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

Abstract

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

Description

MAIZE PLANTS COMPRISING RESISTANCE TO SOUTHERN CORN RUST AND COMPOSITIONS AND METHODS FOR SELECTING AND PRODUCING THE SAMEFIELD

[0001] The present disclosure is related to maize plants comprising resistance to southern corn rust and compositions and methods for selecting and producing the same.REFERENCE TO THE SEQUENCE LISTING

[0002] The official copy of the sequence listing is submitted electronically as an XML file named “108486- WO-S EC- 1 Sequence Listing” created on September 17, 2024. The XML file has a size of 74,908 bytes, and the sequence listing is filed concurrently with this specification. The sequence listing contained in this XML file document is part of the present specification and is incorporated herein by reference in its entirety.BACKGROUND

[0003] Southern rust (“SR”) is a disease of maize plants caused by the fungal pathogen Puccinia polysora. P. polysora does not survive the winter in many corn-growing areas, but, under proper weather conditions, the pathogen can be transported from warmer, more humid regions to regions with colder winters. SR is a concern to farmers and grain producers due to its potential for reducing yields and / or quality of maize crops. SR can be especially damaging to corn due to its potential for rapid development and spread under favorable conditions. While techniques for reducing the impact of SR on maize crops exist, there remains a need for additional development of new techniques for managing and minimizing the potential impact of SR.SUMMARY

[0004] The present disclosure provides maize plants that comprise resistance to southern rust (“SR”). Resistance can be provided via the QTL disclosed herein and NLR01 gene disclosed herein. Breeding and / or genetic engineering techniques can be used to produce plants comprising the resistance provided via the QTL and NLR01 gene. The plants provided herein can improve agricultural yields and / or reduce or eliminate the need for fungicide application when SR is a concern.

[0005] Provided herein is a method for selecting a maize plant comprising the QTL associated with resistance to SR disclosed herein. The method comprises obtaining nucleic acids from one or more maize plants and screening the nucleic acids for the QTL on maize chromosome 10 comprising one or more (e.g., a haplotype) of the marker alleles disclosed in Table 1 andselecting a maize plant comprising the one or more (e.g., haplotype) of marker alleles and thereby selecting a maize plant comprising the QTL.

[0006] Also provided herein is a method for selecting a maize plant comprising a QTL associated with resistance to southern rust. The method comprises obtaining nucleic acid from one or more maize plants. The method comprises screening the nucleic acid for a haplotype on maize chromosome 10 comprising one or more (e.g., a haplotype) of the marker alleles disclosed in Table 1 and / or one or more of (a) an “A” at C01800-1 , corresponding to position 201 of SEQ ID NO: 3; (b) a “G” at C06790-1 , corresponding to position 201 of SEQ ID NO: 4; or (c) a “T” at C00429-801 , corresponding to position 84 of SEQ ID NO: 8. The method comprises selecting a maize plant comprising the haplotype and thereby selecting a maize plant comprising the QTL.

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

[0008] Also provided herein is a method for selecting a maize plant comprising resistance to SR. The method comprises screening a population of maize plants for a gene or the protein encoded by the gene, wherein the protein comprises an amino acid sequence comprising at least 95% sequence identity to SEQ ID NO: 30. The method comprises selecting a maize plant comprising the gene or protein encoded thereby.

[0009] Also provided herein is a method for producing a recombinant maize plant or cell thereof comprising a heterologous gene associated with resistance to SR. The method comprises introducing a heterologous NLR gene sequence that encodes a protein comprising at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO: 30 into one or more cells derived from a first maize plant, thereby producing at least one modified cell comprising the heterologous NLR gene sequence in the modified cell’s genome. The method comprises selecting at least one of the modified maize plant cells and optionally regenerating a recombinant plant comprising the heterologous NLR gene. Also provided herein is a recombinant plant or seed produced from the recombinant plant, whereinthe plant or seed comprises the heterologous gene encoding a protein comprising at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO: 30.

[0010] Also provided herein is a method for producing a recombinant maize plant cell comprising a heterologous gene associated with resistance to SR. The method comprises 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, thereby creating one or more modified plant cells; and selecting a modified plant cell. The NLR gene sequence encodes a protein comprising an amino acid sequence at least 95% identical to SEQ ID NOs: 30.

[0011] Also provided herein is a recombinant plant comprising a heterologous protein comprising at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO: 30. Further, provided herein is a recombinant plant seed comprising a heterologous gene encoding a protein comprising at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO: 30. BRI EF DESCRIPTION OF THE SEQEUNCE LISTING

[0012] The disclosure can be more fully understood from the following detailed description and the sequence listing which forms a part of this application.

[0013] The sequence descriptions and sequence listing attached hereto comply with the rules governing nucleotide and / or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §§1.831-1.834 and in the WIPO ST.26 Standard. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.832. Unless stated otherwise, the case of individual letters representing amino acid or nucleotide residues does not have any significance (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. , reverse complement sequence).

[0014] SEQ ID NO: 1 is the C00422-801 marker reference sequence, with the SNP at position 201 indicated as a “y”, meaning “c or t”. The SNP linked with resistance to SR comprises a “t” at position 51.

[0015] SEQ ID NO: 2 is the C01770-1 marker reference sequence, with the SNP at position 201 indicated as a “y”, meaning “c or t”. The SNP linked with resistance to SR comprises a “c” at position 201.

[0016] SEQ ID NO: 3 is the C01800-1 marker reference sequence, with the SNP at position 201 indicated as a “w”, meaning “a or t”. The SNP linked with resistance to SR comprises an “a” at position 201.

[0017] SEQ ID NO: 4 is the C06790-1 marker reference sequence, with the SNP at position 201 indicated as an “s”, meaning “g or c”. The SNP linked with resistance to SR comprises a “g” at position 201.

[0018] SEQ ID NO: 5 is the C00431-802 marker reference sequence, with the SNP at position 210 indicated as a “k”, meaning “g or t”. The SNP linked with resistance to SR comprises a “t” at position 210.

[0019] SEQ ID NO: 6 is the C002TCN-001 marker reference sequence, with the SNP at position 61 indicated as an “m”, meaning “a or c”. The SNP linked with resistance to SR comprises a “c” at position 61.

[0020] SEQ ID NO: 7 is the C06813-1 marker reference sequence, with the SNP at position 201 indicated as a “w”, meaning “a or t”. The SNP linked with resistance to SR comprises a “t” at position 201.

[0021] SEQ ID NO: 8 is the C00429-801 marker reference sequence, with the SNP at position 84 indicated as a “w”, meaning “a or t”. The SNP linked with resistance to SR comprises a “t” at position 84.

[0022] SEQ ID NO: 9 is the C002TCM-001 marker reference sequence, with the SNP at position 61 indicated as a “k”, meaning “g or t”. The SNP linked with resistance to SR comprises a “t” at position 51.

[0023] SEQ ID NO: 10 is the C06817-1 marker reference sequence, with the SNP at position 201 indicated as an “r”, meaning “a or g”. The SNP linked with resistance to SR comprises an “a” at position 201.

[0024] SEQ ID NO: 11 is the C06824-1 marker reference sequence, with the SNP at position 201 indicated as an T, meaning “a or g”. The SNP linked with resistance to SR comprises a “g” at position 201.

[0025] SEQ ID NO: 12 is the C06834-1 marker reference sequence, with the SNP at position 201 indicated as a “y”, meaning “c or t”. The SNP linked with resistance to SR comprises a “c” at position 201.

[0026] SEQ ID NO: 13 is the C01957-1 marker reference sequence, with the SNP at position 201 indicated as a “y”, meaning “t or c”. The SNP linked with resistance to SR comprises a “t” at position 201.

[0027] SEQ ID NO: 14 is the C06838-1 marker reference sequence, with the SNP at position201 indicated as a “y”, meaning “t or c”. The SNP linked with resistance to SR comprises a “t” at position 201.

[0028] SEQ ID NO: 15 is the C06848-1 marker reference sequence, with the SNP at position 201 indicated as a T, meaning “a or g”. The SNP linked with resistance to SR comprises a “g” at position 201.

[0029] SEQ ID NO: 16 is the PZA9035-19 marker reference sequence, with the SNP at position 211 indicated as an “w”, meaning “a or t”. The SNP linked with resistance to SR comprises an “a” at position 211.

[0030] SEQ ID NO: 17 is the C12422-001 marker reference sequence, with the SNP at position 201 indicated as a “y”, meaning “c or t”. The SNP linked with resistance to SR comprises a “t” at position 201.

[0031] SEQ ID NO: 18 is the PZA10357-20 marker reference sequence, with the SNP at position 121 indicated as a “r”, meaning “a or g”. The SNP linked with resistance to SR comprises an “g” at position 121.

[0032] SEQ ID NO: 19 is the C06839-1 marker reference sequence, with the SNP at position 201 indicated as an “s”, meaning “c or g”. The SNP linked with resistance to SR comprises an “g” at position 201.

[0033] SEQ ID NO: 20 is the C002MY1-001 marker reference sequence, with the SNP at position 61 indicated as an “m”, meaning “a or c”. The SNP linked with resistance to SR comprises an “c” at position 61 .

[0034] SEQ ID NO: 21 is the CSR_2373961 marker reference sequence, with the SNP at position 51 indicated as an “r”, meaning “a or g”. The SNP linked with resistance to SR comprises an “g” at position 51.

[0035] SEQ ID NO: 22 is the CSR_2409943 marker reference sequence, with the SNP at position 51 indicated as a “m”, meaning “a or c”. The SNP linked with resistance to SR comprises an “c” at position 51.

[0036] SEQ ID NO: 23 is the CSR_2643297 marker reference sequence, with the SNP at position 51 indicated as a “k”, meaning “g or t”. The SNP linked with resistance to SR comprises an “g” at position 51.

[0037] SEQ ID NO: 24 is the CSR_2807867 marker reference sequence, with the SNP at position 51 indicated as a “y”, meaning “c or t”. The SNP linked with resistance to SR comprises an “c” at position 51 .

[0038] SEQ ID NO: 25 is the CSR_2837481 marker reference sequence, with the SNP at position 101 indicated as a “y”, meaning “c or t”. The SNP linked with resistance to SR comprises an “c” at position 101.

[0039] SEQ ID NO: 26 is the CSR_2839126 marker reference sequence, with the SNP at position 101 indicated as an “m”, meaning “a or c”. The SNP linked with resistance to SR comprises an “a” at position 101.

[0040] SEQ ID NO: 27 is the promoter sequence driving expression of the NLR01 gene of Inbred A. The promoter includes 1 ,990 base pairs preceding the transcriptional start site.

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

[0042] SEQ ID NO: 29 is the terminator associated with the NLR01 gene of Inbred A. The terminator includes 1 ,500 base pairs that are 3’ of the transcriptional stop site.

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

[0044] SEQ ID NO: 31 is the promoter sequence driving expression of the NLR02 gene of Inbred A. The promoter includes 2,000 base pairs 5’ of the transcriptional start site.

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

[0046] SEQ ID NO: 33 is the terminator associated with the NLR02 gene of Inbred A. The terminator includes 1 ,000 base pairs that are 3’ of the transcriptional stop site.

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

[0048] SEQ ID NO: 35 shows the codons corresponding to the amino acids of SEQ ID NO: 30 (e.g., an NLR01 cDNA sequence).

[0049] SEQ ID NO: 36 shows the codons corresponding to the amino acids of SEQ ID NO: 34 (e.g., an NLR02 cDNA sequence).DETAILED DESCRIPTION

[0050] The present disclosure provides a gene that provides resistance to SR and it also provides markers which have been determined to be genetically linked to a locus (e.g., a QTL) which provides resistance to SR. The present disclosure also provides methods using the gene sequence, the protein encoded by the gene, and the disclosed markers to select resistant plants or to counter-select susceptible plants. Maize plants and methods of making maize plants comprising increased resistance to SR, relative to control plants (e.g., a parental line that is susceptible to SR), are also provided herein. Causal genes which provide the newly conferredor increased resistance when expressed in the plants and plants expressing and / or encoding the same are also provided.

[0051] Definitions

[0052] The following definitions are provided as an aid to understand the present disclosure.

[0053] The disclosure is not limited to particular examples, which can, of course, vary. The terminology and examples used herein are for the purpose of describing particular aspects of the disclosure only and are not intended to be limiting. As used herein, terms in the singular and the singular forms “a”, “an” and “the”, for example, include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “plant”, “the plant” or “a plant” also includes a plurality of plants; also, depending on the context, use of the term “plant” can also include genetically similar or identical progeny of that plant; use of the term “a nucleic acid” optionally includes, as a practical matter, many copies of that nucleic acid molecule; similarly, the term “probe” optionally (and typically) encompasses many similar or identical probe molecules.

[0054] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation. Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer or any non-integer fraction within the defined range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. In describing and claiming the subject matter of the current disclosure, the following terminology will be used in accordance with the definitions set out below and throughout the specification.

[0055] The term “allele” refers to one of two or more different nucleotide sequences that occur at a specific locus.

[0056] “Allele frequency” refers to the frequency (proportion or percentage) at which an allele is present at a locus within an individual, within a line, or within a population of lines. For example, for an allele “A”, diploid individuals of genotype “AA”, “Aa”, or “aa” have allele frequencies of 1.0, 0.5, or 0.0, respectively. One can estimate the allele frequency within a line by averaging the allele frequencies of a sample of individuals from that line. Similarly, one can calculate the allele frequency within a population of lines by averaging the allele frequencies of lines that make up the population. For a population with a finite number of individuals or lines, an allele frequency can be expressed as a count of individuals or lines (or any other specified grouping) containing the allele.

[0057] The term “amplifying” in the context of nucleic acid amplification is any process whereby additional copies of a selected nucleic acid (or a transcribed form thereof) are produced. Typicalamplification methods include various polymerase based replication methods, including the polymerase chain reaction (PCR), ligase mediated methods such as the ligase chain reaction (LCR) and RNA polymerase based amplification (e.g., by transcription) methods.

[0058] An allele is “associated with” a trait when it is part of or linked to a DNA sequence that affects the expression of the trait. The presence of the allele is an indicator of how the trait will be expressed. For example, an allele can be linked to resistance to SR if the allele is part of or linked to a gene that can provide resistance to SR. Similarly, haplotypes or QTLs can be “associated with” a trait.

[0059] A centimorgan (“cM”) is a unit of measure of recombination frequency. One cM is equal to a 1% chance that a marker at one genetic locus will be separated from a marker at a second locus due to crossing over in a single generation.

[0060] As used herein, the term “chromosomal interval” designates a contiguous linear span of genomic DNA that resides in planta on a single chromosome. The genetic elements or genes located on a single chromosomal interval are physically linked. The size of a chromosomal interval is not particularly limited. In some aspects, the genetic elements located within a single chromosomal interval are genetically linked, typically with 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 undergo recombination at a frequency of less than or equal to 20% or 10%.

[0061] A “chromosome” is a single piece of coiled DNA containing many genes that act and move as a unity during cell division and therefore can be said to be linked. It can also be referred to as a “linkage group”.

[0062] The term “contiguous DNA” refers to an uninterrupted stretch of genomic DNA represented by partially overlapping pieces or contigs.

[0063] The term “crossed” or “cross” refers to a sexual cross and involved the fusion of two haploid gametes via pollination to produce diploid progeny (e.g., cells, seeds or plants). The term encompasses both the pollination of one plant by another and selfing (or self-pollination, e.g., when the pollen and ovule are from the same plant).

[0064] A plant referred to herein as “diploid” has two sets of chromosomes.

[0065] A plant referred to herein as a “doubled haploid” is developed by doubling the haploid set of chromosomes (i.e. , half the normal number of chromosomes). A doubled haploid plant has two identical sets of chromosomes, and all loci are considered homozygous.

[0066] An “elite line” is any line that has resulted from breeding and selection for superior agronomic performance.

[0067] An “exotic maize strain” or an “exotic maize germplasm” is a strain derived from a maize plant not belonging to an available elite maize line or strain of germplasm. In the context of a cross between two maize plants or strains of germplasm, an exotic germplasm is not closely related by descent to the elite germplasm with which it is crossed. Most commonly, the exotic germplasm is not derived from any known elite line of maize, but rather is selected to introduce novel genetic elements (typically novel alleles) into a breeding program.

[0068] A “favorable allele” is the allele at a particular locus that confers, or contributes to, an agronomically desirable phenotype, e.g., SR resistance. A favorable allele of a marker is a marker allele that segregates with (e.g., is linked to) the favorable phenotype.

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

[0070] A “genetic map” is a description of genetic linkage relationships among loci on one or more chromosomes (or linkage groups) within a given species, generally depicted in a diagrammatic or tabular form. For each genetic map, distances between loci are measured by how frequently their alleles appear together in a population (their recombination frequencies). Alleles can be detected using DNA or protein markers, or observable phenotypes. A genetic map is a product of the mapping population, types of markers used, and the polymorphic potential of each marker between different populations. Genetic distances between loci can differ from one genetic map to another. However, information can be correlated from one map to another using common markers. One of ordinary skill in the art can use common marker positions to identify positions of markers and other loci of interest on each individual genetic map. The order of loci should not change between maps, although frequently there are small changes in marker orders due to e.g., markers detecting alternate duplicate loci in different populations, differences in statistical approaches used to order the markers, novel mutation or laboratory error.

[0071] A “genetic map location” is a location 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 the process of defining the linkage relationships of loci through the use of genetic markers, populations segregating for the markers, and standard genetic principles of recombination frequency.

[0073] “Genetic markers”, “markers”, or “molecular markers” are nucleic acids that are polymorphic in a population and where the alleles of which can be detected and distinguished by one or more analytic methods, described below. The terms also refer to nucleic acidsequences complementary to the genomic sequences, such as nucleic acids used as probes. Markers corresponding to genetic polymorphisms between members of a population can be detected by analytic methods such as, for example, PCR-based sequence specific amplification methods, detection of restriction fragment length polymorphisms (RFLP), detection of isozyme markers, detection of polynucleotide polymorphisms by allele specific hybridization (ASH), detection of amplified variable sequences of the plant genome, detection of self-sustained sequence replication, detection of simple sequence repeats (SSRs), detection of single nucleotide polymorphisms (SNPs), detection of amplified fragment length polymorphisms (AFLPs), or next generation sequencing techniques. Other appropriate methods include detection of expressed sequence tags (ESTs) and SSR markers derived from EST sequences and randomly amplified polymorphic DNA (RAPD). In addition to nucleic acids, “Genetic markers”, “markers”, or “molecular markers” may also refer to proteins or inherited phenotypes whose causal genes are known or expected to be linked to a favorable allele.

[0074] For example, markers are provided herein, such as the SNPs in Table 1, which are linked to a favorable allele providing SR resistance. As used herein, “Marker allele” refers to the particular sequence at the marker locus that is linked to the favorable allele.

[0075] “Genetic recombination frequency” is the frequency of a crossing over event (recombination) between two genetic loci. Recombination frequency can be observed by following the segregation of markers and / or traits following 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 constitution of an individual (or group of individuals) at one or more genetic loci. Genotype is defined by the allele(s) of one or more known loci that the individual has inherited from its parents. The term genotype can be used to refer to an individual’s genetic constitution at a single locus, at multiple loci, or, more generally, the term genotype can be used to refer to an individual’s genetic make-up for all the genes in its genome.

[0078] “Germplasm” refers to genetic material of or from an individual (e.g., a plant), a group of individuals (e.g., a plant line, variety or family), or a clone derived from a line, variety, species, or culture, or more generally, all individuals within a species or for several species (e.g., maize germplasm collection or Andean germplasm collection). The germplasm can be part of an organism or cell or can be separate from the organism or cell. In general, germplasm provides genetic material with a specific molecular makeup that provides a physical foundation for some or all of the hereditary qualities of an organism or cell culture. As used herein, germplasm includes cells, seed or tissues from which new plants may be grown, or plant parts, such as leaves, stems, pollen, or cells, that can be cultured into a whole plant.

[0079] A plant referred to as “haploid” has a single set (genome) of chromosomes.

[0080] A “haplotype” is the genotype of an individual at a plurality of genetic loci, i.e., a combination of alleles. Typically, the genetic loci described by a haplotype are physically and genetically linked, i.e., on the same chromosome segment.

[0081] The term “heterogeneity” is used to indicate that individuals within the group differ in genotype at one or more specific loci.

[0082] The heterotic response of material, or “heterosis”, can be defined by performance which exceeds the average of the parents (or high parent) when crossed to other dissimilar or unrelated groups.

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

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

[0085] An individual is “homozygous” if the individual has only one type of allele at a given locus (e.g., a diploid individual has a copy of the same allele at a locus for each of two homologous chromosomes).

[0086] The term “hybrid” refers to the progeny obtained between the crossing of at least two genetically dissimilar 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. 0038] The term “hybridize” means to form base pairs between complementary regions of nucleic acid strands.

[0089] The term “inbred” refers to a line that has been bred for genetic homogeneity.

[0090] The term "indel" refers to an insertion or deletion, wherein one line may be referred to as comprising an inserted nucleotide or piece of DNA relative to a second line, or the second line may be referred to as comprising a deletion of a nucleotide or piece of DNA relative to the first line.

[0091] “Introducing”, as used herein, means presenting to the plant or plant cell the polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a cell. The methods of the present disclosure do not depend on a particular method for introducing a polynucleotide or polypeptide into a plant, only that the polynucleotide(s) or polypeptide(s) gains access to the interior of at least one cell of the plant. Methods for introducing polynucleotide(s) or polypeptide(s) into plants include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0092] “Stable transformation” as used herein means that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. “Transient transformation” as used herein means that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant, or a polypeptide is introduced into a plant.

[0093] Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agro bacterium-mediated transformation (US Patent Numbers 5,563,055 and 5,981 ,840), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722) and ballistic (biolistic) particle acceleration (see, for example, US Patent Numbers 4,945,050; 5,879,918;5,886,244 and 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin) and McCabe et al. (1988) Biotechnology 6:923-926) and Led transformation (WO 00 / 28058). For potato transformation see, Tu et al. (1998) Plant Molecular Biology 37:829-838 and Chong et al. (2000) Transgenic Research 9:71-78. Additional transformation procedures can be found in Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean);McCabe et al. (1988) Bio / Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl.Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); US Patent Numbers 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988) Plant Physiol. 91 :440- 444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; US Patent Number 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York), pp.197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992) 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 transmission of a desired allele of a genetic locus from one genetic background to another. For example, introgression of a desired allele at a specified locus can be transmitted to at least one progeny via a sexual cross between two parents of the same species, where at least one of the parents has the desired allele in its genome. Alternatively, for example, transmission of an allele can occur by recombination between two donor genomes, e.g., in a fused protoplast, where at least one of the donor protoplasts has the desired allele in its genome. The desired allele can be, e.g., detected by a marker that is associated with a phenotype, at a QTL, a transgene, or the like. In any case, offspring comprising the desired allele can be repeatedly backcrossed to a line comprising a desired genetic background and selected for the desired allele, to result in the allele becoming fixed in a selected genetic background.

[0095] The process of “introgressing” is often referred to as “backcrossing” when the sexual cross process is repeated two or more times.

[0096] “Backcrossing” refers to the process whereby hybrid progeny are crossed back to one of the parents. In a backcrossing scheme, the “donor” parent refers to the parental plant with the desired gene / genes, locus / loci, or specific phenotype to be introgressed. The “recipient” parent (used one or more times) or “recurrent” parent (used two or more times) refers to the parental plant into which the gene or locus is being backcrossed. Repeated backcrossing can result in “introgression” of the allele 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 gives rise to the Fi generation; the term “BCi” then refers to the second use of the recurrent parent, “BC2” refers to the third use of the recurrent parent, and so on. Similarly, “BC5F2” refers to the second generation produced by the sixth use of the recurrent parent.

[0097] A “line” or “strain” is a group of individuals of identical parentage that are generally inbred to some degree and that are generally homozygous and homogeneous at most loci (isogenic or near isogenic). A “subline” refers to an inbred subset of descendants that are genetically distinct from other similarly inbred subsets descended from the same progenitor.

[0098] As used herein, the term “linkage” is used to describe the degree with which one marker locus is associated with another marker locus, a favorable allele, or some other locus. The linkage relationship between a molecular marker and a locus affecting a phenotype (e.g., a favorable allele) is given as a “probability” or “adjusted probability”. Linkage can be expressedas a desired limit or range. For example, in some examples, any marker is linked (genetically and physically) to any other marker when the markers are separated by less than 50, 40, 30, 25, 20, or 15 map units (or cM) of a single meiosis map (a genetic map based on a population that has undergone one round of meiosis, such as e.g., an F2. In some aspects, it is advantageous to define a bracketed range of linkage, for example, between 10 and 20 cM, between 10 and 30 cM, or between 10 and 40 cM. The more closely a marker is linked to a second locus, the better an indicator for the second locus that marker becomes.

[0099] The term “linkage disequilibrium” refers to a non-random segregation of genetic loci or traits (or both). In either case, linkage disequilibrium implies that the relevant loci are within sufficient physical proximity along a length of a chromosome so that they segregate together with greater than random (i.e., non-random) frequency. Markers that show linkage disequilibrium are considered linked. Linked loci co-segregate more than 50% of the time, e.g., from about 51% to about 100% of the time. In other words, two markers that co-segregate have a recombination frequency of less than 50% (and by definition, are separated by less than 50 cM on the same linkage group.) As used herein, linkage can be between two markers, or alternatively between a marker and a locus affecting a phenotype.

[0100] Linkage disequilibrium is most commonly assessed using the measure r2, which is calculated using the formula described by Hill, W.G. and Robertson, A, Theor. Appl. Genet. 38:226-231(1968). When r2= 1, complete LD exists between the two marker loci, meaning that the markers have not been separated by recombination and have the same allele frequency. The r2value will be dependent on the population used. Values for r2above 1 / 3 indicate sufficiently strong LD to be useful for mapping (Ardlie et al., Nature Reviews Genetics 3:299-309 (2002)). Hence, alleles are in linkage disequilibrium when r2values between pairwise marker loci are greater than or equal to 0.33, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0.

[0101] As used herein, “linkage equilibrium” describes a situation where two markers independently segregate, i.e., sort among progeny randomly. Markers that show linkage equilibrium are considered unlinked (whether or not they lie on the same chromosome).

[0102] The “logarithm of odds (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 linkage is 1000 times more likely than no linkage, while a LOD score of two indicates that linkage is 100 times more likely than no linkage. LOD scores greater than or equal to two may be used to detect linkage. LOD scores can also be used to show the strength of association between marker loci and quantitative traits in “quantitative trait loci” mapping. In this case, the LOD score’s size isdependent on the closeness of the marker locus to the locus affecting the quantitative trait, as well as the size of the quantitative trait effect.

[0103] “Maize” refers to a plant of the Zea mays genus and species and is also known as com .

[0104] The term “maize plant” or “plant” includes whole maize plants and portions of maize plants. Such portions include, for example, maize plant cells, maize plant protoplasts, maize plant cell culture or maize tissue culture from which maize plants can be regenerated, maize plant calli, maize plant clumps or maize plant cells that are part of larger plant structures, maize seeds, maize cobs, maize flowers, maize cotyledons, maize leaves, maize stems, maize buds, maize roots, maize root tips and the like.

[0105] “Marker assisted selection” (of MAS) is a process by which individual plants are selected based on marker genotypes.

[0106] “Marker assisted counter-selection” is a process by which marker genotypes are used to identify plants that will not be selected, allowing them to be removed from a breeding program or planting.

[0107] A “marker haplotype” refers to a combination of alleles at a marker locus.

[0108] A “marker locus” is a specific chromosome location in the genome of a species where a specific marker can be found. A marker locus can be used to track the presence of a second linked locus, e.g., one that affects the expression of a phenotypic trait. For example, a marker locus can be used to monitor segregation of alleles at a genetically or physically linked locus.

[0109] A “marker probe” is a nucleic acid sequence or molecule that can be used to identify the presence of a marker locus, e.g., a nucleic acid probe that is complementary to a marker locus sequence, through nucleic acid hybridization. Marker probes comprising 30 or more contiguous nucleotides of the marker locus (“all or a portion” of the marker locus sequence) may be used for nucleic acid hybridization. Alternatively, in some aspects, a marker probe refers to a probe of any type that is able to distinguish (i.e. , genotype) the particular allele that is present at a marker locus.

[0110] A “nucleotide” is a monomeric unit from which DNA or RNA polymers are constructed, and consists of a purine or pyrimidine base, a pentose, and a phosphoric acid group. Nucleotides (usually found in their 5'-monophosphate form) are referred to by their single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleotide.

[0111] A “polymorphism” is a variation in the DNA between two or more individuals within a population. A polymorphism preferably has a frequency of at least 1% in a population. A useful polymorphism can include an SNP, a simple sequence repeat (SSR), or an insertion / deletion polymorphism, also referred to herein as an “indel”.

[0112] An allele “correlates” with a trait when it is linked to it and when presence of the allele is an indicator that the desired trait or trait form will occur in a plant comprising the allele.

[0113] The term “progeny” refers to the offspring generated from a cross.

[0114] A “progeny plant” is a plant generated from a cross between two plants.

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

[0116] As used herein, a "recombinant" plant, plant cell, or nucleic acid is a plant, plant cell, or nucleic acid comprising a heterologous nucleic acid sequence (which can encode a heterologous protein / peptide or a noncoding RNA).

[0117] As used herein “heterologous” in reference to a nucleotide or amino acid sequence means that the sequence originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and / or its native genomic locus by deliberate human intervention. For example, a promoter operably linked to heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same species, one or both are substantially modified from their original form or genetic locus, or the promoter is not the native promoter of the operably linked polynucleotide. In another example, a heterologous polynucleotide sequence of the present disclosure may be “heterologous” in that the sequence is located at a genomic locus that is different than its native or naturally occurring genomic locus. For example, a heterologous NLR01 gene sequence may be located at a different chromosome or chromosome location compared to the chromosome location of the native NLR01 gene or it may be located between different genes compared to the native NLR01 gene. A heterologous gene can be inserted into a genome via, for example, transformation and / or site-specific nuclease-based methods. Such a heterologous gene can then be transferred into the genome of a plant line by breeding methods such as introgression. A nucleotide construct is “heterologous to” a plant or plant cell if the construct contains nucleotide sequence(s) that, when incorporated into the genome, would be heterologous to the plant / plant cell.

[0118] A "reference sequence" or a “consensus sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence for a marker can be obtained by sequencing a number of lines at the locus, aligning the nucleotide sequences in a sequence alignment program (e.g., Sequencher), and then obtaining the most common nucleotide sequence of the alignment. Polymorphisms found among the individual sequences are annotated within the consensus sequence. A reference sequence is not usually an exact copy of any individual DNA sequence but represents an amalgam of available sequences and is useful for designing primers and probes to polymorphisms within the sequence.

[0119] As used herein, “southern rust (SR) resistance” refers to increased resistance or tolerance to a fungal pathogen that causes SR when compared to a control plant comprising less resistance. Resistance effects may vary from a slight increase in tolerance to the effects of the fungal pathogen (e.g., partial inhibition of pathogenesis) to total resistance such that the plant is unaffected by the presence of the fungal pathogen. The disclosure herein, including Examples, provide materials and methods that provide increased resistance to the fungal pathogen that causes SR.

[0120]

[0121] The term “yield” refers to the productivity per unit area of a particular plant product of commercial value. For example, yield of maize is commonly measured in bushels of seed per acre or metric tons of seed per hectare per season. Yield is affected by both genetic and environmental factors. “Agronomics”, “agronomic traits”, and “agronomic performance” refer to the traits (and underlying genetic elements) of a given plant variety that contribute to yield over the course of growing season. Individual agronomic traits include emergence vigor, vegetative vigor, stress tolerance, disease resistance or tolerance, herbicide resistance, branching, flowering, seed set, seed size, seed density, standability, threshability and the like. Yield is, therefore, the final culmination of all agronomic traits.

[0122] Sequence alignments and percent identity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the MEGALIGN® program of the LASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison, Wl). Unless stated otherwise, multiple alignment of the sequences provided herein were performed using the CLUSTAL V method of alignment (Higgins and Sharp, CABIOS. 5:151 153 (1989)) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the CLUSTAL V method are KTUPLE=1 , GAP PENALTY=3, WIND0W=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2,GAP PENALTY=5, WIND0W=4 and DIAGONALS SAVED=4. After alignment of the sequences, using the CLUSTAL V program, it is possible to obtain “percent identity” and “divergence” values by viewing the “sequence distances” table on the same program; unless stated otherwise, percent identities and divergences provided and claimed herein were calculated in this manner.

[0123] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E.F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter “Sambrook”).

[0124] As used herein, the “heterologous NLR gene sequence” is a nucleotide sequence encoding a protein comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 30 or 34. Alternatively or additionally, the heterologous NLR gene sequence comprises native genomic sequence from Inbred A encoding a protein comprising SEQ ID NO: 30, a protein comprising SEQ ID NO: 34, or both proteins.

[0125] QTL Locations and Causative Genes

[0126] The present disclosure provides a QTL on the short arm of maize chromosome 10 that was identified as being associated with SR resistance (see Examples). Three different inbred lines were used in the work described herein — Inbred A (resistant to SR), Inbred B (susceptible to SR), and Inbred C (susceptible to SR). Markers for the QTL associated with SR resistance that was found on chromosome 10 of Inbred A are located in a chromosomal interval flanked by and including the markers C00422-801 and C002MY1-001. The markers comprise SNPs that were found to vary between Inbred A and at least one of Inbred B and Inbred C. SEQ ID NOs: 27-34 disclose sequences related to two different genes, at least one of which is believed to be the causative gene for Inbred A’s QTL.

[0127] Genetic Mapping

[0128] Specific genetic loci correlating with a particular phenotype, such as disease resistance, can be mapped in an organism’s genome. The plant breeder can advantageously use molecular markers to identify desired individuals by detecting marker alleles that show a statistically significant probability of co-segregation with a desired phenotype, manifested as linkage disequilibrium. By identifying a molecular marker or clusters of molecular markers that co-segregate with a trait of interest, the breeder is able to rapidly select a desired phenotype by selecting for the proper molecular marker allele (a process called marker-assisted selection, or MAS).

[0129] A variety of methods are available for detecting molecular markers or clusters of molecular markers that co-segregate with a trait of interest, such as a disease resistance trait. The basic idea underlying these methods is the detection of markers, for which alternative genotypes (or alleles) have significantly different average phenotypes. Thus, one makes a comparison among marker loci of the magnitude of difference among alternative genotypes (or alleles) or the level of significance of that difference. Trait genes are inferred to be located nearest the marker(s) that have the greatest associated genotypic difference. Two such methods used to detect trait loci of interest are: 1) population-based association analysis (i.e., association mapping) and 2) traditional linkage analysis.

[0130] Association Mapping

[0131] Understanding the extent and patterns of linkage disequilibrium (LD) in the genome is a prerequisite for developing efficient association approaches to identify and map quantitative trait loci (QTL). LD refers to the non-random association of alleles in a collection of individuals. When LD is observed among alleles at linked loci, it is measured as LD decay across a specific region of a chromosome. The extent of the LD reflects the recombinational history of that region. The average rate of LD decay in a genome can help predict the number and density of markers that are required to undertake a genome-wide association study and provides an estimate of the resolution that can be expected.

[0132] Association or LD mapping aims to identify significant genotype-phenotype associations. It has been exploited as a powerful tool for fine mapping in outcrossing species such as humans (Corder et al. (1994) “Protective effect of apolipoprotein-E type-2 allele for late-onset Alzheimer- disease,” Nat Genet 7:180-184; Hastbacka et al. (1992) “Linkage disequilibrium mapping in isolated founder populations: diastrophic dysplasia in Finland,” Nat Genet 2:204-211 ; Kerem et al. (1989) “Identification of the cystic fibrosis gene: genetic analysis,” Science 245:1073-1080) and maize (Remington et al. (2001) “Structure of linkage disequilibrium and phenotype associations in the maize genome,” Proc Natl Acad Sci USA 98:11479-11484; Thornsberry et al. (2001) “Dwarf8 polymorphisms associate with variation in flowering time,” Nat Genet 28:286- 289; reviewed by Flint-Garcia et al. (2003) “Structure of linkage disequilibrium in plants,” Annu Rev Plant Biol. 54:357-374), where recombination among heterozygotes is frequent and results in a rapid decay of LD. In inbreeding species where recombination among homozygous genotypes is not genetically detectable, the extent of LD is greater (i.e., larger blocks of linked markers are inherited together) and this dramatically enhances the detection power of association mapping (Wall and Pritchard (2003) “Haplotype blocks and linkage disequilibrium in the human genome,” Nat Rev Genet 4:587-597).

[0133] The recombinational and mutational history of a population is a function of the mating habit as well as the effective size and age of a population. Large population sizes offer enhanced possibilities for detecting recombination, while older populations are generally associated with higher levels of polymorphism, both of which contribute to observably accelerated rates of LD decay. On the other hand, smaller effective population sizes, e.g., those that have experienced a recent genetic bottleneck, tend to show a slower rate of LD decay, resulting in more extensive haplotype conservation (Flint-Garcia et al. (2003) “Structure of linkage disequilibrium in plants,” Annu Rev Plant Biol. 54:357-374).

[0134] Elite breeding lines provide a valuable starting point for association analyses.Association analyses use quantitative phenotypic scores (e.g., disease tolerance rated from one to nine for each line) in the analysis (as opposed to looking only at tolerant versus resistant allele frequency distributions in intergroup allele distribution types of analysis). The availability of detailed phenotypic performance data collected by breeding programs over multiple years and environments for a large number of elite lines provides a valuable dataset for genetic marker association mapping analyses. This paves the way for a seamless integration between research and application and takes advantage of historically accumulated data sets. However, an understanding of the relationship between polymorphism and recombination is useful in developing appropriate strategies for efficiently extracting maximum information from these resources.

[0135] This type of association analysis neither generates nor requires any map data, but rather is independent of map position. This analysis compares the plants’ phenotypic score with the genotypes at the various loci. Subsequently, any suitable map (for example, a composite map) can optionally be used to help observe distribution of the identified QTL markers and / or QTL marker clustering using previously determined map locations of the markers.

[0136] The same principles underlie traditional linkage analysis; however, LD is generated by creating a population from a small number of founders. The founders are selected to maximize the level of polymorphism within the constructed population, and polymorphic sites are assessed for their level of cosegregation with a given phenotype. A number of statistical methods have been used to identify significant marker-trait associations. One such method is an interval mapping approach (Lander and Botstein, Genetics 121 :185-199 (1989), in which each of many positions along a genetic map (say at 1 cM intervals) is tested for the likelihood that a gene controlling a trait of interest is located at that position. The genotype / phenotype data are used to calculate for each test position a LOD score (log of likelihood ratio). When the LOD score exceeds a threshold value, there is significant evidence for the location of a genecontrolling the trait of interest at that position on the genetic map (which will fall between two particular marker loci).

[0137] Marker loci that demonstrate statistically significant co-segregation with a disease resistance trait, as determined by traditional linkage analysis and by whole genome association analysis, are provided herein. Detection of these loci or additional linked loci can be used in marker assisted breeding programs to produce plants comprising disease resistance.

[0138] Activities in marker assisted breeding programs may include but are not limited to: selecting among new breeding populations to identify which population has the highest frequency of favorable nucleic acid sequences based on historical genotype and agronomic trait associations, selecting favorable nucleic acid sequences among progeny in breeding populations, selecting among parental lines based on prediction of progeny performance, and advancing lines in germplasm improvement activities based on presence of favorable nucleic acid sequences.

[0139] Chromosomal intervals

[0140] Chromosomal intervals that correlate with the disease resistance trait are provided. A variety of methods are available for identifying chromosomal intervals. The boundaries of such chromosomal intervals can be drawn to encompass markers that will be linked to the gene(s) controlling the trait of interest. In other words, the chromosomal interval can be drawn such that any marker that lies within that interval (including the terminal markers that define the boundaries of the interval) can be used as a marker for a disease resistance trait.

[0141] Conversely, e.g., if two markers in close proximity show co-segregation with the desired phenotypic trait, it is sometimes unclear if each of those markers identify the same gene or two different gene or multiple genes. Regardless, knowledge of how many genes are in a particular physical / genomic interval is not necessary to make or practice certain examples of the present disclosure.

[0142] Thus, disclosed herein is an interval on maize chromosome 10. The disclosed interval on chromosome 10 can comprise 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, or 19 of the markers for SR resistance disclosed in Table 1. Optionally, the interval can comprise and be flanked by two of the markers disclosed in Table 1 herein. Other markers from Table 1 can optionally be positioned between the two flanking markers, according to the genome positions shown in Table 1.

[0143] In particular examples, the interval on chromosome 10 can encompass 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the markers for SR resistance disclosed in Table 1 and further encompass C01800-1 , C06790-1 , and / or C00429-801. In particular examples, theinterval on chromosome 10 can encompass 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, or 19 of the markers for SR resistance disclosed in Table 1 and further encompass 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 find use as a marker for SR resistance and can be used in the context of the methods presented herein to select plants that have resistance to SR, compared to a control plant or a plant lacking the SR resistance QTL. Markers disclosed herein are located upstream or downstream of the NLR01 and / or NLR02 gene positions and are closely linked genetically and physically to the genes. Therefore, the markers may be used to select for the NLR01 and / or NLR02 genes for trait introgression and product development.

[0144] Markers and linkage relationships

[0145] A common measure of linkage is the frequency with which traits cosegregate. This can be expressed as a percentage of cosegregation (recombination frequency) or in centiMorgans (cM). The cM is a unit of measure of genetic recombination frequency. One cM is equal to a 1% chance that a trait at one genetic locus will be separated from a trait at another locus due to crossing over in a single generation (meaning the traits segregate together 99% of the time). Because chromosomal distance is approximately proportional to the frequency of crossing over events between traits, there is an approximate physical distance that correlates with recombination frequency.

[0146] Marker loci are themselves traits and can be assessed according to standard linkage analysis by tracking the marker loci during segregation. Thus, one cM is equal to a 1% chance that a marker locus will be separated from another locus, due to crossing over in a single generation.

[0147] The closer a marker is to a gene controlling a trait of interest, the more effective and advantageous that marker is as an indicator for the desired trait. Closely linked loci display an inter-locus cross-over frequency of about 10% or less such as, for example, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, or about 2% or less. In some examples, the relevant loci (e.g., a marker locus and a target locus) display a recombination frequency of about 1% or less, e.g., about 0.75% or less, about 0.5% or less, or about 0.25% or less. Thus, the loci are about 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.75 cM, 0.5 cM or 0.25 cM or less apart. Put another way, two loci that are localized to the same chromosome, and at such a distance that recombination between the two loci occurs at a frequency of less than 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are said to be “proximal to” each other.

[0148] Although particular marker alleles can co-segregate with a disease resistance trait, it is important to note that the marker locus is not necessarily responsible for the expression of the disease resistance phenotype. For example, it is not a requirement that the marker polynucleotide sequence be part of a gene that is responsible for the disease resistant phenotype (for example, is part of the gene open reading frame). The association between a specific marker allele and the disease resistance trait is due to the original “coupling” linkage phase between the marker allele and the allele in the ancestral line from which the allele originated. Eventually, with repeated recombination, crossing over events between the marker and genetic locus can change this orientation. For this reason, the favorable marker allele may change depending on the linkage phase that exists within the parent comprising resistance to the disease that is used to create segregating populations. This does not change the fact that the marker can be used to monitor segregation of the phenotype. It only changes which marker allele is considered favorable in a given segregating population.

[0149] Methods presented herein include detecting the presence of one or more marker alleles associated with disease resistance in a plant and then selecting plants that have favorable alleles at those marker loci. Markers have been identified herein as being associated with the disease resistance trait and hence can be used to predict disease resistance in a plant. Any marker within 50 cM, 40 cM, 30 cM, 20 cM, 15 cM, 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.75 cM, 0.5 cM or 0.25 cM (based on a single meiosis based genetic map) could also be used to predict disease resistance in a plant.

[0150] Marker Assisted Selection

[0151] Molecular markers can be used in a variety of plant breeding applications (e.g., see Staub et al. (1996) Hortscience 31 : 729-741; Tanksley (1983) Plant Molecular Biology Reporter. 1 : 3-8). One area of interest is to increase the efficiency of backcrossing and introgressing genes using marker-assisted selection (MAS). A molecular marker that demonstrates linkage with a locus affecting a desired phenotypic trait provides a useful tool for the selection of the trait in a plant population. This is particularly true where the phenotype is hard to assay. Since DNA marker assays are less laborious and take up less physical space than field phenotyping, much larger populations can be assayed, increasing the chances of finding a recombinant with the target segment from the donor line moved to the recipient line. The closer the linkage, the more useful the marker, as recombination is less likely to occur between the marker and the gene causing the trait, which can result in false positives. Use of flanking markers decreases the chances that false positive selection will occur as a double recombination event would be needed. In some cases, a marker is located within the gene itself, so that recombination cannotoccur between the marker and the gene. In some examples, the methods disclosed herein produce a marker in a disease resistance gene, wherein the gene was identified by inferring genomic location from clustering of conserved domains or a clustering analysis.

[0152] When a gene is introgressed by MAS, it is not only the gene that is introduced but also the flanking regions (Gepts. (2002). Crop Sci; 42: 1780-1790). This is referred to as “linkage drag.” In the case where the donor plant is highly unrelated to the recipient plant, these flanking regions carry additional genes that may code for agronomically undesirable traits. Linkage drag may also result in reduced yield or other negative agronomic characteristics even after multiple cycles of backcrossing into the elite line. This is also sometimes referred to as “yield drag.” The size of the flanking region can be decreased by additional backcrossing, although this is not always successful, as breeders do not have control over the size of the region or the recombination breakpoints (Young et al. (1998) Genetics 120:579-585). In classical breeding it is usually only by chance that recombinations are selected that contribute to a reduction in the size of the donor segment (Tanksley et al. (1989). Biotechnology 7: 257-264). Even after 20 backcrosses in backcrosses of this type, one may expect to find a sizeable piece of the donor chromosome still linked to the gene being selected. With markers however, it is possible to select those rare individuals that have experienced recombination near the gene of interest. In 150 backcross plants, there is a 95% chance that at least one plant will have experienced a crossover within 1 cM of the gene, based on a single meiosis map distance. Markers will allow unequivocal identification of those individuals. With one additional backcross of 300 plants, there would be a 95% chance of a crossover within 1 cM single meiosis map distance of the other side of the gene, generating a segment around the target gene of less than 2 cM based on a single meiosis map distance. This can be accomplished in two generations with markers, while it would have required on average 100 generations without markers (See Tanksley et al., supra). When the exact location of a gene is known, flanking markers surrounding the gene can be utilized to select for recombinations in different population sizes. For example, in smaller population sizes, recombinations may be expected further away from the gene, so more distal flanking markers would be required to detect the recombination.

[0153] Implementation of MAS can include: (i) Defining the population within which the markertrait association will be determined, which can be a segregating population, or a random or structured population; (ii) monitoring the segregation or association of polymorphic markers relative to the trait, and determining linkage or association using statistical methods; (iii) defining a set of desirable markers based on the results of the statistical analysis, and (iv) the use and / or extrapolation of this information to the current set of breeding germplasm to enable marker-based selection decisions to be made. The markers described in this disclosure, as well as other marker types such as SSRs and FLPs, can be used in marker assisted selection protocols.

[0154] SSRs can be defined as relatively short runs of tandemly repeated DNA with lengths of 6 bp or less (Tautz (1989) Nucleic Acid Research 17: 6463-6471 ; Wang et al. (1994) Theoretical and Applied Genetics, 88:1-6). Polymorphisms arise due to variation in the number of repeat units, probably caused by slippage during DNA replication (Levinson and Gutman (1987) Mol Biol Evol 4: 203-221). The variation in repeat length may be detected by designing PCR primers to the conserved non-repetitive flanking regions (Weber and May (1989) Am J Hum Genet. 44:388-396). SSRs are highly suited to mapping and MAS as they are multi-allelic, codominant, reproducible and amenable to high throughput automation (Rafalski et al. (1996) Generating and using DNA markers in plants. In: Non-mammalian genomic analysis: a practical guide. Academic press, pp 75-135).

[0155] Various types of SSR markers can be generated, and SSR profiles can be obtained by gel electrophoresis of the amplification products. Scoring of marker genotype is based on the size of the amplified fragment.

[0156] Various types of FLP markers can also be generated. Most commonly, amplification primers are used to generate fragment length polymorphisms. Such FLP markers are in many ways similar to SSR markers, except that the region amplified by the primers is not typically a highly repetitive region. Still, the amplified region, or amplicon, will have sufficient variability among germplasm, often due to insertions or deletions, such that the fragments generated by the amplification primers can be distinguished among polymorphic individuals, and such indels are known to occur frequently in maize (Bhattramakki et al. (2002). Plant Mol Biol 48, 539-547; Rafalski (2002b), supra).

[0157] SNP markers detect single base pair nucleotide substitutions. Of all the molecular marker types, SNPs are the most abundant, thus potentially providing the highest genetic map resolution (Bhattramakki et al. 2002 Plant Molecular Biology 48:539-547). SNPs can be assayed at an even higher level of throughput than SSRs, in a so-called ultra-high-throughput' fashion, as SNPs do not require large amounts of DNA and automation of the assay may be straightforward. SNPs also have the promise of being relatively low-cost systems. These three factors together make SNPs highly attractive for use in MAS. Several methods are available for SNP genotyping and / or detection, including but not limited to, hybridization, primer extension, oligonucleotide ligation, nuclease cleavage, minisequencing, and coded spheres. Such methods have been reviewed in: Gut (2001) Hum Mutat 17 pp. 475-492; Shi (2001) Clin Chem 47, pp.164-172; Kwok (2000) Pharmacogenomics 1 , pp. 95-100; and Bhattramakki and Rafalski (2001) Discovery and application of single nucleotide polymorphism markers in plants. In: R. J. Henry, Ed, Plant Genotyping: The DNA Fingerprinting of Plants, CABI Publishing, Wallingford. A wide range of commercially available technologies utilize these and other methods to interrogate SNPs including Masscode. TM. (Qiagen), INVADER®. (Third Wave Technologies) and Invader PLUS®, SNAPSHOT®. (Applied Biosystems), TAQMAN®. (Applied Biosystems) and BEADARRAYS®. (Illumina).

[0158] A number of SNPs together within a sequence, or across linked sequences, can be used to describe a haplotype for any particular genotype (Ching et al. (2002), BMC Genet. 3:19 pp Gupta et al. 2001 , Rafalski (2002b), Plant Science 162:329-333). Haplotypes can be more informative than single SNPs and can be more descriptive of any particular genotype. For example, a single SNP may be allele “T' for a specific line or variety with disease resistance, but the allele 'T' might also occur in the breeding population being utilized for recurrent parents. In this case, a haplotype, e.g. a combination of alleles at linked SNP markers, may be more informative. Once a unique haplotype has been assigned to a donor chromosomal region, that haplotype can be used in that population or any subset thereof to determine whether an individual has a particular gene. Using automated high throughput marker detection platforms makes this process highly efficient and effective.

[0159] Markers presented herein can readily be used as single nucleotide polymorphic (SNP) markers to select for the gene or trait of interest. Using PCR, the primers are used to amplify DNA segments from individuals (e.g., an inbred) that represent the diversity in the population of interest. The PCR products are sequenced directly in one or both directions. The resulting sequences are aligned, and polymorphisms are identified. The polymorphisms are not limited to single nucleotide polymorphisms (SNPs), but also include indels, CAPS, SSRs, and VNTRs (variable number of tandem repeats). Specifically, with respect to the fine map information described herein, one can readily use the information provided herein to obtain additional polymorphic SNPs (and other markers) within the region amplified by the primers disclosed herein. Markers within the described map region can be hybridized to BACs or other genomic libraries, or electronically aligned with genome sequences, to find new sequences in the same approximate location as the described markers.

[0160] In addition to SSR's, FLPs and SNPs, as described above, other types of molecular markers are also widely used, including but not limited to expressed sequence tags (ESTs), SSR markers derived from EST sequences, randomly amplified polymorphic DNA (RAPD), and other nucleic acid based markers.

[0161] Isozyme profiles and linked morphological characteristics can, in some cases, also be indirectly used as markers. Even though they do not directly detect DNA differences, they are often influenced by specific genetic differences. However, markers that detect DNA variation are far more numerous and polymorphic than isozyme or morphological markers (Tanksley (1983) Plant Molecular Biology Reporter 1:3-8).

[0162] Sequence alignments or contigs may also be used to find sequences upstream or downstream of the specific markers listed herein. These new sequences, close to the markers described herein, are then used to discover and develop functionally equivalent markers. For example, different physical and / or genetic maps are aligned to locate equivalent markers not described within this disclosure but that are within similar regions. These maps may be within the species, or even across other species that have been genetically or physically aligned.

[0163] In general, MAS uses polymorphic markers that have been identified as displaying a significant likelihood of co-segregation with a trait such as the SR disease resistance trait and / or genes disclosed herein. Such markers are presumed to map near a gene or genes that give the plant its disease resistant phenotype, and are considered indicators for the desired trait, or markers. Plants are tested for the presence of a desired allele in the marker, and plants containing a desired genotype at one or more loci are expected to transfer the desired genotype, along with a desired phenotype, to their progeny. Thus, plants with SR disease resistance may be selected for by detecting one or more marker alleles, and in addition, progeny plants derived from those plants can also be selected. Hence, a plant containing a desired genotype in a given chromosomal region (i.e. a genotype associated with disease resistance) is obtained and then crossed to another plant. The progeny of such a cross would then be evaluated genotypically using one or more markers and the progeny plants with the same genotype in a given chromosomal region would then be selected as comprising disease resistance.

[0164] The SNPs could be used alone or in combination (i.e. a SNP haplotype) to select for a favorable resistant gene allele associated with SR disease resistance. For example, a SNP haplotype can comprise a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, or 19 of the markers for SR resistance in Table 1 of this disclosure.

[0165] The skilled artisan would expect that there might be additional polymorphic sites at marker loci in and around a chromosome marker identified by the methods disclosed herein, wherein one or more polymorphic sites is in linkage disequilibrium (LD) with an allele at one or more of the polymorphic sites in the haplotype and thus could be used in a marker assisted selection program to introgress a gene allele or genomic fragment of interest. Two particular 1alleles at different polymorphic sites are said to be in LD if the presence of the allele at one of the sites tends to predict the presence of the allele at the other site on the same chromosome (Stevens, Mol. Diag. 4:309-17 (1999)). The marker loci can be located within 5 cM, 2 cM, or 1 cM (on a single meiosis based genetic map) of the disease resistance trait QTL.

[0166] Allelic frequency (and hence, haplotype frequency) can differ from one germplasm pool to another. Germplasm pools vary due to maturity differences, heterotic groupings, geographical distribution, etc. As a result, SNPs and other polymorphisms may not be informative in some germplasm pools.

[0167] Methods of Identifying and / or Selecting Plants Comprising SR Resistance

[0168] Provided herein is a method for identifying, and optionally selecting, a maize plant comprising a QTL associated with resistance to SR. The method comprises obtaining a nucleic acid sample from each of one or more maize plants. The method comprises screening each nucleic acid sample for a QTL on maize chromosome 10 (short arm). The method can comprise screening the samples for the presence or absence of one or more of the marker alleles shown in Table 1. The QTL can be the QTL disclosed herein which is associated with resistance to SR and located on maize chromosome 10 (short arm) and is found in Inbred A.

[0169] In some examples, the method comprises screening each nucleic acid sample (e.g., from one or more members of a maize plant population) for at least one marker allele disclosed in Table 1 . If present, the sample (and plant from which it came from) can be identified as comprising the marker allele associated with associated with resistance to SR.

[0170] In some examples, the method comprises screening each nucleic acid for a haplotype comprising at least two marker alleles disclosed in Table 1 , thereby identifying the sample as comprising a haplotype associated with associated with resistance to SR.

[0171] In some examples, the method comprises screening the nucleic acid for all of the marker alleles disclosed in Table 1 , thereby identifying the sample as comprising a haplotype associated with associated with resistance to SR.

[0172] Screening the nucleic acid can comprise any procedure suitable for determining genotypes of the targeted plant(s). For example, screening can comprise PCR, Sanger sequencing, and / or next generation sequencing.

[0173] Marker alleles (e.g., single nucleotide polymorphisms (SNP)) linked to the QTL are provided in Table 1. The right column of Table 1 provides each SNP and flanking genomic sequence for context. In the sequences, the residue associated with resistance to SR is listed before the slash, and the residue associated with susceptibility to SR is listed after the slash. The SNP is shown in bold typeface and within brackets. The sequence of only one strand isprovided, but the disclosed SNPs and the methods disclosed herein encompass all complementary forms (e.g., the SNP in the context of the reverse complement strand).Table 1Table 2

[0174] In some examples, the method comprises screening nucleic acid from the maize plant for one or more additional markers. The additional markers are also located on chromosome 10 and are provided in Table 2. Table 2 is formatted in the same manner as Table 1. Markers C01800-1 , C06790-1 , and / or C00429-801 can be used to differentiate the haplotype of Inbred B (susceptible to SR) from that of Inbred A (resistant to SR). Markers C00431-802, C002TCN- 001 , C002TCM-001 , and / or C06817-1 can be used to differentiate the haplotype of Inbred B (susceptible to SR) from that of Inbred A (resistant to SR).

[0175] The markers C01800-1 , C06790-1 , and / or C00429-801 can be used in combination with any of the markers provided in Table 1.

[0176] The markers C00431-802, C002TCN-001, C002TCM-001, and / or C06817-1 can be used in combination with any of the markers provided in Table 1 .

[0177] Markers CSR_2373961, CSR_2409943, CSR_2643297, CSR_2807867, CSR_2837481 , and CSR_2839126 were determined as a result of fine mapping experiments to locate markerspositioned more closely to the causal gene(s) of the QTL. In some examples, the method can comprise screening for one or more of these markers, exclusively or in combination with other markers in Table 1 or Table 2.

[0178] In some examples, the method comprises selecting a maize plant (e.g., from a population) that comprises the one or more markers. In some examples, selecting the maize plant comprises choosing a plant for further use in, for example breeding activities (e.g., crosses, backcrosses, or introgression). Selecting the plant that comprises the at least one marker can provide a plant that will be an appropriate donor of the SR resistance trait during breeding activities.

[0179] In some examples, the method may further comprise isolating nucleic acid from the maize plant or maize plant population. The isolation of nucleic acid can facilitate the detection of one or more markers. Nucleic acid isolation can comprise any method suitable to provide genomic material for PCR, sequencing procedures, and / or identification of the at least one marker.

[0180] In some examples, plants that are selected and / or screened are members of a population of plants. The population can be known to contain a mixture of plants that do or do not comprise the resistance QTL on chromosome 10 (e.g., a population of progeny plants obtained by crossing a plant comprising the QTL with a plant not comprising the QTL). The method can comprise obtaining a nucleic acid sample from each of a plurality of maize plants in a population and screening each sample for one or more alleles associated with the QTL. The method can comprise selecting the maize plant(s) comprising the screened for marker allele(s) associated with the QTL.

[0181] In some examples, a method for producing a maize plant comprising resistance to SR is provided. The method comprises selecting a plant comprising the QTL on chromosome 10 and crossing the selected plant with a second maize plant. The second maize plant can be from a population of plants used for backcrossing and / or introgression of the SR resistance trait into a population of plants that lacks the trait. In some examples, the second plant lacks the QTL. In some examples, the method comprises obtaining a progeny plant from the cross that comprises at least one marker allele set forth in Table 1 and / or Table 2 and / or that comprises resistance to SR due to the QTL. In some examples, the resistance of the parent and the progeny against SR is increased, compared to the second plant (e.g., parent) which lacks the QTL.

[0182] A maize plant may comprise a portion of the QTL, but nonetheless show the associated SR resistance (e.g., a plant where recombination has removed a portion of the QTL).Accordingly, in some examples, the screened for marker allele is located in a chromosomalinterval comprising the QTL or a portion of the interval that nonetheless comprises the causative gene (e.g., a portion reduced by recombination).

[0183] In some examples, the screened for marker allele is located in a chromosomal interval flanked by and including the markers C06824-1 and C06834-1. The interval can also comprise one or more of the other markers provided in Table 1 and Table 2.

[0184] In some examples, the screened for marker allele is located in a chromosomal interval flanked by and including the markers CSR_2373961 and CSR_2839126. The interval can also comprise one or more of the other markers, CSR_2409943, CSR_2643297, CSR_2807867, or CSR_2837481.

[0185] In some examples, the screened for marker allele is located in a chromosomal interval comprising and flanked by 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 examples, the interval flanked by C00422-801 and C002MY1-001 comprises all of the intervening markers in Table 1.

[0186] In some examples, the screened for marker allele is located in a chromosomal interval comprising and flanked by 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 examples, the interval flanked by C00422-801 and C06839-1 comprises all of the intervening markers in Table 1.

[0187] In some examples, the screened for marker allele is located in a chromosomal interval comprising and flanked by 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 examples, the interval flanked by CSR_2373961 and CSR_2839126 comprises all of the intervening markers in Table 1.

[0188] In some examples, the method for selecting a maize plant comprising a QTL associated with resistance to SR can be employed during performance of a method for producing a maize plant comprising resistance to southern rust. In such examples, the method for producing a maize plant comprising resistance to SR comprises crossing a first parent maize plant with a second parent maize plant to produce one or more progeny plants. The first parent comprises a QTL associated with resistance to SR. The QTL is not found in the second maize parent. The method comprises obtaining a nucleic acid sample from one or more of the progeny plants. Themethod comprises selecting one or more progeny plants comprising the QTL associated with resistance to SR in accordance with the methods disclosed herein.

[0189] In some examples, the method for producing a maize plant comprising resistance to SR comprises performing a backcross. The backcross can provide progeny that comprise agronomic characteristics more similar to the second parent strain (e.g., recurrent parent) while also comprising the SR resistance QTL of the first strain (e.g., QTL donor). The method can comprise crossing the one or more selected progeny plants with the second parent plant to produce one or more backcross progeny plants and obtaining a nucleic acid sample from one or more backcross progeny plants. The method can comprise selecting the one or more backcross progeny plants comprising the QTL associated with resistance to SR.

[0190] In some examples, the method for producing a maize plant comprising resistance to SR comprises one or more subsequent backcrosses. The method can comprise crossing the one or more selected backcross progeny plants with the second parent plant to produce additional backcross progeny plants. The additional backcross progeny plants can more completely recapitulate the agronomic properties of the second parent plant. The method can comprise obtaining a nucleic acid sample from one or more additional backcross progeny plants and selecting the one or more additional backcross progeny plants comprising the QTL associated with resistance to SR. Optionally, the method comprises repeating steps the backcrossing, nucleic acid obtaining, and selecting activities to obtain additional backcross progeny plants comprising the QTL associated with resistance to SR. The additional backcross progeny plants can comprise the agronomic characteristics of the recurrent parent plant when grown in the same environmental conditions.

[0191] Methods of Selecting Maize Plants Encoding SR Resistance Genes

[0192] Provided herein are methods for selecting a maize plant comprising resistance to SR. The method comprises screening a population of maize plants for a protein, or a gene encoding the protein. The protein comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 30. The method also comprises selecting a maize plant comprising the protein or gene encoding the protein.

[0193] In some examples, screening the population of maize plants for the protein or the gene comprises screening for a phenotype associated with the gene, screening for the gene or protein itself, or screening for a genotype linked to the gene. The screening can comprise any procedure suitable for determining genotypes of the targeted plants. For example, screening can comprise PCR, Sanger sequencing, and / or next generation sequencing.

[0194] In some examples, the protein comprises 95% amino acid sequence identity to SEQ ID NO: 30 or 34.

[0195] In some examples, the protein comprises 96% amino acid sequence identity to SEQ ID NO: 30 or 34.

[0196] In some examples, the protein comprises 97% amino acid sequence identity to SEQ ID NO: 30 or 34.

[0197] In some examples, the protein comprises 98% amino acid sequence identity to SEQ ID NO: 30 or 34.

[0198] In some examples, the protein comprises 99% amino acid sequence identity to SEQ ID NO: 30 or 34.

[0199] In some examples, the protein comprises 100% amino acid sequence identity to SEQ ID NO: 30 or 34.

[0200] In some examples, the method comprises crossing the selected maize plant with a second maize plant. In some examples, the method comprises obtaining a progeny plant that comprises the gene encoding the protein. In some examples, the second plant lacks the QTL. In some examples, progeny plants derived from the cross can then be used for further breeding activities and / or as a source of seed. In some examples, the progeny plant displays increased resistance to southern rust, compared to a parent plant (e.g., a parent plant lacking the gene). In some examples, the gene can be introgressed into a plant breeding line (e.g., an elite line) via repeated backcrossing using the progeny plant(s).

[0201] In some examples, the protein is encoded by a gene comprising a nucleotide sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity to SEQ ID NO: 28, 32, 35, or 36.

[0202] In some examples, the protein is encoded by a gene comprising a nucleotide sequence comprising the sequence of SEQ ID NO: 28, 32, 35, or 36.

[0203] In some examples, the protein is encoded by a gene comprising a nucleotide sequence comprising at least 95% sequence identity to the sequence of SEQ ID NO: 28, 32, 35, or 36.

[0204] In some examples, the protein is encoded by a gene comprising a nucleotide sequence comprising at least 96% sequence identity to the sequence of SEQ ID NO: 28, 32, 35, or 36.

[0205] In some examples, the protein is encoded by a gene comprising a nucleotide sequence comprising at least 97% sequence identity to the sequence of SEQ ID NO: 28, 32, 35, or 36.

[0206] In some examples, the protein is encoded by a gene comprising a nucleotide sequence comprising at least 98% sequence identity to the sequence of SEQ ID NO: 28, 32, 35, or 36.

[0207] In some examples, the protein is encoded by a gene comprising a nucleotide sequence comprising at least 99% sequence identity to the sequence of SEQ ID NO: 28, 32, 35, or 36.

[0208] Also provided herein is a method for producing a maize plant comprising resistance to SR. The method comprises screening for a gene and / or protein as described herein and selecting a maize plant comprising the gene and / or protein. The method comprises crossing the selected maize plant with a second maize plant and obtaining a progeny plant that comprises the gene and / or protein. The progeny plant displays increased resistance to SR, compared to the second plant. The second plant can lack the gene and / or protein.

[0209] SR Resistance Genes

[0210] In various aspects of the present disclosure, the NLR01 or NLR02 genes that convey SR resistance are used to produce recombinant plants. Alternatively, plants are screened for said genes. For example, these genes (independently or in combination) can be detected to determine if a given plant comprises resistance to SR, can be transformed into a plant, or can be added into a plant genome using site-specific genome editing techniques (e.g., CRISPR-, TALENs-, meganuclease-, or zinc finger nuclease-based techniques). SR resistance-conferring NLR01 and NLR02 gene sequences were identified in inbred A. Unless noted otherwise, any NLR01 or NLR02 gene sequence presented herein (e.g., any nucleic acid or protein sequence from SEQ ID NOs: 27-34) can be used in any aspect of the present disclosure requiring an NLR01 / NLR02 gene / protein sequence. Additionally, the gene sequences presented herein comprise protein sequences, DNA coding sequences (e.g., cDNA sequences), or genomic DNA sequences that include one or more of promoter, terminator, exon, and intron sequences.

[0211] Methods for Producing Recombinant Maize Plant Cells and / or Plants I

[0212] Provided herein is a method for producing a recombinant maize plant or plant cell comprising a heterologous NLR nucleic acid sequence associated with resistance to SR (e.g., NLR01 disclosed herein). The method comprises introducing a heterologous NLR gene sequence into one or more maize plant cells derived from maize plant that is susceptible to SR (or into the plant itself), thereby producing at least one modified cell comprising the heterologous NLR gene sequence in the modified cell’s genome. The method comprises selecting a modified maize plant cell. The susceptible maize plant can lack the heterologous NLR nucleic acid sequence before the present methods are performed. Alternatively, the method can be used to introduce additional copies of the heterologous NLR nucleic acid sequence.

[0213] In this method, genome editing technology can be used to effect a site-specific modification of at least one target site in the genome of the one or more maize plant cells. Forexample, an SR-susceptible NLR gene allele can be altered by site-specific mutagenesis to encode the SR-resistant NLR gene allele disclosed herein. Alternatively or additionally, the SR- resistant NLR gene allele disclosed herein can be introduced by genome editing at a different location.

[0214] In some examples, the method comprises introducing a polynucleotide modification template comprising the heterologous NLR nucleic acid sequence into the one or more maize plant cells. The template can be integrated into the genome at the site of the site-specific modification via, for example, homologous recombination.

[0215] The heterologous NLR nucleic acid sequence, the polynucleotide modification template, and / or the site specific modification can be introduced successfully into only a portion of the cells. Given that this may be the case, a selection step can be performed to ensure that a given cell has incorporated the various modifications into its genome. The selection can comprise use of a molecular marker and / or sequencing or any other suitable technique to discriminate between modified and unmodified cells.

[0216] In some examples, the site-specific modification is induced by a CRISPR-associated endonuclease. Any CRISPR-associated endonuclease suitable to introduce the site-specific modification can be used. The site-specific modification can comprise, for example, a doublestranded break or any other modification tending to result in the modification of the genome to include the NLR nucleic acid sequence disclosed herein which provides resistance to SR.

[0217] In some examples, the method comprises growing a modified maize plant from the selected maize plant cell. The modified plant can exhibit increased resistance to SR, relative to the first maize plant.

[0218] In some examples, the first maize plant comprises an endogenous locus and the heterologous NLR nucleic acid sequence replaces the endogenous locus. For example, the endogenous locus can comprise an ortholog or paralog of the NLR01 gene disclosed herein or the NLR02 gene disclosed herein.

[0219] In some examples, the heterologous NLR nucleic acid sequence is inserted into a locus that does not naturally comprise an NLR gene, e.g., the long arm of maize chromosome 10 or a locus on chromosome 1 , 2, 3, 4 5, 6, 7, 8, or 9. In some examples, the NLR nucleic acid sequence is inserted into a locus as part of a molecular stack. In some examples, the molecular stack comprises a polynucleotide(s) encoding additional maize disease resistance genes or other genes of interest.

[0220] In some examples, the heterologous NLR nucleic acid sequence comprises a nucleotide sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 28, 32, 35, or 36.

[0221] In some examples, the heterologous NLR nucleic acid sequence comprises a nucleotide sequence comprising at least 95% identity to SEQ ID NO: 28, 32, 35, or 36.

[0222] In some examples, the heterologous NLR nucleic acid sequence comprises a nucleotide sequence comprising at least 96% identity to SEQ ID NO: 28, 32, 35, or 36.

[0223] In some examples, the heterologous NLR nucleic acid sequence comprises a nucleotide sequence comprising at least 97% identity to SEQ ID NO: 28, 32, 35, or 36.

[0224] In some examples, the heterologous NLR nucleic acid sequence comprises a nucleotide sequence comprising at least 98% identity to SEQ ID NO: 28, 32, 35, or 36.

[0225] In some examples, the heterologous NLR nucleic acid sequence comprises a nucleotide sequence comprising at least 99% identity to SEQ ID NO: 28, 32, 35, or 36.

[0226] In some examples, the heterologous NLR nucleic acid sequence comprises the nucleotide sequence of SEQ ID NO: 28, 32, 35, or 36.

[0227] In some examples, the method comprises effecting a site-specific modification of at least one target site into the genome of the maize plant cell and introducing a polynucleotide modification template comprising the heterologous NLR nucleotide sequence into the plant cell.

[0228] In some examples, the site specific modification is effected by a CRISPR-associated endonuclease.

[0229] In some examples, the site-specific modification comprises a single strand break or double strand break (DSB) that is made by a site-specific endonuclease such as, for example, TALENs, meganucleases, zinc finger nucleases, and CRISPR-associated (Cas) protein / guide polynucleotide complexes. In some examples, the introduction of a site-specific modification can be combined with the introduction of the polynucleotide modification template.

[0230] In some examples, a polynucleotide modification template is introduced into a cell by any method suitable to deliver the template into the nucleus of a cell, such as, but not limited to, transient introduction methods, transfection, electroporation, microinjection, particle mediated delivery, topical application, whiskers mediated delivery, delivery via cell-penetrating peptides, or mesoporous silica nanoparticle (MSN)-mediated direct delivery.

[0231] In some examples, the polynucleotide modification template may be introduced into a cell as a single stranded polynucleotide molecule, a double stranded polynucleotide molecule, or as part of a circular DNA (vector DNA). The polynucleotide modification template may also be tethered to the guide polynucleotide and / or the Cas endonuclease. Tethered templates canallow for co-localizing target and template DNA, useful in genome editing and targeted genome regulation, and can also be useful in targeting post-mitotic cells where function of endogenous homologous recombination HR machinery is expected to be highly diminished (Mali et al. 2013 Nature Methods Vol. 10 : 957-963.) The polynucleotide modification template may be present transiently in the cell or it can be introduced via a viral replicon.

[0232] The polynucleotide modification template, as used herein, refers to a polynucleotide that comprises at least one nucleotide modification when compared to the target nucleotide sequence to be edited. A nucleotide modification is at least one nucleotide substitution, addition or deletion. In some examples, the polynucleotide modification template can further comprise homologous nucleotide sequences flanking the at least one nucleotide modification, wherein the flanking homologous nucleotide sequences provide sufficient homology to support the incorporation of the polynucleotide modification template into the genome of the recipient plant cell.

[0233] The process for editing a genomic sequence employing both site-specific modifications and modification templates generally comprises providing to a host cell a site-specific endonuclease (or a nucleic acid encoding the site-specific endonuclease) that recognizes a target sequence in the chromosomal sequence and induction by the site-specific endonuclease of a site-specific modification (e.g., a DSB) in the genomic sequence. The process also comprises providing at least one polynucleotide modification template. The endonuclease can be provided to a cell by any suitable method (e.g., transient introduction methods, transfection, microinjection, topical application, and / or indirectly via recombination constructs). The endonuclease may be provided as a protein or as a guided polynucleotide complex directly to a cell or indirectly via recombination constructs. The endonuclease may be introduced into a cell transiently or can be incorporated into the genome of the host cell. In the case of a CRISPR- Cas system, uptake of the endonuclease and / or the guided polynucleotide into the cell can be facilitated with a Cell Penetrating Peptide (CPP) as described in WO2016073433.

[0234] TAL effector nucleases (TALEN) are a class of sequence-specific nucleases that can be used to make double-strand breaks at specific target sequences in the genome of a plant or other organism. (See Miller et al. (2011) Nature Biotechnology 29:143-148).

[0235] Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Endonucleases include restriction endonucleases, which cleave DNA at specific sites without damaging the bases, and meganucleases, also known as homing endonucleases (HEases), which like restriction endonucleases, bind and cut at a specific recognition site, however the recognition sites for meganucleases are typically longer, about 18bp or more (patent application PCT / US 12 / 30061 , filed on March 22, 2012). Meganucleases have been classified into four families based on conserved sequence motifs, the families are the LAGLIDADG, GIY-YIG, H-N-H, and His-Cys box families. These motifs participate in the coordination of metal ions and hydrolysis of phosphodiester bonds. H Eases are notable for their long recognition sites, and for tolerating some sequence polymorphisms in their DNA substrates. The naming convention for meganuclease is similar to the convention for other restriction endonuclease. Meganucleases are also characterized by prefix F-, I-, or PI- for enzymes encoded by free-standing ORFs, introns, and inteins, respectively. One step in the recombination process involves polynucleotide cleavage at or near the recognition site. The cleaving activity can be used to produce a double-strand break. For reviews of site-specific recombinases and their recognition sites, see, Sauer (1994) Curr Op Biotechnol 5:521-7; and Sadowski (1993) FASEB 7:7Q0-7. In some examples the recombinase is from the Integrase or Resolvase families.

[0236] Zinc finger nucleases (ZFNs) are engineered double-strand break inducing agents comprised of a zinc finger DNA binding domain and a double-strand-break-inducing agent domain. Recognition site specificity is conferred by the zinc finger domain, which typically comprising two, three, or four zinc fingers, for example comprising a C2H2 structure, however other zinc finger structures are known and have been engineered. Zinc finger domains are amenable for designing polypeptides which specifically bind a selected polynucleotide recognition sequence. ZFNs include an engineered DNA-binding zinc finger domain linked to a non-specific endonuclease domain, for example nuclease domain from a Type Ils endonuclease such as Fokl. Additional functionalities can be fused to the zinc-finger binding domain, including transcriptional activator domains, transcription repressor domains, and methylases. In some examples, dimerization of nuclease domain is required for cleavage activity. Each zinc finger recognizes three consecutive base pairs in the target DNA. For example, a 3 finger domain recognized a sequence of 9 contiguous nucleotides, with a dimerization requirement of the nuclease, two sets of zinc finger triplets are used to bind an 18 nucleotide recognition sequence.

[0237] Genome editing using DSB-inducing agents, such as Cas9-gRNA complexes, has been described, for example in U.S. Patent Application US 2015-0082478 A1 , WO2015 / 026886 A1 , WQ2016007347, and WO201625131 , all of which are incorporated by reference herein.

[0238] The term “Cas gene” or “Cas protein” herein refers to a gene that is generally coupled, associated or close to, or in the vicinity of flanking CRISPR loci in bacterial systems. The terms “Cas” and “CRISPR-associated” are used interchangeably herein. The term “Cas endonuclease” herein refers to a protein, or complex of proteins, encoded by a Cas gene. A Casendonuclease as used herein, when in complex with a suitable polynucleotide component, is capable of recognizing, binding to, and optionally nicking or cleaving all or part of a specific DNA target sequence. A Cas endonuclease as described herein comprises one or more nuclease domains. Cas endonucleases of the disclosure includes those comprising a HNH or HNH-like nuclease domain and / or a RuvC or RuvC-like nuclease domain. A Cas endonuclease of the disclosure may include a Cas9 protein, a Cpf1 protein, a C2c1 protein, a C2c2 protein, a C2c3 protein, Cas3, Cas 5, Cas7, Cas8, Casio, or complexes of these.

[0239] As used herein, the terms “guide polynucleotide / Cas endonuclease complex”, “guide polynucleotide / Cas endonuclease system”, “ guide polynucleotide / Cas complex”, “guide polynucleotide / Cas system”, “guided Cas system” are used interchangeably herein and refer to at least one guide polynucleotide and at least one Cas endonuclease that are capable of forming a complex, wherein said guide polynucleotide / Cas endonuclease complex can direct the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single or double strand break) the DNA target site. A guide polynucleotide / Cas endonuclease complex herein can comprise Cas protein(s) and suitable polynucleotide component(s) of any of the four known CRISPR systems (Horvath and Barrangou, 2010, Science 327:167-170) such as a type I, II, or III CRISPR system. A Cas endonuclease unwinds the DNA duplex at the target sequence and optionally cleaves at least one DNA strand, as mediated by recognition of the target sequence by a polynucleotide (such as, but not limited to, a crRNA or guide RNA) that is in complex with the Cas protein. Such recognition and cutting of a target sequence by a Cas endonuclease typically occurs if the correct protospacer-adjacent motif (PAM) is located at or adjacent to the 3' end of the DNA target sequence. Alternatively, a Cas protein herein may lack DNA cleavage or nicking activity but can still specifically bind to a DNA target sequence when complexed with a suitable RNA component. (See also U.S. Patent Application US 2015-0082478 A1 , and US 2015-0059010 A1 , both hereby incorporated in its entirety by reference).

[0240] A guide polynucleotide / Cas endonuclease complex can cleave one or both strands of a DNA target sequence. A guide polynucleotide / Cas endonuclease complex that can cleave both strands of a DNA target sequence typically comprises a Cas protein that has 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). Thus, 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 that can cleave both strands of a DNA target sequence. A Cas9 protein comprising functional RuvC and HNH nuclease domainsis an example of a Cas protein that can cleave both strands of a DNA target sequence. A guide polynucleotide / Cas endonuclease complex that can cleave one strand of a DNA target sequence can be characterized herein as comprising nickase activity (e.g., partial cleaving capability). A Cas nickase typically comprises one functional endonuclease domain that allows the Cas to cleave only one strand (i.e. , make a nick) of a DNA target sequence. For example, a Cas9 nickase may comprise (i) a mutant, dysfunctional RuvC domain and (ii) a functional HNH domain (e.g., wild type HNH domain). As another example, a Cas9 nickase may comprise (i) a functional RuvC domain (e.g., wild type RuvC domain) and (ii) a mutant, dysfunctional HNH domain. Non-limiting examples of Cas9 nickases suitable for use herein are disclosed in U.S. Patent Appl. Publ. No. 2014 / 0189896, which is incorporated herein by reference.

[0241] In some examples, a pair of Cas9 nickases may be used to increase the specificity of DNA targeting. In general, this can be done by providing two Cas9 nickases that, by virtue of being associated with RNA components with different guide sequences, target and nick nearby DNA sequences on opposite strands in the region for desired targeting. Such nearby cleavage of each DNA strand creates a double strand break (i.e., a DSB with single-stranded overhangs), which is then recognized as a substrate for non-homologous-end-joining, NHEJ (prone to imperfect repair leading to mutations) or homologous recombination, HR. Each nick in these examples can 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, for example. One or two Cas9 nickase proteins herein can be used in a Cas9 nickase pair. For example, a Cas9 nickase with a mutant RuvC domain, but functioning HNH domain (i.e., Cas9 HNH+ / RuvC-), could be used (e.g., Streptococcus pyogenes Cas9 HNH+ / RuvC-). Each Cas9 nickase (e.g., Cas9 HNH+ / RuvC-) would be directed to specific DNA sites nearby each other (up to 100 base pairs apart) by using suitable RNA components herein with guide RNA sequences targeting each nickase to each specific DNA site.

[0242] In some examples, a Cas protein may be part of a fusion protein comprising one or more heterologous protein domains (e.g., 1 , 2, 3, or more domains in addition to the Cas protein). Such a fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains, such as between Cas and a first heterologous domain. Examples of protein domains that may be fused to a Cas protein herein include, without limitation, epitope tags (e.g., histidine [His], V5, FLAG, influenza hemagglutinin [HA], myc, VSV- G, thioredoxin [Trx]), reporters (e.g., glutathione-5-transferase [GST], horseradish peroxidase [HRP], chloramphenicol acetyltransferase [CAT], beta-galactosidase, beta-glucuronidase [GUS], luciferase, green fluorescent protein [GFP], HcRed, DsRed, cyan fluorescent protein [CFP],yellow fluorescent protein [YFP], blue fluorescent protein [BFP]), and domains comprising one or more of the following activities: methylase activity, demethylase activity, transcription activation activity (e.g., VP16 or VP64), transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. A Cas protein can also be in fusion with a protein that binds DNA molecules or other molecules, such as maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD), GAL4A DNA binding domain, and herpes simplex virus (HSV) VP16. See PCT patent applications PCT / US16 / 32073, filed May 12, 2016, and PCT / US16 / 32028 filed May 12, 2016 (both applications incorporated herein by reference) for more examples of Cas proteins.

[0243] A guide polynucleotide / Cas endonuclease complex in certain examples may bind to a DNA target site sequence but does not cleave any strand at the target site sequence. Such a complex may comprise a Cas protein in which all of its nuclease domains are mutant, dysfunctional. For example, a Cas9 protein herein that can bind to a DNA target site sequence but does not cleave any strand at the target site sequence, may comprise both a mutant, dysfunctional RuvC domain and a mutant, dysfunctional HNH domain. A Cas protein herein that binds, but does not cleave, a target DNA sequence can be used to modulate gene expression, for example, in which case the Cas protein could be fused with a transcription factor (or portion thereof) (e.g., a repressor or activator, such as any of those disclosed herein). In other aspects, an inactivated Cas protein may be fused with another protein comprising endonuclease activity, such as a Fokl endonuclease.

[0244] “Cas9” (formerly referred to as Cas5, Csn1 , or Csx12) herein refers to a Cas endonuclease of a type II CRISPR system that forms a complex with a crNucleotide and a tracrNucleotide, or with a single guide polynucleotide, for specifically recognizing and cleaving all or part of a DNA target sequence. Cas9 protein comprises a RuvC nuclease domain and an HNH (H-N-H) nuclease domain, each of which can cleave a single DNA strand at a target sequence (the concerted action of both domains leads to DNA double-strand cleavage, whereas activity of one domain leads to a nick). In general, the RuvC domain comprises subdomains I, II and III, where domain I is located near the N-terminus of Cas9 and subdomains II and III are located in the middle of the protein, flanking the HNH domain (Hsu et al, Cell 157:1262-1278). A type II CRISPR system includes a DNA cleavage system utilizing a Cas9 endonuclease in complex with at least one polynucleotide component. For example, a Cas9 can be in complex with a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA). In another example, a Cas9 can be in complex with a single guide RNA.

[0245] In some examples, the Cas endonuclease can comprise a modified form of the Cas9 polypeptide. The modified form of the Cas9 polypeptide can include an amino acid change (e.g., deletion, insertion, or substitution) that reduces the naturally-occurring nuclease activity of the Cas9 protein. For example, in some instances, 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 nuclease activity of the corresponding wild-type Cas9 polypeptide (US patent application US20140068797 A1). In some cases, the modified form of the Cas9 polypeptide has no substantial nuclease activity and is referred to as catalytically “inactivated Cas9” or “deactivated cas9 (dCas9).” Catalytically inactivated Cas9 variants include Cas9 variants that contain 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.

[0246] In some examples, a catalytically inactive Cas9 can be fused to a heterologous sequence (US patent application US20140068797 A1). Suitable fusion partners include, but are not limited to, a polypeptide that provides an activity that indirectly increases transcription by acting directly on the target DNA or on a polypeptide (e.g., a histone or other DNA-binding protein) associated with the target DNA. Additional suitable fusion partners include, but are not limited to, a polypeptide that provides for methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity. Further suitable fusion partners include, but are not limited to, a polypeptide that directly provides for increased transcription of the target nucleic acid (e.g., a transcription activator or a fragment thereof, a protein or fragment thereof that recruits a transcription activator, a small molecule / drug-responsive transcription regulator, etc.). A catalytically inactive Cas9 can also be fused to a Fokl nuclease to generate double strand breaks (Guilinger et al. Nature Biotechnology, volume 32, number 6, June 2014).

[0247] In some examples, the method comprises growing a plant from the plant cell comprising the heterologous NLR nucleic acid sequence. In some examples, the plant exhibits increased resistance to SR. In some examples, the plant derived from the method can then be used for further breeding activities and / or as a source of seed. In some examples, the NLR gene (e.g., NLR01 or NLR02) can be introgressed into a plant breeding line (e.g., an elite line) via repeated backcrossing using the plant produced by the method.

[0248] The heterologous NLR locus can be inserted into any location in the genome of the plant cell that is suitable for the expression of the heterologous NLR gene and, optionally, does not interfere with the expression of endogenous genes. In some examples, the heterologous NLR nucleic acid sequence replaces the endogenous NLR locus. In such examples, at least one allele of the endogenous NLR gene is eliminated or rendered nonfunctional and replaced by the heterologous NLR gene encoded on the polynucleotide modification template. In such examples, at least the coding regions of the endogenous NLR gene are replaced or rendered nonfunctional. Introns, promoter sequences, and terminator sequences may optionally be replaced or rendered nonfunctional as well.

[0249] In some examples, the heterologous NLR nucleic acid sequence is inserted into a locus different from the endogenous NLR locus. In some examples, the heterologous NLR nucleic acid sequence is inserted into a locus different from the endogenous NLR locus, along with other genes which are also inserted at the same locus, forming a molecular stack. In some examples, the molecular stack comprises a stack of disease resistance genes.

[0250] Methods for Producing Recombinant Maize Plant Cells and / or Plants II

[0251] Also provided herein is a method for producing a recombinant maize plant cell comprising a heterologous gene associated with resistance to SR. The method comprises introducing an NLR nucleic acid sequence into one or more plant cells derived from a first maize plant. The first maize plant can lack the NLR nucleic acid sequence before the present methods are performed. The NLR nucleic acid sequence can be heterologous to the first maize plant.

[0252] The method comprises inserting the NLR nucleic acid sequence into the genome of at least one of the plant cells, thereby creating one or more modified plant cells. The method comprises selecting a modified plant cell. The NLR nucleotide can comprise any NLR nucleotide sequence disclosed herein and can encode any of the NLR amino acid sequences disclosed herein. The NLR nucleic acid sequence can encode a protein comprising an amino acid sequence at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 30 or 34. The NLR nucleic acid sequence can encode a protein comprising an amino acid sequence at least 95% identical to SEQ ID NOs: 30 or 34. The NLR nucleic acid sequence can encode a protein comprising the amino acid sequence of SEQ ID NO: 30 or 34.

[0253] In some examples, the selection can comprise use of a molecular marker and / or sequencing or any other suitable technique to discriminate between modified and unmodified cells.

[0254] In some examples, the method comprises growing a modified plant from the selected plant cell. The modified plant can exhibit increased resistance to SR, compared to the first plant.In some examples, the selection can be based on any result indicating that the NLR gene is present. For example, sequencing can be performed to determine if the gene has been inserted and / or plant cells can be selected for using a selectable marker. In some examples, the method comprises crossing the newly grown modified maize plant with a second maize plant (e.g., one lacking the NLR gene) and obtaining a progeny plant that comprises the NLR gene. The progeny plant can then be used for further breeding activities (e.g., backcrosses and / or introgression) and / or as a source of seed.

[0255] In some examples, introducing the NLR nucleotide sequence comprises bacteria- mediated transformation.

[0256] In some examples, introducing the NLR nucleotide sequence comprises biolistic transformation.

[0257] In some examples, the NLR nucleic acid sequence comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity to SEQ ID NO: 28, 32, 35, or 36. In some examples, the NLR nucleic acid sequence comprises at least 95% nucleotide sequence identity to SEQ ID NO: 28, 32, 35, or 36. In some examples, the NLR nucleic acid sequence comprises the sequence of SEQ ID NO: 28, 32, 35, or 36.

[0258] Recombinant Plants and Seeds

[0259] Recombinant maize plants comprising the NLR nucleic acid sequences described herein are provided. Provided herein is a recombinant maize plant comprising a heterologous nucleic acid encoding a protein comprising at least 90 or at least 95% amino acid sequence identity to SEQ ID NO: 30 or 34. In some examples, the heterologous nucleic acid encodes a protein that comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 30 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 maize chromosome 10 or it is located on chromosome 1 , 2, 3, 4 5, 6, 7, 8, or 9. In some examples, the heterologous protein comprises 100% amino acid sequence identity to 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 maize chromosome 10 or it is located on chromosome 1 , 2, 3, 4 5, 6, 7, 8, or 9. In some examples, the heterologous nucleic acid comprises a sequence comprising at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity to 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 locatedon the long arm of maize chromosome 10 or it is located on chromosome 1 , 2, 3, 4 5, 6, 7, 8, or 9.. In some examples, the heterologous protein is encoded by a gene comprising 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 maize chromosome 10 or it is located on chromosome 1 , 2, 3, 4 5, 6, 7, 8, or 9).

[0260] Recombinant maize plant seeds comprising the NLR nucleic acid sequences described herein are also provided. Thus, provided is a recombinant plant seed comprising a heterologous nucleic acid encoding a protein comprising at least 90 or at least 95% amino acid sequence identity to SEQ ID NO: 30, wherein 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 maize chromosome 10 or it is located on chromosome 1 , 2, 3, 4 5, 6, 7, 8, or 9. In some examples, the protein comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 30 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 maize chromosome 10 or it is located on chromosome 1 , 2, 3, 4 5, 6, 7, 8, or 9. In some examples, the protein comprises the amino acid sequence identity to 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 maize chromosome 10 or it is located on chromosome 1, 2, 3, 4 5, 6, 7, 8, or 9. In some examples, the gene comprises a nucleotide sequence comprising at least 90 or at least 95% nucleotide sequence identity to SEQ ID NO: 28 or 35and 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 maize chromosome 10 or it is located on chromosome 1 , 2, 3, 4 5, 6, 7, 8, or 9. In some examples, the gene comprises a nucleotide sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleotide sequence identity to SEQ ID NO: 28, 32, 35, or 36 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 maize chromosome 10 or it is located on chromosome 1 , 2, 3, 4 5, 6, 7, 8, or 9. In some examples, the gene comprises a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 28, 32, 35, or 36 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 maize chromosome 10 or it is located on chromosome 1 , 2, 3, 4 5, 6, 7, 8, or 9.

[0261] Clauses of the Disclosure

[0262] Various aspects of the present disclosure will be understood by reference to the following clauses.1 . A method for identifying a maize plant comprising a sequence associated with resistance to southern rust, the method comprising: a. obtaining a sample comprising nucleic acid from a maize plant, b. screening the sample for a protein, or a nucleic acid encoding the protein, wherein the protein comprises an amino acid sequence comprising at least 95% sequence identity to SEQ ID NO: 30 and c. detecting the protein or nucleic acid encoding the protein and thereby identifying the maize plant as comprising sequence associated with resistance to southern rust.2. The method of clause 1 , wherein the protein comprises 100% amino acid sequence identity to SEQ ID NO: 30.3. The method of clause 1 or 2, wherein the method comprises a. obtaining samples comprising nucleic acid from each plant of a plurality of maize plants, b. screening each sample for the protein or nucleic acid encoding the protein; c. detecting the protein or nucleic acid encoding the protein in at least one sample and thereby identifying at least one maize plant from which the sample was obtained as comprising the sequence associated with resistance to southern rust; and d. selecting the at least one maize plant identified as comprising the sequence associated with resistance to southern rust.4. A method for producing a maize plant comprising a sequence associated with resistance to southern rust, the method comprising selecting a maize plant in accordance with clause 3, and further comprising: c. crossing the at least one maize plant selected in (d) with a second maize plant; and d. obtaining a progeny plant that comprises the sequence associated with resistance to southern rust.5. The method of any one of clauses 1-4, wherein the nucleic acid sequence comprises at least 95% nucleotide sequence identity to SEQ ID NO: 28, or 35.The method of any one of clauses 1-5, wherein the nucleic acid sequence comprises SEQ ID NO: 28 or 35. A method for producing a modified maize plant or modified maize plant cells, the method comprising: providing an unmodified maize plant, or one or more cells thereof, that is susceptible to SR; introducing a heterologous NLR01 nucleic acid sequence that encodes a protein comprising at least 95% amino acid sequence identity to SEQ ID NO: 30 into the genome of the plant, or the one or more cells thereof, thereby producing one or more modified plant, or plant cells thereof, comprising the heterologous NLR01 sequence; and selecting at least one of the modified maize plant or modified maize plant cells. The method of clause 7, wherein the method comprises: a) effecting a site-specific modification of at least one target site in the genome of the susceptible maize plant or one or more cells thereof; and b) introducing a polynucleotide modification template comprising the heterologous NLR01 sequence into the susceptible maize plant or one or more maize plant cells thereof, and thereby producing the modified maize or modified maize plant or modified maize plant cells. The method of clause 7 or 8, wherein the method comprises: a) effecting a site-specific modification to the native NLR01 gene of the susceptible maize plant, or cells thereof, and b) altering the native NLR01 gene sequence to produce the modified maize plant or modified maize plant cells. The method of clause 7, 8, or 9, wherein the site-specific modification is induced by a CRISPR-associated endonuclease. The method of any one of clauses 7-10, wherein the method comprises producing one or more modified maize plant cells and further comprises growing a modified maize plant from the selected maize plant cell, wherein the modified plant exhibits increased resistance to southern rust relative to the unmodified maize plant. The method of any one of clauses 7-11 , wherein the unmodified maize pant comprises an endogenous susceptible NLR01 sequence, and the heterologous NLR01 sequence replaces the endogenous susceptible NLR01 sequence. 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 comprise native NLR01 gene sequence. The method of any one of clauses 7-13, wherein the heterologous NLR gene sequence comprises a nucleotide sequence comprising at least 95% identity to SEQ ID NO: 28 or 35.The method of clause 14, wherein the heterologous NLR gene sequence comprises the nucleotide sequence of SEQ ID NO: 28 or 35. The method of clause 11 , further comprising: crossing the modified maize plant with a maize plant that is susceptible to SR and lacks the heterologous NLR01 sequence and selecting a progeny plant that is more resistant to SR than the susceptible maize plant and comprises the heterologous NLR01 sequence for further breeding or production of maize. Recombinant maize plant seed comprising a heterologous nucleic acid sequence encoding a protein comprising at least 95% amino acid sequence identity to SEQ ID NO: 30. The recombinant maize plant seed of clause 17, wherein the protein comprises 100% amino acid sequence identity to SEQ ID NO: 30. The recombinant maize plant seed of clause 17 or 18, wherein the heterologous nucleic acid sequence comprises at least 95% nucleotide sequence identity to SEQ ID NO: 28 or 35. The recombinant seed of any one of clauses 17-19, wherein the heterologous nucleic acid comprises the nucleotide sequence of SEQ ID NO: 28 or 35. A method for selecting a maize plant comprising a QTL associated with resistance to southern rust, the method comprising: obtaining nucleic acids from one or more maize plants; screening the nucleic acids for a QTL linked to a haplotype on maize chromosome 10 comprising one or more of the following marker alleles: i. a “T” at C00422-801 , corresponding to position 201 of SEQ ID NO: 1 ; ii. a “C” at C01770-1 , corresponding to position 201 of SEQ ID NO: 2; iii. a “T” at C06813-1 , corresponding to position 201 of SEQ ID NO: 7; iv. a “G” at C06824-1 , corresponding to position 201 of SEQ ID NO: 11 ; v. a “C” at C06834-1 , corresponding to position 201 of SEQ ID NO: 12; vi. a “T” at C01957-1 , corresponding to position 201 of SEQ ID NO: 13; vii. a “T” at C06838-1, corresponding to position 201 of SEQ ID NO: 14; viii. a “G” at C06848-1 , corresponding to position 201 of SEQ ID NO: 15; ix. an “A” at PZA9035-19, corresponding to position 211 of SEQ ID NO:16; x. a “T” at C12422-001 , corresponding to position 201 of SEQ ID NO: 17; xi. a “G” at PZA10357-20, corresponding to position 121 of SEQ ID NO:18;xii. a “G” at C06839-1 , corresponding to position 201 of SEQ ID NO: 19; xiii. a “C” at C002 MY 1-001, corresponding to position 61 of SEQ ID NO: 20; xiv. a “G” at CSR_2373961, corresponding to position 51 of SEQ ID NO: 21 ; xv. a “C” at CSR_2409943, corresponding to position 51 of SEQ ID NO: 22; xvi. a “G” at CSR_2643297, corresponding to position 51 of SEQ ID NO: 23; xvii. a “C” at CSR_2807867, corresponding to position 51 of SEQ ID NO: 24; xviii. a “C” at CSR_2837481 , corresponding to position 101 of SEQ ID NO: 25; or xix. an “A” at CSR_2839126, corresponding to position 101 of SEQ ID NO: 26; and selecting a maize plant comprising the haplotype and thereby selecting a maize plant comprising the QTL.The method of clause 21 , further comprising: screening the nucleic acid for one or more of the marker alleles i.-xix. Associated with the QTL.The method of clause 21 or 22, further comprising(A) screening the nucleic acid for one or more of the following marker alleles on chromosome 10: a. an “A” at C01800-1, corresponding to position 201 of SEQ ID NO: 3; b. a “G” at C06790-1 , corresponding to position 201 of SEQ ID NO: 4; and c. a “T” at C00429-801 , corresponding to position 84 of SEQ ID NO: 8 or screening the nucleic acid for one or more of the following marker alleles on chromosome 10: d. a “T” at C00431-802, corresponding to position 210 of SEQ ID NO: 5; e. a “C” at C002TCN-001, corresponding to position 61 of SEQ ID NO: 6; f. a “T” at C002TCM-001 , corresponding to position 61 of SEQ ID NO: 9; andg. an “A” at C06817-1, corresponding to position 201 of SEQ ID NO: 10 and(B) selecting a maize plant comprising one or more marker allele(s) a-g, and thereby selecting a maize plant comprising the QTL. A method for selecting a maize plant comprising a QTL associated with resistance to southern rust, the method comprising: obtaining nucleic acid from one or more maize plants; screening the nucleic acid for a QTL, wherein the QTL is linked to a haplotype on maize chromosome 10 comprising one or more of the following marker alleles: i. a “T” at C00422-801 , corresponding to position 201 of SEQ ID NO: 1 ; ii. a “C” at C01770-1 , corresponding to position 201 of SEQ ID NO: 2; ill. a “T” at C06813-1 , corresponding to position 201 of SEQ ID NO: 7; iv. a “G” at C06824-1 , corresponding to position 201 of SEQ ID NO: 11 ; v. a “C” at C06834-1 , corresponding to position 201 of SEQ ID NO: 12; vi. a “T” at C01957-1 , corresponding to position 201 of SEQ ID NO: 13; vii. a “T” at C06838-1, corresponding to position 201 of SEQ ID NO: 14; viii. a “G” at C06848-1 , corresponding to position 201 of SEQ ID NO: 15; ix. an “A” at PZA9035-19, corresponding to position 211 of SEQ ID NO: 16; x. a “T” at C12422-001 , corresponding to position 201 of SEQ ID NO: 17; xi. a “G” at PZA10357-20, corresponding to position 121 of SEQ ID NO: 18; xii. a “G” at C06839-1 , corresponding to position 201 of SEQ ID NO: 19; xiii. a “C” at C002 MY 1-001, corresponding to position 61 of SEQ ID NO: 20; xiv. a “G” at CSR_2373961, corresponding to position 51 of SEQ ID NO: 21 ; xv. a “C” at CSR_2409943, corresponding to position 51 of SEQ ID NO: 22; xvi. a “G” at CSR_2643297, corresponding to position 51 of SEQ ID NO: 23; xvii. a “C” at CSR_2807867, corresponding to position 51 of SEQ ID NO: 24;xviii. a “C” at CSR_2837481 , corresponding to position 101 of SEQ ID NO: 25; xix. an “A” at CSR_2839126, corresponding to position 101 of SEQ ID NO: 26; a. an “A” at C01800-1, corresponding to position 201 of SEQ ID NO: 3; b. a “G” at C06790-1 , corresponding to position 201 of SEQ ID NO: 4; or c. a “T” at C00429-801 , corresponding to position 84 of SEQ ID NO: 8; and selecting a maize plant comprising the haplotype and thereby selecting a maize plant comprising the QTL. The method of clause 24, further comprising: screening nucleic acid from the maize plant for one or more of the marker alleles i.-xix, a., b., and / or c. associated with the QTL. A method for selecting a maize plant comprising a QTL associated with resistance to southern rust, the method comprising: obtaining nucleic acids from one or more maize plants or germplasm thereof; screening the nucleic acids for a QTL, wherein the QTL is linked to a haplotype on maize chromosome 10 comprising one or more of the following marker alleles: i. a “T” at C00422-801 , corresponding to position 201 of SEQ ID NO: 1 ; ii. a “C” at C01770-1 , corresponding to position 201 of SEQ ID NO: 2; iii. a “T” at C06813-1 , corresponding to position 201 of SEQ ID NO: 7; iv. a “G” at C06824-1 , corresponding to position 201 of SEQ ID NO: 11 ; v. a “C” at C06834-1 , corresponding to position 201 of SEQ ID NO: 12; vi. a “T” at C01957-1 , corresponding to position 201 of SEQ ID NO: 13; vii. a “T” at C06838-1, corresponding to position 201 of SEQ ID NO: 14; viii. a “G” at C06848-1 , corresponding to position 201 of SEQ ID NO: 15; ix. an “A” at PZA9035-19, corresponding to position 211 of SEQ ID NO: 16; x. a “T” at C12422-001 , corresponding to position 201 of SEQ ID NO: 17; xi. a “G” at PZA10357-20, corresponding to position 121 of SEQ ID NO: 18; xii. a “G” at C06839-1 , corresponding to position 201 of SEQ ID NO: 19; xiii. a “C” at C002 MY 1-001, corresponding to position 61 of SEQ ID NO: 20;xiv. a “G” at CSR_2373961, corresponding to position 51 of SEQ ID NO: 21 ; xv. a “C” at CSR_2409943, corresponding to position 51 of SEQ ID NO: 22; xvi. a “G” at CSR_2643297, corresponding to position 51 of SEQ ID NO: 23; xvii. a “C” at CSR_2807867, corresponding to position 51 of SEQ ID NO: 24; xviii. a “C” at CSR_2837481 , corresponding to position 101 of SEQ ID NO: 25; xix. an “A” at CSR_2839126, corresponding to position 101 of SEQ ID NO: 26; d. a “T” at C00431-802, corresponding to position 210 of SEQ ID NO: 5; e. a “C” at C002TCN-001, corresponding to position 61 of SEQ ID NO: 6; f. a “T” at C002TCM-001 , corresponding to position 61 of SEQ ID NO: 9; or g. an “A” at C06817-1, corresponding to position 201 of SEQ ID NO: 10; and selecting a maize plant comprising the haplotype and thereby selecting a maize plant comprising the QTL.27. The method of clause 26, further comprising: screening nucleic acid from the maize plant 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 clauses 21-27, wherein the method comprises screening for one or more of: xiv. a “G” at CSR_2373961, corresponding to position 51 of SEQ ID NO: 21 ; xv. a “C” at CSR_2409943, corresponding to position 51 of SEQ ID NO: 22; xvi. a “G” at CSR_2643297, corresponding to position 51 of SEQ ID NO: 23; xvii. a “C” at CSR_2807867, corresponding to position 51 of SEQ ID NO: 24; xviii. a “C” at CSR_2837481 , corresponding to position 101 of SEQ ID NO: 25; or xix. an “A” at CSR_2839126, corresponding to position 101 of SEQ ID NO: 26.29. The method of any one of clauses 22, 23, 25, or 27, wherein the one or more screened for marker alleles are located in a chromosomal interval flanked by and including:C06824-1 , corresponding to a “G” at position 201 of SEQ ID NO: 11 andC06834-1 , corresponding to a “C” at position 201 of SEQ ID NO: 12. The method of any one of clauses 22, 23, 25, 27, or 29 wherein the one or more screened for marker alleles are located in a chromosomal interval flanked by and including:CSR_2373961 , corresponding to a “G” at position 51 of SEQ ID NO: 21 and CSR_2839126, corresponding to an “A” at position 101 of SEQ ID NO: 26. A method for producing a maize plant comprising resistance to southern rust, the method comprising selecting a maize plant in accordance with any one of clauses 21-30 and further comprising crossing the selected maize plant(s) with a second maize plant; and obtaining a progeny plant that comprises the QTL. A method for producing a maize plant comprising resistance to southern rust, the method comprising: a. crossing a first parent maize plant with a second parent maize plant to produce one or more progeny plants, wherein the first parent comprises a QTL associated with resistance to southern rust that is not found in the second parent; b. obtaining a nucleic acid sample from one or more of the progeny plant(s); and c. screening the sample and selecting one or more progeny plant comprising the QTL associated with resistance to southern rust in accordance with the method of any one of clauses 30-40. The method of clause 32, further comprising: d. crossing the one or more selected progeny plants with the second parent plant to produce one or more backcross progeny plants; e. obtaining a nucleic acid sample from one or more backcross progeny plants; and f. selecting the one or more backcross progeny plants comprising the QTL associated with resistance to southern rust. The method of clause 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. obtaining a nucleic acid sample from one or more additional backcross progeny plants; i. selecting the one or more additional backcross progeny plants comprising the QTL associated with resistance to southern rust; and j. optionally repeating steps (g), (h), and (i) to obtain additional backcross progeny plants comprising the QTL associated with resistance to southern rust.

[0263] EXAMPLES

[0264] The present disclosure will be more completely understood with reference to the following examples. The examples are presented to aid in understanding of the present disclosure and are not intended to limit the scope of the present disclosure.

[0265] Example 1 - Initial mapping of a QTL on maize chromosome 10 associated with resistance to SR

[0266] Using a source of SR resistance (Inbred A) and two susceptible inbreds (Inbred B and Inbred C) two F2 populations were generated for genetic mapping of the SR resistance trait of Inbred A. Assay data generated in greenhouse conditions were used to determine resistance phenotypes. Plants were inoculated with P. polysora in the greenhouse and assessed for SR resistance phenotype. The phenotype data was clear in all parental lines and segregating material in the experiment (Table 3). Segregation ratios matched those expected based on segregation of a single, dominant locus.

[0267] Table 3: Phenotype calls and segregation ratios from Inbred A, Inbred B, Inbred A crossed with Inbred B (Inbred A x Inbred B), Inbred C, and Inbred A crossed with Inbred C (Inbred A x Inbred C) from greenhouse phenotyping.

[0268] The phenotype data with TaqMan SNP marker genotypes from F2 individuals was used for initial mapping to identify a QTL on chromosome 10 (short arm). Based on analysis of 5 key recombination events from Inbred A x Inbred B and 11 events from Inbred A x Inbred C, the flanking markers for the effect were C06824-1 and C06834-1 (Tables 4 and 5).

[0269] Table 4: QTL analysis result of the Inbred A x Inbred B F2 population using greenhouse phenotypes. A total of 162 SNP markers were used covering the entire genome, with a concentration of markers used on chromosome 10. There were no significant markers on chromosomes 1-9, so only chromosome 10 markers are shown. R or S scores were convertedinto a 1 (R) or 2 (S) for the analysis. The p-value for strength of association of marker genotypes to the phenotypes is shown.

[0270] Table 5: QTL analysis result of the Inbred A x Inbred C F2 population analysis using greenhouse phenotypes. A total of 162 SNP markers were used covering the entire genome, with a concentration of markers used on chromosome 10. There were no significant markers on chromosomes 1-9, so only chromosome 10 markers are shown. R or S scores were converted into a 1 (R) or 2 (S) for the analysis. The p-value for strength of association of marker genotypes to the phenotypes is shown. Marker C002TCN-001 was polymorphic in plants resulting from Inbred A x Inbred C but not in plants resulting from Inbred A x Inbred B.

[0271] Table 6: Key recombination events supporting the flanking markers C06824-1 andC06834-1 in the Inbred A x Inbred B population. B=lnbred B genotype, H=heterozygous. Data from plant 437083102 appears to be the result of an incorrect genotype or an incorrect score.

[0272] Table 7: Key recombination events supporting the flanking markers C06824-1 andC06834-1 in the Inbred A x Inbred C population. C=lnbred C, H=heterozygous

[0273] Example 2 - Fine Mapping of the QTL

[0274] Given the strength of association seen on chromosome 10 for these populations and the flanking markers discovered in the first round of mapping, we screened an additional 1840 individuals for recombination events in the region around the C06824-1 and C06834-1 markers using TaqMan SNP markers and DNA from chipped kernels. A new set of 180 individuals were chosen for phenotyping based on genotypes in the region. The selected individuals had similar recombination events to those in Tables 6 and 7.

[0275] The genome of Inbred A was sequenced to identify potential candidate genes in the region. Transcriptome sequencing was done, obtaining approximately 100 million reads from V5 seedling tissue. The genomics data was used to identify candidate genes in the interval and SNPs for additional markers. Markers were designed in the region to identify the most informative recombination events for fine mapping based on locations of previous markers and locations of candidate genes. New recombination events (6 from Inbred C progeny and 2 from Inbred B progeny) gave 2 new flanking markers: CSR_2807867 and CSR_2837481 (Table 8).

[0276] Table 8: Additional recombination events and flanking markers from the Inbred A x Inbred C and Inbred A x Inbred B. B=lnbred B or Inbred C, A=lnbred A and H=heterozygous

[0277] Table 9: Markers and B73 V4 positions used in the Chromosome 10 interval analysis.

[0278] Example 3 - Candidate Gene Identification and Protoplast Assay

[0279] The flanking markers from the fine mapping (CSR_2807867 and CSR_2837481) are 248kb apart on the Inbred A genome sequence. This region contains 2 candidate resistance genes: NLR01 and NLR02. HISAT2 was used to align paired-end RNA-seq reads from an Inbred A leaf tissue library against the sequenced Inbred A genome. Stringtie was then utilized to construct transcript models based on these alignments. Predicted proteins from all proteincoding transcript models within the interval were then assessed using HMMer to identify canonical NLR domains (NB-ARC, coiled-coil, and leucine rich repeats). Two of the genes within the fine mapping interval (NLR01 and NLR02) were found to encode proteins containing canonical NLR domains and were selected as possible candidates for southern corn rust resistance.

[0280] NLR01 was tested in a maize protoplast assay (Deng, C., et al. The RppC-AvrRppC NLR-effector interaction mediates the resistance to southern corn rust in maize. Mol.Plant. 2022; 15: pp. 904-912) for the ability to recognize Southern Rust effector alleles. This assay showed that the NLR01 protein can detect the same P. polysora isolate used in the greenhouse phenotyping and can induce protoplast cell death.

[0281] Example 4 - Transgenic testing of NLR01 and NLR02 candidate genes

[0282] The two NLR genes identified from the Inbred A QTL (NLR01 and NLR02) were tested for efficacy in the same susceptible background(s) used for mapping. The transgenic construct for NLR01 contains the native promoter (1990bp) (SEQ ID NO:27), native coding sequence (7800bp) (SEQ ID NO:28) and the native maize terminator (1500bp) (SEQ ID NO:29). The transgenic construct for NLR 02 contains the native promoter (2000bp) (SEQ ID NO:31), native coding sequence (1097bp) (SEQ ID NO:32) and the native maize terminator (1000bp) (SEQ ID NO:33).

[0283] Transgenic plants expressing either NLR01 or NLR02 were produced via bacteria- mediated transformation. T1 plants (progeny of the initially-produced TO transformants) were grown and infected with P. polysora. Table 10 shows data generated for NLR01 and Table 11 shows data generated for NLR02. The transgenic plants were compared to control plants (i.e. , plants known to be resistant or sensitive) to determine the degree of resistance provided by expression of NLR01 and the degree of resistance provided by expression of NLR02. Control plants are indicated in Event column of indicated in Tables 10 and 11. Transgenic plants were also compared to putatively transformed plants that were isolated during the transformation process but failed to show incorporation of the NLR01 or NLR02 gene. Such plants are referred to as “Null” in Tables 10 and 11 . Southern rust resistance score are provided in the “S. RustScore” column of Tables 10 and 11. Scores are “S” for susceptible, “I” for intermediate, and “R” for resistant.

[0284] Table 10Table 11

[0285] Based on the foregoing results, NLR01 provided plants with resistance to southern rust when incorporated into the genome and expressed. All plants carrying the heterologous NLR01 gene showed at least an 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.

[0286] The results indicate that NLR02 did not provide resistance to southern rust (at least not under the conditions tested).

[0287] Example 5 - Presence of NLR01 in public maize variety genomes.

[0288] A review of public lines found that the NLR01 gene disclosed herein is present in the genomes of public maize varieties W64A Rpp9 (accession Ames 30907) and Boesman Yellow Flint (accession PI 186208). See Sun et al., Phytopathology Research 3:25 (2021) and USDA Annual Report of Cooperative Regional Projects Jan 1- Dec 31 1974.

[0289] For W64A Rpp9, long read re-sequencing data were generated using the Pacific BioSciences (Menlo Park, CA, USA) Sequel2e platform from Pacific BioSciences. PacBio HiFi reads with a read N50 of 16.1 kb and 14x coverage were aligned to the Inbred A reference genome with minimap2-2.28 (r1209), which is available on Github, using default parameters and the map-hifi preset. Aligned reads were visually inspected at the target locus using IGV 2.17.3 with a minimum alignment score of 5000. A range of 6-12x read coverage was observed which showed 100% sequence identity between the NLR01 gene in Inbred A and W64A Rpp9.

[0290] For Boesman Yellow Flint, long read re-sequencing data were generated using the Pacific BioSciences Revio platform. A PacBio HiFi read data set from two cells with a read N50 of 17.2 kb and 70x coverage was aligned to the Inbred A reference genome with minimap2-2.28 (r1209) using default parameters and the map-hifi preset. Aligned reads were visually inspected at the target locus using IGV 2.17.3 with a minimum alignment score of 5000. Due to the seed source being derived from a population, the coverage varied across the genome at a givenlocus for a given allele. A range of 8-11x read coverage was observed which showed 100% sequence identity between the NLR01 gene in Inbred A and Boesman Yellow Flint.

Claims

Claims:1 . A method for identifying a maize plant comprising a sequence associated with resistance to southern rust, the method comprising: a. obtaining a sample comprising nucleic acid from a maize plant, b. screening the sample for a protein, or a nucleic acid encoding the protein, wherein the protein comprises an amino acid sequence comprising at least 95% sequence identity to SEQ ID NO: 30 and c. detecting the protein or nucleic acid encoding the protein and thereby identifying the maize plant as comprising sequence associated with resistance to southern rust.

2. The method of claim 1 , wherein the protein comprises 100% amino acid sequence identity to SEQ ID NO: 30.

3. The method of claim 1 or 2, wherein the method comprises a. obtaining samples comprising nucleic acid from each plant of a plurality of maize plants, b. screening each sample for the protein or nucleic acid encoding the protein; c. detecting the protein or nucleic acid encoding the protein in at least one sample and thereby identifying at least one maize plant from which the sample was obtained as comprising the sequence associated with resistance to southern rust; and d. selecting the at least one maize plant identified as comprising the sequence associated with resistance to southern rust.

4. A method for producing a maize plant comprising a sequence associated with resistance to southern rust, the method comprising selecting a maize plant in accordance with claim 3, and further comprising: c. crossing the at least one maize plant selected in (d) with a second maize plant; and d. obtaining a progeny plant that comprises the sequence associated with resistance to southern rust.

5. The method of any one of claims 1-4, wherein the nucleic acid sequence comprises at least 95% nucleotide sequence identity to 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 a modified maize plant or modified maize plant cells, the method comprising:providing an unmodified maize plant, or one or more cells thereof, that is susceptible to SR; introducing a heterologous NLR01 nucleic acid sequence that encodes a protein comprising at least 95% amino acid sequence identity to SEQ ID NO: 30 into the genome of the plant, or the one or more cells thereof, thereby producing one or more modified plant, or plant cells thereof, comprising the heterologous NLR01 sequence; and selecting at least one of the modified maize plant or modified maize plant cells.

8. The method of claim 7, wherein the method comprises: a) effecting a site-specific modification of at least one target site in the genome of the susceptible maize plant or one or more cells thereof; and b) introducing a polynucleotide modification template comprising the heterologous NLR01 sequence into the susceptible maize plant or one or more maize plant cells thereof, and thereby producing the modified maize or modified maize plant or modified maize plant cells.

9. The method of claim 7 or 8, wherein the method comprises: a) effecting a site-specific modification to the native NLR01 gene of the susceptible maize plant, or cells thereof, and b) altering the native NLR01 gene sequence to produce the modified maize plant or modified maize plant cells.

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 producing one or more modified maize plant cells and further comprises growing a modified maize plant from the selected maize plant cell, wherein the modified plant exhibits increased resistance to southern rust relative to the unmodified maize plant.

12. The method of any one of claims 7-11, wherein the unmodified maize pant comprises 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 comprise native NLR01 gene sequence.

14. The method of any one of claims 7-13, wherein the heterologous NLR gene sequence comprises a nucleotide sequence comprising at least 95% identity to 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:crossing the modified maize plant with a maize plant that is susceptible to SR and lacks the heterologous NLR01 sequence and selecting a progeny plant that is more resistant to SR than the susceptible maize plant and comprises the heterologous NLR01 sequence for further breeding or production of maize.

17. Recombinant maize plant seed comprising a heterologous nucleic acid sequence encoding a protein comprising at least 95% amino acid sequence identity to SEQ ID NO: 30.

18. The recombinant maize plant seed of claim 17, wherein the protein comprises 100% amino acid sequence identity to SEQ ID NO: 30.

19. The recombinant maize plant seed of claim 17 or 18, wherein the heterologous nucleic acid sequence comprises at least 95% nucleotide sequence identity to SEQ ID NO: 28 or 35.

20. The recombinant seed of 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 a maize plant comprising a QTL associated with resistance to southern rust, the method comprising: obtaining nucleic acids from one or more maize plants; screening the nucleic acids for a QTL linked to a haplotype on maize chromosome 10 comprising one or more of the following marker alleles: i. a “T” at C00422-801 , corresponding to position 201 of SEQ ID NO: 1 ; ii. a “C” at C01770-1 , corresponding to position 201 of SEQ ID NO: 2; iii. a “T” at C06813-1 , corresponding to position 201 of SEQ ID NO: 7; iv. a “G” at C06824-1 , corresponding to position 201 of SEQ ID NO: 11 ; v. a “C” at C06834-1 , corresponding to position 201 of SEQ ID NO: 12; vi. a “T” at C01957-1 , corresponding to position 201 of SEQ ID NO: 13; vii. a “T” at C06838-1, corresponding to position 201 of SEQ ID NO: 14; viii. a “G” at C06848-1 , corresponding to position 201 of SEQ ID NO: 15; ix. an “A” at PZA9035-19, corresponding to position 211 of SEQ ID NO: 16; x. a “T” at C12422-001 , corresponding to position 201 of SEQ ID NO: 17; xi. a “G” at PZA10357-20, corresponding to position 121 of SEQ ID NO:18; xii. a “G” at C06839-1 , corresponding to position 201 of SEQ ID NO: 19; xiii. a “C” at C002 MY 1-001, corresponding to position 61 of SEQ ID NO:xiv. a “G” at CSR_2373961, corresponding to position 51 of SEQ ID NO:21 ; xv. a “C” at CSR_2409943, corresponding to position 51 of SEQ ID NO: 22; xvi. a “G” at CSR_2643297, corresponding to position 51 of SEQ ID NO: 23; xvii. a “C” at CSR_2807867, corresponding to position 51 of SEQ ID NO: 24; xviii. a “C” at CSR_2837481 , corresponding to position 101 of SEQ ID NO: 25; or xix. an “A” at CSR_2839126, corresponding to position 101 of SEQ ID NO: 26; and selecting a maize plant comprising the haplotype and thereby selecting a maize plant comprising the QTL.

22. The method of claim 21, further comprising: screening the nucleic acid 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) screening the nucleic acid for one or more of the following marker alleles on chromosome 10: a. an “A” at C01800-1, corresponding to position 201 of SEQ ID NO: 3; b. a “G” at C06790-1 , corresponding to position 201 of SEQ ID NO: 4; and c. a “T” at C00429-801 , corresponding to position 84 of SEQ ID NO: 8 or screening the nucleic acid for one or more of the following marker alleles on chromosome 10: d. a “T” at C00431-802, corresponding to position 210 of SEQ ID NO: 5; e. a “C” at C002TCN-001, corresponding to position 61 of SEQ ID NO: 6; f. a “T” at C002TCM-001 , corresponding to position 61 of SEQ ID NO: 9; and g. an “A” at C06817-1, corresponding to position 201 of SEQ ID NO: 10 and(B) selecting a maize plant comprising one or more marker allele(s) a-g, and thereby selecting a maize plant comprising the QTL.

4. A method for selecting a maize plant comprising a QTL associated with resistance to southern rust, the method comprising: obtaining nucleic acid from one or more maize plants; screening the nucleic acid for a QTL, wherein the QTL is linked to a haplotype on maize chromosome 10 comprising one or more of the following marker alleles: i. a “T” at C00422-801 , corresponding to position 201 of SEQ ID NO: 1 ; ii. a “C” at C01770-1 , corresponding to position 201 of SEQ ID NO: 2; iii. a “T” at C06813-1 , corresponding to position 201 of SEQ ID NO: 7; iv. a “G” at C06824-1 , corresponding to position 201 of SEQ ID NO: 11 ; v. a “C” at C06834-1 , corresponding to position 201 of SEQ ID NO: 12; vi. a “T” at C01957-1 , corresponding to position 201 of SEQ ID NO: 13; vii. a “T” at C06838-1, corresponding to position 201 of SEQ ID NO: 14; viii. a “G” at C06848-1 , corresponding to position 201 of SEQ ID NO: 15; ix. an “A” at PZA9035-19, corresponding to position 211 of SEQ ID NO: 16; x. a “T” at C12422-001 , corresponding to position 201 of SEQ ID NO: 17; xi. a “G” at PZA10357-20, corresponding to position 121 of SEQ ID NO: 18; xii. a “G” at C06839-1 , corresponding to position 201 of SEQ ID NO: 19; xiii. a “C” at C002 MY 1-001, corresponding to position 61 of SEQ ID NO: 20; xiv. a “G” at CSR_2373961, corresponding to position 51 of SEQ ID NO: 21 ; xv. a “C” at CSR_2409943, corresponding to position 51 of SEQ ID NO: 22; xvi. a “G” at CSR_2643297, corresponding to position 51 of SEQ ID NO: 23; xvii. a “C” at CSR_2807867, corresponding to position 51 of SEQ ID NO: 24; xviii. a “C” at CSR_2837481 , corresponding to position 101 of SEQ ID NO: 25; xix. an “A” at CSR_2839126, corresponding to position 101 of SEQ ID NO: 26; a. an “A” at C01800-1, corresponding to position 201 of SEQ ID NO: 3;b. a “G” at C06790-1 , corresponding to position 201 of SEQ ID NO: 4; or c. a “T” at C00429-801 , corresponding to position 84 of SEQ ID NO: 8; and selecting a maize plant comprising the haplotype and thereby selecting a maize plant comprising the QTL.

25. The method of claim 24, further comprising: screening nucleic acid from the maize plant for one or more of the marker alleles i.-xix, a., b., and / or c. associated with the QTL.

26. A method for selecting a maize plant comprising a QTL associated with resistance to southern rust, the method comprising: obtaining nucleic acids from one or more maize plants or germplasm thereof; screening the nucleic acids for a QTL, wherein the QTL is linked to a haplotype on maize chromosome 10 comprising one or more of the following marker alleles: i. a “T” at C00422-801, corresponding to position 201 of SEQ ID NO: 1; ii. a “C” at C01770-1 , corresponding to position 201 of SEQ ID NO: 2; iii. a “T” at C06813-1 , corresponding to position 201 of SEQ ID NO: 7; iv. a “G” at C06824-1 , corresponding to position 201 of SEQ ID NO: 11 ; v. a “C” at C06834-1 , corresponding to position 201 of SEQ ID NO: 12; vi. a “T” at C01957-1 , corresponding to position 201 of SEQ ID NO: 13; vii. a “T” at C06838-1, corresponding to position 201 of SEQ ID NO: 14; viii. a “G” at C06848-1 , corresponding to position 201 of SEQ ID NO: 15; ix. an “A” at PZA9035-19, corresponding to position 211 of SEQ ID NO: 16; x. a “T” at C12422-001 , corresponding to position 201 of SEQ ID NO: 17; xi. a “G” at PZA10357-20, corresponding to position 121 of SEQ ID NO: 18; xii. a “G” at C06839-1 , corresponding to position 201 of SEQ ID NO: 19; xiii. a “C” at C002 MY 1-001, corresponding to position 61 of SEQ ID NO: 20; xiv. a “G” at CSR_2373961, corresponding to position 51 of SEQ ID NO: 21 ; xv. a “C” at CSR_2409943, corresponding to position 51 of SEQ ID NO: 22;xvi. a “G” at CSR_2643297, corresponding to position 51 of SEQ ID NO: 23; xvii. a “C” at CSR_2807867, corresponding to position 51 of SEQ ID NO: 24; xviii. a “C” at CSR_2837481 , corresponding to position 101 of SEQ ID NO: 25; xix. an “A” at CSR_2839126, corresponding to position 101 of SEQ ID NO: 26; d. a “T” at C00431-802, corresponding to position 210 of SEQ ID NO: 5; e. a “C” at C002TCN-001, corresponding to position 61 of SEQ ID NO: 6; f. a “T” at C002TCM-001 , corresponding to position 61 of SEQ ID NO: 9; or g. an “A” at C06817-1, corresponding to position 201 of SEQ ID NO: 10; and selecting a maize plant comprising the haplotype and thereby selecting a maize plant comprising the QTL.

27. The method of claim 26, further comprising: screening nucleic acid from the maize plant 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: xiv. a “G” at CSR_2373961, corresponding to position 51 of SEQ ID NO: 21 ; xv. a “C” at CSR_2409943, corresponding to position 51 of SEQ ID NO: 22; xvi. a “G” at CSR_2643297, corresponding to position 51 of SEQ ID NO: 23; xvii. a “C” at CSR_2807867, corresponding to position 51 of SEQ ID NO: 24; xviii. a “C” at CSR_2837481 , corresponding to position 101 of SEQ ID NO: 25; or xix. an “A” at CSR_2839126, corresponding 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 screened for marker alleles are located in a chromosomal interval flanked by and including:C06824-1 , corresponding to a “G” at position 201 of SEQ ID NO: 11 and C06834-1 , corresponding to a “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 screened for marker alleles are located in a chromosomal interval flanked by and including:CSR_2373961 , corresponding to a “G” at position 51 of SEQ ID NO: 21 andCSR_2839126, corresponding to an “A” at position 101 of SEQ ID NO: 26.

31. A method for producing a maize plant comprising resistance to southern rust, the method comprising: selecting a maize plant in accordance with any one of claims 21-30 and crossing the selected maize plant(s) with a second maize plant; and obtaining a progeny plant that comprises the QTL.

32. A method for producing a maize plant comprising resistance to southern rust, the method comprising: a. crossing a first parent maize plant with a second parent maize plant to produce one or more progeny plants, wherein the first parent comprises a QTL associated with resistance to southern rust that is not found in the second parent; b. obtaining a nucleic acid sample from one or more of the progeny plant(s); and c. screening the sample and selecting one or more progeny plant comprising the QTL associated with resistance to southern rust in accordance with the method of any one of claims 30-40.

33. The method of claim 32, further comprising: d. crossing the one or more selected progeny plants with the second parent plant to produce one or more backcross progeny plants; e. obtaining a nucleic acid sample from one or more backcross progeny plants; and f. selecting the one or more backcross progeny plants comprising the QTL 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. obtaining a nucleic acid sample from one or more additional backcross progeny plants; i. selecting the one or more additional backcross progeny plants comprising the QTL associated with resistance to southern rust; and j. optionally repeating steps (g), (h), and (i) to obtain additional backcross progeny plants comprising the QTL associated with resistance to southern rust.

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