Maize plants comprising resistance against southern leaf blight and compositions and methods for selecting and producing same

Abstract

The present application relates to markers that have been determined to be genetically linked to loci that provide resistance to Southern leaf blight (SLB). Also provided herein are maize plants comprising newly conferred or increased resistance to SLB relative to control plants.

Description

[0001] This application claims the benefit of PCT International Application No. PCT / CN2022 / 0139436, filed on December 16, 2022, the entire disclosure of which is incorporated herein by reference. Technical Field

[0002] The field includes plants, plant genetics, plant breeding, and methods of selecting and producing plants comprising resistance to southern leaf blight. The field also includes disease-resistant plants and related compositions.

[0003] References to electronically submitted sequence listings

[0004] An official copy of the sequence listing is electronically submitted via EFS-Web as an XML file named "117927_WO_SEC_2_sequence_listing" created on December 6, 2023. The XML file is 77,046 bytes in size and the sequence listing is submitted concurrently with this specification. The sequence listing contained in this XML file document is part of this specification and is incorporated herein by reference in its entirety. Background Art

[0005] Southern leaf blight (SLB) is a common fungal disease of maize plants caused by the fungal pathogen Bipolaris maydis (also known as Cochliobolus heterostrophus in its sexual form). SLB has been a major concern for farmers and food producers because of its potential to reduce the yield and / or quality of the maize crop. Subtypes of the pathogen include races O, T, and C. Lesions on the leaves are always present in infected plants, but lesions may also be present on other parts of the plant. SLB can survive on plant debris from one season and spread to newly planted corn the following season. SLB spores can germinate and spread rapidly under favorable conditions. While management and mitigation techniques exist to minimize the impact of SLB on maize crops, new technologies are needed to manage and minimize the potential impacts of SLB. Summary of the Invention

[0006] In one aspect, the present disclosure provides a method of screening maize plants comprising a marker allele associated with resistance to Southern Leaf Blight (SLB). The method includes detecting at least one of the following marker alleles on chromosome 3 in a nucleic acid from a maize plant: a "C" at marker D6, corresponding to position 51 of SEQ ID NO: 1; a "C" at marker C10, corresponding to position 51 of SEQ ID NO: 2; a "C" at marker A10, corresponding to position 51 of SEQ ID NO: 3; a "G" at marker G6, corresponding to position 51 of SEQ ID NO: 4; a "G" at marker H6, corresponding to position 51 of SEQ ID NO: 5; a "T" at marker D10, corresponding to position 51 of SEQ ID NO: 6; a "C" at marker F10, corresponding to position 51 of SEQ ID NO: 7; an "A" at marker K6, corresponding to position 51 of SEQ ID NO: 8; a "C" at marker L6, corresponding to position 51 of SEQ ID NO: 9; a "T" at marker O6, corresponding to position 51 of SEQ ID NO: 10; a "G" at marker Q6, corresponding to position 51 of SEQ ID NO: 11; a "C" at marker G3, corresponding to position 51 of SEQ ID NO: 12; a "T" at marker D7, corresponding to position 51 of SEQ ID NO: "T" at marker L4 corresponds to position 51 of SEQ ID NO:14; "T" at marker E7 corresponds to position 51 of SEQ ID NO:15; "A" at marker G7 corresponds to position 51 of SEQ ID NO:16; "G" at marker H7 corresponds to position 51 of SEQ ID NO:17; "T" at marker I7 corresponds to position 51 of SEQ ID NO:18; "T" at marker J7 corresponds to position 51 of SEQ ID NO:19; "G" at marker K7 corresponds to position 51 of SEQ ID NO:20; "A" at marker L7 corresponds to position 51 of SEQ ID NO:21; "T" at marker M7 corresponds to position 51 of SEQ ID NO:22; "A" at marker N7 corresponds to position 51 of SEQ ID NO:23; or "A" at marker O4 corresponds to position 51 of SEQ ID NO:24. The method can further comprise obtaining a nucleic acid sample from each of the one or more plants, seeds, tissues or germplasm and screening the sample for one or more of the aforementioned marker alleles; wherein the presence of the one or more marker alleles is associated with resistance to SLB.

[0007] In some examples, the aforementioned method includes obtaining a nucleic acid sample from each of a plurality of maize plants (or seeds, tissues, or germplasm thereof); screening each sample and identifying the sample as containing one or more of the aforementioned marker alleles associated with anti-SLB resistance. The method may further include selecting a maize plant (or seeds, tissues, or germplasm thereof) containing at least one marker.

[0008] In another example, the method includes crossing the selected maize plant with a second maize plant; and obtaining a progeny plant comprising at least one of the aforementioned marker alleles associated with SLB resistance. For example, the selected maize plant can comprise a QTL or chromosomal interval flanked by and including: marker D6, corresponding to "C" at position 51 of SEQ ID NO: 1; and G3, corresponding to "C" at position 51 of SEQ ID NO: 12. In some cases, the selected maize plant comprises a QTL allele or chromosomal interval flanked by and including: marker D7, corresponding to "T" at position 51 of SEQ ID NO: 13; and O4, corresponding to "A" at position 51 of SEQ ID NO: 24. In some examples, one or more of markers D6, C10, A10, G6, H6, D10, F10, K6, L6, O6, Q6, and G3 are detected; or one or more of markers D7, L4, E7, G7, H7, I7, J7, K7, L7, M7, N7, and O4 are detected; or each of markers D6, C10, A10, G6, H6, D10, F10, K6, L6, O6, Q6, and G3 are detected; or each of markers D7, L4, E7, G7, H7, I7, J7, K7, L7, M7, N7, and O4 are detected.

[0009] In another aspect, provided herein is a method for producing a maize plant comprising a QTL allele associated with resistance to southern leaf blight. The method includes screening a first population of maize plants for a QTL associated with resistance to southern leaf blight. The QTL comprises one or more of the marker alleles: "C" at D6, corresponding to position 51 of SEQ ID NO: 1; "C" at C10, corresponding to position 51 of SEQ ID NO: 2; "C" at A10, corresponding to position 51 of SEQ ID NO: 3; "G" at G6, corresponding to position 51 of SEQ ID NO: 4; "G" at H6, corresponding to position 51 of SEQ ID NO: 5; "T" at D10, corresponding to position 51 of SEQ ID NO: 6; "C" at F10, corresponding to position 51 of SEQ ID NO: 7; "A" at K6, corresponding to position 51 of SEQ ID NO: 8; "C" at L6, corresponding to position 51 of SEQ ID NO: 9; "T" at O6, corresponding to position 51 of SEQ ID NO: 10; "G" at Q6, corresponding to position 51 of SEQ ID NO: 11; or "C" at G3, corresponding to position 51 of SEQ ID NO: 12. For example, the QTL can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all 12 of each of the aforementioned marker alleles.

[0010] Alternatively, the QTL may include a “T” at D7, corresponding to position 51 of SEQ ID NO:13; a “T” at L4, corresponding to position 51 of SEQ ID NO:14; a “T” at E7, corresponding to position 51 of SEQ ID NO:15; an “A” at G7, corresponding to position 51 of SEQ ID NO:16; a “G” at H7, corresponding to position 51 of SEQ ID NO:17; a “T” at I7, corresponding to position 51 of SEQ ID NO:18; a “T” at J7, corresponding to position 51 of SEQ ID NO:19; a “G” at K7, corresponding to position 51 of SEQ ID NO:20; an “A” at L7, corresponding to position 51 of SEQ ID NO:21; a “T” at M7, corresponding to position 51 of SEQ ID NO:22; an “A” at N7, corresponding to position 51 of SEQ ID NO:23; and an “A” at O4, corresponding to position 51 of SEQ ID NO:24. For example, the QTL can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or all 12 of each of the aforementioned marker alleles.

[0011] The method of producing a maize plant can also include selecting at least one maize plant comprising the QTL allele from the population; crossing the selected maize plant with a second maize plant; and obtaining a progeny plant comprising the QTL allele. In some examples, the method includes introgressing the QTL allele into the second maize plant population. In some examples, the QTL allele is located on a chromosomal interval flanked by and including marker D6, corresponding to "C" at position 51 of SEQ ID NO: 1; and G3, corresponding to "C" at position 51 of SEQ ID NO: 12. In other examples, the QTL allele is located on a chromosomal interval flanked by and including marker D7, corresponding to "T" at position 51 of SEQ ID NO: 13; and O4, corresponding to "A" at position 51 of SEQ ID NO: 24. In some instances, the method includes detecting and selecting plants comprising one or more of the markers D6, C10, A10, G6, H6, D10, F10, K6, L6, O6, Q6, and G3. In some instances, the method includes detecting and selecting plants comprising one or more of the markers D7, L4, E7, G7, H7, I7, J7, K7, L7, M7, N7, and O4. In some instances, the method includes detecting and selecting plants comprising each of the markers D6, C10, A10, G6, H6, D10, F10, K6, L6, O6, Q6, and G3. In some instances, the method includes detecting and selecting plants comprising each of the markers D7, L4, E7, G7, H7, I7, J7, K7, L7, M7, N7, and O4.

[0012] On the other hand, this paper provides a method for selecting a maize plant that comprises resistance to southern leaf blight. The method includes screening a maize plant population for the presence of a gene encoding a protein that comprises at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to one or more of SEQ ID NOs: 27, 30, or 33, and selecting a maize plant that comprises the gene. In some instances, the protein comprises 100% amino acid sequence identity to SEQ ID NOs: 27, 30, or 33. In some instances, the method includes crossing the selected maize plant with a second maize plant, and obtaining a progeny plant that comprises the gene. In some examples, the protein is encoded by a gene comprising a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleotide sequence identity to one or more of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31, or SEQ ID NO: 32. In some examples, the protein is encoded by a gene comprising a nucleotide sequence comprising the sequence of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31, or SEQ ID NO: 32.

[0013] On the other hand, this paper provides a method for producing recombinant maize plant cells. The method includes introducing a heterologous P450 gene sequence into the genome of the maize plant cell, and selecting a plant cell comprising a heterologous P450 gene sequence encoded in the plant cell genome. The method can include introducing site-specific modifications of endogenous P450, thereby introducing a gene-edited P450 nucleotide sequence into the plant cell. The site-specific modifications can be induced by CRISPR-related endonucleases. In some instances, the method further includes growing plants from plant cells comprising gene-edited P450 gene sequences. In certain instances, the plant exhibits increased resistance to southern leaf blight. In some instances, the method includes introducing a heterologous P450 transgenic sequence that replaces an endogenous P450 gene. In other instances, the P450 transgenic sequence is inserted into a locus different from the endogenous P450 locus. The aforementioned gene-edited P450 gene sequence or P450 transgene can encode a protein comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one or more of SEQ ID NOs: 27, 30, or 33; for example, a protein comprising the amino acid sequence of SEQ ID NOs: 27, 30, or 33. In some examples, the heterologous P450 gene sequence or P450 transgene comprises a nucleotide sequence comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one or more of SEQ ID NOs: 25, SEQ ID NOs: 26, SEQ ID NOs: 28, SEQ ID NOs: 29, SEQ ID NOs: 31, or SEQ ID NOs: 32; for example, a nucleotide sequence of SEQ ID NOs: 25, SEQ ID NOs: 26, SEQ ID NOs: 28, SEQ ID NOs: 29, SEQ ID NOs: 31, or SEQ ID NOs: 32. Methods for gene editing and introduction of transgenes are described herein.

[0014] In another aspect, the present disclosure provides a recombinant plant comprising a heterologous gene encoding a protein comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to one or more of SEQ ID NOs: 27, 30, or 33. In some examples, the protein comprises 100% amino acid sequence identity to SEQ ID NOs: 27, 30, or 33. In some examples, the gene comprises a nucleotide sequence comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleotide sequence identity to one or more of SEQ ID NOs: 25, 26, 28, 29, 31, or 32. In some examples, the gene comprises the nucleotide sequence of SEQ ID NOs: 25, 26, 28, 29, 31, or 32.

[0015] In another aspect, the present disclosure provides a recombinant plant seed comprising a heterologous gene encoding a protein comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to one or more of SEQ ID NOs: 27, 30, or 33. In some examples, the protein comprises 100% amino acid sequence identity to SEQ ID NOs: 27, 30, or 33. In some examples, the gene comprises a nucleotide sequence comprising at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleotide sequence identity to one or more of SEQ ID NOs: 25, 26, 28, 29, 31, or 32. In some examples, the gene comprises the nucleotide sequence of SEQ ID NOs: 25, 26, 28, 29, 31, or 32.

[0016] Brief Description of Sequence Listing

[0017] The present disclosure can be more fully understood from the following detailed description and sequence listing, which form a part of this application.

[0018] These sequence descriptions and the accompanying sequence listing follow the rules governing disclosure of nucleotide and / or amino acid sequences in patent applications as set forth in 37 CFR §§ 1.831-1.834 and WIPOST.26 standards. The symbols and formats used for nucleotide and amino acid sequence data follow the regulations set forth in 37 CFR § 1.832. Unless otherwise indicated, single letters representing amino acids or nucleotide residues do not have any meaning (e.g., "atgc" is the same as "ATGC," and "MRYG" is the same as "mryg"). Although only one chain is shown, each disclosed nucleotide sequence should be understood to encompass its complementary chain (i.e., reverse complement sequence).

[0019] SEQ ID NO: 1 is the D6 marker sequence, wherein the SNP at position 51 is represented by "y," meaning "c or t." The SNP associated with anti-SLB resistance includes "c" at position 51.

[0020] SEQ ID NO: 2 is a C10 marker sequence, wherein the SNP at position 51 is represented by "y," meaning "c or t." The SNP associated with anti-SLB resistance includes "c" at position 51.

[0021] SEQ ID NO: 3 is the A10 marker sequence, wherein the SNP at position 51 is represented by "y," meaning "c or t." The SNP associated with anti-SLB resistance includes "c" at position 51.

[0022] SEQ ID NO: 4 is a G6 marker sequence, wherein the SNP at position 51 is represented by "s," meaning "g or c." The SNP associated with anti-SLB resistance contains "g" at position 51.

[0023] SEQ ID NO: 5 is the H6 marker sequence, wherein the SNP at position 51 is represented by "r," meaning "g or a." The SNP associated with anti-SLB resistance includes "g" at position 51.

[0024] SEQ ID NO: 6 is the D10 marker sequence, wherein the SNP at position 51 is represented as "y," meaning "t or c." The SNP associated with anti-SLB resistance includes a "t" at position 51.

[0025] SEQ ID NO: 7 is the F10 marker sequence, wherein the SNP at position 51 is represented as "m," meaning "c or a." The SNP associated with anti-SLB resistance includes "c" at position 51.

[0026] SEQ ID NO: 8 is a K6 marker sequence, wherein the SNP at position 51 is represented as "m," meaning "a or c." The SNP associated with anti-SLB resistance includes "a" at position 51.

[0027] SEQ ID NO: 9 is the L6 marker sequence, wherein the SNP at position 51 is represented as "y," meaning "c or t." The SNP associated with anti-SLB resistance includes "c" at position 51.

[0028] SEQ ID NO: 10 is the O6 marker sequence, wherein the SNP at position 51 is represented by "y," meaning "t or c." The SNP associated with anti-SLB resistance includes "t" at position 51.

[0029] SEQ ID NO: 11 is the Q6 marker sequence, wherein the SNP at position 51 is represented as "k," meaning "g or t." The SNP associated with anti-SLB resistance includes "g" at position 51.

[0030] SEQ ID NO: 12 is a G3 marker sequence, wherein the SNP at position 51 is represented by "y," meaning "c or t." The SNP associated with anti-SLB resistance contains a "C" at position 51.

[0031] SEQ ID NO: 13 is the D7 marker sequence, wherein the SNP at position 51 is represented as "y," meaning "t or c." The SNP associated with anti-SLB resistance includes a "t" at position 51.

[0032] SEQ ID NO: 14 is an L4 marker sequence, wherein the SNP at position 51 is represented as "w," meaning "t or a." The SNP associated with anti-SLB resistance includes a "t" at position 51.

[0033] SEQ ID NO: 15 is the E7 marker sequence, wherein the SNP at position 51 is represented by "y," meaning "t or c." The SNP associated with anti-SLB resistance includes "t" at position 51.

[0034] SEQ ID NO: 16 is a G7 marker sequence, wherein the SNP at position 51 is represented by "r," meaning "a or g." The SNP associated with anti-SLB resistance includes "a" at position 51.

[0035] SEQ ID NO: 17 is the H7 marker sequence, wherein the SNP at position 51 is represented as "k," meaning "g or t." The SNP associated with anti-SLB resistance includes "g" at position 51.

[0036] SEQ ID NO: 18 is the I7 marker sequence, wherein the SNP at position 51 is represented as "y," meaning "t or c." The SNP associated with anti-SLB resistance includes a "t" at position 51.

[0037] SEQ ID NO: 19 is the J7 marker sequence, wherein the SNP at position 51 is represented as "y," meaning "t or c." The SNP associated with anti-SLB resistance includes "t" at position 51.

[0038] SEQ ID NO: 20 is the K7 marker sequence, wherein the SNP at position 51 is represented by "r," meaning "g or a." The SNP associated with anti-SLB resistance includes "g" at position 51.

[0039] SEQ ID NO: 21 is an L7 marker sequence, wherein the SNP at position 51 is represented by "r," meaning "a or g." The SNP associated with anti-SLB resistance includes "a" at position 51.

[0040] SEQ ID NO: 22 is the M7 marker sequence, wherein the SNP at position 51 is represented as "y," meaning "t or c." The SNP associated with anti-SLB resistance includes a "t" at position 51.

[0041] SEQ ID NO: 23 is the N7 marker sequence, wherein the SNP at position 51 is represented by "r," meaning "a or g." The SNP associated with anti-SLB resistance includes "a" at position 51.

[0042] SEQ ID NO: 24 is the O4 marker sequence, wherein the SNP at position 51 is represented by "w," meaning "a or t." The SNP associated with anti-SLB resistance includes "a" at position 51.

[0043] SEQ ID NO: 25 is the nucleic acid genomic sequence of the P450 gene of CIMBL83. The sequence includes the endogenous promoter and terminator regions.

[0044] SEQ ID NO: 26 is a nucleic acid coding sequence (eg, cDNA) encoding the P450 protein of CIMBL83.

[0045] SEQ ID NO: 27 is the amino acid sequence encoded by the P450 gene of CIMBL83.

[0046] SEQ ID NO: 28 is the nucleic acid genomic sequence of the P450 gene of CML304, which includes the endogenous promoter and terminator regions.

[0047] SEQ ID NO: 29 is a nucleic acid coding sequence (eg, cDNA) encoding the P450 protein of CML304.

[0048] SEQ ID NO: 30 is the amino acid sequence encoded by the P450 gene of CML304.

[0049] SEQ ID NO: 31 is the nucleic acid genomic sequence of the P450 gene of K22. The sequence includes the endogenous promoter and terminator regions.

[0050] SEQ ID NO: 32 is a nucleic acid coding sequence (eg, cDNA) encoding the P450 protein of K22.

[0051] SEQ ID NO: 33 is the amino acid sequence encoded by the P450 gene of K22.

[0052] SEQ ID NO: 34 is a G8 marker sequence, wherein the SNP at position 51 is represented by "r," meaning "a or g." The SNP associated with anti-SLB resistance includes "a" at position 51. DETAILED DESCRIPTION

[0053] The present disclosure provides markers that have been identified as genetically linked to a locus that confers resistance to SLB. The present disclosure also provides methods for using the markers to select resistant plants or counterselect susceptible plants. Also provided herein are maize plants that contain newly conferred or increased resistance to SLB relative to control plants. Also provided are causal genes that, when expressed in plants, provide newly conferred or increased resistance and plants expressing these causal genes.

[0054] definition

[0055] The following definitions are provided to aid understanding of this disclosure.

[0056] The present disclosure is not limited to specific examples, which can of course vary. The terms and illustrative examples used herein are for the purpose of describing specific aspects of the present disclosure only and are not intended to be restrictive. As used herein, singular and singular forms of terms such as "a, an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, the term "plant," "the plant," or a plant also includes a variety of plants; also depending on the context, the term "plant" used may also include progeny that are genetically similar or identical to the plant; the term "nucleic acid" used may actually optionally include multiple copies of the nucleic acid molecule; similarly, the term "probe" optionally (and typically) covers many similar or identical probe molecules.

[0057] Unless otherwise indicated, nucleic acids are written from left to right in a 5' to 3' direction. Numerical ranges recited in the specification are inclusive of the numbers defining the range and include each integer or any non-integer fraction within the defined range. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs. In describing and claiming the presently disclosed subject matter, the following terminology will be used in accordance with the definitions set forth below.

[0058] The term "allele" refers to one of two or more different nucleotide sequences occurring at a particular genetic locus.

[0059] " allele frequency " refers to the frequency (ratio or percentage) that allele exists at the locus in individual, strain or the colony of strain.For example, for allele " A", the diploid individual of genotype " AA ", " Aa " or " aa " has an allele frequency of 1.0, 0.5 or 0.0 respectively. The allele frequency in this strain can be estimated by averaging the allele frequency of the individual sample from a strain. Similarly, the allele frequency in this strain colony can be calculated by averaging the allele frequency of the strain that forms a colony.For the colony with a limited number of individual or strain, allele frequency can be expressed as the count of the individual or strain (or any other specified grouping) that comprises this allele.

[0060] The term "amplification" in the context of nucleic acid amplification is any method by which additional copies of a selected nucleic acid (or its transcribed form) are produced. Typical amplification methods include various polymerase-based replication methods, including polymerase chain reaction (PCR), ligase-mediated methods (such as ligase chain reaction (LCR)), and RNA polymerase-based amplification (e.g., by transcription) methods.

[0061] The term "assembly" is applicable to BACs and the tendency of BACs to come together to form a continuous extension of DNA. If BACs are sequenced, or by comparing their BAC fingerprints with the fingerprints of other BACs, the BACs are "assembled" into contigs based on sequence alignments. Public assembly can be found using the publicly available maize genome browser (Maize Genome Browser) on the internet.

[0062] 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 an allele is an indicator of how the trait will be expressed. For example, if an allele is part of or linked to a gene that confers resistance to SLB, then the allele may be linked to resistance to SLB.

[0063] "BAC" or bacterial artificial chromosome is a cloning vector derived from the naturally occurring F factor of E. coli, which itself is a DNA element that can exist as a circular plasmid or can be integrated into the bacterial chromosome. BACs can accept large inserts of DNA sequences. In maize, many BACs (each of which contains large inserts of maize genomic DNA from the maize inbred line B73) have been assembled into contigs (overlapping continuous genetic fragments or "continuous DNA"), and the assembly is publicly available on the Internet.

[0064] Centimorgan ("cM") is a unit of measure for recombination frequency. One cM equals a 1% chance that a marker at one genetic locus will segregate with a marker at a second locus due to crossing over in a single generation.

[0065] As used herein, the term "chromosomal interval" refers to a continuous linear span of genomic DNA present on a single chromosome of a plant. Genetic elements or genes located in a single chromosomal interval are physically linked. The size of a chromosomal interval is not particularly limited. In some aspects, genetic elements located within a single chromosomal interval are genetically linked, typically having a genetic recombination distance of, for example, less than or equal to 20 cM, or alternatively, less than or equal to 10 cM. ​​That is, two genetic elements within a single chromosomal interval recombine at a frequency of less than or equal to 20% or 10%.

[0066] A "chromosome" is a single piece of spiral DNA containing many genes that function and move as a unit during cell division and can therefore be considered linked. It can also be called a "linkage group."

[0067] The term "continuous DNA" refers to an uninterrupted stretch of genomic DNA represented by partially overlapping fragments or contigs.

[0068] The term "hybridized" or "crossing" refers to sexual crossing and involves the fusion of two haploid gametes by pollination to produce diploid progeny (e.g., cells, seeds, or plants). The term encompasses both pollination of one plant by another and selfing (or self-pollination, e.g., when the pollen and ovules are from the same plant).

[0069] Plants referred to herein as "diploid" have two sets of chromosomes.

[0070] Plants referred to herein as "doubled haploids" are developed by doubling the haploid chromosome set (ie, half the normal number of chromosomes). Doubled haploid plants have two identical sets of chromosomes, and all gene loci are considered homozygous.

[0071] An "elite line" is any line resulting from breeding and selection for the performance of superior agronomic traits.

[0072] "Exotic maize varieties" or "exotic maize germplasm" are varieties of maize plants that are not among the available elite maize lines or germplasm varieties. In the case of a cross between two maize plants or germplasm varieties, the offspring of the exotic germplasm are not closely related to the elite germplasm with which it was crossed. Most commonly, the exotic germplasm is not derived from any known elite maize line, but is selected to introduce new genetic elements (usually new alleles) into a breeding program.

[0073] A "favorable allele" is an allele at a particular locus that confers or contributes to an agronomically desirable phenotype (eg, SLB resistance). A favorable allele of a marker is a marker allele that segregates (eg, is linked) with the favorable phenotype.

[0074] "Fragment" is intended to mean a portion of a nucleotide sequence. Fragments can be used as hybridization probes or PCR primers using the methods disclosed herein.

[0075] " Genetic map " is the description of the genetic linkage relationship between the loci on one or more chromosomes (or linkage groups) in a given species, usually described in the form of a chart or table.For each genetic map, the distance between the loci is measured by the frequency (recombination frequency of the loci) of the alleles of these loci occurring together in a colony. DNA or protein markers or observable phenotypes can be used to detect alleles. Genetic map is the product of the polymorphism potential of each marker between the colony, the type of marker used and different colonies used for mapping. For different genetic maps, the genetic distance between the loci can be different. However, universal markers can be used to associate information from a map to another map. Those of ordinary skill in the art can use universal marker positions to identify the position of markers and other target loci on each individual genetic map. Although due to, for example, detecting the marker of alternating repeated loci in different colonies, the difference, new mutation or laboratory error in the statistical method for sorting these markers, there is often a small change in the marker order, the locus order should not change between the maps.

[0076] A "genetic map location" is the location on a genetic map where a specified marker can be found within a given species, relative to surrounding genetic markers on the same linkage group.

[0077] "Genetic mapping" is a method of defining the linkage relationships of genetic loci through the use of genetic markers, population segregation for those markers, and standard genetic principles of recombination frequency.

[0078] "Genetic marker", "mark" or "molecular marker" are nucleic acids that are polymorphic in a colony, and the alleles of this genetic marker can be detected and distinguished by one or more analytical methods described below. These terms also refer to nucleic acid sequences complementary to the genomic sequences (e.g., nucleic acids) used as probes. The mark corresponding to the genetic polymorphism between the colony members can be detected by analytical methods, such as sequence-specific amplification methods based on PCR, restriction fragment length polymorphism detection (RFLP), isozyme marker detection, polynucleotide polymorphism detection by allele-specific hybridization (ASH), amplification variable sequence detection of plant genomes, autonomous sequence replication detection, simple repeat sequence detection (SSR), single nucleotide polymorphism detection (SNP), amplified fragment length polymorphism detection (AFLP) or next generation sequencing technology. Other appropriate methods include detecting expressed sequence tags (ESTs) and SSR markers derived from EST sequences, and randomly amplified polymorphic DNA (RAPD). In addition to nucleic acids, "genetic marker", "mark" or "molecular marker" can also refer to proteins or genetic phenotypes that are known or expected to be linked to favorable alleles by its causal gene.

[0079] For example, provided herein are markers, such as the SNPs in Table 1, that are linked to favorable alleles that provide SLB resistance. As used herein, "marker allele" refers to a specific sequence at a marker locus that is linked to a favorable allele.

[0080] "Genetic recombination frequency" is the frequency of crossover events (recombination) between two genetic loci. Recombination frequency can be observed by following the segregation of markers and / or traits after meiosis.

[0081] "Genome" refers to the total DNA or entire set of genes carried by an individual.

[0082] The term "genotype" refers to the genetic makeup of an individual (or individual group) at one or more genetic loci. Genotype is defined by one or more alleles of one or more known loci of the individual's inheritance from one or more parents. The term genotype can be used to refer to the genetic makeup of an individual at a single locus, a plurality of loci, or more generally, the term genotype can be used to refer to the individual genetic makeup of all genes in its genome.

[0083] " Germplasm " refers to genetic material, and it belongs to or comes from an individual (for example, plant), an individual group (for example, plant strain, variety or family) or the clone derived from strain, variety, species or culture, or more generally, all individuals of a certain species or a plurality of species (for example, maize germplasm collection (maize germplasm collection) or Andean germplasm collection (Andean germplasm collection)). Germplasm can be a part of an organism or cell, or can be separated from this organism or cell. Generally speaking, germplasm provides the genetic material with specific molecular composition, and this specific molecular composition provides the physical basis for some or all of the genetic qualities of an organism or cell culture. As used herein, germplasm comprises the cell, seed or tissue from which new plants can be grown, or the plant part that can be cultured into whole plant, for example leaf, stem, pollen or cell.

[0084] Plants called "haploid" have a single set of chromosomes (genome).

[0085] A "haplotype" is an individual's genotype, ie, a combination of alleles, at a plurality of genetic loci. Typically, the genetic loci described by a haplotype are physically and genetically linked, ie, on the same chromosome segment.

[0086] The term "heterogeneity" is used to indicate that individuals within a group differ in their genotype at one or more specific loci.

[0087] The heterotic response or "heterosis" of a material can be defined by its performance above the average of a parent (or high parent) when crossed to an otherwise dissimilar or unrelated group.

[0088] An individual is "heterozygous" if more than one allele type is present at a given locus (eg, a diploid individual having one copy of each of two different alleles).

[0089] The term "homogeneity" means that members of a group have the same genotype at one or more specific loci.

[0090] An individual is "homozygous" if the individual has only one type of allele at a given locus (eg, a diploid individual has a copy of the same allele at the locus on each of two homologous chromosomes).

[0091] The term "hybrid" refers to the progeny obtained between a cross of at least two genetically different parents.

[0092] "Hybridization" or "nucleic acid hybridization" refers to the pairing of complementary RNA and DNA strands, as well as the pairing of complementary single strands of DNA.

[0093] The term "hybridization" means the formation of base pairs between complementary regions of nucleic acid strands.

[0094] The IBM genetic map is a set of genetic maps that is used to map the genetic information of the maize genotype. The IBM genetic map is a set of genetic maps that is used to map the genetic information of the maize genotype. The IBM genetic map is a set of genetic maps that is used to map the genetic information of the maize genotype. The IBM genetic map is a set of genetic maps that is used to map the genetic information of the maize genotype. The IBM genetic map is a set of genetic maps that is used to map the genetic information of the maize genotype. The IBM genetic map is a set of genetic maps that is used to map the genetic information of the maize genotype. The IBM genetic map is a set of genetic maps that is used to map the genetic information of the maize genotype. The IBM genetic map is a set of genetic maps that is used to map the genetic information of the maize genotype.

[0095] The term "inbred line" refers to a line that has been bred to achieve genetic homogeneity.

[0096] The term "indel" refers to an insertion or deletion, wherein one strain can be referred to as containing an inserted nucleotide or DNA fragment relative to a second strain, or the second strain can be referred to as containing a deleted nucleotide or DNA fragment relative to the first strain.

[0097] As used herein, "introduction" means presenting a polynucleotide or polypeptide to a plant or plant cell in such a manner that the sequence enters the interior of the cell. The methods disclosed herein do not depend on a particular method for introducing a polynucleotide or polypeptide into a plant, as long as the polynucleotide or polypeptide enters the interior of at least one cell of the plant. Methods for introducing a polynucleotide or polypeptide into a plant include, but are not limited to, stable transformation, transient transformation, and viral-mediated methods.

[0098] As used herein, "stable transformation" means that the nucleotide construct introduced into a plant is integrated into the genome of the plant and can be inherited by its progeny. As used herein, "transient transformation" means that a polynucleotide is introduced into a plant and is not integrated into the genome of the plant, or that a polypeptide is introduced into a plant. As used herein, "plant" refers to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, and embryos and progeny thereof. Plant cells can be differentiated or undifferentiated (e.g., callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells, and pollen).

[0099] Transformation protocols, as well as protocols for introducing nucleotide sequences into plants, vary depending on the type of plant or plant cell (ie, monocot or dicot) being targeted for transformation. Suitable methods for introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and 5,981,840), direct gene transfer (Paszkowski et al. (1984) EMBO J 3:2717-2722), and ballistic particle acceleration (see, e.g., U.S. Pat. Nos. 4,945,050; 5,879,918; 5,886,244 and 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Biology). Methods [Plant Cell, Tissue and Organ Culture: Basic Methods], Gamborg and Phillips, eds. (Springer-Verlag, Berlin; and McCabe et al. (1988) Biotechnology 6:923-926); and the Lec1 transformation method (WO 00 / 28058). For potato transformation, see Tu et al. (1998) Plant Molecular Biology 37:829-838 and Chong et al. (2000) Transgenic Research 9:71-78. Additional transformation methods can be found in Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio / Technology 6:923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol.In Vitro Cell and Developmental Biology 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); U.S. Patent Nos. 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) The Experimental Manipulation of Ovule Tissues, Chapman et al., eds. (Longman, New York), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Biology 11:117-119 (pollen); 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).

[0100] The term "infiltration" refers to the phenomenon that the allelomorph of the expectation of the genetic locus is passed to another kind of genetic background from a kind of genetic background.For example, the allelomorphic infiltration of the expectation at the specified locus can be passed to at least one filial generation via the sexual hybridization between two parents of the same species, wherein at least one of these parents has the allelomorph of this expectation in its genome. Alternatively, for example allelomorphic transmission can occur by the reorganization between two donor genomes, and for example, in fusion protoplasts, wherein at least one of the donor protoplasts has the allelomorph of expectation in its genome. The allelomorph of expectation can, for example, detect at QTL, transgenic etc. by the mark associated with phenotype. In any case, the allelomorphic offspring that comprises expectation can repeatedly backcross with the strain that comprises the expected genetic background and select for the allelomorph of expectation, to produce the allelomorph that is fixed in the genetic background of selection.

[0101] When the process of sexual hybridization is repeated two or more times, the process of "introgression" is often called "backcrossing."

[0102] "Backcrossing" refers to a method by which hybrid progeny are backcrossed to one of the parents. In a backcrossing protocol, the "donor" parent refers to the parent plant with the desired gene or genes, loci, or specific phenotype to be infiltrated. The "recipient" parent (used one or more times) or "recurrent" parent (used two or more times) refers to the parent plant to which the gene or locus is backcrossed. Repeated backcrossing can result in the "introgression" of alleles 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 [Marker-assisted backcrossing: a practical example, in Techniques et Utilisations des Marqueurs Moleculaires Les Colloques], Vol. 72, pp. 45-56, and Openshaw et al. (1994) Marker-assisted Selection in Backcross Breeding, Analysis of Molecular Marker Data, pp. 41-43. The initial cross produces an F1 generation; the term "BC1" 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 using the recurrent parent for the sixth time.

[0103] A "line" or "variety" is a group of individuals having the same parents, which are usually inbred to some extent and are usually homozygous and homogeneous (isogenic or near isogenic) at most gene loci. A "subline" refers to a subpopulation of inbred lines that is genetically different from other similar subpopulations of inbred lines that originated from the same ancestor.

[0104] As used herein, term " linkage " is used to describe the degree of association of a kind of marker locus and another kind of marker locus, favorable allelomorph or some other loci.The linkage relationship between molecular marker and the locus (for example, favorable allelomorph) that affects phenotype is represented with " probability " or " probability of adjustment ".Linkage can be expressed as the restriction or scope of expectation.For example, in some instances, when any mark and any other mark are separated less than 50,40,30,25,20 or 15 maps from unit (or cM) on single meiosis map (based on the genetic map of colony (for example, as, F2) that has carried out a round meiosis; IBM2 map is made up of multiple meiosis), these marks are linked (hereditarily or physically).In some respects, it is advantageous to limit the linkage range of bracketing, for example, between 10cM and 20cM, between 10cM and 30cM or between 10cM and 40cM. The tighter the linkage of a marker to a second locus, the better the marker indicates the second locus.

[0105] The term "linkage disequilibrium" refers to the non-random segregation of genetic loci or traits (or both). In either case, linkage disequilibrium means that the related loci are physically close enough along a section of chromosome so that they separate together at a frequency higher than random (i.e., non-random). Markers showing linkage disequilibrium are considered to be linked. Linked loci have a chance of more than 50% (e.g., about 51% to about 100%) of co-segregation. In other words, two co-segregating markers have a recombination frequency of less than 50% (and, by definition, are separated by less than 50 cM on the same linkage group). As used herein, linkage can be present between two markers, or alternatively, between a marker and a locus that affects the phenotype.

[0106] Linkage disequilibrium is most commonly measured using r 2 The metric was calculated using the formula in Hill, WG and Robertson, A, Theor. Appl. Genet. 38: 226-231 (1968). 2 =1, there is complete LD between the two marker loci, meaning that these markers have not yet undergone recombination and have the same allele frequency. 2 The value will depend on the population used. 2A value greater than 1 / 3 indicates sufficiently strong LD for mapping (Ardlie et al., Nature Reviews Genetics 3:299-309 (2002)). 2 When the value is greater than or equal to 0.33, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0, the alleles are in linkage disequilibrium.

[0107] As used herein, "linkage equilibrium" describes a situation in which two markers segregate independently, i.e., randomly distribute among progeny. Markers that exhibit linkage equilibrium are considered unlinked (regardless of whether they are located on the same chromosome).

[0108] " logarithm of advantage (LOD) value " or " LOD score " (Risch, Science [science] 255:803-804 (1992)) are used for genetic interval mapping to describe the linkage degree between two marker loci. Between two marks, LOD score is three, indicating that the linkage probability is 1000 times that of no linkage probability, while LOD score is two, indicating that the linkage probability is 100 times that of no linkage probability. An LOD score greater than or equal to two can be used to detect linkage. LOD score can also be used to display the strength of association between marker locus and the quantitative trait in " quantitative trait locus " mapping. In this case, the LOD score size depends on the compactness between this marker locus and the locus that affects this quantitative trait, and the size of this quantitative trait effect.

[0109] "Maize" refers to the Zea mays plant, also known as "corn."

[0110] The term "maize plant" includes whole maize plants, maize plant cells, maize plant protoplasts, maize plant cells or maize tissue cultures from which maize plants can be regenerated, maize plant callus, maize plant clumps, and intact maize plant cells or maize plant parts within a maize plant, such as maize seeds, maize cobs, maize flowers, maize cotyledons, maize leaves, maize stems, maize sprouts, maize roots, maize root tips, and the like.

[0111] "Marker-assisted selection" (MAS) is a method of selecting individual plants based on their marker genotype.

[0112] "Marker-assisted counter-selection" is a method whereby marker genotypes are used to identify plants that are not to be selected, allowing these plants to be removed from the breeding program or planting.

[0113] A "marker haplotype" refers to the combination of alleles at a marker locus.

[0114] "Marker locus" is a specific chromosomal location in a species genome where specific markers can be found. The marker locus can be used to track the presence of a second linked locus (e.g., a linked locus that affects phenotypic trait expression). For example, the marker locus can be used to monitor the separation of alleles at a genetically or physically linked locus.

[0115] " marker probe " is a nucleic acid sequence or molecule that can be used to identify the presence of a marker locus by nucleic acid hybridization, such as a nucleic acid probe complementary to a marker locus sequence. A marker probe comprising 30 or more continuous nucleotides of the marker locus (" all or part " of the marker locus sequence) can be used for nucleic acid hybridization. Alternatively, in some aspects, a marker probe refers to a probe that can distinguish any type (i.e., genotype) of a specific allele present at the marker locus.

[0116] An allele is "negatively" associated with a trait when the allele is linked to the trait and when the presence of the allele is an indication that the desired trait or form of the trait will not appear in a plant containing the allele.

[0117] "Nucleotide" is the monomeric unit that makes up the DNA or RNA polymer and is composed of a purine or pyrimidine base, a pentose sugar, and a phosphate group. Nucleotides (usually found in their 5'-monophosphate form) are represented by their single-letter names as follows: "A" represents adenylic acid or deoxyadenylic acid (for RNA or DNA, respectively), "C" represents cytidylic acid or deoxycytidylic acid, "G" represents guanylic acid or deoxyguanylic acid, "U" represents uridylic acid, "T" represents deoxythymidylic acid, "R" represents a purine (A or G), "Y" represents a pyrimidine (C or T), "K" represents G or T, "H" represents A or C or T, "I" represents inosine, and "N" represents any nucleotide.

[0118] A "physical map" of a genome is a map showing the linear order of identifiable markers (including genes, markers, etc.) on chromosomal DNA. However, in contrast to a genetic map, the distances between markers are absolute (e.g., measured in base pairs or isolated and overlapping contiguous genetic segments) and are not based on genetic recombination (which can vary in different populations).

[0119] A "plant" may be a whole plant, any part thereof, or a cell or tissue culture derived from a plant.

[0120] A "polymorphism" is a variation in the DNA between two or more individuals within a population. A polymorphism preferably has a frequency of at least 1% in a population. Useful polymorphisms can include SNPs, simple sequence repeats (SSRs), or insertion / deletion polymorphisms (also referred to herein as "indels").

[0121] An allele is "positively" associated with a trait when the allele is linked to the trait and when the presence of the allele is an indicator that the desired trait or form of the trait will appear in a plant containing the allele.

[0122] The term "progeny" refers to the offspring produced by a cross.

[0123] A "progeny plant" is a plant produced by a cross between two plants.

[0124] 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 tightly linked to a nucleic acid sequence (e.g., one or more genes) that affects the trait in question.

[0125] As used herein, "recombinant" plants or plant cells or "recombinant" nucleic acids or proteins / peptides are plants, plant cells, nucleic acids or proteins / peptides comprising heterologous nucleic acid sequences (which encode heterologous proteins / peptides or non-coding RNAs). As used herein, "heterologous" means that a given nucleic acid sequence has been located in a genome, locus or construct by human intervention. A heterologous sequence can be naturally occurring, but is now located in a genome, locus or construct where the sequence is not naturally found. Alternatively, a sequence can be "heterologous" because it is located in a genome, locus or construct and is non-naturally occurring. For example, a P450 gene can be naturally occurring in a plant, but is "heterologous" because the gene is now encoded in a plant genome that did not previously encode the gene or encodes a different allele of the gene. A heterologous gene can be inserted into the genome via, for example, conversion and / or a method based on site-specific nucleases.

[0126] A "reference sequence" or "consensus sequence" is a defined sequence used as the basis for sequence alignment. A labeled reference sequence can be obtained by sequencing multiple lines at the locus, aligning these nucleotide sequences in a sequence alignment program (e.g., Sequencher), and then obtaining the most common nucleotide sequence for the alignment. Polymorphisms found in these individual sequences are annotated in the consensus sequence. A reference sequence is generally not an exact copy of any individual DNA sequence, but rather represents a mix of available sequences and is used to design primers and probes for polymorphisms within the sequence.

[0127] As used herein, "Southern leaf blight (SLB) resistance" refers to increased resistance or tolerance to a fungal pathogen that causes SLB when compared to a control plant containing less resistance. The effects of resistance can vary from a slight increase in tolerance to a fungal pathogen (e.g., partial inhibition of pathogenesis) to complete resistance, rendering the plant unaffected by the presence of the fungal pathogen. Examples of the present disclosure provide materials and methods for increasing resistance to fungal pathogens that cause SLB.

[0128] A "topcross test" is a test performed by crossing each individual (eg, a selection, inbred line, clone, or progeny individual) to the same pollen parent or "tester" (usually a homozygous line).

[0129] The phrase "under stringent conditions" refers to conditions under which a probe or polynucleotide will hybridize to a specific nucleic acid sequence, typically in a complex mixture of nucleic acids, but essentially without other sequences. Stringent conditions are sequence-dependent and will vary in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally speaking, stringent conditions are typically selected to be about 5°C-10°C lower than the thermal melting point (Tm) for a specific sequence at a defined ionic strength and pH. The Tm is the temperature at which, under defined ionic strength, pH, and nucleic acid concentration, 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (because there is an excess of target sequence, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions should be those at a pH of 7.0 to 8.3, a salt concentration of less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts), and a temperature of at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., more than 50 nucleotides). Stringent conditions can also be achieved by adding destabilizing agents such as formamide. For selective hybridization or specific hybridization, a positive signal is at least twice the background, preferably 10 times the background hybridization. Exemplary stringent hybridization conditions are typically: 50% formamide, 5x SSC, and 1% SDS, incubated at 42°C, or 5x SSC, 1% SDS, incubated at 65°C, and washed with 0.2x SSC and 0.1% SDS at 65°C. For PCR, a temperature of about 36°C is typical for low stringency amplification, while the annealing temperature depends on the primer length and can vary between about 32°C and 48°C. Additional guidelines for determining hybridization parameters are provided in multiple references.

[0130] An "unfavorable allele" of a marker is a marker allele that segregates with the unfavorable plant phenotype, thus providing the benefit of identifying plants that can be removed from a breeding program or planting.

[0131] The term "yield" refers to the productivity per unit area of ​​a specific plant product with commercial value. For example, maize yield is generally measured in bushels of seeds per season / acre or metric tons of seeds / hectare. Yield is affected by both heredity and environmental factors." agronomy," "agronomic traits" and "agronomic trait performance" refer to the proterties (and potential genetic elements) of a given plant variety, which contribute to yield during the vegetative period. Individual agronomic traits include vigor of emergence, nutritional potential, stress tolerance, disease resistance or tolerance, herbicide resistance, branching, flowering, seed formation, seed size, seed density, lodging resistance, threshing ability, etc. Therefore, yield is the ultimate apex of all agronomic traits.

[0132] Sequence alignments and percent identity calculations can be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, LASERGENE Bioinformatics computing package (DNASTAR MEGALIGN, Inc., Madison, Wisconsin Program. Unless otherwise stated, the multiple alignments of the sequences provided herein were performed using the CLUSTAL V method of alignment (Higgins and Sharp, CABIOS. [Computers in Biology] 5: 151153 (1989)) and default parameters (gap penalty = 10, gap length penalty = 10). The default parameters for calculating the percent identity of each pair and protein sequence using the CLUSTAL V method were KTUPLE = 1, gap penalty = 3, window (WINDOW) = 5, and diagonal (DIAGONALS SAVED) = 5. For nucleic acids, these parameters were KTUPLE = 2, gap penalty = 5, window = 4, and diagonal (DIAGONALS SAVED) = 4. After aligning the sequences using the CLUSTAL V program, it was possible to obtain "percent identity" and "divergence" values ​​by viewing the "sequence distance" table in the same program. Unless otherwise stated, the percent identity and divergence provided herein and required are calculated in this manner.

[0133] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, EF, and Maniatis, T., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter "Sambrook").

[0134] QTL location and causative genes

[0135] The present disclosure provides a QTL on maize chromosome 3 that has been identified as being associated with SLB resistance (see Example 1). The QTL is named qSLB3.1. In the work described herein, three different inbred lines were used as donor plants: CIMBL83, CML304, and K22. The markers for the QTLs for CIMBL83 and CML304 are located in an interval on maize chromosome 3 that is flanked by and includes the following: D6 marker and G3 marker. The marker for the QTL for K22 is located in an interval on maize chromosome 3 that is flanked by and includes the following: D7 marker and O4 marker. The markers overlap significantly, with the endpoints of each QTL corresponding to positions 30, 542, 688 and 36, 339, 580 of the B73 genome or positions 30, 685, 578 and 36, 015, 815 of the B73 genome. Without wishing to be bound by theory, it is believed that the K22 QTL and the CIMBL83 and CML304 QTL comprise the same causal gene. SEQ ID NOs: 25-33 disclose the sequences of the genes believed to be the causal genes for the corresponding QTL for each inbred line.

[0136] Genetic mapping

[0137] The specific genetic loci associated with a particular phenotype (such as disease resistance) can be mapped in the genome of an organism. Plant breeders can advantageously use molecular markers to identify desired individuals by detecting marker alleles that show statistically significant probability of cosegregation with the desired phenotype, exhibiting linkage disequilibrium. By identifying molecular markers or molecular marker clusters that cosegregate with the desired trait, breeders can rapidly select the desired phenotype by selecting suitable molecular marker alleles (a method known as marker-assisted selection or MAS).

[0138] Various methods can be used for detecting molecular markers or molecular marker clusters that are co-segregated with the purpose proterties (for example disease resistance proterties).The basic concept of these methods is to detect the markers of alternative genotypes (or allelotrope) with significantly different average phenotypes.Therefore, the difference size between the alternative genotypes (or allelotrope) between the comparative marker loci or the significance level of this difference.Infer that the proterties gene is located at the position of one or more markers of the genotypic difference with maximum correlation closest.Two methods for detecting the purpose proterties loci like this are: 1) association analysis (i.e. association mapping) based on colony and 2) traditional linkage analysis.

[0139] Association mapping

[0140] Understanding the degree and pattern of linkage disequilibrium (LD) in the genome is a prerequisite for developing an effective, in order to identify and draw an association method for quantitative trait loci (QTL). LD refers to the non-random association of alleles in an individual collection. When observing LD in the allele at the locus of a linkage, LD is measured as the LD decay in the specific region across chromosome. The degree of LD reflects the recombination history in this region. The average rate of LD decay in the genome can help predict the quantity and density of the markers required for whole genome association studies, and provides an estimated value of the resolution that can be expected.

[0141] Association or LD mapping aims to identify significant genotype-phenotype associations. It has been developed as a powerful tool for fine mapping in outcrossing species such as humans (Corder et al. (1994) "Protective effect of apolipoprotein-E type-2 allele for late-onset Alzheimer-disease," Nat Genet 7:180-184; Hastbacka et al. (1992) "Linkage disequilibrium mapping in isolated founder populations: diastrophic dysplasia in Finland," Nat Genet 2:204-211; Kerem et al. (1989) "Identification of the cystic fibrosis gene: genetic analysis [Identification of cystic fibrosis genes: genetic analysis], Science 245:1073-1080) and maize (Remington et al. (2001) “Structure of linkage disequilibrium and phenotype associations in the maize genome,” Proc Natl Acad Sci USA 98:11479-11484; Thornsberry et al. (2001) “Dwarf8 polymorphisms associate with variation in flowering time,” Nat Genet 28:286-289; reviewed by Flint-Garcia et al. (2003) “Structure of linkage disequilibrium in plants,” Annu Rev Plant Biol. [Annual Review of Plant Biology] 54:357-374), in which recombination between heterozygotes is frequent and leads to rapid decay of LD.In inbred species, where recombination between homozygous genotypes is not genetically detectable, the extent of LD is greater (i.e., larger blocks of linked markers are inherited together) and this greatly increases the detective power of association mapping (Wall and Pritchard, (2003) "Haplotype blocks and linkage disequilibrium in the human genome", Nat Rev Genet 4:587-597).

[0142] The reorganization of colony and sudden change history are the function of the effective size of mating habit and colony and age.Bigger colony size provides the possibility of enhancing for detecting reorganization, and older colony is usually relevant to the polymorphism of higher level, and both of these cause the remarkable acceleration of LD decay rate.On the other hand, smaller effective colony size, for example those colonies that have experienced the recent genetic bottleneck, tend to show slower LD decay rate, cause more widely haplotype conservatism (people such as Flint-Garcia, (2003) " Structure of linkage disequilibrium in plants [structure of plant linkage disequilibrium] ", Annu Rev Plant Biol. [plant biology annual review] 54:357-374).

[0143] Elite breeding lines provide a valuable starting point for association analyses. Association analyses use quantitative phenotypic scores (e.g., a disease tolerance rating from one to nine for each line) in the analysis (rather than considering only the tolerance vs. resistance allele frequency distribution in the type of allele distribution between groups analyzed). The availability of detailed phenotypic performance data collected over many years through breeding programs and the environment of a large number of elite lines provide valuable data sets for genetic marker association mapping analyses. This paves the way for seamless integration between research and application, and takes advantage of historically accumulated data sets. However, understanding the relationship between polymorphism and recombination is useful for developing appropriate strategies for efficiently extracting maximum information from these resources.

[0144] This type of association analysis neither generates nor requires any map data, but is independent of map position. This analysis compares the phenotypic scores of plants with the genotypes at different loci. Subsequently, using the previously determined map locations of these markers, any suitable map (e.g., a composite map) can optionally be used to help visualize the distribution of identified QTL markers and / or QTL marker clusters.

[0145] The present invention relates to the method for the identification of the polymorphism of target gene and the phenotypic association of target gene.Traditional linkage analysis is based on the same principle; However, LD is generated by creating a colony from a small amount of founders. Founders are selected to maximize the polymorphism level in the structured colony, and the co-segregation level of the polymorphic sites and a given phenotype is assessed. A large number of statistical methods have been used to identify significant marker-trait associations. One such method is the interval mapping method (Lander and Botstein, Genetics [heredity] 121:185-199 (1989), wherein each position in many positions along the genetic map (for example, with an interval of 1 cM) is tested for the probability of the gene for controlling the target proterties being positioned at this position. Genotype / phenotypic data are used to calculate the LOD scores (logarithm of the probability ratio) for each test position. When the LOD score is greater than a threshold value, there is significant evidence (to be positioned between two specific marker loci) at this location of the gene for controlling the target proterties being positioned at the genetic map.

[0146] Provided herein are marker loci that show statistically significant cosegregation with disease resistance traits as determined by traditional linkage analysis and genome-wide association analysis. Detection of these loci or additional linked loci can be used in marker-assisted breeding programs to produce plants that include disease resistance.

[0147] Activities in a marker-assisted breeding program can include, but are not limited to, selecting among new breeding populations based on historical genotype and agronomic trait associations to identify which population has the highest frequency of favorable nucleic acid sequences, selecting among progeny in a breeding population for favorable nucleic acid sequences, selecting among parental lines based on predictions of progeny performance, and advancing lines in germplasm improvement activities based on the presence of favorable nucleic acid sequences.

[0148] Chromosome interval

[0149] Chromosome intervals associated with disease resistance traits are provided. Various methods can be used to identify chromosomal intervals. The boundaries of such chromosomal intervals are expanded to encompass markers that will be linked to one or more genes controlling the target trait. In other words, the chromosomal interval is expanded so that any marker within the interval (including the end markers that define the boundaries of the interval) can be used as a marker for the disease resistance trait.

[0150] Conversely, if, for example, two markers that are very close together show co-segregation with a desired phenotypic trait, it is sometimes unclear whether each of those markers identifies the same gene or two different genes or genes. In any case, knowledge of how many genes are within a particular physical / genomic interval is not necessary for formulating or practicing certain examples of the present disclosure.

[0151] Thus, disclosed herein is an interval on maize chromosome 3. The interval on chromosome 3 of the present disclosure can encompass 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the SLB resistance markers disclosed in Table 1. In a specific example, the interval on chromosome 3 can encompass 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of markers D6, C10, A10, G6, H6, D10, F10, K6, L6, O6, Q6, and G3. In a specific example, the interval on chromosome 3 can encompass 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of markers D7, L4, E7, G7, H7, I7, J7, K7, L7, M7, N7, and O4. Any marker located within these intervals can be used as a marker for SLB resistance and can be used in the context of the methods presented herein to select plants that are resistant to SLB compared to control plants. In some instances, markers located upstream and downstream of the P450 gene location are closely linked genetically and physically and can therefore be used to select P450 genes for trait introgression and product development.

[0152] Tags and chain relationships

[0153] A common measure of linkage is the frequency of co-segregation of traits. This can be expressed as a percentage of co-segregation (recombination frequency), or in centimorgans (cM). cM is a unit of measure for genetic recombination frequency. One cM is equivalent to a 1% chance that a trait at one genetic locus will separate from a trait at another locus due to a crossover in a single generation (meaning there is a 99% chance that these traits will separate altogether). Since chromosomal distance is roughly proportional to the frequency of crossover events between traits, there is an approximate physical distance associated with recombination frequency.

[0154] The marker locus itself is a trait and can be assessed according to standard linkage analysis by tracking the marker locus during segregation. Thus, one cM equals a 1% chance that a marker locus will segregate with another locus due to crossing over in a single generation.

[0155] The gene of marker distance control purpose proterties is nearer, and then this mark is more effective and favourable as the instruction of this desired proterties.The locus of tight linkage shows about 10% or lower, for example as about 9% or lower, about 8% or lower, about 7% or lower, about 6% or lower, about 5% or lower, about 4% or lower, about 3% or lower or about 2% or lower locus between crossover frequency.In some instances, related gene seat (for example, marker gene seat and target gene seat) shows about 1% or lower, for example about 0.75% or lower, about 0.5% or lower or about 0.25% or lower recombination frequency.Therefore, these loci are at a distance of about 10cM, 9cM, 8cM, 7cM, 6cM, 5cM, 4cM, 3cM, 2cM, 1cM, 0.75cM, 0.5cM or 0.25cM or lower. In other words, two loci that are located on the same chromosome and have a distance such that recombination between the two loci occurs with a frequency of less than 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25% or less) are considered to be "adjacent" to each other.

[0156] Although specific marker alleles can be co-segregated with disease resistance traits, it is important to note that the marker locus does not necessarily cause the expression of the disease resistance phenotype. For example, it is not a requirement that the marker polynucleotide sequence is a part of a gene that produces a disease resistance phenotype (for example, a part of a gene open reading frame). The association between a specific marker allele and a disease resistance trait is due to the initial "coupling" between the marker allele and the allele in the ancestral strain from which the allele originated. Finally, through repeated recombination, the crossover event between the marker and the genetic locus can change this orientation. For this reason, favorable marker alleles can change according to the linkage phase for creating a segregating population that is present in the parent comprising disease resistance. This does not change the fact that markers can be used to monitor phenotypic separation. It only changes which marker allele is considered favorable in a given segregating population.

[0157] The method proposed herein comprises the existence of one or more marker allelotrope associated with disease resistance in the detection plant, and then selects in those marker loci places, there is the plant of favorable allele.Mark has been accredited as and is associated with disease resistance proterties in this article, and therefore can be used for predicting the disease resistance in the plant.Any mark (based on the genetic map of single meiosis) in 50cM, 40cM, 30cM, 20cM, 15cM, 10cM, 9cM, 8cM, 7cM, 6cM, 5cM, 4cM, 3cM, 2cM, 1cM, 0.75cM, 0.5cM or 0.25cM also can be used for predicting the disease resistance of plant.

[0158] Marker-assisted selection

[0159] 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 the use of marker-assisted selection (MAS) to increase the efficiency of backcrossing and gene introgression. Molecular markers that exhibit linkage to loci that affect desired phenotypic traits provide a useful tool for selecting traits in plant populations. This is especially true when phenotypes are difficult to determine. Because DNA marker assays are more labor-efficient and take up less physical space than field phenotyping, larger populations can be assayed, increasing the probability of finding recombinants with target segments that have moved from the donor line to the recipient line. The tighter the linkage, the more useful the marker, because recombination is less likely to occur between the marker and the gene responsible for the trait, which could result in false positives. The use of flanking markers reduces the probability of false positive selections due to the need for double recombination events. In some cases, the marker is located within the gene itself, such that recombination between the marker and the gene cannot occur. In some examples, the methods disclosed herein generate a marker in a disease resistance gene, wherein the gene is identified by inferring the genomic location from clustering or cluster analysis of conserved domains.

[0160] When genes are introduced by MAS, not only genes but also flanking regions are introduced (Gepts. (2002). Crop Sci [Crop Science]; 42: 1780-1790). This is called "linkage drag". In the case where the donor plant is extremely unrelated to the recipient plant, these flanking regions carry additional genes that can encode agronomically undesirable traits. Even after multiple cycles of backcrossing with improved lines, linkage drag may also lead to yield decline or other negative agronomic characteristics. This is sometimes also referred to as "yield drag". The size of the flanking regions can be reduced by additional backcrossing, although this is not always successful because breeders cannot control the size of the region or recombination breakpoints (Young et al., (1998) Genetics [Genetics] 120: 579-585). In classical breeding, it is usually only by chance that recombination that helps reduce the size of the donor segment is selected (Tanksley et al. (1989). Biotechnology [Biotechnology] 7: 257-264). Even after 20 backcrosses in this type of backcross, it is still possible to find that a large region on the donor chromosome remains linked to the selected gene. However, if markers are used, it is possible to select rare individuals that have undergone recombination near the target gene. In 150 backcross plants, there is a 95% probability that at least one plant will experience a crossover within 1 cM (based on a single meiotic division map distance) of the gene. Markers make it possible to clearly identify these individuals. Using another backcross of 300 plants, there is a 95% probability of crossover within a single meiotic division map distance on the other side of the gene, thereby producing a segment near the target gene that is less than 2 cM based on a single meiotic division map distance. This can be achieved in two generations using markers, while without markers it would take an average of 100 generations (see Tanksley et al., supra). When the exact location of a gene is known, flanking markers around the gene can be used to select for recombination in different population sizes. For example, in smaller populations, recombination may be expected to occur further away from the gene, thus requiring more distal flanking markers to detect the recombination.

[0161] The implementation of MAS can include: (i) defining a population in which marker-trait associations are to be determined, which can be a segregating population, or a random or structured population; (ii) monitoring the segregation or association of polymorphic markers with respect to the trait and using statistical methods to determine linkage or association; (iii) defining a set of desired markers based on the results of the statistical analysis, and (iv) using and / or extrapolating this information to the current breeding germplasm set to enable marker-based selection decisions. The markers described in this disclosure, as well as other marker types, such as SSRs and FLPs, can be used in marker-assisted selection schemes.

[0162] SSRs can be defined as relatively short sequences of tandemly repeated DNA that are 6 bp or less in length (Tautz (1989) Nucleic Acid Research 17:6463-6471; Wang et al. (1994) Theoretical and Applied Genetics, 88:1-6). Polymorphisms arise from variations in the number of repeat units, which can be caused by slippage during DNA replication (Levinson and Gutman (1987) Mol Biol Evol 4:203-221). Variations in repeat length can be detected by designing PCR primers to conserved, non-repeat flanking regions (Weber and May (1989) Am J Hum Genet. 44:388-396). Because SSRs are multiallelic, codominant, regenerable, and amenable to high-throughput automation, they are well suited for mapping and MAS (Rafalski et al. (1996) Generating and using DNA markers in plants. In: Non-mammalian genomic analysis: a practical guide. Academic press. pp. 75-135).

[0163] Various types of SSR markers can be generated, and SSR profiles can be obtained by gel electrophoresis of the amplified products. The scoring of marker genotypes is based on the size of the amplified fragments.

[0164] Various types of FLP markers can also be generated. Most commonly, amplification primers are used to generate fragment length polymorphisms. Such FLP markers are similar to SSR markers in many respects, except that the regions amplified by the primers are generally not highly repetitive regions. Usually due to insertions or deletions, the amplified regions or amplicons still have enough variability between germplasms so that the fragments produced by the amplification primers can be distinguished in polymorphic individuals, and such insertions and deletions are known to often occur in maize (Bhattramakki et al. (2002). Plant Mol Biol [Plant Molecular Biology] 48, 539-547; Rafalski (2002b), ibid).

[0165] SNP markers detect single base pair nucleotide substitutions. Among all molecular marker types, SNP is the most abundant and therefore potentially provides the highest genetic map resolution (Bhattramakki et al., 2002 Plant Molecular Biology [Plant Molecular Biology] 48: 539-547). Since SNP does not require a large amount of DNA and the automation of the assay can be direct, SNP can be measured in a so-called "ultra-high throughput" manner with a throughput level even higher than that of SSR. SNP is also likely to become a relatively low-cost system. These three factors together make it highly attractive to use SNP in MAS. Several methods can be used to perform SNP genotyping and / or detection, including but not limited to: hybridization, primer extension, oligonucleotide ligation, nuclease cleavage, microsequencing, and coded spheres. These methods have been reviewed in Gut (2001) Hum Mutat 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: RJ Henry, ed., Plant Genotyping: The DNA Fingerprinting of Plants, CABI Publishing, Wallingford. A wide range of commercially available technologies utilize these and other methods to detect SNPs, including: Masscode.TM. (Qiagen), INVADER (Third Wave Technologies) and Invader PLUS SNAPSHOT (Applied Biosystems), TAQMAN (Applied Biosystems) and BEADARRAYS (Illumina).

[0166] Many SNPs within or across linked sequences can be used to describe the haplotype of any particular genotype (Ching et al. (2002), BMC Genet. [BMC genetics] 3: 19pp Gupta et al. 2001, Rafalski (2002b), Plant Science [plant science] 162: 329-333). Haplotypes can be more informative than single SNPs and can describe any particular genotype in more detail. For example, a single SNP may be the allele "T" of a particular strain or variety with disease resistance, but allele "T" may also occur in the breeding population for the recurrent parent. In this case, a haplotype (such as a combination of alleles at a linked SNP marker) may be more informative. Once a unique haplotype is assigned to a donor chromosome region, the haplotype can be used in the population or any subpopulation thereof to determine whether an individual has a specific gene. Using an automated high-throughput marker detection platform makes this method efficient and effective.

[0167] Many marks proposed herein can be easily used as single nucleotide polymorphism (SNP) marks to select P450 gene.Utilize PCR, primer is used to increase the DNA segment of the individual (such as inbred line) representing the diversity of target population.PCR product is directly ordered in one or two directions.The sequence obtained is compared and identified polymorphism.Polymorphism is not limited to single nucleotide polymorphism (SNP), and comprises insertion and deletion, CAPS, SSR and VNTR (variable number of tandem repeats).Especially, for fine atlas information as herein described, people can be easy to use the information provided herein to obtain other polymorphic SNP (and other marks) in the region amplified by primer disclosed herein.The mark in the described atlas region can be hybridized with BAC or other genomic libraries, or carries out electronic comparison with genomic sequence, to find new sequence in the roughly location identical with described mark.

[0168] In addition to the above-mentioned SSR, FLP and SNP, other types of molecular markers are also widely used, including but not limited to: expressed sequence tags (ESTs), SSR markers derived from EST sequences, randomly amplified polymorphic DNA (RAPD) and other nucleic acid-based markers.

[0169] Isozyme profiles and linked morphological features can also be used indirectly as markers in some cases. Although they do not directly detect DNA differences, they are often affected by specific genetic differences. However, markers for detecting DNA variations are much more numerous and more polymorphic than isozyme or morphological markers (Tanksley (1983) Plant Molecular Biology Reporter [Plant Molecular Biology Guide] 1: 3-8).

[0170] Sequence alignment or overlapping group can also be used to find the sequence of the upstream or downstream of the specific marker listed herein.Then use these new sequences close to mark as herein described to find and develop functionally equivalent marks.For example, different physics and / or genetic maps are compared to locate the equivalent markers that are not described in the present disclosure but are positioned at similar regions.These maps may be in species, or even span other species that carry out heredity or physically compare.

[0171] In general, MAS uses polymorphic markers that have been identified as exhibiting a significant likelihood of co-segregation with traits such as SLB disease resistance traits and / or genes disclosed herein. Such markers are presumed to be located near one or more genes that confer a plant disease resistance phenotype on the map and are considered to be indicative of the desired trait or marker. Plants are tested for the presence of the desired allele in the marker, and it is expected that plants containing the desired genotype at one or more loci will transfer the desired genotype, along with the desired phenotype, to their progeny. Therefore, plants with SLB disease resistance can be selected by detecting one or more marker alleles, and further, progeny plants derived from these plants can also be selected. Thus, a plant containing the desired genotype (i.e., a genotype associated with disease resistance) in a given chromosomal region is obtained and then hybridized with another plant. The progeny of such a hybrid is then genotyped using one or more markers, and progeny plants with the same genotype in the given chromosomal region are then selected as containing disease resistance.

[0172] SNPs (i.e., SNP haplotypes) can be used alone or in combination to select for favorable resistance gene alleles associated with GLS disease resistance. For example, the SNP haplotype can include a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the SLB resistance markers in Table 1 of the present disclosure. The 2-12 markers can be (1) K22 markers or (2) CIMBL83 / CML304 markers.

[0173] Those skilled in the art will anticipate that there may be other polymorphic sites at the marker locus in and near the chromosomal marker identified by method disclosed herein, wherein the allele at one or more polymorphic sites and the polymorphic sites in the haplotype is in linkage disequilibrium (LD), and therefore can be used in marker-assisted selection program to infiltrate target gene allele or target genome fragment. If the existence of the allele at one of these sites tends to predict the existence of the allele at other sites on the same chromosome, it is believed that two specific alleles at different polymorphic sites are in LD (Stevens, Mol.Diag. [molecular diagnosis] 4:309-17 (1999)). The marker locus can be located within 5cM, 2cM or 1cM of the disease resistance trait QTL (on a genetic map based on a single meiosis).

[0174] Allele frequencies (and therefore haplotype frequencies) can vary between germplasm pools. Germplasm pools vary due to differences in maturity, heterosis grouping, geographical distribution, etc. Therefore, SNPs and other polymorphisms may not be informative in some germplasm pools.

[0175] Methods for selecting plants comprising resistance to SLB

[0176] This article provides a method for identifying a maize plant that contains a marker allele associated with resistance to southern leaf blight. The method includes screening a nucleic acid from the maize plant for at least one of the marker alleles on chromosome 3 disclosed herein. The plant can be a member of a maize plant population, and the nucleic acid can be isolated from a plurality of plants to screen for plants that contain at least one marker.

[0177] Marker alleles (e.g., single nucleotide polymorphisms (SNPs)) are provided in Table 1. The right column of Table 1 provides each SNP and flanking genomic sequence for context. Residues associated with anti-SLB resistance are listed before the slash, and residues associated with SLB susceptibility are listed after the slash. SNPs are shown in bold font and brackets. The sequence of only one strand is provided, but the disclosed SNPs and methods of use encompass all complementary forms (e.g., in the context of reverse complementary strands).

[0178] Table 1

[0179]

[0180]

[0181]

[0182] In some instances, the method includes selecting a maize plant (e.g., from a population) that includes at least one marker. In some instances, selecting a maize plant includes selecting a plant for further use in, for example, breeding activities (e.g., hybridization, backcrossing, or introgression). Selecting a plant that includes at least one marker can provide a plant that will be a suitable donor of the SLB resistance trait during a breeding activity.

[0183] In some examples, methods of selecting maize plants comprising resistance to southern leaf blight include obtaining nucleic acid samples from a plurality of plants, seeds, tissues, or germplasm in a population; screening each sample for at least one marker allele located on chromosome 3; and selecting one or more plants, seeds, tissues, or germplasm comprising at least one of the marker alleles associated with resistance to SLB.

[0184] In some instances, the method for selecting a maize plant comprising resistance to southern leaf blight comprises hybridizing the selected plant with a second maize plant. The second maize plant can be from a plant population that has been backcrossed for the SLB resistance trait and / or infiltrated into a plant population that lacks the trait. In some instances, the method comprises obtaining a progeny plant comprising at least one marker allele listed in Table 1 from the hybridization.

[0185] In some instances, the method can further comprise isolating nucleic acid from maize plant or maize plant colony.The separation of nucleic acid can promote the detection of this at least one mark.Nucleic acid separation can comprise any method that is suitable for providing genomic material to be used for the evaluation of PCR, sequencing procedure and / or at least one mark.

[0186] In some examples, the marker alleles to be screened are located in a chromosomal interval containing the QTLs for CIMBL83 and CML304 or a portion of the interval that still contains the causative gene (e.g., a portion reduced by recombination). In some examples, the marker alleles to be screened are located in a chromosomal interval flanked by and including: marker D6, corresponding to "C" at position 51 of SEQ ID NO: 1; and G3, corresponding to "C" at position 51 of SEQ ID NO: 12. This interval may also include one or more of markers C10, A10, G6, H6, D10, F10, K6, L6, O6, and Q6.

[0187] In some examples, the marker alleles to be screened are located in a portion of the chromosomal interval containing the QTL for K22 or the interval that still contains the causative gene (e.g., the portion reduced by recombination). In some examples, the marker alleles to be screened are located in a chromosomal interval flanked by and including: marker D7, corresponding to "T" at position 51 of SEQ ID NO: 13; and O4, corresponding to "A" at position 51 of SEQ ID NO: 24. The interval may also include one or more of markers L4, E7, G7, H7, I7, J7, K7, L7, M7, and N7.

[0188] In some instances, one or more of markers D6, C10, A10, G6, H6, D10, F10, K6, L6, O6, Q6, and G3 are detected. These markers have been found to be linked to the SLB resistance QTLs of CIMBL83 and CML304, and are also linked to each other. Therefore, the presence and detection of one or more of these markers is associated with SLB resistance. In some instances, each of D6, C10, A10, G6, H6, D10, F10, K6, L6, O6, Q6, and G3 are detected. In some instances, one or more of markers G6, H6, D10, F10, K6, and L6 are detected. In some instances, markers G6 and L6 are detected. In some instances, markers K6 and L6 are detected.

[0189] In some instances, one or more of markers D7, L4, E7, G7, H7, I7, J7, K7, L7, M7, N7, and O4 are detected. These markers have been found to be linked to the SLB resistance QTL of K22 and are also linked to each other. Therefore, the presence or detection of one or more of these markers is associated with SLB resistance. In some instances, each of markers D7, L4, E7, G7, H7, I7, J7, K7, L7, M7, N7, and O4 is detected. In some instances, one or more of markers I7, J7, K7, and L7 are detected. In some instances, markers I7 and L7 are detected.

[0190] Methods for producing maize plants comprising resistance to SLB

[0191] The present invention provides a method for producing a maize plant comprising a QTL allele associated with SLB resistance. The method comprises screening a maize plant population for the QTL allele associated with SLB resistance. The QTL allele comprises one or more of the following marker alleles: (1) D6, C10, A10, G6, H6, D10, F10, K6, L6, O6, Q6, and G3 or (2) one or more of D7, L4, E7, G7, H7, I7, J7, K7, L7, M7, N7, and O4. The method comprises selecting at least one maize plant comprising the QTL allele from the population. The selection can be based on any result indicating the presence of the QTL. For example, sequencing of a genomic region can be performed to determine whether one or more markers are present in the test plant. The method comprises hybridizing the selected maize plant with a second maize plant, and obtaining a progeny plant comprising the QTL allele. The progeny plants can then be used in further breeding activities and / or as a seed source.

[0192] In some examples, the method includes introgressing the QTL allele into a second population of maize plants. Introgression can be accomplished via repeated backcrossing and can be used to produce plants having, for example, one or more desired agronomic characteristics and increased SLB resistance due to the presence of the QTL allele.

[0193] In some examples, the QTL allele is located on a chromosomal interval flanked by and including marker D6, corresponding to "C" at position 51 of SEQ ID NO: 1; and G3, corresponding to "C" at position 51 of SEQ ID NO: 12.

[0194] In some examples, the QTL allele is located on a chromosomal interval flanked by and including marker D7, corresponding to "T" at position 51 of SEQ ID NO: 13; and O4, corresponding to "A" at position 51 of SEQ ID NO: 24.

[0195] In some instances, one or more of markers D6, C10, A10, G6, H6, D10, F10, K6, L6, O6, Q6, and G3 are detected. These markers have been found to be linked to the SLB resistance QTLs of CIMBL83 and CML304, and are also linked to each other. Therefore, the presence and detection of one or more of these markers is associated with SLB resistance. In some instances, each of markers D6, C10, A10, G6, H6, D10, F10, K6, L6, O6, Q6, and G3 are detected. In some instances, one or more of markers G6, H6, D10, F10, K6, and L6 are detected. In some instances, markers G6 and L6 are detected. In some instances, markers K6 and L6 are detected.

[0196] In some instances, one or more of markers D7, L4, E7, G7, H7, I7, J7, K7, L7, M7, N7, and O4 are detected. These markers have been found to be linked to the SLB resistance QTL of K22 and are also linked to each other. Therefore, the presence or detection of one or more of these markers is associated with SLB resistance. In some instances, each of markers D7, L4, E7, G7, H7, I7, J7, K7, L7, M7, N7, and O4 is detected. In some instances, one or more of markers I7, J7, K7, and L7 are detected. In some instances, markers I7 and L7 are detected.

[0197] Methods for selecting and / or producing maize plants encoding SLB resistance genes

[0198] Provided herein is a method for selecting maize plants that are resistant to southern leaf blight. The method comprises screening a population of maize plants for the presence of a gene encoding a protein that has at least 95% amino acid sequence identity to one or more of SEQ ID NOs: 27, 30, or 33. The method also comprises selecting maize plants that contain the gene.

[0199] In some instances, the method comprises the maize plant of selection and the hybridization of the second maize plant. In some instances, the method comprises the progeny plant that acquisition comprises this gene. In some instances, the progeny plant derived from hybridization can be used for further breeding activities and / or as seed source then. In some instances, can use progeny plant to infiltrate gene in plant breeding system (for example, improved seed system) via repeating backcrossing.

[0200] SLB resistance gene

[0201] In various aspects of the present disclosure, the P450 genes that confer SLB resistance are used to produce recombinant plants or for screening plants for the genes. For example, these genes can be detected to determine whether a given plant comprises anti-SLB resistance, transformed into a plant, or added to a plant genome using site-specific genome editing techniques (e.g., technology based on CRISPR, TALEN, meganucleases, or zinc finger nucleases). P450 gene sequences that confer SLB resistance were identified in inbred lines K22, CIMBL83, and CML304. Unless otherwise indicated, any P450 gene sequence presented herein (e.g., any nucleic acid or protein sequence from SEQ ID NO: 25-33) (see Table 2) can be used for any aspect of the present disclosure requiring a specific P450 gene / protein sequence. In addition, the P450 gene sequence presented herein comprises a protein sequence, a DNA coding sequence (e.g., a cDNA sequence), or a genomic DNA sequence, including a promoter, terminator, exon, and intron sequence. Therefore, these sequences are described in more detail below.

[0202] In some examples, the protein encoded by the gene comprises at least 90% amino acid sequence identity to one or more of SEQ ID NOs: 27, 30, or 33. For example, the gene can comprise at least 95% amino acid sequence identity to one or more of SEQ ID NOs: 27, 30, or 33. In another example, the gene can comprise at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to one or more of SEQ ID NOs: 27, 30, or 33. In another example, the gene can comprise at least 85% amino acid sequence identity to one or more of SEQ ID NOs: 27, 30, or 33. In another example, the gene can comprise 100% amino acid sequence identity to one or more of SEQ ID NOs: 27, 30, or 33.

[0203] In some instances, the gene is encoded by a specific nucleotide sequence. The sequence may include only a coding sequence (e.g., a cDNA sequence) or both a coding sequence and a non-coding sequence (e.g., exons and a promoter, terminator, and / or introns). In some instances, the promoter is naturally present in the gene or is heterologous to the gene. In some instances, the terminator is naturally present in the gene or is heterologous to the gene.

[0204] In some examples, the gene comprises a nucleotide sequence comprising at least 95% identity to one or more of SEQ ID NOs: 25, 26, 28, 29, 31, or 32. In some examples, the gene comprises a nucleotide sequence comprising at least 90% identity to one or more of SEQ ID NOs: 25, 26, 28, 29, 31, or 32. In some examples, the gene comprises a nucleotide sequence comprising at least 96% identity to one or more of SEQ ID NOs: 25, 26, 28, 29, 31, or 32. In some examples, the gene comprises a nucleotide sequence comprising at least 97% identity to one or more of SEQ ID NOs: 25, 26, 28, 29, 31, or 32. In some examples, the gene comprises a nucleotide sequence comprising at least 98% identity to one or more of SEQ ID NOs: 25, 26, 28, 29, 31, or 32. In some examples, the gene comprises a nucleotide sequence comprising at least 99% identity to one or more of SEQ ID NOs: 25, 26, 28, 29, 31, or 32. In some examples, the gene comprises a nucleotide sequence comprising 100% identity to one or more of SEQ ID NOs: 25, 26, 28, 29, 31, or 32.

[0205] Table 2

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220] Methods for producing recombinant maize plant cells and / or plants

[0221] This article provides methods for producing recombinant maize plants / cells. These recombinant plants include anti-SLB resistance. These methods include introducing a heterologous P450 gene sequence into the genome of a maize plant cell and selecting a plant cell that is included in the heterologous P450 gene sequence encoded in the plant cell genome.

[0222] In some examples, the method comprises introducing a site-specific modification of at least one target site into the genome of a maize plant cell, and introducing a polynucleotide modification template comprising a heterologous P450 nucleotide sequence into the plant cell.

[0223] In some examples, the site-specific modification is induced by a CRISPR-associated endonuclease.

[0224] In some instances, the site-specific modification comprises a single-strand break or a double-strand break (DSB) produced by a site-specific endonuclease, such as, for example, TALEN, a meganuclease, a zinc finger nuclease, and a CRISPR-associated (Cas) protein / guide polynucleotide complex. In some instances, the introduction of a site-specific modification can be combined with the introduction of a polynucleotide modification template.

[0225] In some examples, a polynucleotide modified template is introduced into a cell by any method suitable for delivering the template to the cell nucleus, such as, but not limited to, transient introduction methods, transfection, electroporation, microinjection, particle-mediated delivery, topical application, whisker-mediated delivery, delivery via cell-penetrating peptides, or direct delivery mediated by mesoporous silica nanoparticles (MSNs).

[0226] In some instances, polynucleotide modification templates can be introduced into cells as single-stranded polynucleotide molecules, double-stranded polynucleotide molecules or as a part of circular DNA (vector DNA). Polynucleotide modification templates can also be tethered with guidance polynucleotides and / or Cas endonucleases. The tethered template can allow for co-localization target and template DNA, can be used for genome editing and targeted genome regulation, and can also be used for targeting late mitotic cells, in which the function of endogenous homologous recombination HR mechanism is expected to be greatly reduced (Mali et al. 2013Nature Methods [natural methods] Vol. 10: 957-963). Polynucleotide modification templates can be transiently present in cells, or can be introduced via viral replicons.

[0227] As used herein, a polynucleotide modification template refers to a polynucleotide comprising at least one nucleotide modification when compared to a target nucleotide sequence to be edited. A nucleotide modification is a substitution, addition, or deletion of at least one nucleotide. In some examples, the polynucleotide modification template may further comprise a homologous nucleotide sequence flanking at least one nucleotide modification, wherein the flanking homologous nucleotide sequence provides sufficient homology to support incorporation of the polynucleotide modification template into the genome of a recipient plant cell.

[0228] Methods for editing genomic sequences that combine site-specific modifications and modification templates generally include providing a site-specific endonuclease or a nucleic acid encoding a site-specific endonuclease that recognizes a target sequence in a chromosomal sequence to a host cell, and wherein the site-specific endonuclease induces site-specific modifications (e.g., DSBs) in the genomic sequence; and providing at least one polynucleotide modification template. The endonuclease can be provided to the cell by any method known in the art, such as, but not limited to, transient introduction methods, transfection, microinjection, and / or topical application, or indirectly via a recombinant construct. The endonuclease can be provided directly to the cell as a protein or as a guide polynucleotide complex or indirectly via a recombinant construct. The endonuclease can be transiently introduced into the cell, or can be incorporated into the genome of the host cell. In the case of a CRISPR-Cas system, as described in WO 2016073433, a cell-penetrating peptide (CPP) can be used to promote the uptake of endonucleases and / or guide polynucleotides into the cell.

[0229] TAL effector nucleases (TALENs) are a class of sequence-specific nucleases that can be used to create double-strand breaks at specific target sequences in the genomes of plants or other organisms (see Miller et al. (2011) Nature Biotechnology 29:143–148).

[0230] Endonucleases are enzymes that cut phosphodiester bonds within polynucleotide chains. Endonucleases include restriction endonucleases, which cut DNA at specific sites without damaging bases; and include meganucleases, also known as homing endonucleases (HE enzymes), which are similar to restriction endonucleases, bind and cut at specific recognition sites, but for meganucleases, the recognition sites are typically longer, about 18 bp or longer (patent application PCT / US12 / 30061 filed on March 22, 2012). Meganucleases are classified into four families based on conserved sequence motifs, which are LAGLIDADG, GIY-YIG, HNH, and His-Cys box families. These motifs are involved in the coordination of metal ions and the hydrolysis of phosphodiester bonds. The notable feature of HE enzymes is their long recognition sites and their tolerance to some sequence polymorphisms in their DNA substrates. The naming convention for meganucleases is similar to that for other restriction endonucleases. Meganucleases are also characterized by the prefixes F-, I-, or PI- for enzymes encoded by independent ORFs, introns, and inteins, respectively. One step of the recombination method involves cleavage of a polynucleotide at or near a recognition site. The cleavage activity can be used to produce double-strand breaks. For a review of site-specific recombinases and their recognition sites, see Sauer (1994) Curr Op Biotechnol [New Views in Biotechnology] 5:521-7; and Sadowski (1993) FASEB [Journal of the Federation of American Societies for Experimental Biology] 7:760-7. In some instances, the recombinase is from the integrase or resolvase family.

[0231] Zinc finger nuclease (ZFN) is an engineered double-strand break inducing agent consisting of a zinc finger DNA binding domain and a double-strand break-inducing agent domain. Recognition site specificity is given by a zinc finger domain, which typically comprises two, three, or four zinc fingers, such as a C2H2 structure, but other zinc finger structures are known and have been engineered. The zinc finger domain is suitable for designing polypeptides that specifically bind to selected polynucleotide recognition sequences. ZFN includes an engineered DNA binding zinc finger domain connected to a non-specific endonuclease domain (e.g., a nuclease domain from a type IIs endonuclease such as FokI). Additional functionality can be fused to the zinc finger binding domain, including a transcription activator domain, a transcription repressor domain, and a methylase. In some instances, dimerization of the nuclease domain is required for cleavage activity. Each zinc finger recognizes three consecutive base pairs in the target DNA. For example, the 3-finger domain recognizes a sequence of 9 consecutive nucleotides, and because the nuclease requires dimerization, two sets of zinc finger triplets are used to bind to the 18-nucleotide recognition sequence.

[0232] Genome editing using DSB-inducing agents (e.g., Cas9-gRNA complexes) has been described, for example, in U.S. patent applications US2015-0082478 A1, WO 2015 / 026886 A1, WO 2016007347, and WO 201625131, all of which are incorporated herein by reference.

[0233] The term "Cas gene" or "Cas protein" herein refers to a gene that is typically coupled, associated, or close to or in proximity to a flanking CRISPR locus in a bacterial system. The terms "Cas" and "CRISPR-associated" are used interchangeably herein. The term "Cas endonuclease" herein refers to a protein or protein complex encoded by a Cas gene. When complexed with a suitable polynucleotide component, the Cas endonuclease disclosed herein is capable of recognizing, binding to all or part of a specific DNA target sequence, and optionally nicking or cutting all or part of a specific DNA target sequence. The Cas endonuclease as described herein comprises one or more nuclease domains. The Cas endonucleases disclosed herein include those comprising an HNH or HNH-like nuclease domain and / or a RuvC or RuvC-like nuclease domain. The Cas endonucleases disclosed herein may include Cas9 protein, Cpf1 protein, C2c1 protein, C2c2 protein, C2c3 protein, Cas3, Cas 5, Cas7, Cas8, Cas10, or a complex of these.

[0234] As used herein, the terms "guide polynucleotide / Cas endonuclease complex," "guide polynucleotide / Cas endonuclease system," "guide polynucleotide / Cas complex," "guide polynucleotide / Cas system," and "guide Cas system" are used interchangeably herein and refer to at least one guide polynucleotide and at least one Cas endonuclease capable of forming a complex, wherein the guide polynucleotide / Cas endonuclease complex can guide the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave the DNA target site (introducing single-strand or double-strand breaks). The guide polynucleotide / Cas endonuclease complex herein can comprise one or more Cas proteins and one or more suitable polynucleotide components of any of the four known CRISPR systems (Horvath and Barrangou, 2010, Science 327: 167-170) (e.g., type I, type II, or type III CRISPR systems). Cas endonuclease unwinds the DNA duplex at the target sequence and optionally cuts at least one DNA chain, as mediated by the recognition of the target sequence by a polynucleotide (such as, but not limited to, crRNA or guide RNA) compounded with the Cas protein. If the correct pre-spacer adjacent motif (PAM) is located at or adjacent to the 3' end of the DNA target sequence, such recognition and cutting of the target sequence by the Cas endonuclease typically occurs. Alternatively, the Cas protein herein may lack DNA cutting or nicking activity, but when compounded with a suitable RNA component, it can still specifically bind to the DNA target sequence. (See also U.S. Patent Application US2015-0082478 A1 and US2015-0059010 A1, both of which are hereby incorporated by reference in their entirety).

[0235] Instruction polynucleotide / Cas endonuclease complex can cut one or two chains of DNA target sequence.The guidance polynucleotide / Cas endonuclease complex that can cut two chains of DNA target sequence typically includes the Cas protein (such as wild-type endonuclease domain or its variant that retains some or all activity in each endonuclease domain) with all its endonuclease domains in functional state.Therefore, wild-type Cas protein or its variant (retaining some or all activity in each endonuclease domain of Cas protein) is the suitable example of the Cas endonuclease that can cut two chains of DNA target sequence.The Cas9 protein comprising functional RuvC and HNH nuclease domain is the example of the Cas protein that can cut two chains of DNA target sequence.The guidance polynucleotide / Cas endonuclease complex that can cut a chain of DNA target sequence can be characterized as comprising nickase activity (for example, partial cutting ability) in this article.Cas nickase typically includes a functional endonuclease domain, and this domain allows Cas to only cut a chain (that is, form nick) of DNA target sequence. For example, the Cas9 nickase may comprise (i) a mutated, dysfunctional RuvC domain and (ii) a functional HNH domain (e.g., a wild-type HNH domain). As another example, the Cas9 nickase may comprise (i) a functional RuvC domain (e.g., a wild-type RuvC domain) and (ii) a mutated dysfunctional HNH domain. Non-limiting examples of Cas9 nickases suitable for use herein are disclosed in U.S. Patent Application Publication No. 2014 / 0189896, which is incorporated herein by reference.

[0236] In some instances, a pair of Cas9 nickases can be used to increase the specificity of DNA targeting.In general, this can be carried out by providing two Cas9 nickases, which, by associating with RNA components having different guide sequences, carry out targeting and nick near the DNA sequence on the opposite chain in the region of the target. Such nearby cutting of each DNA chain produces double-strand breaks (that is, DSBs with single-stranded overhangs), which are then identified as substrates for non-homologous end joining (NHEJ) (tending to produce the imperfect repair causing mutations) or homologous recombination (HR). Each nick in these examples can, for example, be at least about 5,10,15,20,30,40,50,60,70,80,90 or 100 (or any integer between 5 and 100) bases apart from each other. One or two Cas9 nickase proteins herein can be used for Cas9 nickase pairs. For example, a Cas9 nickase (i.e., Cas9 HNH+ / RuvC-) with a mutated RuvC domain but a functional HNH domain (e.g., Streptococcus pyogenes Cas9 HNH+ / RuvC-) can be used. By using a suitable RNA component herein (with a guide RNA sequence that targets each nickase to each specific DNA site), each Cas9 nickase (e.g., Cas9 HNH+ / RuvC-) is guided to a specific DNA site adjacent to each other (up to 100 base pairs apart).

[0237] In some examples, the Cas protein can be part of a fusion protein comprising one or more heterologous protein domains (e.g., 1, 2, 3, or more domains other than the Cas protein). Such a fusion protein can include any additional protein sequences, and optionally a linker sequence between any two domains (e.g., between the Cas and the first heterologous domain). Examples of protein domains that can be fused to the Cas proteins herein include, but are not limited to, 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], β-galactosidase, β-glucuronidase [GUS], luciferase, green fluorescent protein [GFP], HcRed, DsRed, cyan fluorescent protein [CFP], yellow fluorescent protein [YFP], blue fluorescent protein [BFP]); and domains comprising one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity (e.g., VP16 or VP64), transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Cas proteins can also be fused to proteins that bind to 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. For more examples of Cas proteins, see PCT patent applications PCT / US16 / 32073 filed on May 12, 2016 and PCT / US16 / 32028 filed on May 12, 2016 (both applications are incorporated herein by reference).

[0238] In some instances, the guidance polynucleotide / Cas endonuclease complex can bind to the DNA target site sequence, but does not cut any chain at the target site sequence. Such a complex can include Cas proteins in which all nuclease domains are mutations, dysfunctions. For example, the Cas9 protein herein that can be bound to the DNA target site sequence but does not cut any chain at the target site sequence can include mutations, dysfunctions, RuvC domains and mutations, dysfunctions, HNH domains. The Cas protein herein that is bound but does not cut the target DNA sequence can be used to regulate gene expression, for example, in this case, the Cas protein can be fused to a transcription factor (or part thereof) (such as a repressor or activator, such as any of those disclosed herein). In other aspects, the inactivated Cas protein can be fused to another protein such as FokI endonuclease comprising endonuclease activity.

[0239] "Cas9" herein (formerly known as Cas5, Csn1, or Csx12) refers to the Cas endonuclease of the type II CRISPR system that forms a complex with cr nucleotides and tracr nucleotides or with a single guide polynucleotide, which is used to specifically recognize and cut all or part of a DNA target sequence. The Cas9 protein includes a RuvC nuclease domain and an HNH (HNH) nuclease domain, each of which can cut a single DNA chain at the target sequence (the synergistic action of the two domains results in double-stranded DNA cutting, and the activity of one domain results in a nick). Typically, the RuvC domain includes subdomains I, II, and III, wherein domain I is located near the N-terminus of Cas9, and subdomains II and III are located in the middle of the protein, i.e., flanking the HNH domain (Hsu et al., Cell [cell], 157: 1262-1278). Type II CRISPR systems include DNA cutting systems that utilize a Cas9 endonuclease complexed with at least one polynucleotide component. For example, Cas9 can be complexed with CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA).In another example, Cas9 can be complexed with a single guide RNA.

[0240] In some instances, the Cas endonuclease may comprise a modified form of a Cas9 polypeptide. The modified form of the Cas9 polypeptide may include amino acid changes (e.g., deletions, insertions, or substitutions) that reduce the naturally occurring nuclease activity of the Cas9 protein. For example, in some cases, the modified form of the Cas9 protein has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1% of the nuclease activity of the corresponding wild-type Cas9 polypeptide (U.S. patent application US20140068797 A1). In some cases, the modified form of the Cas9 polypeptide does not have substantial nuclease activity and is referred to as catalytically “inactivated Cas9” or “inactivated cas9 (dCas9)”. Catalytically inactivated Cas9 variants include Cas9 variants comprising mutations in the HNH and RuvC nuclease domains. These catalytically inactivated Cas9 variants are able to interact with sgRNA and bind to the target site in vivo but cannot cut either chain of the target DNA.

[0241] In some instances, the catalytically inactivated Cas9 can be fused to a heterologous sequence (U.S. patent application US20140068797 A1). Suitable fusion partners include, but are not limited to, polypeptides that provide activity that indirectly increases transcription by acting directly on the target DNA or on a polypeptide associated with the target DNA (e.g., histone or other DNA-binding proteins). Other suitable fusion partners include, but are not limited to, polypeptides that provide methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating enzyme activity, adenylation activity, deadenylation activity, sumoylation (SUMOylating) activity, desumoylation (deSUMOylating) activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity. Further suitable fusion partners include, but are not limited to, polypeptides that directly provide increased transcription of the target nucleic acid (e.g., transcriptional activators or fragments thereof, proteins or fragments thereof that recruit transcriptional activators, small molecule / drug-responsive transcriptional regulators, etc.). Catalytically inactive Cas9 can also be fused to the FokI nuclease to generate double-strand breaks (Guilinger et al. Nature Biotechnology, Vol. 32, No. 6, June 2014).

[0242] In some instances, the method comprises growing plants from plant cells comprising heterologous P450 gene sequences. In some instances, the plants exhibit increased resistance to southern leaf blight. In some instances, the plants derived from this method can then be used for further breeding activities and / or as a seed source. In some instances, the plants produced by this method can be used to infiltrate P450 genes into plant breeding lines (e.g., improved lines) via repeated backcrossing.

[0243] The heterologous P450 locus can be inserted into any position in the plant cell genome that is suitable for heterologous P450 gene expression and optionally does not interfere with endogenous gene expression. In some instances, the heterologous P450 gene sequence replaces the endogenous P450 locus. In such instances, at least one allele of the endogenous P450 gene is eliminated and replaced by the heterologous P450 gene encoded on the polynucleotide modified template. In such instances, at least the coding region of the endogenous P450 gene is replaced. Introns, promoter sequences, and terminator sequences also can be optionally replaced.

[0244] In some instances, a heterologous P450 gene sequence is inserted into a locus different from the endogenous P450 locus. In some instances, a heterologous P450 gene sequence is inserted into a locus different from the endogenous P450 locus along with other genes that are also inserted into the same locus to form a molecular stack. In some instances, the molecular stack comprises a stack of disease resistance genes.

[0245] This article also provides a method for producing a recombinant maize plant cell. The method includes introducing a P450 nucleotide sequence into one or more plant cells; inserting the P450 nucleotide sequence into the genome of at least one of the one or more plant cells; and selecting a plant cell in which the P450 nucleotide sequence has been inserted into the genome of the plant cell. The P450 nucleotide sequence can comprise any P450 nucleotide sequence disclosed herein and can encode any P450 amino acid sequence disclosed herein.

[0246] In some instances, the method comprises the plant cell growth plant from the P450 nucleotide sequence inserted into the plant cell genome. In some instances, plant shows the anti-southern leaf blight resistance that increases. In some instances, selection can be based on any result of indicating the presence of the P450 gene. For example, order-checking can be performed to determine whether the gene has been inserted and / or plant cell can be selected to use a selective marker. In some instances, the method comprises hybridizing the newly grown maize plant with the second maize plant, and obtaining the progeny plant that comprises this P450 gene. Then, this progeny plant can be used for further breeding activities (for example, backcrossing and / or infiltration) and / or be used as a seed source.

[0247] In some examples, introduction of the P450 nucleotide sequence is accomplished via bacteria-mediated transformation.

[0248] In some examples, introduction of the P450 nucleotide sequence is accomplished via biolistic transformation.

[0249] Recombinant plants and seeds

[0250] Provided are recombinant plants comprising a P450 gene sequence as described herein. Provided herein are recombinant plants comprising a heterologous gene encoding a protein comprising at least 95% amino acid sequence identity to one or more of SEQ ID NOs: 27, 30, or 33. In some examples, the gene comprises a nucleotide sequence comprising at least 95% nucleotide sequence identity to one or more of SEQ ID NOs: 25, 26, 28, 29, 31, or 32.

[0251] Provided are recombinant plant seeds comprising a P450 gene sequence as described herein. Provided herein are recombinant plant seeds comprising a heterologous gene encoding a protein comprising at least 95% amino acid sequence identity to one or more of SEQ ID NOs: 27, 30, or 33. In some examples, the gene comprises a nucleotide sequence comprising at least 95% nucleotide sequence identity to one or more of SEQ ID NOs: 25, 26, 28, 29, 31, or 32.

[0252] Terms of this Disclosure

[0253] Various aspects of this disclosure will be understood by reference to the following clauses.

[0254] 1. A method for identifying a maize plant comprising a marker allele associated with resistance to southern leaf blight, the method comprising:

[0255] Nucleic acids from maize plants are screened for at least one of the following marker alleles on chromosome 3:

[0256] i. "C" at D6, corresponding to position 51 of SEQ ID NO: 1;

[0257] ii. "C" at C10, corresponding to position 51 of SEQ ID NO: 2;

[0258] iii. "C" at A10, corresponding to position 51 of SEQ ID NO: 3;

[0259] iv. "G" at G6, corresponding to position 51 of SEQ ID NO: 4;

[0260] v. "G" at H6, corresponding to position 51 of SEQ ID NO: 5;

[0261] vi. "T" at D10, corresponding to position 51 of SEQ ID NO: 6;

[0262] vii. "C" at F10, corresponding to position 51 of SEQ ID NO: 7;

[0263] viii. "A" at K6, corresponding to position 51 of SEQ ID NO: 8;

[0264] ix. "C" at L6, corresponding to position 51 of SEQ ID NO:9;

[0265] "T" at x.06, corresponding to position 51 of SEQ ID NO: 10;

[0266] xi. "G" at Q6, corresponding to position 51 of SEQ ID NO: 11;

[0267] xii. "C" at G3, corresponding to position 51 of SEQ ID NO: 12;

[0268] xiii. "T" at D7, corresponding to position 51 of SEQ ID NO: 13;

[0269] xiv. "T" at L4, corresponding to position 51 of SEQ ID NO: 14;

[0270] xv. "T" at E7, corresponding to position 51 of SEQ ID NO: 15;

[0271] xvi. "A" at G7, corresponding to position 51 of SEQ ID NO: 16;

[0272] xvii. "G" at H7, corresponding to position 51 of SEQ ID NO: 17;

[0273] xviii. "T" at I7, corresponding to position 51 of SEQ ID NO: 18;

[0274] xix. "T" at J7, corresponding to position 51 of SEQ ID NO: 19;

[0275] "G" at xx.K7, corresponding to position 51 of SEQ ID NO:20;

[0276] xxi. "A" at L7, corresponding to position 51 of SEQ ID NO: 21;

[0277] xxii. "T" at M7, corresponding to position 51 of SEQ ID NO: 22;

[0278] xxiii. "A" at N7, corresponding to position 51 of SEQ ID NO: 23; or

[0279] xxiv. "A" at O4 corresponds to position 51 of SEQ ID NO: 24,

[0280] The presence of the marker allele is associated with resistance to southern leaf blight.

[0281] 2. The method according to clause 1, further comprising

[0282] a. obtaining nucleic acid samples from multiple plants, seeds, tissues or germplasm in a population;

[0283] b. screening each sample for at least one of the nucleic acid marker alleles on chromosome 3; and

[0284] c. Selecting one or more of plants, seeds, tissues, or germplasm comprising at least one of said marker alleles associated with resistance to southern leaf blight.

[0285] 3. A method as described in claim 1 or 2, wherein the marker alleles screened are located on the chromosome interval flanking and including: marker D6, corresponding to "C" at position 51 of SEQ ID NO: 1; and G3, corresponding to "C" at position 51 of SEQ ID NO: 12.

[0286] 4. A method as described in claim 1 or 2, wherein the marker alleles screened are located on the chromosomal interval flanking and including: marker D7, corresponding to "T" at position 51 of SEQ ID NO:13; and O4, corresponding to "A" at position 51 of SEQ ID NO:24.

[0287] 5. The method of any one of clauses 1 to 4, wherein one or more of markers i.-xii. are detected.

[0288] 6. The method of any one of clauses 1 to 4, wherein one or more of markers xiii.-xxiv. are detected.

[0289] 7. The method of any one of clauses 1 to 6, wherein each of markers i.-xii. is detected.

[0290] 8. The method of any one of clauses 1 to 6, wherein each of markers xiii.-xxiv. is detected.

[0291] 9. The method of any one of clauses 1 to 8, wherein one or more of markers iv.-ix. are detected, markers iv. and ix. are detected, or markers viii. and ix. are detected.

[0292] 10. The method of any one of clauses 1 to 8, wherein one or more of markers xviii.-xxi., or markers xviii. and xxi. are detected.

[0293] 11. A method of producing a maize plant comprising a QTL associated with resistance to southern leaf blight, the method comprising:

[0294] a. Screening a population of maize plants for a QTL associated with resistance to southern leaf blight, wherein the QTL comprises one or more marker alleles selected from the group consisting of:

[0295] i. "C" at D6, corresponding to position 51 of SEQ ID NO: 1;

[0296] ii. "C" at C10, corresponding to position 51 of SEQ ID NO: 2;

[0297] iii. "C" at A10, corresponding to position 51 of SEQ ID NO: 3;

[0298] iv. "G" at G6, corresponding to position 51 of SEQ ID NO: 4;

[0299] v. "G" at H6, corresponding to position 51 of SEQ ID NO: 5;

[0300] vi. "T" at D10, corresponding to position 51 of SEQ ID NO: 6;

[0301] vii. "C" at F10, corresponding to position 51 of SEQ ID NO: 7;

[0302] viii. "A" at K6, corresponding to position 51 of SEQ ID NO: 8;

[0303] ix. "C" at L6, corresponding to position 51 of SEQ ID NO:9;

[0304] "T" at x.06, corresponding to position 51 of SEQ ID NO: 10;

[0305] xi. "G" at Q6, corresponding to position 51 of SEQ ID NO: 11; and

[0306] xii. "C" at G3, corresponding to position 51 of SEQ ID NO: 12;

[0307] or one or more marker alleles selected from the group consisting of:

[0308] xiii. "T" at D7, corresponding to position 51 of SEQ ID NO: 13;

[0309] xiv. "T" at L4, corresponding to position 51 of SEQ ID NO: 14;

[0310] xv. "T" at E7, corresponding to position 51 of SEQ ID NO: 15;

[0311] xvi. "A" at G7, corresponding to position 51 of SEQ ID NO: 16;

[0312] xvii. "G" at H7, corresponding to position 51 of SEQ ID NO: 17;

[0313] xviii. "T" at I7, corresponding to position 51 of SEQ ID NO: 18;

[0314] xix. "T" at J7, corresponding to position 51 of SEQ ID NO: 19;

[0315] "G" at xx.K7, corresponding to position 51 of SEQ ID NO:20;

[0316] xxi. "A" at L7, corresponding to position 51 of SEQ ID NO: 21;

[0317] xxii. "T" at M7, corresponding to position 51 of SEQ ID NO: 22;

[0318] xxiii. "A" at N7, corresponding to position 51 of SEQ ID NO: 23; and

[0319] xxiv. "A" at O4, corresponding to position 51 of SEQ ID NO: 24;

[0320] b. selecting at least one maize plant comprising the QTL from the population;

[0321] c. hybridizing the maize plant selected in (b) with a second maize plant; and

[0322] d. Obtaining progeny plants comprising the QTL.

[0323] 12. The method of clause 11, further comprising introgressing the QTL into a second population of maize plants.

[0324] 13. A method as described in clause 11 or 12, wherein the QTL is located on a chromosome interval flanked by and including: marker D6, corresponding to "C" at position 51 of SEQ ID NO: 1; and G3, corresponding to "C" at position 51 of SEQ ID NO: 12.

[0325] 14. A method as described in claim 11 or 12, wherein the QTL allele is located on the chromosome interval flanked by and including: marker D7, corresponding to "T" at position 51 of SEQ ID NO: 13; and O4, corresponding to "A" at position 51 of SEQ ID NO: 24.

[0326] 15. The method of any one of clauses 11 to 14, wherein the selected plants and progeny plants comprise one or more of markers i.-xii.

[0327] 16. The method of any one of clauses 11 to 14, wherein the selected plants and progeny plants comprise one or more of markers xiii.-xxiv.

[0328] 17. The method of any one of clauses 11 to 16, wherein the selected plants and progeny plants comprise each of markers i.-xii.

[0329] 18. The method of any one of clauses 11 to 16, wherein the selected plants and progeny plants comprise each of markers xiii.-xxiv.

[0330] 19. The method of any one of clauses 11 to 18, wherein the selected plants and progeny plants comprise one or more of markers iv.-ix., both markers iv. and ix., or both markers viii. and ix.

[0331] 20. The method of any one of clauses 11 to 18, wherein the selected plants and progeny plants comprise one or more of markers xviii.-xxi., or both markers xviii. and xxi.

[0332] 21. A method of selecting maize plants comprising resistance to southern leaf blight, the method comprising:

[0333] a. screening a population of maize plants for the presence of a gene encoding a protein comprising at least 95% amino acid sequence identity to one or more of SEQ ID NOs: 27, 30, or 33, and

[0334] b. Selecting maize plants comprising the gene.

[0335] 22. The method of clause 21, wherein the protein comprises 100% amino acid sequence identity to SEQ ID NO: 27, 30 or 33.

[0336] 23. The method according to clause 21 or 22, further comprising:

[0337] c. hybridizing the maize plant selected in (b) with a second maize plant; and

[0338] d. obtaining progeny plants comprising the gene.

[0339] 24. The method of any one of clauses 21 to 23, wherein the protein is encoded by a gene comprising a nucleotide sequence having at least 95% nucleotide sequence identity to one or more of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID NO: 32.

[0340] 25. The method of any one of clauses 21 to 24, wherein the protein is encoded by a gene comprising a nucleotide sequence comprising the sequence of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID NO: 32.

[0341] 26. A method for producing a recombinant maize plant cell, the method comprising:

[0342] introducing a heterologous P450 gene sequence into the genome of a maize plant cell, and

[0343] A plant cell is selected that comprises the heterologous P450 gene sequence encoded in the genome of the plant cell.

[0344] 27. The method of clause 26, further comprising:

[0345] a) introducing a site-specific modification of at least one target site into the genome of said maize plant cell, and

[0346] b) introducing a polynucleotide modification template comprising the heterologous P450 nucleotide sequence into the plant cell.

[0347] 28. The method of clause 27, wherein the site-specific modification is induced by a CRISPR-associated endonuclease.

[0348] 29. The method of any one of clauses 26 to 28, further comprising growing a plant from a plant cell comprising the heterologous P450 gene sequence, wherein the plant exhibits increased resistance to southern leaf blight.

[0349] 30. The method of any one of clauses 26 to 29, wherein the heterologous P450 gene sequence replaces an endogenous P450 locus.

[0350] 31. The method of any one of clauses 26 to 29, wherein the heterologous P450 gene sequence is inserted into a locus different from the endogenous P450 gene locus.

[0351] 32. The method of any one of clauses 26 to 31, wherein the P450 gene sequence encodes a protein comprising an amino acid sequence at least 95% identical to one or more of SEQ ID NOs: 27, 30 or 33.

[0352] 33. The method of any one of clauses 26 to 32, wherein the P450 gene sequence encodes a protein comprising the amino acid sequence of SEQ ID NO: 27, 30 or 33.

[0353] 34. The method of any one of clauses 26 to 33, wherein the P450 gene sequence comprises a nucleotide sequence comprising at least 95% identity to one or more of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID NO: 32.

[0354] 35. The method of any one of clauses 26 to 34, wherein the P450 gene sequence comprises the nucleotide sequence of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID NO: 32.

[0355] 36. A method for producing a recombinant maize plant cell, the method comprising:

[0356] introducing a P450 nucleotide sequence into one or more plant cells;

[0357] inserting the P450 nucleotide sequence into the genome of at least one of the one or more plant cells; and

[0358] A plant cell is selected in which the P450 nucleotide sequence has been inserted into the genome of the plant cell.

[0359] 37. The method of clause 36, wherein the P450 nucleotide sequence encodes a protein comprising an amino acid sequence that is at least 95% identical to one or more of SEQ ID NOs: 27, 30, or 33.

[0360] 38. The method of clause 36 or 37, wherein the P450 nucleotide sequence encodes a protein comprising the amino acid sequence of SEQ ID NO: 27, 30 or 33.

[0361] 39. The method of any one of clauses 36 to 38, wherein the P450 nucleotide sequence comprises at least 95% identity to one or more of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID NO: 32.

[0362] 40. The method of any one of clauses 36 to 39, wherein the P450 nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID NO: 32.

[0363] 41. The method of any one of clauses 36 to 40, further comprising growing a plant from the plant cell into which the P450 nucleotide sequence has been inserted into the genome of the plant cell, wherein the plant exhibits increased resistance to southern leaf blight.

[0364] 42. The method of any one of clauses 36 to 41, wherein introducing the P450 nucleotide sequence comprises bacteria-mediated transformation.

[0365] 43. The method of any one of clauses 36 to 41, wherein the introduction of the P450 nucleotide sequence is accomplished via biolistic transformation.

[0366] 44. A recombinant plant comprising a heterologous gene encoding a protein comprising at least 95% amino acid sequence identity to one or more of SEQ ID NO: 27, 30, or 33.

[0367] 45. The recombinant plant of Clause 44, wherein the protein comprises 100% amino acid sequence identity to SEQ ID NO: 27, 30 or 33.

[0368] 46. ​​The recombinant plant of clause 44 or 45, wherein the gene comprises a nucleotide sequence comprising at least 95% nucleotide sequence identity to one or more of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID NO: 32.

[0369] 47. The recombinant plant of any one of clauses 44 to 46, wherein the gene comprises the nucleotide sequence of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID NO: 32.

[0370] 48. A recombinant plant seed comprising a heterologous gene encoding a protein comprising at least 95% amino acid sequence identity to one or more of SEQ ID NO: 27, 30, or 33.

[0371] 49. The recombinant seed of clause 48, wherein the protein comprises 100% amino acid sequence identity to SEQ ID NO: 27, 30 or 33.

[0372] 50. The recombinant seed of clause 48 or 49, wherein the gene comprises a nucleotide sequence comprising at least 95% nucleotide sequence identity to one or more of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID NO: 32.

[0373] 51. The recombinant seed of any one of clauses 48 to 50, wherein the gene comprises the nucleotide sequence of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID NO: 32.

[0374] Examples

[0375] The following examples are provided to illustrate but not limit various aspects of the present disclosure. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and those skilled in the art will recognize that various reagents or parameters can be changed without departing from the spirit of the present disclosure or the scope of the appended claims.

[0376] Example 1

[0377] To map the genomic loci responsible for SLB resistance, three SLB-resistant inbred lines (CML304, CIMBL83, and K22) were used to generate recombinant inbred line (RIL) populations, with GEMS41 serving as the SLB-susceptible parent. The RIL populations were grown, inoculated with southern leaf spot pathogen B. maydis, and collected in Henan, China for SLB scores and genotyped using genome-wide SNP markers. QTL analysis identified a major SLB resistance QTL within the same chromosome 3 interval (between SNP markers D6 and G3) in all three RIL populations. This QTL was designated qSLB3.1.

[0378] Example 2

[0379] CML304 and GEMS41 were used to develop a large BC5F2 population for fine mapping of qSLB3.1. Approximately 3,000 BC5F2 individuals were genotyped using markers D6 and G3 to identify recombinants within the QTL interval. Recombinants were scored as SLB-resistant or susceptible and genotyped using additional single-nucleotide polymorphism (SNP) markers within the interval. qSLB3.1 was defined as a 51 kb interval (B73 v4 reference sequence) flanked by SNP markers K6 and G8.

[0380] Initial markers determined for qSLB3.1 in the CML304 and GEMS41 genomes include those described in Table 3. The sequences in Table 3 are presented in the same manner as Table 1.

[0381] Table 3

[0382]

[0383] A similar process was performed to generate BC5F2 populations of K22 and GEMS41. After identifying and genotyping the recombinants, additional SNP markers for the QTL were found in the K22 parent. Without wishing to be bound by theory, it is believed that both QTLs (one in the CML304 and CIMBL83 parents and one in the K22 parent) contain the same causal gene. The markers represented by SEQ ID NOs: 1-12 in Table 1 are derived from CML304 and GEMS41. The markers represented by SEQ ID NOs: 13-24 in Table 1 are derived from K22.

[0384] Example 3

[0385] The genomes of three SLB-resistant lines (CML304, CIMBL83, and K22) and the susceptible parent (GEMS41) were sequenced, and RNA-seq data were generated to facilitate gene annotation. Two annotated genes were identified within the qSLB3.1 interval, the first of which encodes a cytochrome P450 protein and the second encodes an apoptosis-antagonizing transcription factor. Comparative analysis of the qSLB3.1 interval between resistant and susceptible lines revealed that the transcription factor was absent in two of the three resistant lines. The P450 gene was identical or nearly identical (97.2%-100% identical) in the three resistant alleles, whereas it was truncated and likely nonfunctional in the susceptible allele. It was concluded that the P450 gene was the only plausible candidate gene for qSLB3.1.

[0386] Example 4 - Transgenic Validation of qSLB3.1 Candidate P450 Gene

[0387] Transgenic plants were generated using constructs containing the CDS of P450 genes (identical to SEQ ID NOs: 27 and 30), where expression was driven by a constitutive maize promoter. T1 plants isolated from three different events were subjected to a greenhouse SLB assay. The results showed that transgenic-positive plants had significantly enhanced levels of SLB resistance when compared to transgenic-negative (null) plants. These results confirm that P450 genes are the causal resistance genes underlying qSLB3.1.

[0388] In all cases, SLB scoring was based on a scale of 1-9, with 1 being the most susceptible to SLB and 9 being the most resistant to SLB.

[0389] Table 4

[0390]

[0391] Example 4 - Transgenic Validation of qSLB3.1 Candidate P450 Gene

[0392] Transgenic plants were generated using constructs containing the CDS of P450 genes (consistent with SEQ ID NO: 27, 30, and / or 33), where expression was driven by a constitutive maize promoter. T1 plants isolated from each of the different events were subjected to greenhouse SLB assays. The expected results showed that transgenic positive plants had significantly enhanced levels of SLB resistance when compared to transgenic negative (null) plants.

Claims

1. A method for identifying a maize plant comprising a marker allele associated with resistance to southern leaf blight, the method comprising: Nucleic acids from maize plants are screened for at least one of the following marker alleles on chromosome 3: i. "C" at D6, corresponding to position 51 of SEQ ID NO: 1; ii. "C" at C10, corresponding to position 51 of SEQ ID NO: 2; iii. "C" at A10, corresponding to position 51 of SEQ ID NO: 3; iv. "G" at G6, corresponding to position 51 of SEQ ID NO: 4; v. "G" at H6, corresponding to position 51 of SEQ ID NO:5; vi. "T" at D10, corresponding to position 51 of SEQ ID NO: 6; vii. "C" at F10, corresponding to position 51 of SEQ ID NO: 7; viii. "A" at K6, corresponding to position 51 of SEQ ID NO: 8; ix. "C" at L6, corresponding to position 51 of SEQ ID NO:9; "T" at x.06 corresponds to position 51 of SEQ ID NO: 10; xi. "G" at Q6, corresponding to position 51 of SEQ ID NO: 11; xii. "C" at G3, corresponding to position 51 of SEQ ID NO: 12; xiii. "T" at D7 corresponds to position 51 of SEQ ID NO: 13; xiv. "T" at L4, corresponding to position 51 of SEQ ID NO: 14; xv. "T" at E7, corresponding to position 51 of SEQ ID NO: 15; xvi. "A" at G7, corresponding to position 51 of SEQ ID NO: 16; xvii. "G" at H7, corresponding to position 51 of SEQ ID NO: 17; xviii. "T" at I7 corresponds to position 51 of SEQ ID NO: 18; xix. "T" at J7, corresponding to position 51 of SEQ ID NO: 19; "G" at xx.K7, corresponding to position 51 of SEQ ID NO:20; xxi. "A" at L7, corresponding to position 51 of SEQ ID NO: 21; xxii. "T" at M7, corresponding to position 51 of SEQ ID NO: 22; xxiii. "A" at N7, corresponding to position 51 of SEQ ID NO: 23; or xxiv. "A" at O4 corresponds to position 51 of SEQ ID NO: 24, The presence of the marker allele is associated with resistance to southern leaf blight.

2. The method of claim 1, further comprising a. obtaining nucleic acid samples from multiple plants, seeds, tissues or germplasm in a population; b. screening each sample for at least one of the nucleic acid marker alleles on chromosome 3; as well as c. Selecting one or more of plants, seeds, tissues, or germplasm comprising at least one of said marker alleles associated with resistance to southern leaf blight.

3. The method of claim 1 or 2, wherein the marker alleles screened are located flanking and including the following chromosomal intervals: marker D6, corresponding to "C" at position 51 of SEQ ID NO: 1; and G3, corresponding to "C" at position 51 of SEQ ID NO:

12.

4. The method of claim 1 or 2, wherein the marker alleles screened are located flanking and including the following chromosomal intervals: marker D7, corresponding to "T" at position 51 of SEQ ID NO: 13; and O4, corresponding to "A" at position 51 of SEQ ID NO:

24.

5. The method of any one of claims 1 to 4, wherein one or more of markers i.-xii. are detected.

6. The method of any one of claims 1 to 4, wherein one or more of markers xiii.-xxiv. are detected.

7. The method of any one of claims 1 to 6, wherein each of markers i.-xii. is detected.

8. The method of any one of claims 1 to 6, wherein each of markers xiii.-xxiv. is detected.

9. A method of producing a maize plant comprising a QTL associated with resistance to southern leaf blight, the method comprising: a. Screening a population of maize plants for a QTL associated with resistance to southern leaf blight, wherein the QTL comprises one or more marker alleles selected from the group consisting of: i. "C" at D6, corresponding to position 51 of SEQ ID NO: 1; ii. "C" at C10, corresponding to position 51 of SEQ ID NO: 2; iii. "C" at A10, corresponding to position 51 of SEQ ID NO: 3; iv. "G" at G6, corresponding to position 51 of SEQ ID NO: 4; v. "G" at H6, corresponding to position 51 of SEQ ID NO:5; vi. "T" at D10, corresponding to position 51 of SEQ ID NO: 6; vii. "C" at F10, corresponding to position 51 of SEQ ID NO: 7; viii. "A" at K6, corresponding to position 51 of SEQ ID NO: 8; ix. "C" at L6, corresponding to position 51 of SEQ ID NO:9; "T" at x.06 corresponds to position 51 of SEQ ID NO: 10; xi. "G" at Q6, corresponding to position 51 of SEQ ID NO: 11; as well as xii. "C" at G3, corresponding to position 51 of SEQ ID NO: 12; or one or more marker alleles selected from the group consisting of: xiii. "T" at D7 corresponds to position 51 of SEQ ID NO: 13; xiv. "T" at L4, corresponding to position 51 of SEQ ID NO: 14; xv. "T" at E7, corresponding to position 51 of SEQ ID NO: 15; xvi. "A" at G7, corresponding to position 51 of SEQ ID NO: 16; xvii. "G" at H7, corresponding to position 51 of SEQ ID NO: 17; xviii. "T" at I7 corresponds to position 51 of SEQ ID NO: 18; xix. "T" at J7, corresponding to position 51 of SEQ ID NO: 19; "G" at xx.K7, corresponding to position 51 of SEQ ID NO:20; xxi. "A" at L7, corresponding to position 51 of SEQ ID NO: 21; xxii. "T" at M7, corresponding to position 51 of SEQ ID NO: 22; xxiii. "A" at N7, corresponding to position 51 of SEQ ID NO: 23; as well as xxiv. "A" at O4, corresponding to position 51 of SEQ ID NO: 24; b. selecting at least one maize plant comprising the QTL from the population; c. hybridizing the maize plant selected in (b) with a second maize plant; and d. Obtaining progeny plants comprising the QTL.

10. The method of claim 9, further comprising introgressing the QTL into a second population of maize plants.

11. The method of claim 9 or 10, wherein the QTL is located on a chromosomal interval flanked by and including: marker D6, corresponding to "C" at position 51 of SEQ ID NO: 1; and G3, corresponding to "C" at position 51 of SEQ ID NO:

12.

12. The method of claim 9 or 10, wherein the QTL allele is located on a chromosomal interval flanked by and including: marker D7, corresponding to "T" at position 51 of SEQ ID NO: 13; and O4, corresponding to "A" at position 51 of SEQ ID NO:

24.

13. The method of any one of claims 9 to 12, wherein the selected plants and progeny plants comprise one or more of markers i.-xii.

14. The method of any one of claims 9-12, wherein the selected plants and progeny plants comprise one or more of markers xiii.-xxiv.

15. The method of any one of claims 9 to 14, wherein the selected plants and progeny plants comprise each of markers i.-xii.

16. The method of any one of claims 9-14, wherein the selected plants and progeny plants comprise each of markers xiii.-xxiv.

17. A method of selecting maize plants comprising resistance to southern leaf blight, the method comprising: a. screening a population of maize plants for the presence of a gene encoding a protein comprising at least 95% amino acid sequence identity to one or more of SEQ ID NOs: 27, 30, or 33, and b. Selecting maize plants comprising the gene.

18. The method of claim 17, wherein the protein comprises 100% amino acid sequence identity to SEQ ID NO: 27, 30, or 33.

19. The method of claim 17 or 18, further comprising: c. hybridizing the maize plant selected in (b) with a second maize plant; as well as d. obtaining progeny plants comprising the gene.

20. The method of any one of claims 17-19, wherein the protein is encoded by a gene comprising a nucleotide sequence having at least 95% nucleotide sequence identity to one or more of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31, or SEQ ID NO:

32.

21. The method of any one of claims 17-20, wherein the protein is encoded by a gene comprising a nucleotide sequence comprising the sequence of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID NO:

32.

22. A method for producing a recombinant maize plant cell, the method comprising: introducing a heterologous P450 gene sequence into the genome of a maize plant cell, and A plant cell is selected that comprises the heterologous P450 gene sequence encoded in the genome of the plant cell.

23. The method of claim 22, further comprising: a) introducing a site-specific modification of at least one target site into the genome of said maize plant cell, and b) introducing a polynucleotide modification template comprising the heterologous P450 nucleotide sequence into the plant cell.

24. The method of claim 23, wherein the site-specific modification is induced by a CRISPR-associated endonuclease.

25. The method of any one of claims 22-24, further comprising growing a plant from a plant cell comprising the heterologous P450 gene sequence, wherein the plant exhibits increased resistance to southern leaf blight.

26. The method of any one of claims 22-25, wherein the heterologous P450 gene sequence replaces an endogenous P450 locus.

27. The method of any one of claims 22-25, wherein the heterologous P450 gene sequence is inserted into a locus different from the endogenous P450 gene locus.

28. The method of any one of claims 22-27, wherein the P450 gene sequence encodes a protein comprising an amino acid sequence at least 95% identical to one or more of SEQ ID NOs: 27, 30, or 33.

29. The method of any one of claims 22 to 28, wherein the P450 gene sequence encodes a protein comprising the amino acid sequence of SEQ ID NO: 27, 30 or 33.

30. The method of any one of claims 22-29, wherein the P450 gene sequence comprises a nucleotide sequence comprising at least 95% identity to one or more of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31, or SEQ ID NO:

32.

31. The method of any one of claims 22-30, wherein the P450 gene sequence comprises the nucleotide sequence of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31 or SEQ ID NO:

32.

32. A method for producing a recombinant maize plant cell, the method comprising: introducing a P450 nucleotide sequence into one or more plant cells; inserting the P450 nucleotide sequence into the genome of at least one of the one or more plant cells; as well as A plant cell is selected in which the P450 nucleotide sequence has been inserted into the genome of the plant cell.

33. The method of claim 32, wherein the P450 nucleotide sequence encodes a protein comprising an amino acid sequence at least 95% identical to one or more of SEQ ID NOs: 27, 30, or 33.

34. The method of claim 32 or 33, wherein the P450 nucleotide sequence encodes a protein comprising the amino acid sequence of SEQ ID NO: 27, 30 or 33.

35. The method of any one of claims 32-34, wherein the P450 nucleotide sequence comprises at least 95% identity to one or more of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31 or SEQ ID NO:

32.

36. The method of any one of claims 32-35, wherein the P450 nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:31 or SEQ ID NO:

32.

37. The method of any one of claims 34-36, further comprising growing a plant from the plant cell in which the P450 nucleotide sequence has been inserted into the genome of the plant cell, wherein the plant exhibits increased resistance to southern leaf blight.

38. The method of any one of claims 34-37, wherein introducing the P450 nucleotide sequence comprises bacteria-mediated transformation.

39. The method of any one of claims 34 to 38, wherein the introduction of the P450 nucleotide sequence is accomplished via biolistic transformation.

40. A recombinant plant comprising a heterologous gene encoding a protein comprising at least 95% amino acid sequence identity to one or more of SEQ ID NO: 27, 30, or 33.

41. The recombinant plant of claim 40, wherein the protein comprises 100% amino acid sequence identity to SEQ ID NO: 27, 30, or 33.

42. The recombinant plant of claim 40 or 41, wherein the gene comprises a nucleotide sequence comprising at least 95% nucleotide sequence identity to one or more of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31, or SEQ ID NO:

32.

43. The recombinant plant of any one of claims 40-42, wherein the gene comprises the nucleotide sequence of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID NO:

32.

44. A recombinant plant seed comprising a heterologous gene encoding a protein comprising at least 95% amino acid sequence identity to one or more of SEQ ID NO: 27, 30, or 33.

45. The recombinant seed of claim 44, wherein the protein comprises 100% amino acid sequence identity to SEQ ID NO: 27, 30, or 33.

46. ​​The recombinant seed of claim 44 or 45, wherein the gene comprises a nucleotide sequence comprising at least 95% nucleotide sequence identity to one or more of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31, or SEQ ID NO:

32.

47. The recombinant seed of any one of claims 44-46, wherein the gene comprises the nucleotide sequence of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31 or SEQ ID NO: 32.

Images