heteronucleotide is located at a chromosomal locus other than the natural NLR01 locus on the short arm of chromosome 10 (e.g., the heteronucleotide is located on the long arm of chromosome 10 or on chromosomes 1, 2, 3, 4, 5, 6, 7, 8, or 9). In some instances, the gene contains a nucleotide sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity with SEQ ID NO: 28, 32, 35, or 36, and the heteronucleotide is located at a chromosomal locus other than the natural NLR01 locus on the short arm of chromosome 10 (e.g., the heteronucleotide is located on the long arm of chromosome 10 or on chromosomes 1, 2, 3, 4, 5, 6, 7, 8, or 9). In some instances, the gene contains nucleotide sequences including SEQ ID NO:28, 32, 35, or 36, wherein the heteronucleotide is located at a chromosomal locus other than the native NLR01 locus on the short arm of chromosome 10 (e.g., the heteronucleotide is located on the long arm of corn chromosome 10 or on chromosomes 1, 2, 3, 4, 5, 6, 7, 8, or 9).

[0264] Terms of this disclosure

[0265] The various aspects of this disclosure will be understood by referring to the following terms.

[0266] 1. A method for identifying corn plants containing sequences associated with resistance to southern rust, the method comprising:

[0267] a. Obtaining samples containing nucleic acids from corn plants.

[0268] b. Screening the sample for the protein or the nucleic acid encoding the protein, wherein the protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 30, and

[0269] c. Detect the protein or the nucleic acid encoding the protein to identify the corn plant as containing a sequence associated with resistance to southern rust.

[0270] 2. The method as described in Clause 1, wherein the protein has 100% amino acid sequence identity with SEQ ID NO: 30.

[0271] 3. The method as described in Clause 1 or 2, wherein the method includes

[0272] a. Obtain samples containing nucleic acids from each of several corn plant species.

[0273] b. Screen each sample for the protein or the nucleic acid encoding the protein;

[0274] c. Detecting the protein or nucleic acid encoding the protein in at least one sample, thereby identifying at least one corn plant from which the sample was obtained as containing the sequence associated with resistance to southern rust; and

[0275] d. Select at least one of the corn plants identified as containing the sequence associated with resistance to southern rust.

[0276] 4. A method for producing a corn plant containing a sequence associated with resistance to southern rust, the method comprising selecting the corn plant according to clause 3, and further comprising:

[0277] c. Hybridizing at least one of the corn plants selected in (d) with a second corn plant; and

[0278] d. Obtain progeny plants containing the sequences associated with resistance to southern rust.

[0279] 5. The method of any one of Clauses 1-4, wherein the nucleic acid sequence has at least 95% nucleotide sequence identity with SEQ ID NO: 28 or 35.

[0280] 6. The method of any one of Clauses 1-5, wherein the nucleic acid sequence comprises SEQ ID NO: 28 or 35.

[0281] 7. A method for producing modified maize plants or modified maize plant cells, the method comprising:

[0282] Provide unmodified corn plants or one or more cells thereof that are susceptible to SR;

[0283] Introducing a heterologous NLR01 nucleic acid sequence encoding a protein having at least 95% amino acid sequence identity with SEQ ID NO: 30 into the genome of the plant or one or more cells thereof, thereby producing one or more modified plants or plant cells thereof containing the heterologous NLR01 sequence; and

[0284] Select at least one of the modified corn plants or modified corn plant cells.

[0285] 8. The method as described in Clause 7, wherein the method comprises:

[0286] a) Site-specific modification of at least one target site in the genome of the susceptible corn plant or one or more cells thereof; and

[0287] b) Introducing a polynucleotide modified template containing the heterologous NLR01 sequence into the susceptible corn plant or one or more corn plant cells to produce the modified corn, or modified corn plant, or modified corn plant cells.

[0288] 9. The method as described in Clause 7 or 8, wherein the method comprises:

[0289] a) Site-specific modification of the natural NLR01 gene in the susceptible corn plant or its cells, and

[0290] b) Altering the natural NLR01 gene sequence to produce the modified corn plant or modified corn plant cell.

[0291] 10. The method as described in clauses 7, 8 or 9, wherein the site-specific modification is induced by a CRISPR-associated endonuclease.

[0292] 11. The method of any one of Clauses 7-10, wherein the method comprises generating one or more modified corn plant cells, and further comprises growing modified corn plants from the selected corn plant cells, wherein the modified plants exhibit increased resistance to southern rust relative to the unmodified corn plants.

[0293] 12. The method of any one of Clauses 7-11, wherein the unmodified corn plant contains an endogenous susceptible NLR01 sequence, and the heterologous NLR01 sequence replaces the endogenous susceptible NLR01 sequence.

[0294] 13. The method of any one of clauses 7, 8, 10 and 11, wherein the heterologous NLR01 sequence is inserted into a locus that does not naturally contain a natural NLR01 gene sequence.

[0295] 14. The method of any one of clauses 7-13, wherein the heterologous NLR gene sequence comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 28 or 35.

[0296] 15. The method of claim 14, wherein the heterologous NLR gene sequence comprises the nucleotide sequence of SEQ ID NO: 28 or 35.

[0297] 16. The method as described in Clause 11, further comprising:

[0298] The modified maize plants were crossed with maize plants susceptible to SR and lacking the heterologous NLR01 sequence, and

[0299] Progeny plants that are more resistant to SR than the susceptible corn plants and contain the heterologous NLR01 sequence are selected for further breeding or corn production.

[0300] 17. A recombinant corn seed comprising a heterologous nucleic acid sequence encoding a protein having at least 95% amino acid sequence identity with SEQ ID NO: 30.

[0301] 18. The recombinant corn plant seed as described in Clause 17, wherein the protein has 100% amino acid sequence identity with SEQ ID NO: 30.

[0302] 19. Recombinant corn plant seeds as described in Clause 17 or 18, wherein the heterologous nucleic acid sequence has at least 95% nucleotide sequence identity with SEQ ID NO: 28 or 35.

[0303] 20. The recombinant seed as described in any one of clauses 17-19, wherein the heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO:28 or 35.

[0304] 21. A method for selecting corn plants containing QTLs associated with resistance to southern rust, the method comprising:

[0305] Nucleic acids obtained from one or more corn plants;

[0306] The nucleic acid was screened for QTLs linked to a haplotype on chromosome 10 of maize, the haplotype comprising one or more of the following marker alleles:

[0307] i. The “T” at C00422-801 corresponds to position 201 of SEQ ID NO: 1;

[0308] ii. The “C” at C01770-1 corresponds to position 201 of SEQ ID NO: 2;

[0309] iii. The “T” at C06813-1 corresponds to position 201 of SEQ ID NO: 7;

[0310] iv. The “G” at C06824-1 corresponds to position 201 of SEQ ID NO: 11;

[0311] The “C” at C06834-1 corresponds to position 201 of SEQ ID NO: 12;

[0312] vi. The “T” at C01957-1 corresponds to position 201 of SEQ ID NO: 13;

[0313] vii. The “T” at C06838-1 corresponds to position 201 of SEQ ID NO: 14;

[0314] viii. The “G” at C06848-1 corresponds to position 201 of SEQ ID NO: 15;

[0315] ix. The “A” at PZA9035-19 corresponds to position 211 in SEQ ID NO: 16;

[0316] x. The “T” at C12422-001 corresponds to position 201 of SEQ ID NO: 17;

[0317] The “G” at position xi. PZA10357-20 corresponds to position 121 of SEQ ID NO: 18;

[0318] xii. The “G” at C06839-1 corresponds to position 201 of SEQ ID NO: 19;

[0319] xiii. The “C” at C002MY1-001 corresponds to position 61 of SEQ ID NO: 20;

[0320] xiv. The “G” at CSR_2373961 corresponds to position 51 of SEQ ID NO: 21;

[0321] The “C” at xv. CSR_2409943 corresponds to position 51 of SEQ ID NO: 22;

[0322] xvi. The “G” at CSR_2643297 corresponds to position 51 of SEQ ID NO: 23;

[0323] xvii. The “C” at CSR_2807867 corresponds to position 51 of SEQ ID NO: 24;

[0324] xviii. The “C” at CSR_2837481 corresponds to position 101 of SEQ ID NO: 25; or

[0325] xix. The “A” at CSR_2839126 corresponds to position 101 of SEQ ID NO: 26; and

[0326] Select corn plants containing the haplotype, thereby selecting corn plants containing the QTL.

[0327] 22. The method as described in Clause 21, further comprising:

[0328] The nucleic acids are screened for one or more of the marker alleles i.-xix. associated with the QTL.

[0329] 23. The method as described in Clause 21 or 22, further comprising:

[0330] (A) Screen the nucleic acid for one or more of the following marker alleles on chromosome 10:

[0331] a. The “A” at C01800-1 corresponds to position 201 of SEQ ID NO: 3;

[0332] b. The “G” at C06790-1 corresponds to position 201 of SEQ ID NO: 4; and

[0333] c. The “T” at C00429-801 corresponds to position 84 of SEQ ID NO: 8.

[0334] Alternatively, the nucleic acid may be screened against one or more of the following marker alleles on chromosome 10:

[0335] d. The “T” at C00431-802 corresponds to position 210 of SEQ ID NO: 5;

[0336] e. The “C” at C002TCN-001 corresponds to position 61 of SEQ ID NO: 6;

[0337] f. The “T” at C002TCM-001 corresponds to position 61 of SEQ ID NO: 9; and

[0338] g. The “A” at C06817-1 corresponds to position 201 of SEQ ID NO: 10, and

[0339] (B) Select corn plants containing one or more marker alleles ag, thereby selecting corn plants containing the QTL.

[0340] 24. A method for selecting corn plants containing QTLs associated with resistance to southern rust, the method comprising:

[0341] Nucleic acids obtained from one or more corn plants;

[0342] The nucleic acids are screened against QTLs, wherein the QTLs are linked to haplotypes on chromosome 10 of maize, and the haplotypes contain one or more of the following marker alleles:

[0343] i. The “T” at C00422-801 corresponds to position 201 of SEQ ID NO: 1;

[0344] ii. The “C” at C01770-1 corresponds to position 201 of SEQ ID NO: 2;

[0345] iii. The “T” at C06813-1 corresponds to position 201 of SEQ ID NO: 7;

[0346] iv. The “G” at C06824-1 corresponds to position 201 of SEQ ID NO: 11;

[0347] The “C” at C06834-1 corresponds to position 201 of SEQ ID NO: 12;

[0348] vi. The “T” at C01957-1 corresponds to position 201 of SEQ ID NO: 13;

[0349] vii. The “T” at C06838-1 corresponds to position 201 of SEQ ID NO: 14;

[0350] viii. The “G” at C06848-1 corresponds to position 201 of SEQ ID NO: 15;

[0351] ix. The “A” at PZA9035-19 corresponds to position 211 in SEQ ID NO: 16;

[0352] x. The “T” at C12422-001 corresponds to position 201 of SEQ ID NO: 17;

[0353] The “G” at position xi. PZA10357-20 corresponds to position 121 of SEQ ID NO: 18;

[0354] xii. The “G” at C06839-1 corresponds to position 201 of SEQ ID NO: 19;

[0355] xiii. The “C” at C002MY1-001 corresponds to position 61 of SEQ ID NO: 20;

[0356] xiv. The “G” at CSR_2373961 corresponds to position 51 of SEQ ID NO: 21;

[0357] The “C” at xv. CSR_2409943 corresponds to position 51 of SEQ ID NO: 22;

[0358] xvi. The “G” at CSR_2643297 corresponds to position 51 of SEQ ID NO: 23;

[0359] xvii. The “C” at CSR_2807867 corresponds to position 51 of SEQ ID NO: 24;

[0360] xviii. The “C” at CSR_2837481 corresponds to position 101 in SEQ ID NO: 25;

[0361] xix. The “A” at CSR_2839126 corresponds to position 101 of SEQ ID NO: 26;

[0362] a. The “A” at C01800-1 corresponds to position 201 of SEQ ID NO: 3;

[0363] b. The “G” at C06790-1 corresponds to position 201 of SEQ ID NO: 4; or

[0364] c. The “T” at C00429-801 corresponds to position 84 of SEQ ID NO: 8;

[0365] as well as

[0366] Select corn plants containing the haplotype, thereby selecting corn plants containing the QTL.

[0367] 25. The method as described in Clause 24, further comprising:

[0368] Nucleic acids from the corn plant are screened for one or more of the marker alleles i.-xix, a., b. and / or c. associated with the QTL.

[0369] 26. A method for selecting corn plants containing QTLs associated with resistance to southern rust, the method comprising:

[0370] Nucleic acids are obtained from one or more corn plants or their germplasm;

[0371] The nucleic acids are screened against QTLs, wherein the QTLs are linked to haplotypes on chromosome 10 of maize, and the haplotypes contain one or more of the following marker alleles:

[0372] i. The “T” at C00422-801 corresponds to position 201 of SEQ ID NO: 1;

[0373] ii. The “C” at C01770-1 corresponds to position 201 of SEQ ID NO: 2;

[0374] iii. The “T” at C06813-1 corresponds to position 201 of SEQ ID NO: 7;

[0375] iv. The “G” at C06824-1 corresponds to position 201 of SEQ ID NO: 11;

[0376] The “C” at C06834-1 corresponds to position 201 of SEQ ID NO: 12;

[0377] vi. The “T” at C01957-1 corresponds to position 201 of SEQ ID NO: 13;

[0378] vii. The “T” at C06838-1 corresponds to position 201 of SEQ ID NO: 14;

[0379] viii. The “G” at C06848-1 corresponds to position 201 of SEQ ID NO: 15;

[0380] ix. The “A” at PZA9035-19 corresponds to position 211 in SEQ ID NO: 16;

[0381] x. The “T” at C12422-001 corresponds to position 201 of SEQ ID NO: 17;

[0382] The “G” at position xi. PZA10357-20 corresponds to position 121 of SEQ ID NO: 18;

[0383] xii. The “G” at C06839-1 corresponds to position 201 of SEQ ID NO: 19;

[0384] xiii. The “C” at C002MY1-001 corresponds to position 61 of SEQ ID NO: 20;

[0385] xiv. The “G” at CSR_2373961 corresponds to position 51 of SEQ ID NO: 21;

[0386] The “C” at xv. CSR_2409943 corresponds to position 51 of SEQ ID NO: 22;

[0387] xvi. The “G” at CSR_2643297 corresponds to position 51 of SEQ ID NO: 23;

[0388] xvii. The “C” at CSR_2807867 corresponds to position 51 of SEQ ID NO: 24;

[0389] xviii. The “C” at CSR_2837481 corresponds to position 101 in SEQ ID NO: 25;

[0390] xix. The “A” at CSR_2839126 corresponds to position 101 of SEQ ID NO: 26;

[0391] d. The “T” at C00431-802 corresponds to position 210 of SEQ ID NO: 5;

[0392] e. The “C” at C002TCN-001 corresponds to position 61 of SEQ ID NO: 6;

[0393] f. The “T” at C002TCM-001 corresponds to position 61 of SEQ ID NO: 9; or

[0394] g. The “A” at C06817-1 corresponds to position 201 in SEQ ID NO: 10;

[0395] as well as

[0396] Select corn plants containing the haplotype, thereby selecting corn plants containing the QTL.

[0397] 27. The method as described in Clause 26, further comprising:

[0398] Nucleic acids from the corn plant are screened for one or more of the marker alleles i.-xix, d., e., f. and / or g. associated with the QTL.

[0399] 28. The method of any one of clauses 21-27, wherein the method comprises screening for one or more of the following:

[0400] xiv. The “G” at CSR_2373961 corresponds to position 51 of SEQ ID NO: 21;

[0401] The “C” at xv. CSR_2409943 corresponds to position 51 of SEQ ID NO: 22;

[0402] xvi. The “G” at CSR_2643297 corresponds to position 51 of SEQ ID NO: 23;

[0403] xvii. The “C” at CSR_2807867 corresponds to position 51 of SEQ ID NO: 24;

[0404] xviii. The “C” at CSR_2837481 corresponds to position 101 of SEQ ID NO: 25; or

[0405] The “A” at xix. CSR_2839126 corresponds to position 101 of SEQ ID NO: 26.

[0406] 29. The method of any one of clauses 22, 23, 25, or 27, wherein the one or more selected marker alleles are located in a lateral chromosomal region comprising:

[0407] C06824-1, corresponding to the "G" at position 201 of SEQ ID NO: 11 and

[0408] C06834-1 corresponds to the "C" at position 201 of SEQ ID NO: 12.

[0409] 30. The method of any one of clauses 22, 23, 25, 27, or 29, wherein the one or more selected marker alleles are located in a lateral chromosomal region comprising:

[0410] CSR_2373961 corresponds to the "G" at position 51 of SEQ ID NO: 21 and

[0411] CSR_2839126 corresponds to “A” at position 101 of SEQ ID NO: 26.

[0412] 31. A method for producing maize plants containing resistance to southern rust, the method comprising selecting maize plants according to any one of claims 21-30, and further comprising...

[0413] Hybridize one or more selected corn plants with a second corn plant; and

[0414] Obtain offspring plants containing the QTL.

[0415] 32. A method for producing corn plants containing resistance to southern rust, the method comprising:

[0416] a. Crossing a first parental maize plant with a second parental maize plant to produce one or more progeny plants, wherein the first parental plant contains a QTL associated with resistance to southern rust, the QTL being absent in the second parental plant;

[0417] b. Obtaining nucleic acid samples from one or more of the said progeny plants; and

[0418] c. The sample is screened according to the method of any one of Clauses 30-40 and one or more progeny plants containing the QTL associated with resistance to southern rust are selected.

[0419] 33. The method as described in Clause 32, further comprising:

[0420] d. Cross one or more selected progeny plants with the second parent plant to produce one or more backcross progeny plants;

[0421] e. Obtaining nucleic acid samples from one or more backcross progeny plants; and

[0422] f. Select one or more backcross progeny plants that contain the QTLs associated with resistance to southern rust.

[0423] 34. The method as described in Clause 33, further comprising:

[0424] g. Crossing the one or more selected backcross progeny plants with the second parent plant to produce additional backcross progeny plants;

[0425] h. Obtain nucleic acid samples from one or more other backcross progeny plants;

[0426] i. Select one or more additional backcross progeny plants containing the QTLs associated with resistance to southern rust; and

[0427] j. Optionally repeat steps (g), (h), and (i) to obtain additional backcross progeny plants containing the QTLs associated with resistance to southern rust.

[0428] Example

[0429] The following examples will provide a more comprehensive understanding of this disclosure. These examples are presented to aid in understanding the disclosure and are not intended to limit its scope.

[0430] Example 1 - Initial mapping of QTLs associated with anti-SR resistance on chromosome 10 of corn.

[0431] Two F2 populations were generated using an SR-resistant source (inbred line A) and two susceptible inbred lines (inbred lines B and C) for genetic mapping of the SR resistance trait in inbred line A. The resistance phenotype was determined using assay data generated under greenhouse conditions. Plants were inoculated with *Pseudomonas aeruginosa* in a greenhouse and evaluated for the SR resistance phenotype. Phenotypic data for all parental lines and segregating materials were clear in the experiment (Table 3). Segregation rates matched the expected rates based on segregation at a single dominant locus.

[0432] Table 3: Phenotype calls and segregation rates for inbred lines A, B, inbred line A (crossed with inbred line B), C, and inbred line A (crossed with inbred line C) from greenhouse phenotypic analysis.

[0433]

[0434] Phenotypic data from TaqMan SNP marker genotypes of F2 individuals were used for initial mapping to identify QTLs on chromosome 10 (short arm). Based on analysis of 5 key recombination events from inbred line A x inbred line B and 11 events from inbred line A x inbred line C, the flanking markers for this effect were C06824-1 and C06834-1 (Tables 4 and 5).

[0435] Table 4: QTL analysis results for the inbred line A x inbred line B F2 population using the greenhouse phenotype. A total of 162 SNP markers were used, covering the entire genome, with the markers used concentrated on chromosome 10. No significant markers were found on chromosomes 1–9, therefore only markers on chromosome 10 are shown. R or S scores were converted to 1 (R) or 2 (S) for analysis. p-values ​​of the association strength between marker genotype and phenotype are shown.

[0436]

[0437] Table 5: QTL analysis results of the F2 population of inbred line A x inbred line C using the greenhouse phenotype. A total of 162 SNP markers were used, covering the entire genome, with the markers used concentrated on chromosome 10. No significant markers were found on chromosomes 1–9, so only markers on chromosome 10 are shown. R or S scores were converted to 1 (R) or 2 (S) for analysis. p-values ​​of the association strength between marker genotype and phenotype are shown. The marker C002TCN-001 is polymorphic in plants produced from inbred line A x inbred line C, but not in plants produced from inbred line A x inbred line B.

[0438]

[0439] Table 6: Key recombination events supporting flanking markers C06824-1 and C06834-1 in the A x B population of inbred lines. B = genotype of inbred line B, H = heterozygous. Data from plant 437083102 appear to be the result of inappropriate genotypes or inappropriate scoring.

[0440]

[0441] Table 7: Key recombination events supporting flanking markers C06824-1 and C06834-1 in inbred line A x inbred line C populations. C = inbred line C, H = heterozygous.

[0442]

[0443] Example 2 - Fine-grained plotting of QTL

[0444] Given the strong associations observed on chromosome 10 in these populations and the flanking markers identified in the first round of mapping, we screened an additional 1840 individuals using TaqMan SNP markers and DNA from broken grains for recombination events in the regions surrounding the C06824-1 and C06834-1 markers. Based on the genotypes in this region, a new set of 180 individuals was selected for phenotypic analysis. The selected individuals had recombination events similar to those in Tables 6 and 7.

[0445] The genome of inbred line A was sequenced to identify potential candidate genes in the regions. Transcriptome sequencing was performed, yielding approximately 100 million reads from V5 seedling tissue. Genomic data were used to identify candidate genes and additional marker SNPs within the regions. Markers were designed within the regions to identify the most informative recombination events based on the positions of previously marked and candidate genes for fine mapping. New recombination events (6 from progeny of inbred line C and 2 from progeny of inbred line B) yielded two new flanking markers: CSR_2807867 and CSR_2837481 (Table 8).

[0446] Table 8: Additional recombination events and flank markers for inbred lines A x C and A x B. B = inbred line B or C, A = inbred line A, and H = heterozygous.

[0447]

[0448] Table 9: Markers and B73 V4 positions used in the analysis of chromosome 10 regions.

[0449]

[0450] Example 3 - Candidate Gene Identification and Protoplast Determination

[0451] Flanking markers from the fine mapping (CSR_2807867 and CSR_2837481) are 248 kb apart in the genome sequence of inbred line A. This region contains two candidate resistance genes: NLR01 and NLR02. Paired-terminal RNA-seq reads from a leaf tissue library of inbred line A were aligned with the sequenced genome of inbred line A using HISAT2. Transcript models were then constructed using Stringtie based on these alignments. Predicted proteins from all protein-coding transcript models within the region were then evaluated using HMMer to identify typical NLR domains (NB-ARC, coiled helices, and leucine-rich repetitive sequences). Two genes (NLR01 and NLR02) within the fine mapping region were found to encode proteins containing typical NLR domains, and these two genes were selected as potential candidates for resistance to southern maize rust.

[0452] The ability of NLR01 to recognize southern corn rust effector alleles was tested in a protoplast assay of maize (Deng, C. et al. The RppC-AvrRppC NLR-effector interaction mediates the resistance to southern corn rust in maize. Mol. Plant. [Molecular Plant] 2022; 15: 904-912). This assay showed that the NLR01 protein could detect the same *Rhizoctonia solani* isolates used in the greenhouse phenotypic analysis and could induce protoplast cell death.

[0453] Example 4 - Transgenic testing of NLR01 and NLR02 candidate genes

[0454] The efficacy of two NLR genes (NLR01 and NLR02) identified from inbred line A QTL was tested under one or more identical susceptible backgrounds used for mapping. The transgenic construct of NLR01 contained a natural promoter (1990 bp) (SEQ ID NO:27), a natural coding sequence (7800 bp) (SEQ ID NO:28), and a natural corn terminator (1500 bp) (SEQ ID NO:29). The transgenic construct of NLR02 contained a natural promoter (2000 bp) (SEQ ID NO:31), a natural coding sequence (1097 bp) (SEQ ID NO:32), and a natural corn terminator (1000 bp) (SEQ ID NO:33).

[0455] Transgenic plants expressing NLR01 or NLR02 were produced via bacterial-mediated transformation. T1 plants (the progeny of the initially produced T0 transformants) were grown and infected with *Symplocos rubrum*. Table 10 shows data on NLR01 production, and Table 11 shows data on NLR02 production. Transgenic plants were compared to control plants (i.e., plants known to be resistant or susceptible) to determine the extent of resistance provided by NLR01 expression and the extent of resistance provided by NLR02 expression. Control plants are indicated in the event column in Tables 10 and 11. Transgenic plants were also compared to presumed transformed plants that segregated during the transformation process but failed to show NLR01 or NLR02 gene incorporation. Such plants were designated “null” in Tables 10 and 11. Southern rust resistance scores are provided in the “Southern Rust Score” column of Tables 10 and 11. Susceptibility is scored as “S”, intermediate as “I”, and resistance as “R”.

[0456] Table 10

[0457]

[0458] Table 11

[0459]

[0460] Based on the foregoing results, NLR01, when incorporated into the genome and expressed, provides resistance to southern rust in plants. All plants carrying the heterologous NLR01 gene showed at least the intermediate phenotype (except for plants 10, 11, and 34), while null plants were all susceptible. Positive and negative control plants showed the expected positive and negative phenotypes, respectively.

[0461] The results indicate that NLR02 does not provide resistance to southern rust (at least not under the conditions tested).

[0462] Example 5 - The presence of NLR01 in the genome of a common corn variety.

[0463] A review of public strains revealed that the NLR01 gene disclosed in this paper is present in the genomes of the public corn cultivars W64A Rpp9 (accession number Ames 30907) and Boesman Yellow Flint (accession number PI 186208). See Sun et al., Phytopathology Research 3:25 (2021) and USDA Annual Report of Cooperative Regional Projects, January 1, 1974 – December 31, 1974.

[0464] For W64A Rpp9, long-read resequencing data were generated using the Sequel2e platform from Pacific Biosciences (Menlo Park, California, USA). PacBio HiFi reads with a read length of 16.1 kb and 14x coverage were aligned to the reference genome of inbred line A using minimap2-2.28 (r1209) (available on GitHub) with default parameters and map-hifi presets. Visual inspection of aligned reads at target loci was performed using IGV 2.17.3, with a minimum alignment score of 5000. Read coverage ranging from 6 to 12x was observed, indicating 100% sequence identity between the NLR01 gene in inbred line A and W64A Rpp9.

[0465] For Boesman Yellow Flint, long-read resequencing data were generated using the Revio platform from Pacific Biosciences. PacBio HiFi read datasets from two cell lines, with a read length of 17.2 kb and N50 and 70x coverage, were aligned to the reference genome of inbred line A using minimap2-2.28 (r1209) with default parameters and map-hifi presets. Visual inspection of aligned reads at target loci was performed using IGV 2.17.3, with a minimum alignment score of 5000. Due to the seed source being derived from the population, coverage at a given locus varied for a given allele. Read coverage ranges of 8–11x were observed, indicating 100% sequence identity between the NLR01 gene in inbred line A and Boesman Yellow Flint.

Claims

1. A method for identifying corn plants containing sequences associated with resistance to southern rust, the method comprising: a. Obtaining samples containing nucleic acids from corn plants. b. Screening the sample for the protein or the nucleic acid encoding the protein, wherein the protein comprises an amino acid sequence having at least 95% sequence identity with SEQ ID NO: 30, and c. Detect the protein or the nucleic acid encoding the protein to identify the corn plant as containing a sequence associated with resistance to southern rust.

2. The method of claim 1, wherein the protein has 100% amino acid sequence identity with SEQ ID NO:

30.

3. The method of claim 1 or 2, wherein the method includes a. Obtain samples containing nucleic acids from each of several corn plant species. b. Screen each sample for the protein or the nucleic acid encoding the protein; c. Detect the protein or nucleic acid encoding the protein in at least one sample, thereby identifying at least one corn plant from which the sample was obtained as containing the sequence associated with resistance to southern rust; as well as d. Select at least one of the corn plants identified as containing the sequence associated with resistance to southern rust.

4. A method for producing a corn plant comprising a sequence associated with resistance to southern rust, the method comprising selecting the corn plant according to claim 3, and further comprising: c. Hybridize at least one of the corn plants selected in (d) with a second corn plant; as well as d. Obtain progeny plants containing the sequences associated with resistance to southern rust.

5. The method according to any one of claims 1-4, wherein the nucleic acid sequence has at least 95% nucleotide sequence identity with SEQ ID NO: 28 or 35.

6. The method of any one of claims 1-5, wherein the nucleic acid sequence comprises SEQ ID NO: 28 or 35.

7. A method for producing modified maize plants or modified maize plant cells, the method comprising: Provide unmodified corn plants or one or more cells thereof that are susceptible to SR; Introducing a heterologous NLR01 nucleic acid sequence encoding a protein having at least 95% amino acid sequence identity with SEQ ID NO: 30 into the genome of the plant or one or more cells thereof, thereby producing one or more modified plants or plant cells thereof containing the heterologous NLR01 sequence; as well as Select at least one of the modified corn plants or modified corn plant cells.

8. The method of claim 7, wherein the method comprises: a) Site-specific modification of at least one target site in the genome of the susceptible corn plant or one or more cells thereof; as well as b) Introducing a polynucleotide modified template containing the heterologous NLR01 sequence into the susceptible corn plant or one or more corn plant cells to produce the modified corn, or modified corn plant, or modified corn plant cells.

9. The method of claim 7 or 8, wherein the method comprises: a) Site-specific modification of the natural NLR01 gene in the susceptible corn plant or its cells, and b) Altering the natural NLR01 gene sequence to produce the modified corn plant or modified corn plant cell.

10. The method of claim 7, 8 or 9, wherein the site-specific modification is induced by a CRISPR-associated endonuclease.

11. The method of any one of claims 7-10, wherein the method comprises generating one or more modified corn plant cells, and further comprises growing modified corn plants from the selected corn plant cells, wherein the modified plants exhibit increased resistance to southern rust relative to the unmodified corn plants.

12. The method of any one of claims 7-11, wherein the unmodified corn plant contains an endogenous susceptible NLR01 sequence, and the heterologous NLR01 sequence replaces the endogenous susceptible NLR01 sequence.

13. The method of any one of claims 7, 8, 10 and 11, wherein the heterologous NLR01 sequence is inserted into a locus that does not naturally contain a natural NLR01 gene sequence.

14. The method of any one of claims 7-13, wherein the heterologous NLR gene sequence comprises a nucleotide sequence having at least 95% identity with SEQ ID NO: 28 or 35.

15. The method of claim 14, wherein the heterologous NLR gene sequence comprises the nucleotide sequence of SEQ ID NO: 28 or 35.

16. The method of claim 11, further comprising: The modified maize plants were crossed with maize plants susceptible to SR and lacking the heterologous NLR01 sequence, and Progeny plants that are more resistant to SR than the susceptible corn plants and contain the heterologous NLR01 sequence are selected for further breeding or corn production.

17. A recombinant corn seed comprising a heterologous nucleic acid sequence encoding a protein having at least 95% amino acid sequence identity with SEQ ID NO:

30.

18. The recombinant corn seed of claim 17, wherein the protein has 100% amino acid sequence identity with SEQ ID NO:

30.

19. The recombinant corn seed as claimed in claim 17 or 18, wherein the heterologous nucleic acid sequence has at least 95% nucleotide sequence identity with SEQ ID NO: 28 or 35.

20. The recombinant seed according to any one of claims 17-19, wherein the heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO:28 or 35.

21. A method for selecting corn plants containing QTLs associated with resistance to southern rust, the method comprising: Nucleic acids obtained from one or more corn plants; The nucleic acid was screened for QTLs linked to a haplotype on chromosome 10 of maize, the haplotype comprising one or more of the following marker alleles: i. The "T" at C00422-801 corresponds to position 201 of SEQ ID NO: 1; ii. The "C" at C01770-1 corresponds to position 201 of SEQ ID NO: 2; iii. The "T" at C06813-1 corresponds to position 201 of SEQ ID NO: 7; iv. The "G" at C06824-1 corresponds to position 201 of SEQ ID NO: 11; The "C" at C06834-1 corresponds to position 201 in SEQ ID NO: 12; vi. The "T" at C01957-1 corresponds to position 201 of SEQ ID NO: 13; vii. The "T" at C06838-1 corresponds to position 201 of SEQ ID NO: 14; viii. The "G" at C06848-1 corresponds to position 201 of SEQ ID NO: 15; ix. The "A" at PZA9035-19 corresponds to position 211 in SEQ ID NO: 16; x. The "T" at C12422-001 corresponds to position 201 of SEQ ID NO: 17; The "G" at position xi. PZA10357-20 corresponds to position 121 of SEQ ID NO: 18; xii. The "G" at C06839-1 corresponds to position 201 of SEQ ID NO: 19; xiii. The "C" at C002MY1-001 corresponds to position 61 of SEQ ID NO: 20; xiv. The "G" at CSR_2373961 corresponds to position 51 of SEQ ID NO: 21; The "C" at xv. CSR_2409943 corresponds to position 51 of SEQ ID NO: 22; xvi. The "G" at CSR_2643297 corresponds to position 51 of SEQ ID NO: 23; xvii. The "C" at CSR_2807867 corresponds to position 51 of SEQ ID NO: 24; xviii. The "C" at CSR_2837481 corresponds to position 101 of SEQ ID NO: 25; or The "A" at xix. CSR_2839126 corresponds to position 101 of SEQ ID NO: 26; and Select corn plants containing the haplotype, thereby selecting corn plants containing the QTL.

22. The method of claim 21, further comprising: The nucleic acids are screened for one or more of the marker alleles i.-xix. associated with the QTL.

23. The method of claim 21 or 22, further comprising: (A) Screen the nucleic acid for one or more of the following marker alleles on chromosome 10: a. The "A" at C01800-1 corresponds to position 201 of SEQ ID NO: 3; b. The "G" at C06790-1 corresponds to position 201 of SEQ ID NO: 4; and c. The "T" at C00429-801 corresponds to position 84 of SEQ ID NO:

8. Alternatively, the nucleic acid may be screened against one or more of the following marker alleles on chromosome 10: d. The "T" at C00431-802 corresponds to position 210 of SEQ ID NO: 5; e. The "C" at C002TCN-001 corresponds to position 61 of SEQ ID NO: 6; f. The "T" at C002TCM-001 corresponds to position 61 of SEQ ID NO: 9; and g. The "A" at C06817-1 corresponds to position 201 of SEQ ID NO: 10, and (B) Select corn plants containing one or more marker alleles ag, thereby selecting corn plants containing the QTL.

24. A method for selecting corn plants containing QTLs associated with resistance to southern rust, the method comprising: Nucleic acids obtained from one or more corn plants; The nucleic acids are screened against QTLs, wherein the QTLs are linked to haplotypes on chromosome 10 of maize, and the haplotypes contain one or more of the following marker alleles: i. The "T" at C00422-801 corresponds to position 201 of SEQ ID NO: 1; ii. The "C" at C01770-1 corresponds to position 201 of SEQ ID NO: 2; iii. The "T" at C06813-1 corresponds to position 201 of SEQ ID NO: 7; iv. The "G" at C06824-1 corresponds to position 201 of SEQ ID NO: 11; The "C" at C06834-1 corresponds to position 201 in SEQ ID NO: 12; vi. The "T" at C01957-1 corresponds to position 201 of SEQ ID NO: 13; vii. The "T" at C06838-1 corresponds to position 201 of SEQ ID NO: 14; viii. The "G" at C06848-1 corresponds to position 201 of SEQ ID NO: 15; ix. The "A" at PZA9035-19 corresponds to position 211 in SEQ ID NO: 16; x. The "T" at C12422-001 corresponds to position 201 of SEQ ID NO: 17; The "G" at position xi. PZA10357-20 corresponds to position 121 of SEQ ID NO: 18; xii. The "G" at C06839-1 corresponds to position 201 of SEQ ID NO: 19; xiii. The "C" at C002MY1-001 corresponds to position 61 of SEQ ID NO: 20; xiv. The "G" at CSR_2373961 corresponds to position 51 of SEQ ID NO: 21; The "C" at xv. CSR_2409943 corresponds to position 51 of SEQ ID NO: 22; xvi. The "G" at CSR_2643297 corresponds to position 51 of SEQ ID NO: 23; xvii. The "C" at CSR_2807867 corresponds to position 51 of SEQ ID NO: 24; xviii. The "C" at CSR_2837481 corresponds to position 101 in SEQ ID NO: 25; xix. The "A" at CSR_2839126 corresponds to position 101 of SEQ ID NO: 26; a. The "A" at C01800-1 corresponds to position 201 of SEQ ID NO: 3; b. The "G" at C06790-1 corresponds to position 201 of SEQ ID NO: 4; or c. The "T" at C00429-801 corresponds to position 84 of SEQ ID NO: 8; as well as Select corn plants containing the haplotype, thereby selecting corn plants containing the QTL.

25. The method of claim 24, further comprising: Nucleic acids from the corn plant are screened for one or more of the marker alleles i.-xix, a., b. and / or c. associated with the QTL.

26. A method for selecting corn plants containing QTLs associated with resistance to southern rust, the method comprising: Nucleic acids are obtained from one or more corn plants or their germplasm; The nucleic acids are screened against QTLs, wherein the QTLs are linked to haplotypes on chromosome 10 of maize, and the haplotypes contain one or more of the following marker alleles: i. The "T" at C00422-801 corresponds to position 201 of SEQ ID NO: 1; ii. The "C" at C01770-1 corresponds to position 201 of SEQ ID NO: 2; iii. The "T" at C06813-1 corresponds to position 201 of SEQ ID NO: 7; iv. The "G" at C06824-1 corresponds to position 201 of SEQ ID NO: 11; The "C" at C06834-1 corresponds to position 201 in SEQ ID NO: 12; vi. The "T" at C01957-1 corresponds to position 201 of SEQ ID NO: 13; vii. The "T" at C06838-1 corresponds to position 201 of SEQ ID NO: 14; viii. The "G" at C06848-1 corresponds to position 201 of SEQ ID NO: 15; ix. The "A" at PZA9035-19 corresponds to position 211 in SEQ ID NO: 16; x. The "T" at C12422-001 corresponds to position 201 of SEQ ID NO: 17; The "G" at position xi. PZA10357-20 corresponds to position 121 of SEQ ID NO: 18; xii. The "G" at C06839-1 corresponds to position 201 of SEQ ID NO: 19; xiii. The "C" at C002MY1-001 corresponds to position 61 of SEQ ID NO: 20; xiv. The "G" at CSR_2373961 corresponds to position 51 of SEQ ID NO: 21; The "C" at xv. CSR_2409943 corresponds to position 51 of SEQ ID NO: 22; xvi. The "G" at CSR_2643297 corresponds to position 51 of SEQ ID NO: 23; xvii. The "C" at CSR_2807867 corresponds to position 51 of SEQ ID NO: 24; xviii. The "C" at CSR_2837481 corresponds to position 101 in SEQ ID NO: 25; xix. The "A" at CSR_2839126 corresponds to position 101 of SEQ ID NO: 26; d. The "T" at C00431-802 corresponds to position 210 of SEQ ID NO: 5; e. The "C" at C002TCN-001 corresponds to position 61 of SEQ ID NO: 6; f. The "T" at C002TCM-001 corresponds to position 61 of SEQ ID NO: 9; or g. The "A" at C06817-1 corresponds to position 201 in SEQ ID NO: 10; as well as Select corn plants containing the haplotype, thereby selecting corn plants containing the QTL.

27. The method of claim 26, further comprising: Nucleic acids from the corn plant are screened for one or more of the marker alleles i.-xix, d., e., f. and / or g. associated with the QTL.

28. The method of any one of claims 21-27, wherein the method comprises screening for one or more of the following: xiv. The "G" at CSR_2373961 corresponds to position 51 of SEQ ID NO: 21; The "C" at xv. CSR_2409943 corresponds to position 51 of SEQ ID NO: 22; xvi. The "G" at CSR_2643297 corresponds to position 51 of SEQ ID NO: 23; xvii. The "C" at CSR_2807867 corresponds to position 51 of SEQ ID NO: 24; xviii. The "C" at CSR_2837481 corresponds to position 101 of SEQ ID NO: 25; or The "A" at xix. CSR_2839126 corresponds to position 101 of SEQ ID NO:

26.

29. The method of any one of claims 22, 23, 25, or 27, wherein the one or more selected marker alleles are located in lateral chromosomal regions comprising: C06824-1, corresponding to the "G" at position 201 of SEQ ID NO: 11 and C06834-1 corresponds to the "C" at position 201 of SEQ ID NO:

12.

30. The method of any one of claims 22, 23, 25, 27, or 29, wherein the one or more selected marker alleles are located in a lateral chromosomal region comprising: CSR_2373961 corresponds to the "G" at position 51 of SEQ ID NO: 21 and CSR_2839126 corresponds to "A" at position 101 of SEQ ID NO:

26.

31. A method for producing corn plants containing resistance to southern rust, the method comprising: Choose a maize plant according to any one of claims 21-30, and Hybridize one or more selected corn plants with a second corn plant; and Obtain offspring plants containing the QTL.

32. A method for producing corn plants containing resistance to southern rust, the method comprising: a. Crossing a first parental maize plant with a second parental maize plant to produce one or more progeny plants, wherein the first parental plant contains a QTL associated with resistance to southern rust, the QTL being absent in the second parental plant; b. Obtain nucleic acid samples from one or more of the said progeny plants; as well as c. The sample is screened using the method of any one of claims 30-40, and one or more progeny plants containing the QTL associated with resistance to southern rust are selected.

33. The method of claim 32, further comprising: d. Cross one or more selected progeny plants with the second parent plant to produce one or more backcross progeny plants; e. Obtaining nucleic acid samples from one or more backcross progeny plants; and f. Select one or more backcross progeny plants that contain the QTLs associated with resistance to southern rust.

34. The method of claim 33, further comprising: g. Crossing the one or more selected backcross progeny plants with the second parent plant to produce additional backcross progeny plants; h. Obtain nucleic acid samples from one or more other backcross progeny plants; i. Select one or more additional backcross progeny plants containing the QTLs associated with resistance to southern rust; and j. Optionally repeat steps (g), (h), and (i) to obtain additional backcross progeny plants containing the QTLs associated with resistance to southern rust.

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