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WO2025184759A1 - Plantes de maïs présentant une résistance aux taches grises des feuilles, compositions et procédés de sélection et de production associés - Google Patents

Plantes de maïs présentant une résistance aux taches grises des feuilles, compositions et procédés de sélection et de production associés

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Publication number
WO2025184759A1
WO2025184759A1 PCT/CN2024/079799 CN2024079799W WO2025184759A1 WO 2025184759 A1 WO2025184759 A1 WO 2025184759A1 CN 2024079799 W CN2024079799 W CN 2024079799W WO 2025184759 A1 WO2025184759 A1 WO 2025184759A1
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WO
WIPO (PCT)
Prior art keywords
plant
protein
polynucleotide
seq
heterologous
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WO2025184759A8 (fr
Inventor
Zhibing LAI
Zhikang DAI
Long HU
Qianyu PI
Bailin Li
Girma M. TABOR
Shawn Thatcher
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Huazhong Agricultural University
Pioneer Hi Bred International Inc
Original Assignee
Huazhong Agricultural University
Pioneer Hi Bred International Inc
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Priority to PCT/CN2024/079799 priority Critical patent/WO2025184759A1/fr
Publication of WO2025184759A1 publication Critical patent/WO2025184759A1/fr
Publication of WO2025184759A8 publication Critical patent/WO2025184759A8/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

Definitions

  • the present disclosure is related to maize plants comprising resistance to gray leaf spot and compositions and methods for selecting and producing the same.
  • Gray leaf spot is a disease of maize plants caused by the fungal pathogens Cercospora zeae-maydis and Cercospora zeina. GLS is one of the most significant diseases of maize worldwide. GLS can cause significant reduction in yield, grain weight and quality. Yield losses occur from premature plant death that interrupts filling of the grain and from stalk breakage and lodging that causes ears to be lost in the field. Gray leaf spot occurs in all corn growing areas and can result in 10%to 20%losses.
  • GLS is a concern to farmers and grain producers due to its potential for reducing yields and/or quality of maize crops. While there are several techniques for reducing the impact of GLS on maize crops exist, these can impact the pathogens and/or maize differently. There remains a need for additional development of new options for managing and minimizing the potential impact of GLS.
  • the present disclosure provides maize plants, methods, and other compositions that provide resistance to gray leaf spot (GLS) .
  • Resistance can be provided via the WAK01 gene disclosed herein. Breeding and/or genetic engineering techniques can be used to produce plants comprising the resistance provided via the WAK01 gene.
  • the plants provided herein can improve agricultural yields and/or reduce or eliminate the need for fungicide application when GLS is a concern.
  • the method comprises screening a population of maize plants for a protein, or a gene encoding the protein.
  • the protein comprises an amino acid sequence comprising at least 95%sequence identity to SEQ ID NO: 3 and selecting a maize plant comprising the gene.
  • the method comprises introducing a heterologous gene sequence into one or more maize plant cells derived from a first maize plant, thereby producing at least one modified cell comprising the heterologous gene sequence in the modified cell’s genome and selecting a modified maize plant cell.
  • the heterologous gene sequence encodes a protein comprising at least 95%amino acid sequence identity to SEQ ID NO: 3.
  • Also provided herein is a method for producing a recombinant maize plant cell comprising a heterologous gene associated with resistance to GLS.
  • the method comprises introducing a gene sequence into one or more plant cells derived from a first plant; inserting the gene sequence into the genome of at least one of the plant cells, thereby creating one or more modified plant cells; and selecting a modified plant cell.
  • the gene sequence encodes a protein comprising an amino acid sequence at least 95%identical to SEQ ID NO: 3.
  • Also provided herein is a recombinant plant.
  • Also provided herein is a recombinant plant seed.
  • sequence descriptions and sequence listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. ⁇ 1.831-1.834 and in the WIPO ST. 26 Standard.
  • the symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. ⁇ 1.832.
  • the case of individual letters representing amino acid or nucleotide residues does not have any significance (e.g., “atgc” is the same as “ATGC” , and “MRYG” is the same as “mryg” .
  • each disclosed nucleotide sequence should be understood to encompass its complementary strand (i.e., reverse complement sequence) .
  • SEQ ID NO: 1 is the WAK01 genomic gene sequence, comprising promoter regions, coding regions, and terminator regions.
  • SEQ ID NO: 2 is the WAK01 coding sequence (e.g., a cDNA sequence) , which codes for the 960 amino acid residues of the WAK01 protein.
  • the stop codon (encoded as TAA) is not included in SEQ ID NO: 2.
  • SEQ ID NO: 3 is the WAK01 protein, as encoded by SEQ ID NO: 1 and SEQ ID NO: 2.
  • SEQ ID NO: 4 is the TD209 flanking marker reference sequence, with the SNP at position 28 indicated as a “y” , meaning “c or t” .
  • the SNP linked with resistance to GLS comprises a “c” at position 28.
  • SEQ ID NO: 5 is the TD380 flanking marker reference sequence, with the SNP at position 30 indicated as an “s” , meaning “g or c” .
  • the SNP linked with resistance to GLS comprises a “g” at position 30.
  • the present disclosure provides a gene which has been determined to be a source of genetic resistance to GLS.
  • the present disclosure also provides methods of selecting resistant plants or to counter-select susceptible plants.
  • Maize plants comprising increased resistance to GLS, relative to control plants (e.g., a parental line) are also provided herein.
  • the WAK01 causal gene which provides the newly conferred or increased resistance when expressed in the plants, is provided herein.
  • Plants expressing and/or encoding the WAK01 gene are also provided herein.
  • plant also includes a plurality of plants; also, depending on the context, use of the term “plant” can also include genetically similar or identical progeny of that plant; use of the term “anucleic acid” optionally includes, as a practical matter, many copies of that nucleic acid molecule; similarly, the term “probe” optionally (and typically) encompasses many similar or identical probe molecules.
  • nucleic acids are written left to right in 5' to 3' orientation.
  • Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer or any non-integer fraction within the defined range.
  • all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. In describing and claiming the subject matter of the current disclosure, the following terminology will be used in accordance with the definitions set out below and throughout the specification.
  • allele refers to one of two or more different nucleotide sequences that occur at a specific locus.
  • Allele frequency refers to the frequency (proportion or percentage) at which an allele is present at a locus within an individual, within a line, or within a population of lines.
  • allele frequencies 1.0, 0.5, or 0.0, respectively.
  • One can estimate the allele frequency within a line by averaging the allele frequencies of a sample of individuals from that line.
  • one can calculate the allele frequency within a population of lines by averaging the allele frequencies of lines that make up the population. For a population with a finite number of individuals or lines, an allele frequency can be expressed as a count of individuals or lines (or any other specified grouping) containing the allele.
  • amplifying in the context of nucleic acid amplification is any process whereby additional copies of a selected nucleic acid (or a transcribed form thereof) are produced.
  • Typical amplification methods include various polymerase based replication methods, including the polymerase chain reaction (PCR) , ligase mediated methods such as the ligase chain reaction (LCR) and RNA polymerase based amplification (e.g., by transcription) methods.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • RNA polymerase based amplification e.g., by transcription
  • An allele is “associated with” a trait when it is part of or linked to a DNA sequence that affects the expression of the trait.
  • the presence of the allele is an indicator of how the trait will be expressed. For example, an allele can be linked to resistance to SR if the allele is part of or linked to a gene that can provide resistance to SR.
  • haplotypes or QTLs can be “associated with” a trait.
  • a centimorgan ( “cM” ) is a unit of measure of recombination frequency.
  • One cM is equal to a 1%chance that a marker at one genetic locus will be separated from a marker at a second locus due to crossing over in a single generation.
  • chromosomal interval designates a contiguous linear span of genomic DNA that resides in planta on a single chromosome.
  • the genetic elements or genes located on a single chromosomal interval are physically linked.
  • the size of a chromosomal interval is not particularly limited.
  • the genetic elements located within a single chromosomal interval are genetically linked, typically with a genetic recombination distance of, for example, less than or equal to 20 cM, or alternatively, less than or equal to 10 cM. That is, two genetic elements within a single chromosomal interval undergo recombination at a frequency of less than or equal to 20%or 10%.
  • a “chromosome” is a single piece of coiled DNA containing many genes that act and move as a unity during cell division and therefore can be said to be linked. It can also be referred to as a “linkage group” .
  • contiguous DNA refers to an uninterrupted stretch of genomic DNA represented by partially overlapping pieces or contigs.
  • crossed refers to a sexual cross and involved the fusion of two haploid gametes via pollination to produce diploid progeny (e.g., cells, seeds or plants) .
  • diploid progeny e.g., cells, seeds or plants.
  • the term encompasses both the pollination of one plant by another and selfing (or self-pollination, e.g., when the pollen and ovule are from the same plant) .
  • a plant referred to herein as “diploid” has two sets of chromosomes.
  • a plant referred to herein as a “doubled haploid” is developed by doubling the haploid set of chromosomes (i.e., half the normal number of chromosomes) .
  • a doubled haploid plant has two identical sets of chromosomes, and all loci are considered homozygous.
  • An “elite line” is any line that has resulted from breeding and selection for superior agronomic performance.
  • a “favorable allele” is the allele at a particular locus that confers, or contributes to, an agronomically desirable phenotype, e.g., SR resistance.
  • a favorable allele of a marker is a marker allele that segregates with (e.g., is linked to) the favorable phenotype.
  • Fragments is intended to mean a portion of a nucleotide sequence. Fragments can be used as hybridization probes or PCR primers using methods disclosed herein.
  • a “genetic map” is a description of genetic linkage relationships among loci on one or more chromosomes (or linkage groups) within a given species, generally depicted in a diagrammatic or tabular form. For each genetic map, distances between loci are measured by how frequently their alleles appear together in a population (their recombination frequencies) . Alleles can be detected using DNA or protein markers, or observable phenotypes.
  • a genetic map is a product of the mapping population, types of markers used, and the polymorphic potential of each marker between different populations. Genetic distances between loci can differ from one genetic map to another. However, information can be correlated from one map to another using common markers.
  • One of ordinary skill in the art can use common marker positions to identify positions of markers and other loci of interest on each individual genetic map.
  • the order of loci should not change between maps, although frequently there are small changes in marker orders due to e.g., markers detecting alternate duplicate loci in different populations, differences in statistical approaches used to order the markers, novel mutation or laboratory error.
  • a “genetic map location” is a location on a genetic map relative to surrounding genetic markers on the same linkage group where a specified marker can be found within a given species.
  • Genetic mapping is the process of defining the linkage relationships of loci through the use of genetic markers, populations segregating for the markers, and standard genetic principles of recombination frequency.
  • Gene markers are nucleic acids that are polymorphic in a population and where the alleles of which can be detected and distinguished by one or more analytic methods, described below. The terms also refer to nucleic acid sequences complementary to the genomic sequences, such as nucleic acids used as probes.
  • Markers corresponding to genetic polymorphisms between members of a population can be detected by analytic methods such as, for example, PCR-based sequence specific amplification methods, detection of restriction fragment length polymorphisms (RFLP) , detection of isozyme markers, detection of polynucleotide polymorphisms by allele specific hybridization (ASH) , detection of amplified variable sequences of the plant genome, detection of self-sustained sequence replication, detection of simple sequence repeats (SSRs) , detection of single nucleotide polymorphisms (SNPs) , detection of amplified fragment length polymorphisms (AFLPs) , or next generation sequencing techniques.
  • analytic methods such as, for example, PCR-based sequence specific amplification methods, detection of restriction fragment length polymorphisms (RFLP) , detection of isozyme markers, detection of polynucleotide polymorphisms by allele specific hybridization (ASH) , detection of amplified variable sequences of the
  • ESTs expressed sequence tags
  • RAPD randomly amplified polymorphic DNA
  • markers are provided herein, such as the SNPs in Table 1, which are linked to a favorable allele providing SR resistance.
  • Marker allele refers to the particular sequence at the marker locus that is linked to the favorable allele.
  • Geneetic recombination frequency is the frequency of a crossing over event (recombination) between two genetic loci. Recombination frequency can be observed by following the segregation of markers and/or traits following meiosis.
  • Gene refers to the total DNA, or the entire set of genes, carried by an individual.
  • genotype is the genetic constitution of an individual (or group of individuals) at one or more genetic loci. Genotype is defined by the allele (s) of one or more known loci that the individual has inherited from its parents.
  • genotype can be used to refer to an individual’s genetic constitution at a single locus, at multiple loci, or, more generally, the term genotype can be used to refer to an individual’s genetic make-up for all the genes in its genome.
  • germplasm refers to genetic material of or from an individual (e.g., a plant) , a group of individuals (e.g., a plant line, variety or family) , or a clone derived from a line, variety, species, or culture, or more generally, all individuals within a species or for several species (e.g., maize germplasm collection or Andean germplasm collection) .
  • the germplasm can be part of an organism or cell or can be separate from the organism or cell.
  • germplasm provides genetic material with a specific molecular makeup that provides a physical foundation for some or all of the hereditary qualities of an organism or cell culture.
  • germplasm includes cells, seed or tissues from which new plants may be grown, or plant parts, such as leaves, stems, pollen, or cells, that can be cultured into a whole plant.
  • a plant referred to as “haploid” has a single set (genome) of chromosomes.
  • haplotype is the genotype of an individual at a plurality of genetic loci, i.e., a combination of alleles.
  • genetic loci described by a haplotype are physically and genetically linked, i.e., on the same chromosome segment.
  • heterogeneity is used to indicate that individuals within the group differ in genotype at one or more specific loci.
  • heterotic response of material can be defined by performance which exceeds the average of the parents (or high parent) when crossed to other dissimilar or unrelated groups.
  • An individual is “heterozygous” if more than one allele type is present at a given locus (e.g., a diploid individual with one copy each of two different alleles) .
  • homogeneity indicates that members of a group have the same genotype at one or more specific loci.
  • An individual is “homozygous” if the individual has only one type of allele at a given locus (e.g., a diploid individual has a copy of the same allele at a locus for each of two homologous chromosomes) .
  • hybrid refers to the progeny obtained between the crossing of at least two genetically dissimilar parents.
  • Hybridization or “nucleic acid hybridization” refers to the pairing of complementary RNA and DNA strands as well as the pairing of complementary DNA single strands.
  • hybridize means to form base pairs between complementary regions of nucleic acid strands.
  • inbred refers to a line that has been bred for genetic homogeneity.
  • the term "indel” refers to an insertion or deletion, wherein one line may be referred to as comprising an inserted nucleotide or piece of DNA relative to a second line, or the second line may be referred to as comprising a deletion of a nucleotide or piece of DNA relative to the first line.
  • “Introducing” means presenting to the plant or plant cell the polynucleotide or polypeptide in such a manner that the sequence gains access to the interior of a cell.
  • the methods of the present disclosure do not depend on a particular method for introducing a polynucleotide or polypeptide into a plant, only that the polynucleotide (s) or polypeptide (s) gains access to the interior of at least one cell of the plant.
  • Methods for introducing polynucleotide (s) or polypeptide (s) into plants include, but are not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.
  • “Stable transformation” as used herein means that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. “Transient transformation” as used herein means that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant, or a polypeptide is introduced into a plant.
  • Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al. (1986) Biotechniques 4: 320-334) , electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606) , Agrobacterium-mediated transformation (US Patent Numbers 5,563,055 and 5,981,840) , direct gene transfer (Paszkowski et al. (1984) EMBO J.
  • introgression refers to the transmission of a desired allele of a genetic locus from one genetic background to another.
  • introgression of a desired allele at a specified locus can be transmitted to at least one progeny via a sexual cross between two parents of the same species, where at least one of the parents has the desired allele in its genome.
  • transmission of an allele can occur by recombination between two donor genomes, e.g., in a fused protoplast, where at least one of the donor protoplasts has the desired allele in its genome.
  • the desired allele can be, e.g., detected by a marker that is associated with a phenotype, at a QTL, a transgene, or the like.
  • offspring comprising the desired allele can be repeatedly backcrossed to a line comprising a desired genetic background and selected for the desired allele, to result in the allele becoming fixed in a selected genetic background.
  • Backcrossing refers to the process whereby hybrid progeny are crossed back to one of the parents.
  • the “donor” parent refers to the parental plant with the desired gene/genes, locus/loci, or specific phenotype to be introgressed.
  • the “recipient” parent (used one or more times) or “recurrent” parent (used two or more times) refers to the parental plant into which the gene or locus is being backcrossed. Repeated backcrossing can result in “introgression” of the allele into the recipient or recurrent parent line. For example, see Ragot, M. et al.
  • a “line” or “strain” is a group of individuals of identical parentage that are generally inbred to some degree and that are generally homozygous and homogeneous at most loci (isogenic or near isogenic) .
  • a “subline” refers to an inbred subset of descendants that are genetically distinct from other similarly inbred subsets descended from the same progenitor.
  • linkage is used to describe the degree with which one marker locus is associated with another marker locus, a favorable allele, or some other locus.
  • the linkage relationship between a molecular marker and a locus affecting a phenotype (e.g., a favorable allele) is given as a “probability” or “adjusted probability” .
  • Linkage can be expressed as a desired limit or range.
  • any marker is linked (genetically and physically) to any other marker when the markers are separated by less than 50, 40, 30, 25, 20, or 15 map units (or cM) of a single meiosis map (agenetic map based on a population that has undergone one round of meiosis, such as e.g., an F 2 ; the IBM2 maps consist of multiple meioses) .
  • it is advantageous to define a bracketed range of linkage for example, between 10 and 20 cM, between 10 and 30 cM, or between 10 and 40 cM. The more closely a marker is linked to a second locus, the better an indicator for the second locus that marker becomes.
  • linkage disequilibrium refers to a non-random segregation of genetic loci or traits (or both) . In either case, linkage disequilibrium implies that the relevant loci are within sufficient physical proximity along a length of a chromosome so that they segregate together with greater than random (i.e., non-random) frequency. Markers that show linkage disequilibrium are considered linked. Linked loci co-segregate more than 50%of the time, e.g., from about 51%to about 100%of the time. In other words, two markers that co-segregate have a recombination frequency of less than 50% (and by definition, are separated by less than 50 cM on the same linkage group. ) As used herein, linkage can be between two markers, or alternatively between a marker and a locus affecting a phenotype.
  • Linkage disequilibrium is most commonly assessed using the measure r 2 , which is calculated using the formula described by Hill, W. G. and Robertson, A, Theor. Appl. Genet. 38:226-231 (1968) .
  • r 2 1
  • complete LD exists between the two marker loci, meaning that the markers have not been separated by recombination and have the same allele frequency.
  • the r 2 value will be dependent on the population used. Values for r 2 above 1/3 indicate sufficiently strong LD to be useful for mapping (Ardlie et al., Nature Reviews Genetics 3: 299-309 (2002) ) .
  • alleles are in linkage disequilibrium when r 2 values between pairwise marker loci are greater than or equal to 0.33, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0.
  • linkage equilibrium describes a situation where two markers independently segregate, i.e., sort among progeny randomly. Markers that show linkage equilibrium are considered unlinked (whether or not they lie on the same chromosome) .
  • LOD score The “logarithm of odds (LOD) value” or “LOD score” (Risch, Science 255: 803-804 (1992) ) is used in genetic interval mapping to describe the degree of linkage between two marker loci.
  • a LOD score of three between two markers indicates that linkage is 1000 times more likely than no linkage, while a LOD score of two indicates that linkage is 100 times more likely than no linkage.
  • LOD scores greater than or equal to two may be used to detect linkage.
  • LOD scores can also be used to show the strength of association between marker loci and quantitative traits in “quantitative trait loci” mapping. In this case, the LOD score’s size is dependent on the closeness of the marker locus to the locus affecting the quantitative trait, as well as the size of the quantitative trait effect.
  • Maize refers to a plant of the Zea mays genus and species and is also known as “corn” .
  • hybrid plant includes whole maize plants and portions of maize plants. Such portions include, for example, maize plant cells, maize plant protoplasts, maize plant cell culture or maize tissue culture from which maize plants can be regenerated, maize plant calli, maize plant clumps or maize plant cells that are part of larger plant structures, maize seeds, maize cobs, maize flowers, maize cotyledons, maize leaves, maize stems, maize buds, maize roots, maize root tips and the like.
  • Marker assisted selection (of MAS) is a process by which individual plants are selected based on marker genotypes.
  • Marker assisted counter-selection is a process by which marker genotypes are used to identify plants that will not be selected, allowing them to be removed from a breeding program or planting.
  • a “marker haplotype” refers to a combination of alleles at a marker locus.
  • a “marker locus” is a specific chromosome location in the genome of a species where a specific marker can be found.
  • a marker locus can be used to track the presence of a second linked locus, e.g., one that affects the expression of a phenotypic trait.
  • a marker locus can be used to monitor segregation of alleles at a genetically or physically linked locus.
  • a “marker probe” is a nucleic acid sequence or molecule that can be used to identify the presence of a marker locus, e.g., a nucleic acid probe that is complementary to a marker locus sequence, through nucleic acid hybridization. Marker probes comprising 30 or more contiguous nucleotides of the marker locus ( “all or a portion” of the marker locus sequence) may be used for nucleic acid hybridization. Alternatively, in some aspects, a marker probe refers to a probe of any type that is able to distinguish (i.e., genotype) the particular allele that is present at a marker locus.
  • An allele “negatively” correlates with a trait when it is linked to it and when presence of the allele is an indicator that a desired trait or trait form will not occur in a plant comprising the allele.
  • a “nucleotide” is a monomeric unit from which DNA or RNA polymers are constructed, and consists of a purine or pyrimidine base, a pentose, and a phosphoric acid group. Nucleotides (usually found in their 5'monophosphate form) are referred to by their single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively) , “C” for cytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate, “T” for deoxythymidylate, “R” for purines (A or G) , “Y” for pyrimidines (C or T) , “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleotide.
  • a “physical map” of the genome is a map showing the linear order of identifiable landmarks (including genes, markers, etc. ) on chromosome DNA.
  • the distances between landmarks are absolute (for example, measured in base pairs or isolated and overlapping contiguous genetic fragments) and not based on genetic recombination (that can vary in different populations) .
  • a “polymorphism” is a variation in the DNA between two or more individuals within a population.
  • a polymorphism preferably has a frequency of at least 1%in a population.
  • a useful polymorphism can include an SNP, a simple sequence repeat (SSR) , or an insertion/deletion polymorphism, also referred to herein as an “indel” .
  • An allele “positively” correlates with a trait when it is linked to it and when presence of the allele is an indicator that the desired trait or trait form will occur in a plant comprising the allele.
  • progeny refers to the offspring generated from a cross.
  • a “progeny plant” is a plant generated from a cross between two plants.
  • QTL quantitative trait locus
  • a "recombinant" plant or plant cell, or a “recombinant” nucleic acid or protein/peptide is a plant, plant cell, nucleic acid, or protein/peptide comprising a heterologous nucleic acid sequence (which encodes a heterologous protein/peptide or a noncoding RNA) .
  • heterologous in reference to a nucleotide or amino acid sequence means that the sequence originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • a promoter operably linked to heterologous polynucleotide is from a species different from the species from which the polynucleotide was derived, or, if from the same/analogous species, one or both are substantially modified from their original form or genetic locus, or the promoter is not the native promoter of the operably linked polynucleotide.
  • a heterologous polynucleotide sequence of the present disclosure may be “heterologous” in that the sequence is located at a genomic locus that is different than the locus observed in the native host genome.
  • the heterologous polynucleotide sequence may be located on a different chromosome compared to the native genome or may be located between different genes compared to the native genome.
  • a heterologous gene can be inserted into a genome via, for example, transformation and/or site-specific nuclease-based methods.
  • a heterologous gene can be transferred into the genome of a plant line by breeding methods such as introgression.
  • a nucleotide construct can be heterologous to a plant or plant cell if the construct contains nucleotide sequence (s) that, when incorporated into the genome, would be heterologous to the plant/plant cell.
  • a "reference sequence” or a “consensus sequence” is a defined sequence used as a basis for sequence comparison.
  • a reference sequence for a marker can be obtained by sequencing a number of lines at the locus, aligning the nucleotide sequences in a sequence alignment program (e.g., Sequencher) , and then obtaining the most common nucleotide sequence of the alignment. Polymorphisms found among the individual sequences are annotated within the consensus sequence.
  • a reference sequence is not usually an exact copy of any individual DNA sequence but represents an amalgam of available sequences and is useful for designing primers and probes to polymorphisms within the sequence.
  • GLS resistance refers to increased resistance or tolerance to a pathogen that causes GLS when compared to a control plant comprising less resistance. Resistance effects may vary from a slight increase in tolerance to the effects of the fungal pathogen (e.g., partial inhibition of pathogenesis) to total resistance such that the plant is unaffected by the presence of the fungal pathogen.
  • the examples of the disclosure provide materials and methods that will increase resistance to the pathogen that causing GLS.
  • a “topcross test” is a test performed by crossing each individual (e.g., a selection, inbred line, clone or progeny individual) with the same pollen parent or “tester” , usually a homozygous line.
  • 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 to essentially no other sequences.
  • Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5-10°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH.
  • the Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50%of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50%of the probes are occupied at equilibrium) .
  • Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is 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., greater than 50 nucleotides) .
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • a positive signal is at least two times background, preferably 10 times background hybridization.
  • Exemplary stringent hybridization conditions are often: 50%formamide, 5x SSC, and 1%SDS, incubating at 42°C, or 5x SSC, 1%SDS, incubating at 65°C, with wash in 0.2x SSC, and 0.1%SDS at 65°C.
  • a temperature of about 36°C is typical for low stringency amplification, although annealing temperatures may vary between about 32°C and 48°C, depending on primer length. Additional guidelines for determining hybridization parameters are provided in numerous references.
  • an “unfavorable allele” of a marker is a marker allele that segregates with the unfavorable plant phenotype, therefore providing the benefit of identifying plants that can be removed from a breeding program or planting.
  • yield refers to the productivity per unit area of a particular plant product of commercial value. For example, yield of maize is commonly measured in bushels of seed per acre or metric tons of seed per hectare per season. Yield is affected by both genetic and environmental factors. “Agronomics” , “agronomic traits” , and “agronomic performance” refer to the traits (and underlying genetic elements) of a given plant variety that contribute to yield over the course of growing season. Individual agronomic traits include emergence vigor, vegetative vigor, stress tolerance, disease resistance or tolerance, herbicide resistance, branching, flowering, seed set, seed size, seed density, standability, threshability and the like. Yield is, therefore, the final culmination of all agronomic traits.
  • Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter “Sambrook” ) .
  • the “heterologous WAK gene sequence” is a nucleotide sequence encoding a protein comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%amino acid sequence identity to SEQ ID NO: 3.
  • the heterologous NLR gene sequence comprises native genomic sequence from Inbred A.
  • the native genomic sequence comprises a gene encoding a protein comprising SEQ ID NO: 3.
  • Specific genetic loci correlating with a particular phenotype, such as disease resistance, can be mapped in an organism’s genome.
  • the plant breeder can advantageously use molecular markers to identify desired individuals by detecting marker alleles that show a statistically significant probability of co-segregation with a desired phenotype, manifested as linkage disequilibrium.
  • the breeder is able to rapidly select a desired phenotype by selecting for the proper molecular marker allele (aprocess called marker-assisted selection, or MAS) .
  • a variety of methods are available for detecting molecular markers or clusters of molecular markers that co-segregate with a trait of interest, such as a disease resistance trait.
  • the basic idea underlying these methods is the detection of markers, for which alternative genotypes (or alleles) have significantly different average phenotypes.
  • Trait genes are inferred to be located nearest the marker (s) that have the greatest associated genotypic difference.
  • Two such methods used to detect trait loci of interest are: 1) population-based association analysis (i.e., association mapping) and 2) traditional linkage analysis.
  • LD linkage disequilibrium
  • QTL quantitative trait loci
  • association or LD mapping aims to identify significant genotype-phenotype associations. It has been exploited as a powerful tool for fine mapping in outcrossing species such as humans (Corder et al. (1994) “Protective effect of apolipoprotein-E type-2 allele for late-onset Alzheimer-disease, ” Nat Genet 7: 180-184; Hastbacka et al. (1992) “Linkage disequilibrium mapping in isolated founder populations: diastrophic dysplasia in Finland, ” Nat Genet 2: 204-211; Kerem et al. (1989) “Identification of the cystic fibrosis gene: genetic analysis, ” Science 245: 1073-1080) and maize (Remington et al.
  • the recombinational and mutational history of a population is a function of the mating habit as well as the effective size and age of a population.
  • Large population sizes offer enhanced possibilities for detecting recombination, while older populations are generally associated with higher levels of polymorphism, both of which contribute to observably accelerated rates of LD decay.
  • smaller effective population sizes e.g., those that have experienced a recent genetic bottleneck, tend to show a slower rate of LD decay, resulting in more extensive haplotype conservation (Flint-Garcia et al. (2003) “Structure of linkage disequilibrium in plants, ” Annu Rev Plant Biol. 54: 357-374) .
  • Association analyses use quantitative phenotypic scores (e.g., disease tolerance rated from one to nine for each line) in the analysis (as opposed to looking only at tolerant versus resistant allele frequency distributions in intergroup allele distribution types of analysis) .
  • the availability of detailed phenotypic performance data collected by breeding programs over multiple years and environments for a large number of elite lines provides a valuable dataset for genetic marker association mapping analyses. This paves the way for a seamless integration between research and application and takes advantage of historically accumulated data sets. However, an understanding of the relationship between polymorphism and recombination is useful in developing appropriate strategies for efficiently extracting maximum information from these resources.
  • This type of association analysis neither generates nor requires any map data, but rather is independent of map position.
  • This analysis compares the plants’ phenotypic score with the genotypes at the various loci. Subsequently, any suitable map (for example, a composite map) can optionally be used to help observe distribution of the identified QTL markers and/or QTL marker clustering using previously determined map locations of the markers.
  • LD is generated by creating a population from a small number of founders.
  • the founders are selected to maximize the level of polymorphism within the constructed population, and polymorphic sites are assessed for their level of cosegregation with a given phenotype.
  • a number of statistical methods have been used to identify significant marker-trait associations.
  • One such method is an interval mapping approach (Lander and Botstein, Genetics 121: 185-199 (1989) , in which each of many positions along a genetic map (say at 1 cM intervals) is tested for the likelihood that a gene controlling a trait of interest is located at that position.
  • the genotype/phenotype data are used to calculate for each test position a LOD score (log of likelihood ratio) . When the LOD score exceeds a threshold value, there is significant evidence for the location of a gene controlling the trait of interest at that position on the genetic map (which will fall between two particular marker loci) .
  • Marker loci that demonstrate statistically significant co-segregation with a disease resistance trait are provided herein. Detection of these loci or additional linked loci can be used in marker assisted breeding programs to produce plants comprising disease resistance.
  • Activities in marker assisted breeding programs may include but are not limited to: selecting among new breeding populations to identify which population has the highest frequency of favorable nucleic acid sequences based on historical genotype and agronomic trait associations, selecting favorable nucleic acid sequences among progeny in breeding populations, selecting among parental lines based on prediction of progeny performance, and advancing lines in germplasm improvement activities based on presence of favorable nucleic acid sequences.
  • Chromosomal intervals can correlate with the disease resistance traits.
  • a variety of methods are available for identifying chromosomal intervals.
  • the boundaries of such chromosomal intervals can be drawn to encompass markers that will be linked to the gene (s) controlling the trait of interest.
  • the chromosomal interval can be drawn such that any marker that lies within that interval (including the terminal markers that define the boundaries of the interval) can be used as a marker for a disease resistance trait.
  • a common measure of linkage is the frequency with which traits cosegregate. This can be expressed as a percentage of cosegregation (recombination frequency) or in centiMorgans (cM) .
  • the cM is a unit of measure of genetic recombination frequency.
  • One cM is equal to a 1%chance that a trait at one genetic locus will be separated from a trait at another locus due to crossing over in a single generation (meaning the traits segregate together 99%of the time) . Because chromosomal distance is approximately proportional to the frequency of crossing over events between traits, there is an approximate physical distance that correlates with recombination frequency.
  • Marker loci are themselves traits and can be assessed according to standard linkage analysis by tracking the marker loci during segregation. Thus, one cM is equal to a 1%chance that a marker locus will be separated from another locus, due to crossing over in a single generation.
  • Closely linked loci display an inter-locus cross-over frequency of about 10%or less such as, for example, about 9%or less, about 8%or less, about 7%or less, about 6%or less, about 5%or less, about 4%or less, about 3%or less, or about 2%or less.
  • the relevant loci e.g., a marker locus and a target locus
  • the loci are about 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.75 cM, 0.5 cM or 0.25 cM or less apart.
  • two loci that are localized to the same chromosome, and at such a distance that recombination between the two loci occurs at a frequency of less than 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are said to be “proximal to” each other.
  • marker alleles can co-segregate with a disease resistance trait
  • the marker locus is not necessarily responsible for the expression of the disease resistance phenotype.
  • the marker polynucleotide sequence be part of a gene that is responsible for the disease resistant phenotype (for example, is part of the gene open reading frame) .
  • the association between a specific marker allele and the disease resistance trait is due to the original “coupling” linkage phase between the marker allele and the allele in the ancestral line from which the allele originated. Eventually, with repeated recombination, crossing over events between the marker and genetic locus can change this orientation.
  • the favorable marker allele may change depending on the linkage phase that exists within the parent comprising resistance to the disease that is used to create segregating populations. This does not change the fact that the marker can be used to monitor segregation of the phenotype. It only changes which marker allele is considered favorable in a given segregating population.
  • Molecular markers can be used in a variety of plant breeding applications (e.g., see Staub et al. (1996) Hortscience 31: 729-741; Tanksley (1983) Plant Molecular Biology Reporter. 1: 3-8) .
  • One area of interest is to increase the efficiency of backcrossing and introgressing genes using marker-assisted selection (MAS) .
  • MAS marker-assisted selection
  • a molecular marker that demonstrates linkage with a locus affecting a desired phenotypic trait provides a useful tool for the selection of the trait in a plant population. This is particularly true where the phenotype is hard to assay.
  • DNA marker assays are less laborious and take up less physical space than field phenotyping, much larger populations can be assayed, increasing the chances of finding a recombinant with the target segment from the donor line moved to the recipient line.
  • Use of flanking markers decreases the chances that false positive selection will occur as a double recombination event would be needed.
  • a marker is located within the gene itself, so that recombination cannot occur between the marker and the gene.
  • the methods disclosed herein produce a marker in a disease resistance gene, wherein the gene was identified by inferring genomic location from clustering of conserved domains or a clustering analysis.
  • flanking regions When a gene is introgressed by MAS, it is not only the gene that is introduced but also the flanking regions (Gepts. (2002) . Crop Sci; 42: 1780-1790) . This is referred to as “linkage drag. ” In the case where the donor plant is highly unrelated to the recipient plant, these flanking regions carry additional genes that may code for agronomically undesirable traits. Linkage drag may also result in reduced yield or other negative agronomic characteristics even after multiple cycles of backcrossing into the elite line. This is also sometimes referred to as “yield drag.
  • the size of the flanking region can be decreased by additional backcrossing, although this is not always successful, as breeders do not have control over the size of the region or the recombination breakpoints (Young et al. (1998) Genetics 120: 579-585) . In classical breeding it is usually only by chance that recombinations are selected that contribute to a reduction in the size of the donor segment (Tanksley et al. (1989) . Biotechnology 7: 257-264) . Even after 20 backcrosses in backcrosses of this type, one may expect to find a sizeable piece of the donor chromosome still linked to the gene being selected. With markers however, it is possible to select those rare individuals that have experienced recombination near the gene of interest.
  • flanking markers surrounding the gene can be utilized to select for recombinations in different population sizes. For example, in smaller population sizes, recombinations may be expected further away from the gene, so more distal flanking markers would be required to detect the recombination.
  • Implementation of MAS can include: (i) Defining the population within which the marker-trait association will be determined, which can be a segregating population, or a random or structured population; (ii) monitoring the segregation or association of polymorphic markers relative to the trait, and determining linkage or association using statistical methods; (iii) defining a set of desirable markers based on the results of the statistical analysis, and (iv) the use and/or extrapolation of this information to the current set of breeding germplasm to enable marker-based selection decisions to be made.
  • the markers described in this disclosure, as well as other marker types such as SSRs and FLPs, can be used in marker assisted selection protocols.
  • SSRs can be defined as relatively short runs of tandemly repeated DNA with lengths of 6 bp or less (Tautz (1989) Nucleic Acid Research 17: 6463-6471; Wang et al. (1994) Theoretical and Applied Genetics, 88: 1-6) . Polymorphisms arise due to variation in the number of repeat units, probably caused by slippage during DNA replication (Levinson and Gutman (1987) Mol Biol Evol 4: 203-221) . The variation in repeat length may be detected by designing PCR primers to the conserved non-repetitive flanking regions (Weber and May (1989) Am J Hum Genet. 44:388-396) .
  • SSRs are highly suited to mapping and MAS as they are multi-allelic, codominant, reproducible and amenable to high throughput automation (Rafalski et al. (1996) Generating and using DNA markers in plants. In: Non-mammalian genomic analysis: a practical guide. Academic press. pp 75-135) .
  • SSR markers can be generated, and SSR profiles can be obtained by gel electrophoresis of the amplification products. Scoring of marker genotype is based on the size of the amplified fragment.
  • FLP markers can also be generated. Most commonly, amplification primers are used to generate fragment length polymorphisms. Such FLP markers are in many ways similar to SSR markers, except that the region amplified by the primers is not typically a highly repetitive region. Still, the amplified region, or amplicon, will have sufficient variability among germplasm, often due to insertions or deletions, such that the fragments generated by the amplification primers can be distinguished among polymorphic individuals, and such indels are known to occur frequently in maize (Bhattramakki et al. (2002) . Plant Mol Biol 48, 539-547; Rafalski (2002b) , supra) .
  • SNP markers detect single base pair nucleotide substitutions. Of all the molecular marker types, SNPs are the most abundant, thus potentially providing the highest genetic map resolution (Bhattramakki et al. 2002 Plant Molecular Biology 48: 539-547) . SNPs can be assayed at an even higher level of throughput than SSRs, in a so-called ⁇ ultra-high-throughput ⁇ fashion, as SNPs do not require large amounts of DNA and automation of the assay may be straight-forward. SNPs also have the promise of being relatively low-cost systems. These three factors together make SNPs highly attractive for use in MAS.
  • a number of SNPs together within a sequence, or across linked sequences, can be used to describe a haplotype for any particular genotype (Ching et al. (2002) , BMC Genet. 3: 19 pp Gupta et al. 2001, Rafalski (2002b) , Plant Science 162: 329-333) .
  • Haplotypes can be more informative than single SNPs and can be more descriptive of any particular genotype.
  • a single SNP may be allele “T ⁇ for a specific line or variety with disease resistance, but the allele ⁇ T ⁇ might also occur in the breeding population being utilized for recurrent parents.
  • a haplotype e.g.
  • a combination of alleles at linked SNP markers may be more informative.
  • that haplotype can be used in that population or any subset thereof to determine whether an individual has a particular gene. Using automated high throughput marker detection platforms makes this process highly efficient and effective.
  • SNP single nucleotide polymorphic
  • the primers are used to amplify DNA segments from individuals (e.g., an inbred) that represent the diversity in the population of interest.
  • the PCR products are sequenced directly in one or both directions.
  • the resulting sequences are aligned, and polymorphisms are identified.
  • the polymorphisms are not limited to single nucleotide polymorphisms (SNPs) , but also include indels, CAPS, SSRs, and VNTRs (variable number of tandem repeats) .
  • markers within the described map region can be hybridized to BACs or other genomic libraries, or electronically aligned with genome sequences, to find new sequences in the same approximate location as the described markers.
  • ESTs expressed sequence tags
  • RAPD randomly amplified polymorphic DNA
  • Isozyme profiles and linked morphological characteristics can, in some cases, also be indirectly used as markers. Even though they do not directly detect DNA differences, they are often influenced by specific genetic differences. However, markers that detect DNA variation are far more numerous and polymorphic than isozyme or morphological markers (Tanksley (1983) Plant Molecular Biology Reporter 1: 3-8) .
  • Sequence alignments or contigs may also be used to find sequences upstream or downstream of the specific markers listed herein. These new sequences, close to the markers described herein, are then used to discover and develop functionally equivalent markers. For example, different physical and/or genetic maps are aligned to locate equivalent markers not described within this disclosure but that are within similar regions. These maps may be within the species, or even across other species that have been genetically or physically aligned.
  • MAS uses polymorphic markers that have been identified as displaying a significant likelihood of co-segregation with a trait such as the SR disease resistance trait and/or genes disclosed herein. Such markers are presumed to map near a gene or genes that give the plant its disease resistant phenotype, and are considered indicators for the desired trait, or markers. Plants are tested for the presence of a desired allele in the marker, and plants containing a desired genotype at one or more loci are expected to transfer the desired genotype, along with a desired phenotype, to their progeny. Thus, plants with SR disease resistance may be selected for by detecting one or more marker alleles, and in addition, progeny plants derived from those plants can also be selected.
  • a plant containing a desired genotype in a given chromosomal region i.e. a genotype associated with disease resistance
  • a desired genotype in a given chromosomal region i.e. a genotype associated with disease resistance
  • the progeny of such a cross would then be evaluated genotypically using one or more markers and the progeny plants with the same genotype in a given chromosomal region would then be selected as comprising disease resistance.
  • polymorphic sites at marker loci in and around a chromosome marker identified by the methods disclosed herein wherein one or more polymorphic sites is in linkage disequilibrium (LD) with an allele at one or more of the polymorphic sites in the haplotype and thus could be used in a marker assisted selection program to introgress a gene allele or genomic fragment of interest.
  • LD linkage disequilibrium
  • Two particular alleles at different polymorphic sites are said to be in LD if the presence of the allele at one of the sites tends to predict the presence of the allele at the other site on the same chromosome (Stevens, Mol. Diag. 4: 309-17 (1999) ) .
  • the marker loci can be located within 5 cM, 2 cM, or 1 cM (on a single meiosis based genetic map) of the disease resistance trait QTL.
  • Allelic frequency can differ from one germplasm pool to another.
  • Germplasm pools vary due to maturity differences, heterotic groupings, geographical distribution, etc. As a result, SNPs and other polymorphisms may not be informative in some germplasm pools.
  • the method comprises screening one or more maize plants for a protein, or a polynucleotide encoding the protein (e.g., a WAK01 gene or protein of the present disclosure) .
  • the protein comprises an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%amino acid sequence identity to SEQ ID NO: 3.
  • the polynucleotide can comprise a gene, a coding sequence, a construct, or the like.
  • the method also comprises selecting one or more maize plant comprising the polynucleotide.
  • the polynucleotide can comprise a heterologous polynucleotide or a native (e.g., naturally occurring) polynucleotide. Selecting the maize plant can be performed based on the results of the screening procedure.
  • screening the population of maize plants for the protein or the polynucleotide encoding the protein comprises screening for a phenotype associated with the gene (e.g., GLS resistance) , screening for the gene or polynucleotide itself, and/or screening for a genotype linked to the polynucleotide.
  • the screening can comprise any procedure suitable for determining genotypes of the targeted plants.
  • screening can comprise an amplification step (e.g., PCR) , use of markers, nucleotide sequencing, next generation sequencing and/or mass spectrometry
  • the screening can be for a polynucleotide encoding a protein or the protein itself, and the protein can comprise at least 95%amino acid sequence identity to SEQ ID NO: 3, at least 96%amino acid sequence identity to SEQ ID NO: 3, at least 97%amino acid sequence identity to SEQ ID NO: 3, at least 98%amino acid sequence identity to SEQ ID NO: 3, at least 99%amino acid sequence identity to SEQ ID NO: 3, or 100%amino acid sequence identity to SEQ ID NO: 3.
  • the method comprises crossing the one or more selected maize plant with a second parent maize plant and obtaining a progeny plant that comprises the polynucleotide encoding the protein.
  • the second parent plant lacks the QTL.
  • progeny plants derived from the cross can then be used for further breeding activities and/or as a source of seed.
  • the progeny plant displays increased resistance to GLS, compared to a control plant (e.g., a parent plant lacking the polynucleotide, the second parent plant, an otherwise isogenic plant lacking the polynucleotide, and/or any progeny thereof lacking the polynucleotide) .
  • the polynucleotide can be introgressed into a plant breeding line (e.g., an elite line) via repeated backcrossing with the second parent plant using the progeny plant (s) .
  • the protein is encoded by a polynucleotide (e.g., a WAK01 gene) comprising a nucleotide sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%nucleotide sequence identity to SEQ ID NO: 1 or 2.
  • a polynucleotide e.g., a WAK01 gene
  • the protein is encoded by a gene comprising a nucleotide sequence comprising 100%sequence identity to the sequence of SEQ ID NO: 1 or 2.
  • the method comprises crossing a first parent maize plant with a second parent maize plant to produce one or more progeny plants.
  • the first parent comprises a protein or a polynucleotide encoding the protein (e.g., a WAK01 gene encoding a WAK01 protein as described herein) .
  • the protein can comprise an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%amino acid sequence identity to SEQ ID NO: 3.
  • the polynucleotide can comprise a nucleotide sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%nucleotide sequence identity to SEQ ID NO: 1 or 2.
  • the method comprises obtaining a nucleic acid sample from the progeny plant (s) ; and selecting one or more progeny plant comprising the polynucleotide and/or protein described herein.
  • the progeny plant can display increased resistance to GLS, compared to the second plant.
  • the second plant can lack the polynucleotide and/or protein.
  • the method can comprise crossing the one or more selected progeny plants with the second parent plant, or an individual isogenic to the second parent plant, to produce one or more backcross progeny plants; obtaining a nucleic acid sample from one or more backcross progeny plants; and selecting the one or more backcross progeny plants comprising the polynucleotide and/or protein described herein.
  • the method comprises crossing the one or more selected backcross progeny plants with the second parent plant, or an individual isogenic to the second parent plant, to produce additional backcross progeny plants; obtaining a nucleic acid sample from one or more additional backcross progeny plants; selecting the one or more additional backcross progeny plants comprising the polynucleotide and/or protein described herein; and optionally obtaining further additional backcross progeny plants comprising the polynucleotide and/or protein described herein.
  • a WAK01 gene (encoding a WAK01 protein) capable of conveying GLS resistance is used to produce recombinant plants.
  • recombinant plants are screened for the gene or encoded protein.
  • a WAK01 gene or protein can be detected to determine if a given plant comprises resistance to GLS, a WAK01 gene can be transformed into a plant, or a WAK01 gene can be added into a plant genome using site-specific genome editing techniques (e.g., CRISPR-, TALENs-, meganuclease-, or zinc finger nuclease-based techniques) .
  • site-specific genome editing techniques e.g., CRISPR-, TALENs-, meganuclease-, or zinc finger nuclease-based techniques
  • any WAK01 gene sequence presented herein e.g., any nucleic acid or protein sequence from SEQ ID NOs: 1-3
  • WAK01 sequences presented herein comprise protein sequences, DNA coding sequences (e.g., cDNA sequences) , or genomic DNA sequences that can include promoter, terminator, exon, and intron sequences.
  • a WAK01 gene sequence can comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the WAK01 genomic sequence of SEQ ID NO: 1.
  • a WAK01 gene sequence can comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the WAK01 coding sequence of SEQ ID NO: 2.
  • a WAK01 protein sequence can comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%sequence identity to the WAK01 protein sequence of SEQ ID NO: 3.
  • the method comprises introducing a heterologous gene sequence into one or more maize plant cells derived from a first maize plant, thereby producing at least one modified cell comprising the heterologous polynucleotide in the modified cell’s genome.
  • the method comprises selecting a modified maize plant cell. The selection can be based on screening one or more nucleic acid sample obtained from the at least one modified plant cell and/or can be based on use of a selectable marker that is introduced along with the heterologous polynucleotide.
  • the first maize plant can lack the heterologous polynucleotide before the present methods are performed.
  • the first maize plant can comprise a different allele of the heterologous polynucleotide or lack any alleles thereof
  • the heterologous polynucleotide can comprise a WAK01 gene sequence.
  • the heterologous polynucleotide can encode a protein comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%amino acid sequence identity to SEQ ID NO: 3.
  • the heterologous polynucleotide can comprise a sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%nucleotide sequence identity to SEQ ID NO: 1 or 2.
  • the method comprises effecting a site-specific modification of at least one target site in the genome (s) of the one or more maize plant cell (s) .
  • the method can comprise introducing a polynucleotide modification template comprising the heterologous polynucleotide into the one or more maize plant cells.
  • the introduction of the heterologous polynucleotide , the polynucleotide modification template, and/or the site specific modification can be introduced into only a portion of the cells. Given that this may be the case, the selection step is performed to ensure that a given cell has incorporated the various modifications into its genome.
  • the selection step can comprise use of a molecular marker and/or sequencing methods or any other technique suitable to discriminate between modified and unmodified cells.
  • the site-specific modification is induced by a CRISPR-associated endonuclease.
  • a CRISPR-associated endonuclease Any CRISPR-associated endonuclease suitable to introduce the site-specific modification can be used.
  • the site-specific modification can comprise, for example, a double-stranded break or any other modification tending to result in the heterologous gene sequence inserting into the modified cell’s genome.
  • the method comprises regenerating a modified maize plant from the selected modified maize plant cell.
  • the modified plant can exhibit increased resistance to GLS, relative to the first maize plant.
  • the first maize plant comprises an endogenous allele of the heterologous polynucleotide and the heterologous polynucleotide replaces the endogenous allele.
  • the endogenous locus can comprise an ortholog or paralog of the WAK01 gene disclosed herein.
  • the heterologous polynucleotide sequence is inserted into a locus that does not naturally comprise a WAK01 gene or an allele thereof. In some examples, the heterologous polynucleotide sequence is inserted into a locus as part of a molecular stack.
  • the method comprises effecting a site-specific modification of at least one target site into the genome of the maize plant cell and introducing a polynucleotide modification template comprising the heterologous nucleotide sequence into the plant cell.
  • the site-specific modification comprises a single strand break or double strand break (DSB) that is made by a site-specific endonuclease such as, for example, TALENs, meganucleases, zinc finger nucleases, or CRISPR-associated (Cas) protein/guide polynucleotide complexes.
  • a site-specific endonuclease such as, for example, TALENs, meganucleases, zinc finger nucleases, or CRISPR-associated (Cas) protein/guide polynucleotide complexes.
  • the introduction of a site-specific modification can be combined with the introduction of the polynucleotide modification template.
  • a polynucleotide modification template comprising the heterologous nucleotide sequence is introduced into a cell by any method suitable to deliver the template into the nucleus of a cell, such as, but not limited to, transient introduction methods, transfection, electroporation, microinjection, particle mediated delivery, topical application, whiskers mediated delivery, delivery via cell-penetrating peptides, mesoporous silica nanoparticle (MSN) -mediated direct delivery, or transformation.
  • transient introduction methods such as, but not limited to, transient introduction methods, transfection, electroporation, microinjection, particle mediated delivery, topical application, whiskers mediated delivery, delivery via cell-penetrating peptides, mesoporous silica nanoparticle (MSN) -mediated direct delivery, or transformation.
  • the polynucleotide modification template comprising the heterologous nucleotide sequence may be introduced into a cell as a single stranded polynucleotide molecule, a double stranded polynucleotide molecule, or as part of a circular DNA (vector DNA) .
  • the polynucleotide modification template may also be tethered to the guide polynucleotide and/or the Cas endonuclease. Tethered templates can allow for co-localizing target and template DNA, useful in genome editing and targeted genome regulation, and can also be useful in targeting post-mitotic cells where function of endogenous homologous recombination HR machinery is expected to be highly diminished (Mali et al. 2013 Nature Methods Vol. 10 : 957-963. )
  • the polynucleotide modification template may be present transiently in the cell or it can be introduced via a viral replicon.
  • the polynucleotide modification template refers to a polynucleotide that comprises at least one nucleotide modification when compared to the target nucleotide sequence to be edited.
  • a nucleotide modification is at least one nucleotide substitution, addition or deletion.
  • the polynucleotide modification template can further comprise homologous nucleotide sequences flanking the at least one nucleotide modification, wherein the flanking homologous nucleotide sequences provide sufficient homology to support the incorporation of the polynucleotide modification template into the genome of the recipient plant cell.
  • the process for editing a genomic sequence employing both site-specific modifications and modification templates generally comprises providing to a host cell a site-specific endonuclease (or a nucleic acid encoding the site-specific endonuclease) that recognizes a target sequence in the chromosomal sequence and induction by the site-specific endonuclease of a site-specific modification (e.g., a DSB) in the genomic sequence.
  • the process also comprises providing at least one polynucleotide modification template.
  • the endonuclease can be provided to a cell by any suitable method (e.g., transient introduction methods, transfection, microinjection, topical application, and/or indirectly via recombination constructs) .
  • the endonuclease may be provided as a protein or as a guided polynucleotide complex directly to a cell or indirectly via recombination constructs.
  • the endonuclease may be introduced into a cell transiently or can be incorporated into the genome of the host cell.
  • uptake of the endonuclease and/or the guided polynucleotide into the cell can be facilitated with a Cell Penetrating Peptide (CPP) as described in WO2016073433.
  • CCPP Cell Penetrating Peptide
  • TAL effector nucleases are a class of sequence-specific nucleases that can be used to make double-strand breaks at specific target sequences in the genome of a plant or other organism. (See Miller et al. (2011) Nature Biotechnology 29: 143–148) .
  • Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Endonucleases include restriction endonucleases, which cleave DNA at specific sites without damaging the bases, and meganucleases, also known as homing endonucleases (HEases) , which like restriction endonucleases, bind and cut at a specific recognition site, however the recognition sites for meganucleases are typically longer, about 18 bp or more (patent application PCT/US12/30061, filed on March 22, 2012) .
  • restriction endonucleases which cleave DNA at specific sites without damaging the bases
  • meganucleases also known as homing endonucleases (HEases) , which like restriction endonucleases, bind and cut at a specific recognition site, however the recognition sites for meganucleases are typically longer, about 18 bp or more (patent application PCT/US12/
  • Meganucleases have been classified into four families based on conserved sequence motifs, the families are the LAGLIDADG, GIY-YIG, H-N-H, and His-Cys box families. These motifs participate in the coordination of metal ions and hydrolysis of phosphodiester bonds. HEases are notable for their long recognition sites, and for tolerating some sequence polymorphisms in their DNA substrates. The naming convention for meganuclease is similar to the convention for other restriction endonuclease. Meganucleases are also characterized by prefix F-, I-, or PI-for enzymes encoded by free-standing ORFs, introns, and inteins, respectively.
  • One step in the recombination process involves polynucleotide cleavage at or near the recognition site.
  • the cleaving activity can be used to produce a double-strand break.
  • site-specific recombinases and their recognition sites see, Sauer (1994) Curr Op Biotechnol 5: 521-7; and Sadowski (1993) FASEB 7: 760-7.
  • the recombinase is from the Integrase or Resolvase families.
  • Zinc finger nucleases are engineered double-strand break inducing agents comprised of a zinc finger DNA binding domain and a double-strand-break-inducing agent domain. Recognition site specificity is conferred by the zinc finger domain, which typically comprising two, three, or four zinc fingers, for example comprising a C2H2 structure, however other zinc finger structures are known and have been engineered. Zinc finger domains are amenable for designing polypeptides which specifically bind a selected polynucleotide recognition sequence. ZFNs include an engineered DNA-binding zinc finger domain linked to a non-specific endonuclease domain, for example nuclease domain from a Type IIs endonuclease such as FokI.
  • Additional functionalities can be fused to the zinc-finger binding domain, including transcriptional activator domains, transcription repressor domains, and methylases.
  • dimerization of nuclease domain is required for cleavage activity.
  • Each zinc finger recognizes three consecutive base pairs in the target DNA. For example, a 3 finger domain recognized a sequence of 9 contiguous nucleotides, with a dimerization requirement of the nuclease, two sets of zinc finger triplets are used to bind an 18 nucleotide recognition sequence.
  • Genome editing using DSB-inducing agents such as Cas9-gRNA complexes, has been described, for example in U.S. Patent Application US 2015-0082478 A1, WO2015/026886 A1, WO2016007347, and WO201625131, all of which are incorporated by reference herein.
  • Cas gene or “Cas protein” herein refers to a gene that is generally coupled, associated or close to, or in the vicinity of flanking CRISPR loci in bacterial systems.
  • Cas and “CRISPR-associated” are used interchangeably herein.
  • Cas endonuclease herein refers to a protein, or complex of proteins, encoded by a Cas gene.
  • a Cas endonuclease as used herein, when in complex with a suitable polynucleotide component, is capable of recognizing, binding to, and optionally nicking or cleaving all or part of a specific DNA target sequence.
  • a Cas endonuclease as described herein comprises one or more nuclease domains.
  • Cas endonucleases of the disclosure includes those comprising a HNH or HNH-like nuclease domain and /or a RuvC or RuvC-like nuclease domain.
  • a Cas endonuclease of the disclosure may include a Cas9 protein, a Cpf1 protein, a C2c1 protein, a C2c2 protein, a C2c3 protein, Cas3, Cas 5, Cas7, Cas8, Cas10, or complexes of these.
  • guide polynucleotide/Cas endonuclease complex As used herein, the terms “guide polynucleotide/Cas endonuclease complex” , “guide polynucleotide/Cas endonuclease system” , “guide polynucleotide/Cas complex” , “guide polynucleotide/Cas system” , “guided Cas system” are used interchangeably herein and refer to at least one guide polynucleotide and at least one Cas endonuclease that are capable of forming a complex, wherein said guide polynucleotide/Cas endonuclease complex can direct the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single or double strand break) the DNA target site.
  • a guide polynucleotide/Cas endonuclease complex herein can comprise Cas protein (s) and suitable polynucleotide component (s) of any of the four known CRISPR systems (Horvath and Barrangou, 2010, Science 327: 167-170) such as a type I, II, or III CRISPR system.
  • a Cas endonuclease unwinds the DNA duplex at the target sequence and optionally cleaves at least one DNA strand, as mediated by recognition of the target sequence by a polynucleotide (such as, but not limited to, a crRNA or guide RNA) that is in complex with the Cas protein.
  • a Cas endonuclease typically occurs if the correct protospacer-adjacent motif (PAM) is located at or adjacent to the 3' end of the DNA target sequence.
  • a Cas protein herein may lack DNA cleavage or nicking activity but can still specifically bind to a DNA target sequence when complexed with a suitable RNA component. (See also U.S. Patent Application US 2015-0082478 A1, and US 2015-0059010 A1, both hereby incorporated in its entirety by reference) .
  • a guide polynucleotide/Cas endonuclease complex can cleave one or both strands of a DNA target sequence.
  • a guide polynucleotide/Cas endonuclease complex that can cleave both strands of a DNA target sequence typically comprises a Cas protein that has all of its endonuclease domains in a functional state (e.g., wild type endonuclease domains or variants thereof retaining some or all activity in each endonuclease domain) .
  • a wild-type Cas protein, or a variant thereof, retaining some or all activity in each endonuclease domain of the Cas protein is a suitable example of a Cas endonuclease that can cleave both strands of a DNA target sequence.
  • a Cas9 protein comprising functional RuvC and HNH nuclease domains is an example of a Cas protein that can cleave both strands of a DNA target sequence.
  • a guide polynucleotide/Cas endonuclease complex that can cleave one strand of a DNA target sequence can be characterized herein as comprising nickase activity (e.g., partial cleaving capability) .
  • a Cas nickase typically comprises one functional endonuclease domain that allows the Cas to cleave only one strand (i.e., make a nick) of a DNA target sequence.
  • a Cas9 nickase may comprise (i) a mutant, dysfunctional RuvC domain and (ii) a functional HNH domain (e.g., wild type HNH domain) .
  • a Cas9 nickase may comprise (i) a functional RuvC domain (e.g., wild type RuvC domain) and (ii) a mutant, dysfunctional HNH domain.
  • Non-limiting examples of Cas9 nickases suitable for use herein are disclosed in U. S. Patent Appl. Publ. No. 2014/0189896, which is incorporated herein by reference.
  • a pair of Cas9 nickases may be used to increase the specificity of DNA targeting. In general, this can be done by providing two Cas9 nickases that, by virtue of being associated with RNA components with different guide sequences, target and nick nearby DNA sequences on opposite strands in the region for desired targeting. Such nearby cleavage of each DNA strand creates a double strand break (i.e., a DSB with single-stranded overhangs) , which is then recognized as a substrate for non-homologous-end-joining, NHEJ (prone to imperfect repair leading to mutations) or homologous recombination, HR.
  • NHEJ non-homologous-end-joining
  • HR homologous recombination
  • Each nick in these examples can be at least about 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 (or any integer between 5 and 100) bases apart from each other, for example.
  • One or two Cas9 nickase proteins herein can be used in a Cas9 nickase pair.
  • a Cas9 nickase with a mutant RuvC domain, but functioning HNH domain i.e., Cas9 HNH+/RuvC-
  • could be used e.g., Streptococcus pyogenes Cas9 HNH+/RuvC-
  • Each Cas9 nickase (e.g., Cas9 HNH+/RuvC-) would be directed to specific DNA sites nearby each other (up to 100 base pairs apart) by using suitable RNA components herein with guide RNA sequences targeting each nickase to each specific DNA site.
  • a Cas protein may be part of a fusion protein comprising one or more heterologous protein domains (e.g., 1, 2, 3, or more domains in addition to the Cas protein) .
  • a fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains, such as between Cas and a first heterologous domain.
  • protein domains that may be fused to a Cas protein herein include, without limitation, epitope tags (e.g., histidine [His] , V5, FLAG, influenza hemagglutinin [HA] , myc, VSV-G, thioredoxin [Trx] ) , reporters (e.g., glutathione-5-transferase [GST] , horseradish peroxidase [HRP] , chloramphenicol acetyltransferase [CAT] , beta-galactosidase, beta-glucuronidase [GUS] , luciferase, green fluorescent protein [GFP] , HcRed, DsRed, cyan fluorescent protein [CFP] , yellow fluorescent protein [YFP] , blue fluorescent protein [BFP] ) , and domains comprising one or more of the following activities: methylase activity, demethylase activity, transcription activation activity (e.g.
  • a Cas protein can also be in fusion with a protein that binds DNA molecules or other molecules, such as maltose binding protein (MBP) , S-tag, Lex A DNA binding domain (DBD) , GAL4A DNA binding domain, and herpes simplex virus (HSV) VP16.
  • MBP maltose binding protein
  • DBD Lex A DNA binding domain
  • GAL4A DNA binding domain GAL4A DNA binding domain
  • HSV herpes simplex virus
  • a guide polynucleotide/Cas endonuclease complex in certain examples may bind to a DNA target site sequence but does not cleave any strand at the target site sequence.
  • Such a complex may comprise a Cas protein in which all of its nuclease domains are mutant, dysfunctional.
  • a Cas9 protein herein that can bind to a DNA target site sequence but does not cleave any strand at the target site sequence may comprise both a mutant, dysfunctional RuvC domain and a mutant, dysfunctional HNH domain.
  • a Cas protein herein that binds, but does not cleave, a target DNA sequence can be used to modulate gene expression, for example, in which case the Cas protein could be fused with a transcription factor (or portion thereof) (e.g., a repressor or activator, such as any of those disclosed herein) .
  • a transcription factor or portion thereof
  • an inactivated Cas protein may be fused with another protein comprising endonuclease activity, such as a FokI endonuclease.
  • Cas9 (formerly referred to as Cas5, Csn1, or Csx12) herein refers to a Cas endonuclease of a type II CRISPR system that forms a complex with a crNucleotide and a tracrNucleotide, or with a single guide polynucleotide, for specifically recognizing and cleaving all or part of a DNA target sequence.
  • Cas9 protein comprises a RuvC nuclease domain and an HNH (H-N-H) nuclease domain, each of which can cleave a single DNA strand at a target sequence (the concerted action of both domains leads to DNA double-strand cleavage, whereas activity of one domain leads to a nick) .
  • the RuvC domain comprises subdomains I, II and III, where domain I is located near the N-terminus of Cas9 and subdomains II and III are located in the middle of the protein, flanking the HNH domain (Hsu et al, Cell 157: 1262-1278) .
  • a type II CRISPR system includes a DNA cleavage system utilizing a Cas9 endonuclease in complex with at least one polynucleotide component.
  • a Cas9 can be in complex with a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA) .
  • a Cas9 can be in complex with a single guide RNA.
  • the Cas endonuclease can comprise a modified form of the Cas9 polypeptide.
  • the modified form of the Cas9 polypeptide can include an amino acid change (e.g., deletion, insertion, or substitution) that reduces the naturally-occurring nuclease activity of the Cas9 protein.
  • the modified form of the Cas9 protein has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5%, or less than 1%of the nuclease activity of the corresponding wild-type Cas9 polypeptide (US patent application US20140068797 A1) .
  • the modified form of the Cas9 polypeptide has no substantial nuclease activity and is referred to as catalytically “inactivated Cas9” or “deactivated cas9 (dCas9) . ”
  • Catalytically inactivated Cas9 variants include Cas9 variants that contain mutations in the HNH and RuvC nuclease domains. These catalytically inactivated Cas9 variants are capable of interacting with sgRNA and binding to the target site in vivo but cannot cleave either strand of the target DNA.
  • a catalytically inactive Cas9 can be fused to a heterologous sequence (US patent application US20140068797 A1) .
  • Suitable fusion partners include, but are not limited to, a polypeptide that provides an activity that indirectly increases transcription by acting directly on the target DNA or on a polypeptide (e.g., a histone or other DNA-binding protein) associated with the target DNA.
  • Additional suitable fusion partners include, but are not limited to, a polypeptide that provides for methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribosylation activity, myristoylation activity, or demyristoylation activity.
  • fusion partners include, but are not limited to, a polypeptide that directly provides for increased transcription of the target nucleic acid (e.g., a transcription activator or a fragment thereof, a protein or fragment thereof that recruits a transcription activator, a small molecule/drug-responsive transcription regulator, etc. ) .
  • a catalytically inactive Cas9 can also be fused to a FokI nuclease to generate double strand breaks (Guilinger et al. Nature Biotechnology, volume 32, number 6, June 2014) .
  • the method comprises regenerating a plant from the plant cell comprising the heterologous polynucleotide sequence.
  • the plant exhibits increased resistance to GLS.
  • the plant derived from the method can then be used for further breeding activities and/or as a source of seed.
  • the WAK01 gene e.g., located within the heterologous polynucleotide sequence
  • a plant breeding line e.g., an elite line
  • the heterologous polynucleotide sequence can be inserted into any location in the genome of the plant cell that is suitable for the expression of the heterologous polynucleotide sequence and, optionally, does not interfere with the expression of endogenous genes.
  • the heterologous polynucleotide sequence replaces an endogenous gene.
  • at least one allele of the endogenous gene is eliminated or rendered nonfunctional and replaced by the heterologous polynucleotide sequence encoded on the polynucleotide modification template.
  • at least the coding regions of the endogenous gene are replaced or rendered nonfunctional. Introns, promoter sequences, and terminator sequences may optionally be replaced or rendered nonfunctional as well.
  • the endogenous gene can be an allele or ortholog of the WAK01 gene disclosed herein.
  • the heterologous polynucleotide sequence is inserted into a locus different from any endogenous WAK01 allele or ortholog. In some examples, the heterologous polynucleotide sequence is inserted into a locus different from any endogenous WAK01 allele or ortholog, along with other genes which are also inserted at the same locus, forming a molecular stack. In some examples, the molecular stack comprises a stack of disease resistance genes.
  • the method comprises introducing the heterologous polynucleotide into one or more plant cells derived from a first maize plant.
  • the first maize plant (and cells thereof) can lack the heterologous polynucleotide before the present methods are performed.
  • the first maize plant can comprise a different allele of the heterologous polynucleotide or lack any alleles thereof.
  • the method comprises inserting the heterologous polynucleotide into the genome of at least one of the plant cells, thereby creating one or more modified plant cells.
  • the method comprises selecting a modified plant cell.
  • the heterologous polynucleotide sequence can comprise any of SEQ ID NOs: 1-3.
  • the heterologous polynucleotide sequence can encode a WAK01 protein.
  • the heterologous polynucleotide sequence can encode a protein comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 3.
  • the heterologous polynucleotide sequence comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%nucleotide sequence identity to SEQ ID NO: 1 or 2.
  • the selection can comprise use of a molecular marker and/or sequencing or any other suitable technique to discriminate between modified and unmodified cells.
  • the method comprises regenerating a modified plant from the selected plant cell.
  • the modified plant can exhibit increased resistance to GLS, compared to the first plant.
  • the selection can be based on any result indicating that the heterologous polynucleotide is present. For example, sequencing can be performed to determine if the heterologous polynucleotide has been inserted and/or plant cells can be selected for using a selectable marker.
  • the method comprises crossing the newly grown modified maize plant with a second maize plant (e.g., one lacking the gene sequence) and obtaining a progeny plant that comprises the gene sequence. The progeny plant can then be used for further breeding activities (e.g., backcrosses and/or introgression) and/or as a source of seed.
  • introducing the heterologous polynucleotide comprises bacteria-mediated transformation of the heterologous polynucleotide.
  • introducing the heterologous polynucleotide comprises biolistic transformation (e.g., use of a gene gun) of the heterologous polynucleotide.
  • Recombinant plants comprising the gene sequences described herein are provided.
  • a recombinant plant comprising a heterologous protein comprising at least 95%amino acid sequence identity to SEQ ID NO: 3.
  • the heterologous protein comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%amino acid sequence identity to SEQ ID NO: 3.
  • the heterologous protein is encoded by a polynucleotide comprising a nucleotide sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%nucleotide sequence identity to SEQ ID NO: 1 or 2.
  • Recombinant plant seeds comprising the gene sequences described herein are provided.
  • a recombinant plant seed comprising a heterologous gene encoding a protein comprising at least at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%amino acid sequence identity to SEQ ID NO: 3.
  • the polynucleotide comprises a nucleotide sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%nucleotide sequence identity to SEQ ID NO: 1 or 2.
  • a method for selecting a maize plant comprising resistance to gray leaf spot comprising:
  • a method for producing a maize plant comprising resistance to gray leaf spot comprising:
  • a method for producing a recombinant maize plant cell comprising a heterologous polynucleotide associated with resistance to gray leaf spot comprising:
  • heterologous polynucleotide into one or more maize plant cells derived from a first maize plant, thereby producing at least one modified cell comprising the heterologous polynucleotide in the modified cell’s genome
  • heterologous polynucleotide encodes a protein comprising at least 95%amino acid sequence identity to SEQ ID NO: 3.
  • heterologous polynucleotide comprises a nucleotide sequence comprising at least 95%identity to SEQ ID NO: 1 or 2.
  • heterologous polynucleotide comprises a nucleotide sequence comprising at least 96%identity to SEQ ID NO: 1 or 2.
  • heterologous polynucleotide comprises a nucleotide sequence comprising at least 97%identity to SEQ ID NO: 1 or 2.
  • heterologous polynucleotide comprises a nucleotide sequence comprising at least 98%identity to SEQ ID NO: 1 or 2.
  • heterologous polynucleotide comprises a nucleotide sequence comprising at least 99%identity to SEQ ID NO: 1 or 2.
  • heterologous polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1 or 2.
  • a method for producing a recombinant maize plant cell comprising a heterologous polynucleotide associated with resistance to gray leaf spot comprising:
  • heterologous polynucleotide into one or more plant cells derived from a first plant
  • heterologous polynucleotide into the genome of at least one of the plant cells, thereby creating one or more modified plant cells
  • heterologous polynucleotide encodes a protein comprising an amino acid sequence at least 95%identical to SEQ ID NO: 3.
  • heterologous polynucleotide encodes a protein comprising the amino acid sequence of SEQ ID NO: 3.
  • heterologous polynucleotide encodes a protein comprising an amino acid sequence at least 99%identical to SEQ ID NO: 3.
  • heterologous polynucleotide sequence comprises at least 95%nucleotide sequence identity to SEQ ID NO: 1 or 2.
  • heterologous polynucleotide sequence comprises at least 96%nucleotide sequence identity to SEQ ID NO: 1 or 2.
  • heterologous polynucleotide sequence comprises at least 97%nucleotide sequence identity to SEQ ID NO: 1 or 2.
  • heterologous polynucleotide sequence comprises at least 98%nucleotide sequence identity to SEQ ID NO: 1 or 2.
  • heterologous polynucleotide sequence comprises at least 99%nucleotide sequence identity to SEQ ID NO: 1 or 2.
  • heterologous polynucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 1 or 2.
  • a recombinant plant comprising a heterologous protein comprising at least 95%amino acid sequence identity to SEQ ID NO: 3.
  • a recombinant plant seed comprising a heterologous protein comprising at least 95%amino acid sequence identity to SEQ ID NO: 3.
  • a BC1F1 population was generated with B11 (aGLS resistant inbred line) and B206 (aGLS susceptible inbred line) , with B206 as the recurrent parent.
  • B11 aGLS resistant inbred line
  • B206 aGLS susceptible inbred line
  • One hundred eighty-three BC1F1 individuals were phenotyped for GLS resistance.
  • GLS resistance was scored in five scales: 1, 3, 5, 7 and 9, with 1 being the most resistant and 9 the most susceptible. More specifically, the GLS severity was scored based on the percentage of lesion area: 1: 0-5%; 3: 5-10%; 5: 10-30%; 7: 30-70%; 9: 70-100% (See Zhang, Y., et al. (2012) . QTL mapping of resistance to gray leaf spot in maize. Theor Appl Genet 125 (8) , 1797-1808) .
  • GLS scores were collected 2-3 weeks after pollination.
  • the same 183 BC1F1 individuals were genotyped by a genotyping-by-sequencing (GBS) protocol.
  • GLS genotyping-by-sequencing
  • a major QTL on chromosome 1 (bins 1.05-1.07) was identified and named as qRgs1-B11. It explained 30.2%of the phenotypic variation.
  • a sequential fine mapping approach was taken for qRgs1-B11 with a BC2F1 population and additional backcross populations derived from the BC2F1 population.
  • a progeny test was conducted to obtain the GLS resistance phenotype of each recombinant.
  • the B11 genome was sequenced and assembled to facilitate marker development and candidate gene identification.
  • qRgs1-B11 was delimited to a 1.76 Mb interval in the B11 genome, between markers TD209 and TD380.
  • Five annotated genes, three wall-associated kinases (WAK01, WAK02 and WAK03) and two B-lectin kinases (PRR1 and PRR2) were identified as candidate genes for qRgs1-B11.
  • the B11 genomic sequence (comprising a promoter and terminator region) of each candidate gene was used in a binary vector to generate transgenic plants.
  • the constructs were transformed into maize inbred lines PH1V69, PHHHD and KN5585, all of them are susceptible to GLS.
  • the transgenic events in PH1V69 and PHHHD were tested in greenhouse for GLS resistance, while transgenic events in KN5585 were field tested for GLS resistance.
  • Expression of WAK01 significantly enhanced GLS resistance, while the other candidate genes did not have significant impacts. Therefore, WAK01 is the causal gene underlying qRgs1-B11 and is responsible for the GLS resistance.

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Abstract

L'invention concerne des procédés de sélection d'une plante de maïs comprenant un polynucléotide associé à la résistance aux taches grises des feuilles. L'invention concerne également des procédés de production de plantes ou de cellules végétales comprenant un polynucléotide associé à la résistance aux taches grises des feuilles. L'invention concerne également les plantes résistantes ou les cellules végétales elles-mêmes, conjointement avec des graines de plantes résistantes.
PCT/CN2024/079799 2024-03-04 2024-03-04 Plantes de maïs présentant une résistance aux taches grises des feuilles, compositions et procédés de sélection et de production associés Pending WO2025184759A1 (fr)

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