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WO2025153582A1 - Plants de maïs présentant une résistance aux maladies améliorée - Google Patents

Plants de maïs présentant une résistance aux maladies améliorée

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Publication number
WO2025153582A1
WO2025153582A1 PCT/EP2025/050969 EP2025050969W WO2025153582A1 WO 2025153582 A1 WO2025153582 A1 WO 2025153582A1 EP 2025050969 W EP2025050969 W EP 2025050969W WO 2025153582 A1 WO2025153582 A1 WO 2025153582A1
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Prior art keywords
sequence
seq
set forth
nos
identity
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PCT/EP2025/050969
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English (en)
Inventor
Daniela SCHEUERMANN
Bettina KESSEL
Thomas PRESTERL
Daniel STIRNWEIS
Jan BETTGENHÄUSER
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KWS SAAT SE and Co KGaA
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KWS SAAT SE and Co KGaA
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Publication of WO2025153582A1 publication Critical patent/WO2025153582A1/fr
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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
    • A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • A01H1/1245—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance
    • A01H1/1255—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or 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
    • 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

Definitions

  • the present invention relates to maize plants with improved disease resistance. Specifically, the invention concerns methods for identifying or generating maize plants and parts or cells thereof, with disease resistance. Further encompassed by the present invention are maize plants with improved disease resistance produced by methods of the invention.
  • Disease resistance is an important agronomic trait, particularly for the production of food crops. Although some disease resistance alleles have been identified, efforts to combine several disease resistance traits in a single plant line have been hindered by tightly linked or even allelic loci conferring resistance to different pathogens or pathogen isolates. This is further complicated by high densities of repeated sequences in regions of plant genomes controlling disease resistance, which can greatly reduce the possibility of developing useful genetic markers.
  • NCLB neuronorthern corn leaf blight
  • NCLB resistance in corn has a complex genetic architecture comprising the use of the HT 1 gene located on the long arm of chromosome 2 together with a partial quantitative resistance which has been used to control helminthosporiosis in maize (Welz, 1998).
  • Different races of pathogen, mutation rates and pathogen population dynamic further complicate the situation.
  • Globally, races 0 and 1 of H. turcicum are most prevalent (approximately 55%) (Lipps et al., 1997), while other races such as 2N and 23N are rare and occur in geographically restricted areas only (Welz, 1998). For example, in Brazil the H. turcicum population already appears to be substantially more diverse with regard to the race composition than in North America.
  • HTN1 introgression lines exhibit a gene mapping on the long arm of chromosome 8.
  • HTN1 confers resistance by delaying the onset of sporulation, and thus combats the development of lesions. As a result, fewer, smaller lesions as well as reduced sporulation zones are formed (Simcox & Bennetzen, 1993, "The use of molecular markers to study Setospaeria turcica resistance in maize.” Phytopathology 83: 1326-1330).
  • Chlorotic-necrotic lesions such as those which occur with HT 1 , HT2 or HT3-conferred resistance, are not formed (Gevers, 1975). However, in the 1990s it was reported that H. turcicum races had broken the resistance conferred by the HT1 gene. Furthermore, instability of the resistance genes is observed in response to certain environmental factors, such as temperature and light intensity in some climate zones (Thakur et al., 1989, "Effects of temperature and light on virulence of Exserohilum turcicum on corn.” Phytopathology 1989, 79: 631-635).
  • WO2015/032494 discloses the identification of the causative gene, RLK1, which confers the “Pepitilla” resistance phenotype on bin 8.06 in corn, and describes molecular markers which are suitable to benefit from this resistance locus without close-linked, undesired linkage drag leading to a negative impact on the yield potential.
  • WO2011/163590 discloses the genotypes PH99N and PH26N as alternative sources for NCLB resistance on chromosome 8 bin 5.
  • the present invention relates to maize plants with improved disease resistance. Specifically, the invention concerns methods for identifying or generating maize plants, parts thereof, or cells, with disease resistance.
  • the invention provides a method for identifying a maize plant having resistance and/or tolerance to a fungal pathogen, comprising screening for the presence of at least one QTL located on chromosome 5 in a maize plant or plant part, wherein said QTL is located on a chromosomal interval flanked by markers M1 and M26, wherein markers M1 and M26 are SNPs which are respectively guanine (G) at the position 53948820 bp and cytosine (C) at the position 61159296 bp, on reference genome B73_AGPvO4.
  • a method for identifying a maize plant or plant part comprising screening for the presence of one or more markers selected from M1 to M26 as defined herein, or one or more marker regions, wherein said marker regions comprise a sequence selected from the group consisting of SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, and 51.
  • a method for generating a maize plant comprising introducing into the genome of a plant or a plant part: a) a QTL as defined herein; b) one or more markers selected from M2 to M25 as defined herein; c) one or more sequences selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, and 49; d) a polynucleic acid encoding exogenous Zm00001d014641 comprising one or more, preferably both, of the markers M15 and/or M16; preferably having a sequence as set forth in any of SEQ ID NOs: 59 and 60, or a sequence having an identity of at least 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%
  • a method for generating a maize plant comprising: a) mutating endogenous Zm00001d014641 to a sequence as set forth in any of SEQ ID NOs: 59 and 60, or a sequence having an identity of at least 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% to a sequence as set forth in any of SEQ ID NOs: 59 and 60, preferably over the entire length of the sequence; or to a sequence encoding a polypeptide sequence as set forth in SEQ ID NO: 61 or a sequence having an identity of at least 80%, 85%, 90%, 95%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99
  • a method of creating a population of maize plants, corn seeds or corn cells comprising: a) crossing a first resistant maize plant comprising a resistant QTL located on chromosome 5, wherein said QTL is flanked by markers M1 and M26, wherein markers M1 and M26 are SNPs which are respectively guanine (G) at the position 53948820 bp and cytosine (C) at the position 61159296 bp, on reference genome B73_AGPvO4, with a second maize plant to generate a first population of maize plants, or maize cells; b) genotyping said first population of maize plants, maize seeds or maize cells at one or more loci are linked to, and within 10 centimorgans (cM) of, said markers M1 and M26; and c) selecting from said first population one or more maize plants, maize seeds, or maize cells having resistance to a fungal pathogen comprising one or more markers selected from the group
  • M2 is a SNP which is cytosine (C) at position 54422803 when referenced to the B73 reference genome AGPv4;
  • M3 is a SNP which is guanine (G) at position 54917958 when referenced to the B73 reference genome AGPv4;
  • M6 is a SNP which is thymine (T) at position 56925593 when referenced to the B73 reference genome AGPv4;
  • M8 is a SNP which is guanine (G) at position 57859040 when referenced to the B73 reference genome AGPv4;
  • M12 is a SNP which is guanine (G) at position 59905013 when referenced to the B73 reference genome AGPv4;
  • M 14 is a SNP which is thymine (T) at position 60843932 when referenced to the B73 reference genome AGPv4;
  • M15 is a SNP which is guanine (G) at position 57056956 when referenced to the B73 reference genome AGPv4;
  • M 16 is a SNP which is adenine (A) at position 57057094 when referenced to the B73 reference genome AGPv4;
  • M17 is a SNP which is cytosine (C) at position 57191086 when referenced to the B73 reference genome AGPv4;
  • M 19 is a SNP which is adenine (A) at position 57311744 when referenced to the B73 reference genome AGPv4;
  • an isolated polynucleic acid comprising: a) a QTL as defined herein; b) one or more markers selected from M2 to M25 as defined herein; c) one or more sequences selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, and 49; d) one or more sequences selected from the group consisting of SEQ I D NOs: 59 and 60; 68 and 69; 77 and 78; 86 and 87; 95 and 96; 104 and 105; and 113 and 114; and/or e) one or more sequences with an identity of at least 80%, 85%, 90%, 95%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%
  • an isolated polynucleic acid specifically hybridizing with the polynucleic acid according to the preceding aspect, the complement thereof, or the reverse complement thereof, preferably wherein said polynucleic acid is a primer or a probe.
  • the present invention is based on the identification of a gene selected from any one of Zm00001d014641, Zm00001d014649, Zm00001d014650, Zm00001d014652, Zm00001d014654, Zm00001d014657, and/or Zm00001d014687, which confer (or increase) resistance of a plant against a fungal pathogen, such as Helminthosporium turcicum.
  • nucleic acid molecules comprising or consisting of a nucleotide sequence encoding a gene selected from any one or more of Zm00001d014641, Zm00001d014649, Zm00001d014650, Zm00001d014654, and/or Zm00001d014687.
  • Zm00001d014641 is located at position 57055394 to 57057761 on the B73_AGPv4 genome.
  • Zm00001d014641 comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 59 and 60.
  • Zm00001d014641 encodes a polypeptide with a sequence selected from the group consisting of SEQ ID NO: 61.
  • Zm00001d014641 comprises markers M15 and/or M16.
  • Zm00001d014649 comprises markers M17 and/or M18.
  • Zm00001d014650 is located at position 57311562 to 57315887 on the B73_AGPv4 genome.
  • Zm00001d014650 comprises or consists of a sequence selected from the group consisting of: 77 and 78.
  • Zm00001d014650 encodes a polypeptide with a sequence selected from the group consisting of SEQ ID NO: 79.
  • Zm00001d014650 comprises markers M19, M20, and/or M21.
  • Zm00001d014652 is located at position 57321789 to 57336205 on the B73_AGPv4 genome.
  • Zm00001d014652 comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 86 and 87.
  • Zm00001d014652 encodes a polypeptide with a sequence selected from the group consisting of SEQ ID NO: 88.
  • Zm00001d014654 is located at position 57560758 to 57565194 on the B73_AGPv4 genome.
  • Zm00001d014654 comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 95 and 96.
  • Zm00001d014654 encodes a polypeptide with a sequence selected from the group consisting of SEQ ID NO: 97.
  • Zm00001d014654 comprises marker M22.
  • Zm00001d014657 is located at position 57745010 to 57746548 on the B73_AGPv4 genome.
  • Zm00001d014657 comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 104 and 105.
  • Zm00001d014657 encodes a polypeptide with a sequence selected from the group consisting of SEQ ID NO: 106.
  • Zm00001d014687 is located at position 59017351 to 59024982 on the B73_AGPv4 genome.
  • Zm00001d014687 comprises or consists of a sequence selected from the group consisting of SEQ ID NO: 113 and 114.
  • Zm00001d014687 encodes a polypeptide with a sequence selected from the group consisting of SEQ ID NO: 115.
  • Zm00001d014687 comprises markers M23, M24, and/or M25.
  • nucleic acid molecules according to the invention encode a polypeptide capable of conferring to a plant (or increasing in a plant) resistance against northern corn leaf blight (NCLB).
  • nucleic acid molecules according to the invention encode a polypeptide capable of conferring to a plant (or increasing in a plant) resistance against Helminthosporium turcicum, in particular resistance against the currently known Helminthosporium turcicum races 0, 1, 2, 3, N, 12, 23, 2N, 12N, 23N and/or 123N, or resistance against Helminthosporium turcicum races 0, 1 and/or N.
  • identifying and/or generating maize plants that have resistance such as increased resistance and/or tolerance to a pathogen or pathogen infection. Further provided are maize plants that have said resistance to pathogen or pathogen infection.
  • pathogen infection should be understood to mean the earliest time at which a pathogen interacts with a plant host tissue.
  • pathogens are fungi or fungi-like organisms such as ascomycetes or oomycetes
  • interactions include the growth of hyphae or the formation of specific infection structures such as penetration hyphae and the appressorium.
  • Helminthosporium turcicum may be investigated using various stain techniques (for example trypan blue) (Chung et al., BMC Plant Biology 10 (2010), 103; Walsh et al. (2008), Poster presentation P192, 50th Maize Genetics Conference in Washington D.C.).
  • the present invention provides methods for identifying and/or generating maize plants that have resistance, such as increased resistance and/or tolerance to a fungal pathogen. Further provided by the invention are maize plants that have said resistance to a fungal pathogen.
  • the fungal pathogen against which resistance is conferred belongs to the division of Ascomycota or Basidiomycota.
  • the fungal pathogen may belong to family Pleosporaceae, Pucciniaceae or Botryosphaeriaceae.
  • the fungal pathogen belongs to the genus of Setosphaeria, Bipolaris, Puccinia or Diplodia, more preferably is the species of Helminthosporium turcicum, Setosphaeria rostrata, Setosphaeria glycinea, Setosphaeria holmii, Setosphaeria khartoumensis, Setosphaeria minor, Setosphaeria monoceras, Setosphaeria pedicellata, Setosphaeria prolata, Bipolaris australis, Bipolaris brizae, Bipolaris buchloes, Bipolaris cactivora, Bipolaris clavata, Bipolaris coicis, Bipolaris colocasiae, Bipolaris crotonis, Bipolaris crustacean, Bipolaris cylindrical, Bipolaris euchlaenae, Bipolaris halepensis, Bipolaris heveae, Bipolaris incurvata, Bipolaris indica, Bipolaris cylindrical
  • NCLB non-northern corn leaf blight
  • resistance or “resistant” as regards a pathogen should be understood to mean the ability of a plant or plant cell to resist the damaging effects of the pathogen. This includes a delay in the development of disease to complete suppression of the development of the disease.
  • a plant or plant cell is resistant, or has a resistance to the pathogen Helminthosporium turcicum (H. turcicum or HT), i.e., to the leaf disease northern corn leaf blight (NCLB).
  • the resistance may be complete or partial and may be specific or nonspecific to the pathogen race.
  • the virulent currently known races of Helminthosporium turcicum may, for example, include N, 1 N, 2N, 23N or 123N; the avirulent races may, for example, include 0, 1 , 2, 3, 12, 23 or 123.
  • a conferred resistance may be a newly inherited resistance or an increase in a partial resistance which is already existent.
  • Plants in the context of the invention may be any species of monocotyledon, dicotyledon, or gymnosperm plant.
  • the plants described herein are monocotyledon plants and are of interest in agriculture or horticulture or for the production of bioenergy (bioethanol, biogas, etc), such as Zea mays, Sorghum sp., Triticum sp., Hordeum vulgare, Secale cereale, Avena sp., Oryza sativa, Musa sp., Saccharum officinarum, Gossypium sp., Brachypodium distachyon, turf grass and forage grass.
  • bioenergy bioethanol, biogas, etc
  • Zea mays such as Zea mays, Sorghum sp., Triticum sp., Hordeum vulgare, Secale cereale, Avena sp., Oryza sativa, Musa sp., Saccharum officinarum, Gossypium sp., Brachypodium distachyon, turf grass and forage grass.
  • bioenergy bioethanol, biogas,
  • the plant according to the invention is Sorghum bicolor.
  • the plant according to the invention is selected from the group consisting of: Hordeum vulgare, Hordeum bulbusom, Sorghum bicolor, Saccharum officinarium, Zea mays, Setaria italica, Oryza minuta, Oriza sativa, Oryza australiensis, Oryza nie, Triticum aestivum, Triticum durum, Secale cereale, Triticale, Malus domestica, Brachypodium distachyon, Hordeum marinum, Aegilops tauschii, Daucus glochidiatus, Beta vulgaris, Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Nicotiana sylvestris, Nicotiana tomentosiformis, Nicotiana tabacum, Nicotiana benthamiana, Solanum lycopersicum, Solanum tuberosum, Coffea canephora, Vitis vinifera,
  • the plant is a maize plant.
  • a “maize plant” should be understood to mean a plant from the species Zea mays as well as its subspecies such as, for example, Zea mays ssp. mays, Zea mays ssp. mexicana or Zea mays ssp. parviglumis.
  • a maize plant comprising: a) a QTL as defined herein; b) one or more markers selected from M2 to M25 as defined herein; c) one or more sequences selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, and 49; d) a polynucleic acid encoding exogenous Zm00001d014641 comprising one or more, preferably both, of the markers M15 and/or M16; preferably having a sequence as set forth in any of SEQ ID NOs: 59 and 60, or a sequence having an identity of at least 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% to a sequence as
  • the maize plant or plant part is transgenic, gene-edited, or mutagenized.
  • Chemical- or radiation-induced mutagenesis is well established in the art and may be performed by any known suitable method, such as but not limited to EMS (ethyl methanesulfonate) mutagenesis or UV mutagenesis.
  • chemical- or radiation-induced mutagenesis is a random mutagenesis approach
  • functional expression of a gene of interest may be measured as disclosed herein or by any suitable method known in the art.
  • one or more plants having altered functional expression of a gene of interest can be identified and/or selected after chemical- or radiation-induced mutagenesis.
  • expression of a gene may be measured in a number of ways, dependent upon the level at which gene regulation is being assessed. For example, the type/amount of protein may be measured (therefore assessing expression at the translational level), the type/amount of mRNA may be measured (therefore assessing expression at the transcriptional level), or the nature of the genomic DNA itself may be assessed.
  • the introduction is of the at least one functional copy of a gene or nucleotide sequence (such as a fragment of a gene), wherein said functional copy of a gene or nucleotide sequence is integrated into the chromosomal plant genome and/or the chloroplast genome.
  • Transformation may be performed by any method known in the art, such as but not limited to agrobacterium-mediated transformation, particle bombardment, PEG-mediated transformation, particle uptake or electroporation.
  • Introduction through genome-modification technology may be performed by any site-specific genome modification technique known in the art using an SDN-3 approach, wherein the introduced sequence may be inserted by replacing at least on endogenous copy of a gene of interest or may be introduced elsewhere in the genome.
  • Embodiments using a) introduction and/or b) mutagenesis by genome modification rely on a genome modification system, wherein a genome modification system refers to any DNA, RNA and/or amino acid sequence introduced into the cell, on a suitable vector and/or coated on a particles and/or directly introduced, wherein the genome modification system causes the modification of the genome of the cell in which it has been introduced, wherein the genome modification system comprises at least one site-directed nuclease, nickase or inactivated variant thereof, and optionally at least one further molecule, such as a guide molecule.
  • a genome modification system refers to any DNA, RNA and/or amino acid sequence introduced into the cell, on a suitable vector and/or coated on a particles and/or directly introduced, wherein the genome modification system causes the modification of the genome of the cell in which it has been introduced, wherein the genome modification system comprises at least one site-directed nuclease, nickase or inactivated variant thereof, and optionally at least one further molecule
  • a “site-directed nuclease” herein refers to a nuclease or an active fragment thereof, which is capable of specifically recognizing and cleaving DNA at a certain location, the target sequence.
  • Such nucleases typically produce a double-strand break (DSB), which is then repaired by non-homologous end-joining (NHEJ) or homologous recombination (HR).
  • Sitespecific nucleases include meganucleases, homing endonucleases, zinc finger nucleases, transcription activator-like nucleases and CRISPR nucleases, or variants including nickases or nuclease-dead variants thereof.
  • CRISPR nuclease is a specific form of a site-directed nuclease and refers to any nucleic acid guided nuclease which has been identified in a naturally occurring CRISPR system, which has subsequently been isolated from its natural context, and which preferably has been modified or combined into a recombinant construct of interest to be suitable as tool for targeted genome engineering.
  • Any CRISPR nuclease can be used and optionally reprogrammed or additionally mutated to be suitable for the various embodiments according to the present invention as long as the original wild-type CRISPR nuclease provides for DNA recognition, i.e., binding properties.
  • CRISPR nucleases also comprise mutants or catalytically active fragments or fusions of a naturally occurring CRISPR effector sequences, or the respective sequences encoding the same.
  • a CRISPR nuclease may in particular also refer to a CRISPR nickase or even a nuclease-dead variant of a CRISPR polypeptide having endonucleolytic function in its natural environment.
  • CRISPR nucleases/systems and variants thereof are meanwhile known to the skilled person and include, inter alia, CRISPR/Cas systems, including CRISPR/Cas9 systems (EP2771468), CRISPR/Cpf1 systems (EP3009511 B1), CRISPR/C2C2 systems, CRISPR/CasX systems, CRISPR/CasY systems, CRISPR/Cmr systems, CRISPR/MAD systems, including, for example, CRISPR/MAD7 systems (WO2018236548A1) and CRISPR/MAD2 systems, CRISPR/CasZ systems and/or any combination, variant, or 30 catalytically active fragment thereof.
  • a nuclease may be a DNAse and/or an RNAse, in particular taking into consideration that certain CRISPR effector nucleases have RNA cleavage activity alone, or in addition to the DNA cleavage activity.
  • the "guide molecule” or “guide nucleic acid sequence” (usually called and abbreviated as guide RNA, crRNA, crRNA+tracrRNA, gRNA, sgRNA, depending on the corresponding CRISPR system representing a prototypic nucleic acid-guided site-directed nuclease system), which recognizes a target sequence to be cut by the nuclease.
  • the at least one "guide nucleic acid sequence” or “guide molecule” comprises a “scaffold region” and a "target region".
  • the "scaffold region” is a sequence, to which the nucleic acid guided nuclease binds to form a targetable nuclease complex.
  • the scaffold region may comprise direct repeats, which are recognized and processed by the nucleic acid guided nuclease to provide mature crRNA.
  • a pegRNAs may comprise a further region within the guide molecule, the so-called "primerbinding site".
  • the "target region” defines the complementarity to the target site, which is intended to be cleaved.
  • a crRNA as used herein may thus be used interchangeably herein with the term guide RNA in case it unifies the effects of meanwhile well-established CRISPR nuclease guide RNA functionalities.
  • CRISPR nucleases may be used by providing two individual guide nucleic acid sequences in the form of a tracrRNA and a crRNA, which may be provided separately, or linked via covalent or non-covalent bonds/interactions.
  • the guide RNA may also be a pegRNA of a Prime Editing system.
  • the at least one guide molecule may be provided in the form of one coherent molecule, or the sequence encoding the same, or in the form of two individual molecules, e.g., crRNA and tracrRNA, or the sequences encoding the same.
  • nucleotide sequence is inserted, such as a sequence encoding a gene of interest
  • the insertion is performed in a manner that allows functional expression of the gene of interest.
  • the inserted nucleotide sequence comprises or consists of: a) a QTL as defined herein; b) one or more markers selected from M2 to M25 as defined herein; c) one or more sequences selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, and 49; d) one or more polynucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 59 and 60; 68 and 69; 77 and 78; 86 and 87; 95 and 96; 104 and 105; and 113 and 114; and/or e) one or more polynucleic acid having a sequence with an identity of at least 80%, 85%, 90%, 95%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99
  • polypeptides selected from the group consisting of SEQ ID NOs: 61 , 70, 79, 88, 97, 106, and 115; and/or g) one or more polynucleic acids encoding a polypeptide with an identity of at least 80%, 85%, 90%, 95%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% to a sequence selected from the group consisting of SEQ ID NOs: 61 , 70, 79, 88, 97, 106, and 115.
  • the inserted nucleotide sequence may further be selected from a nucleotide sequence that comprises or consists of: a) one or more sequences selected from the group consisting of SEQ ID NOs: 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, and 49; b) one or more polynucleic acid having a sequence selected from the group consisting of SEQ ID NOs: 59 and 60; 68 and 69; 77 and 78; 95 and 96; and 113 and 114; and/or c) one or more polynucleic acid having a sequence with an identity of at least 80%, 85%, 90%, 95%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% to a sequence selected from the group of SEQ ID NOs: 59 and 60;
  • polynucleic acids encoding a polypeptide selected from the group consisting of SEQ ID NOs: 61 , 70, 79, 97, and 115; and/or e) one or more polynucleic acids encoding a polypeptide with an identity of at least 80%, 85%, 90%, 95%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% to a sequence selected from the group consisting of SEQ ID NOs: 61 , 70, 79, 97, and 115.
  • the inserted sequence may comprise all regulatory sequences necessary for efficient transcription and translation operably linked to the nucleotide sequence to be expressed.
  • the nucleotide sequence to be expressed may be inserted without all regulatory sequences necessary for efficient transcription and translation if the insertion leads to an operably linked position relative to regulatory sequences present in the target genome and thereby the efficient transcription and translation are achieved.
  • the skilled person is well aware of suitable regulatory sequences and the particulars of functional expression in plants of the genus Zea.
  • At least one repair template may be delivered with the at least one genome modification or editing system simultaneously or subsequently with the proviso that it will be active, i.e., present and readily available at the site of a genomic target sequence in the plant cell to be modified together with the at least one further tools of interest.
  • the at least one site-directed nuclease optionally, the at least one guide molecule and (for SDN2) the at least one repair template as RNA and/or Polypeptide in order to avoid the introduction of transgenic DNA.
  • Plants or parts thereof may have their genomes edited by tiling approaches.
  • mutants such as single point mutants, are generated across the entire length of a target region (i.e., producing one or more single point mutant for each position within a region of interest).
  • Such tiling approaches will be known to the skilled person and may include, for example CRISPR tiling (Yang, L., Chan, A.K.N., Miyashita, K. et al. High-resolution characterization of gene function using single-cell CRISPR tiling screen. Nat Commun 12, 4063 (2021)).
  • the region of interest is the QTL as defined herein and/or any one or more of the genes selected from the group consisting of Zm00001d014641 , Zm00001d014649, Zm00001d014650, Zm00001d014652, Zm00001d014654, Zm00001d014657, and
  • the region of interest is the QTL as defined herein and/or any one or more of the genes selected from the group consisting of Zm00001d014641 , Zm00001d014649, Zm00001d014650, Zm00001d014654, and/or Zm00001d014687.
  • a maize plant that is obtainable or obtained by any method according to the invention.
  • the plant according to the invention is a transgenic plant. In one embodiment, the plant according to the invention is a transgenic maize plant.
  • the plant according to the invention is a transgenic plant from the genus Zea, in particular the species Zea mays, or Sorghum.
  • a “transgenic plant” is a plant into the genome of which at least one polynucleotide, preferably a heterologous polynucleotide, has been integrated.
  • the polynucleotide has been integrated in a stable manner, which means that the integrated polynucleotide remains stable in the plant, is expressed and can also be stably inherited to descendants.
  • the stable introduction of a polynucleotide into the genome of a plant also includes integration into the genome of a plant of the previous parental generation, whereby the polynucleotide can be further inherited in a stable manner.
  • heterologous means that the introduced polynucleotide originates, for example, from a cell or an organism with another genetic background of the same species or from another species or is homologous with the prokaryotic or eukaryotic host cell, but then is localized in a different genetic environment and thus is different from any possible corresponding naturally occurring polynucleotide.
  • a heterologous polynucleotide can be present in addition to a corresponding endogenous gene.
  • the plant according to the invention is a plant into which one at least one heterologous polynucleotide has been integrated.
  • Plants may be modified by targeted gene regulation that does not involve stable and/or heritable changes to the genome of a cell of said plant.
  • RNA mediated gene regulation e.g., using microRNAs or RNA interference (RNAi).
  • a gene of interest confers resistance as a result of reduced or abrogated protein function or guantity
  • the effect of increased resistance may be recreated without the need to introduce the mutation into the genome of the plant for which resistance is to be conferred.
  • reducing or removing a protein e.g., by reducing mRNA levels
  • RNAi RNAi
  • an isolated polynucleic acid that specifically hybridizes with a sequence as set forth in any one or more of SEQ ID NOs: 1 to 52, preferably 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, and 51 ; or a sequence having an identity of at least 80%, 85%, 90%, 95%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% thereto.
  • an isolated polynucleic acid that specifically hybridizes with a sequence as set forth in any one or more of SEQ ID NOs: 59 and 60; 68 and 69; 77 and 78; 86 and 87; 95 and 96; 104 and 105; and 113 and 114; or a sequence having an identity of at least 80%, 85%, 90%, 95%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% thereto.
  • an isolated polynucleic acid that specifically hybridizes with a sequence as set forth in any one or more of SEQ ID NOs: 59 and 60; 68 and 69; 77 and 78; 95 and 96; and 113 and 114; or a sequence having an identity of at least 80%, 85%, 90%, 95%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% thereto.
  • polynucleic acids may bind only to a short segment of their target sequence (i.e., the sequence with which they hybridize). Hybridization may occur between fully complementary sequences (i.e., 100% complementary) or partially complementary sequences (i.e., ⁇ 100% complementary).
  • Reference to plants herein also includes subdivisions of plants, that is, parts of plants or plant cells.
  • the plant is a maize plant or a part or cell thereof.
  • Plants may be described at different levels of organisation, from the cellular level to the organismal level.
  • Plant “parts” means a fusion of several organs, for example a flower or a seed or a part of an organ, for example a cross segment from the stem.
  • organs are leaves, plant stems, stems, roots, vegetative buds, meristems, embryos, anthers, ovulae or fruit.
  • tissues are callus tissue, soft tissue, meristem tissue, leaf tissue, bud tissue, root tissue, plant tumour tissue or reproductive tissue.
  • cells should be understood to mean isolated plant cells with a cell wall or aggregates thereof or protoplasts, for example.
  • the invention provides for polynucleic acids comprising or consisting of sequences according to the invention.
  • polynucleic acid will be understood to mean any polymer of nucleic acids, such as DNA or RNA. It may be referred to other common names in the art, such as nucleic acid molecule, nucleic acid, or polynucleotide.
  • Polynucleic acids may be single or double stranded.
  • Polynucleic acids may be naturally occurring or artificial. Further, polynucleic acids may be naturally derived or synthetic.
  • the polynucleic acid of the invention is a DNA.
  • an isolated polynucleic acid comprising: a) a QTL as defined herein; b) one or more markers selected from M2 to M25 as defined herein; c) one or more sequences selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41 , 43, 45, 47, and 49; d) one or more sequences selected from the group consisting of SEQ ID NOs: 59 and 60; 68 and 69; 77 and 78; 86 and 87; 95 and 96; 104 and 105; and 113 and 114; and/or e) one or more sequences with an identity of at least 80%, 85%, 90%, 95%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.
  • an isolated polynucleic acid comprising: a) a QTL as defined herein; b) one or more markers selected from M15 to M25 as defined herein; c) one or more sequences selected from the group consisting of SEQ ID NOs: 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, and 49; d) one or more sequences selected from the group consisting of SEQ ID NOs: 59 and 60; 68 and 69; 77 and 78; 86 and 87; 95 and 96; 104 and 105; and 113 and 114; and/or e) one or more sequences with an identity of at least 80%, 85%, 90%, 95%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% to a sequence selected from
  • the isolated polynucleic comprises one or more markers selected from M15 to M25.
  • an isolated polynucleic acid comprising: a) a QTL as defined herein; b) one or more markers selected from M15 to M25 as defined herein; c) one or more sequences selected from the group consisting of SEQ ID NOs: 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, and 49; d) one or more sequences selected from the group consisting of SEQ ID NOs: 59 and 60; 68 and 69; 77 and 78; 95 and 96; and 113 and 114; and/or e) one or more sequences with an identity of at least 80%, 85%, 90%, 95%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% to a sequence selected from the group consisting of SEQ ID NOs:
  • a polynucleic acid that specifically hybridizes with another polynucleic acid according to the invention, the complement thereof, or the reverse complement thereof.
  • the polynucleic acid that specifically hybridizes with a polynucleic acid according to the invention is a primer or a probe.
  • isolated nucleic acid molecule or “isolate polynucleotide” should be understood to mean a nucleic acid molecule or polynucleotide removed from its natural or original environment. The term also encompasses a synthetically produced nucleic acid molecule.
  • a vector comprising the nucleic acid molecule of the present invention.
  • the vector may be a plasmid, a cosmid, a phage or an expression vector, a transformation vector, shuttle vector or cloning vector, it may be double or single stranded, linear or circular, or it may be a prokaryotic or eukaryotic host, either by integration into its genome or by extrachromosomal transformation.
  • an expression cassette comprising the nucleic acid molecule of the invention.
  • the nucleotide sequence of the invention is operably linked to regulatory element allowing expression of the nucleotide sequence in a plant cell.
  • the plant cell may be infectable by a fungal pathogen or infected by a fungal pathogen.
  • the plant cell is located in a leaf or a leaf tissue.
  • the regulatory element may be a promoter (native, synthetic, core promoter or chimeric promoter), a terminator, an enhancer or a cis-acting element.
  • the regulatory element may be heterologous to the nucleotide sequence operably linked to regulatory element.
  • the nucleotide sequence of the invention is operably linked in an expression vector to one or more regulatory sequences, which allow transcription and optionally expression in a prokaryotic or eukaryotic host cell.
  • the nucleotide sequence may be under the control of a suitable promoter or a terminator.
  • Suitable promoters may be promoters which are constitutively induced (see, for example, the 35S promoter from the “cauliflower mosaic virus” (Odell JT, Nagy F, Chua N-H (1985) “Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter.” Nature 313, 810 - 812 1985); other examples are the Actin promoter of Oryza sativa or the EF1 promoter of Brachypodium distachyon.
  • promoters are those promoters which are pathogen-inducible (see, for example, the PR1 promoter from parsley (Rushton PJ, Torres JT, Parniske M, Wernert P, Hahlbrock K und Somssich IE (1996) Interaction of elicitor- induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes. EMBO J. 15(20): 5690-5700).
  • Particularly suitable pathogen-inducible promoters are synthetic or chimeric promoters which do not occur in nature, are composed of several elements, and contain a minimum promoter as well as, upstream of the minimum promoter, at least one cis-regulatory element which act as the binding site for special transcription factors. Chimeric promoters are custom-designed and are induced by various factors or re-primed. Examples of such promoters can be found in W02000/29592 and W02007/147395.
  • An example of a suitable terminator is the nos-terminator (Depicker A, Stachel S, Dhaese P, Zambryski P, Goodman HM (1982) Nopaline synthase: transcript mapping and DNA sequence. J Mol Appl Genet. 1(6): 561-73).
  • “Operatively linked” means linked in a common nucleic acid molecule in a manner such that the linked elements are positioned and orientated with respect to each other such that transcription of the nucleic acid molecule can take place.
  • a DNA which is operatively linked with a promoter is under the transcriptional control of this promoter.
  • the invention provides for a cell comprising the nucleic acid molecule of the invention, or the vector of the invention, or the expression cassette of the invention.
  • the vector or the expression cassette may, for example, be introduced into the host cell by conjugation, mobilization, biolistic transformation, agrobacterium-conferred transformation, transfection, transduction, vacuum infiltration or electroporation.
  • Methods of this type as well as methods for the preparation of the vectors described are familiar to the person skilled in the art (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory, 3rd Ed., 2001).
  • the host cell may be a prokaryotic cell (for example, a bacterial cell).
  • the host cell may be a eukaryotic cell (for example, a plant cell or a yeast cell).
  • a eukaryotic cell for example, a plant cell or a yeast cell.
  • Particularly preferred bacterial host cells are Agrobacterium tumefaciens, A. rhizogenes, and E. coli.
  • the cell comprises markers M15 to M25. In one embodiment the cell is a transgenic cell that comprises markers M15 to M25.
  • the cell comprises: a) a Zm00001d014641 sequence comprising one or more, preferably both, of the markers M15 and/or M16; preferably having a sequence as set forth in any of SEQ ID NOs: 59 and 60, or a sequence having an identity of at least 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% to a sequence as set forth in any of SEQ ID NOs: 59 and 60, preferably over the entire length of the sequence; or a polynucleic acid encoding a polypeptide sequence as set forth in SEQ ID NO: 61 or a sequence having an identity of at least 80%, 85%, 90%, 95%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%,
  • the cell comprises: a) a Zm00001d014641 sequence comprising one or more, preferably both, of the markers M15 and/or M16; preferably having a sequence as set forth in any of SEQ ID NOs: 59 and 60, or a sequence having an identity of at least 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% to a sequence as set forth in any of SEQ ID NOs: 59 and 60, preferably over the entire length of the sequence; or a polynucleic acid encoding a polypeptide sequence as set forth in SEQ ID NO: 61 or a sequence having an identity of at least 80%, 85%, 90%, 95%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%,
  • the cell comprises a polynucleotide sequence encoding, and/or is capable of expressing any one or more of a: a) polypeptide sequence as set forth in SEQ ID NO: 61 ora sequence having an identity of at least 80%, 85%, 90%, 95%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% to a sequence as set forth in SEQ ID NO: 61 , preferably over the entire length of the sequence; b) polypeptide sequence as set forth in SEQ ID NO: 70 ora sequence having an identity of at least 80%, 85%, 90%, 95%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.6%,
  • the invention provides a method for identifying a maize plant having resistance and/or tolerance to a fungal pathogen, comprising screening for the presence of at least one QTL located on chromosome 5 in a maize plant or plant part, wherein said QTL is located on a chromosomal interval flanked by markers M1 and M26, wherein markers M1 and M26 are SNPs which are respectively guanine (G) at the position 53948820 bp and cytosine (C) at the position 61159296 bp, on reference genome B73_AGPvO4.
  • a method for identifying a maize plant having resistance and/or tolerance to a fungal pathogen wherein said QTL comprises the marker M2, wherein M2 is a SNP which is cytosine (C) at position 54422803 when referenced to the B73 reference genome AGPv4.
  • a method for identifying a maize plant having resistance and/or tolerance to a fungal pathogen wherein said QTL comprises the marker M4, wherein M4 is a SNP which is thymine (T) at position 55314620 when referenced to the B73 reference genome AGPv4.
  • a method for identifying a maize plant having resistance and/or tolerance to a fungal pathogen wherein said QTL comprises the marker M5, wherein M5 is a SNP which is cytosine (C) at position 56109835 when referenced to the B73 reference genome AGPv4.
  • a method for identifying a maize plant having resistance and/or tolerance to a fungal pathogen wherein said QTL comprises the marker M6, wherein M6 is a SNP which is thymine (T) at position 56925593 when referenced to the B73 reference genome AGPv4.
  • a method for identifying a maize plant having resistance and/or tolerance to a fungal pathogen wherein said QTL comprises the marker M8, wherein M8 is a SNP which is guanine (G) at position 57859040 when referenced to the B73 reference genome AGPv4.
  • a method for identifying a maize plant having resistance and/or tolerance to a fungal pathogen wherein said QTL comprises the marker M10, wherein M10 is a SNP which is cytosine (C) at position 59015277 when referenced to the B73 reference genome AGPv4.
  • a method for identifying a maize plant having resistance and/or tolerance to a fungal pathogen wherein said QTL comprises the marker M12, wherein M 12 is a SNP which is guanine (G) at position 59905013 when referenced to the B73 reference genome AGPv4.
  • a method for identifying a maize plant having resistance and/or tolerance to a fungal pathogen wherein said QTL comprises the marker M15, wherein M 15 is a SN P which is guanine (G) at position 57056956 when referenced to the B73 reference genome AGPv4.
  • a method for identifying a maize plant having resistance and/or tolerance to a fungal pathogen wherein said QTL comprises the marker M17, wherein M17 is a SNP which is cytosine (C) at position 57191086 when referenced to the B73 reference genome AGPv4.
  • a method for identifying a maize plant having resistance and/or tolerance to a fungal pathogen wherein said QTL comprises the marker M19, wherein M 19 is a SN P which is adenine (A) at position 57311744 when referenced to the B73 reference genome AGPv4.
  • a method for identifying a maize plant having resistance and/or tolerance to a fungal pathogen wherein said QTL comprises the marker M22, wherein M22 is a SN P which is guanine (G) at position 57563791 when referenced to the B73 reference genome AGPv4.
  • a method for identifying a maize plant having resistance and/or tolerance to a fungal pathogen wherein said QTL comprises the marker M23, wherein M23 is a SNP which is thymine (T) at position 59024339 when referenced to the B73 reference genome AGPv4.
  • a method for identifying a maize plant having resistance and/or tolerance to a fungal pathogen wherein said QTL comprises the marker M24, wherein M24 is a SNP which is adenine (A) at position 59024546 when referenced to the B73 reference genome AGPv4.
  • a method for identifying a maize plant having resistance and/or tolerance to a fungal pathogen wherein said QTL comprises any one or more of the markers M1 to M26.
  • a method for identifying a maize plant having resistance and/or tolerance to a fungal pathogen wherein said QTL comprises any one or more of the markers M2 to M25.
  • a method for identifying a maize plant having resistance and/or tolerance to a fungal pathogen wherein said QTL comprises any one or more of the markers M15 to M25.
  • a method for identifying a maize plant or plant part comprising screening for the presence of one or more markers selected from M1 to M26 as defined herein, or one or more marker regions, wherein said marker regions comprise a sequence selected from the group consisting of SEQ ID NOs: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, and 51.
  • a method for identifying a maize plant or plant part comprising screening for the presence of one or more markers selected from M2 to M25 as defined herein, or one or more marker regions, wherein said marker regions comprise a sequence selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, and 49.
  • a method for identifying a maize plant or plant part comprising screening for the presence of one or more markers selected from M15 to M25 as defined herein, or one or more marker regions, wherein said marker regions comprise a sequence selected from the group consisting of SEQ ID NOs: 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, and 49.
  • a method for identifying a maize plant having resistance and/or tolerance to a fungal pathogen comprising one or more markers selected from the group comprising M2 to M25, wherein: a) M2 is a SNP which is cytosine (C) at position 54422803 when referenced to the B73 reference genome AGPv4; b) M3 is a SNP which is guanine (G) at position 54917958 when referenced to the B73 reference genome AGPv4; c) M4 is a SNP which is thymine (T) at position 55314620 when referenced to the B73 reference genome AGPv4; d) M5 is a SNP which is cytosine (C) at position 56109835 when referenced to the B73 reference genome AGPv4; e) M6 is a SNP which is thymine (T) at position 56925593 when referenced to the B73 reference genome AGPv4; f)
  • a method for identifying a maize plant having resistance and/or tolerance to a fungal pathogen comprising one or more markers selected from the group comprising M15 to M25, wherein: a) M15 is a SNP which is guanine (G) at position 57056956 when referenced to the B73 reference genome AGPv4; b) M 16 is a SNP which is adenine (A) at position 57057094 when referenced to the B73 reference genome AGPv4; c) M 17 is a SNP which is cytosine (C) at position 57191086 when referenced to the B73 reference genome AGPv4; d) M18 is a SNP which is thymine (T) at position 57192412 when referenced to the B73 reference genome AGPv4; e) M 19 is a SNP which is adenine (A) at position 57311744 when referenced to the B73 reference genome AGPv4;
  • a method for identifying a maize plant having resistance and/or tolerance to a fungal pathogen wherein the maize plant comprises one or more genes selected from the group consisting of:
  • Zm00001d014641 comprising a sequence as set forth in SEQ ID NO: 59 or 60;
  • Zm00001d014650 comprising a sequence as set forth in SEQ ID NO: 77 or 78;
  • Zm00001d014641 comprising a sequence as set forth in SEQ ID NO: 59 or 60;
  • M25 is a SNP which is guanine (G) at position 59024651 when referenced to the B73 reference genome AGPv4.
  • said fungal pathogen is selected from the group consisting of Exserohilum sp, Cercospora zeae-maydis or Puccinia polysora, preferably said pathogen is Exserohilum turcicum.
  • M18 is a SNP which is thymine (T) at position 57192412 when referenced to the B73 reference genome AGPv4;
  • M 19 is a SNP which is adenine (A) at position 57311744 when referenced to the B73 reference genome AGPv4;
  • M21 is a SNP which is cytosine (C) at position 57313785 when referenced to the B73 reference genome AGPv4;
  • M22 is a SNP which is guanine (G) at position 57563791 when referenced to the B73 reference genome AGPv4;
  • M24 is a SNP which is adenine (A) at position 59024546 when referenced to the B73 reference genome AGPv4; and/or
  • cM centimorgans
  • one, two or more markers capable of detecting the presence of one, two, three or more of the QTL as defined herein, one or more markers as defined herein, one or more marker regions as described herein, and/or one or more gene as described herein, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity thereto, or a functional ortholog, homolog or paralog thereof, for identifying a maize plant having resistance and/or tolerance to Exserohilum sp, Cercospora zeae-maydis or Puccinia polysora, preferably Exserohilum turcicum
  • one, two or more markers capable of detecting the presence of one, two, three or more of the QTL as defined herein, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, and/or 49, or a functional ortholog, homolog or paralog thereof, for identifying a maize plant having resistance and/or tolerance to Exserohilum sp, Cercospora zeae-maydis or Puccinia polysora, preferably Exserohilum turcicum.
  • one, two or more markers capable of detecting the presence of one, two, three or more of the QTL as defined herein, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, and/or 49, or a functional ortholog, homolog or paralog thereof, for identifying a maize plant having resistance and/or tolerance to Exserohilum sp, Cercospora zeae-maydis or Puccinia polysora, preferably Exserohilum turcicum.
  • one, two or more markers capable of detecting the presence of one, two, three or more of the QTL as defined herein, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 59 and 60; 68 and 69; 77 and 78; 86 and 87; 95 and 96; 104 and 105; and 113 and 114, or a functional ortholog, homolog or paralog thereof, for identifying a maize plant having resistance and/or tolerance to Exserohilum sp, Cercospora zeae-maydis or Puccinia polysora, preferably Exserohilum turcicum.
  • one, two or more markers capable of detecting the presence of one, two, three or more of the QTL as defined herein, or a sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any of SEQ ID NOs: 59 and 60; 68 and 69; 77 and 78; 95 and 96; and 113 and 114, or a functional ortholog, homolog or paralog thereof, for identifying a maize plant having resistance and/or tolerance to Exserohilum sp, Cercospora zeae-maydis or Puccinia polysora, preferably Exserohilum turcicum.
  • one or more markers capable of detecting the presence of one or more of the QTL as defined herein, for identifying a maize plant having resistance and/or tolerance to Exserohilum sp, Cercospora zeae-maydis or Puccinia polysora, preferably Exserohilum turcicum.
  • one or more markers having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to one or more of SEQ ID NO: 1 , 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, 49, and/or 51 , or a functional ortholog, homolog or paralog thereof, for identifying a maize plant having resistance and/or tolerance to Exserohilum sp, Cercospora zeae-maydis or Puccinia polysora, preferably Exserohilum turcicum.
  • one or more markers having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to one or more of SEQ ID NO: 3, 5, 7, 9, 11 , 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, and/or 49, or a functional ortholog, homolog or paralog thereof, for identifying a maize plant having resistance and/or tolerance to Exserohilum sp, Cercospora zeae-maydis or Puccinia polysora, preferably Exserohilum turcicum.
  • one or more markers having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to one or more of SEQ ID NO: 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47, and/or 49, or a functional ortholog, homolog or paralog thereof, for identifying a maize plant having resistance and/or tolerance to Exserohilum sp, Cercospora zeae-maydis or Puccinia polysora, preferably Exserohilum turcicum.
  • a method as described herein comprises that said at least one plant cell, tissue, organ, plant, or seed is not obtained by an essentially biological process. Instead, said at least one plant cell, tissue, organ, plant, or seed is obtained by at least one step of artificial human intervention as such not occurring in nature and influencing the plant cell by modifying and/or introducing a step of technical nature influencing sexually crossing and selecting.
  • a step may include a step of genome editing, e.g., to exchange a base or nucleotide of interest, a chemical treatment, e.g. for chromosome doubling an agent or gene or gene product including chromosome elimination, the introduction of an exogenous gene or genetic material into a plant genome (nuclear, mitochondrial or plastid genome) and the like, or any combination thereof.
  • a quantitative trait locus is a region of DNA associated with a specific phenotype or trait that varies within a population.
  • a QTL may herein also be referred to as a locus, wherein a "locus” is a position on a chromosome where one or more genes are found which cause an agronomic feature or influence one.
  • a “marker” is a nucleotide sequence which is used as a reference or orientation point.
  • a marker for recognizing a recombination event should be suitable for monitoring differences or polymorphisms in a plant population.
  • these differences are on a DNA level and, for example, are polynucleotide sequence differences such as, for example, SSRs (simple sequence repeats), RFLPs (restriction fragment length polymorphisms), FLPs (fragment length polymorphisms) or SNPs (single nucleotide polymorphisms).
  • a “marker region” is a nucleotide that comprises a “marker” as defined above as well as its local context, i.e., sequences immediately 3’ and 5’ of said marker. It will be understood that said marker regions may have any start and end point and are simply used to illustrate the local context of marker, for example a SNP.
  • the markers may be derived from genomic or expressed nucleic acids such as spliced RNA, cDNA or ESTs and may be based on nucleic acids which are used as probes or primer pairs and as such are suitable for amplifying a sequence fragment using PCR-based methods.
  • Markers which concern genetic polymorphisms between parts of a population can be detected using established methods from the prior art (An Introduction to Genetic Analysis. 7th Edition, Griffiths, Miller, Suzuki et al., 2000). These include, for example: DNA sequencing, PCR-based, sequence-specific amplification, assaying of RFLPs, assaying of polynucleotide polymorphisms using allele-specific hybridization (ASH), detection of SSRs, SNPs or AFLPs. Methods for detecting ESTs (expressed sequence tags) and RAPD (randomly amplified polymorphic DNA) are also known.
  • the term “marker” in the description may also mean a specific chromosome position in the genome of a species where a specific marker (for example SNP) can be found.
  • a marker position of this type can be used in order to monitor the presence of a coupled locus, for example a coupled locus which contributes to the expression of a specific phenotypical feature (e.g., linkage).
  • the marker locus may also be used to observe the segregation of alleles at a locus (QTL or individual gene) which are genetically or physically closely coupled with the marker position.
  • allele refers to one or two or more nucleotide sequences at a specific locus in the genome.
  • a first allele is on a chromosome, a second on a second chromosome at the same position. If the two alleles are different, they are heterozygous, and if they are the same, they are homozygous.
  • Various alleles of a gene differ in at least one SNP or an indel. Depending on the context of the description, an allele also means a single SNP which, for example, allows for a distinction between the donor and recurrent parent.
  • chromosome fragment means a specific chromosomal DNA segment of a specific chromosome which comprises at least one gene.
  • An integrated chromosome fragment derives from a donor source.
  • the sequential succession of the genes within an integrated chromosome fragment corresponds to that sequence as it is present in the original chromosome fragment of the donor source.
  • the integrated chromosome fragment may be present over the whole length unchanged compared with the corresponding chromosome fragment in the donor source.
  • a chromosome fragment or a part thereof may constitute a specific “haplotype”, wherein the chromosome fragment may comprise specific SNPs through which the haplotype can also be unequivocally specified and identified.
  • regulatory sequence means a nucleotide sequence which influences the specificity and/or strength of expression, for example insofar as the regulatory sequence confers a specific tissue specificity.
  • a regulatory sequence of this type may be localized upstream of the transcription initiation point of a minimum promoter, but also downstream thereof, for example in a transcribed but not translated leader sequence or within an intron.
  • introduction refers to the provision of specific polynucleotide sequences to a plant or part thereof; such provision not being limited to providing exogenous sequences but also including altering endogenous sequences.
  • distal and proximal describe the position of a chromosomal interval or a genetic segment in relation to a specific reference point (for example a specific polynucleotide, another chromosomal interval or a gene) on a whole chromosome; “distal” means that the interval or the segment is localized on the side of the reference point distant from the chromosome centromere, and “proximal” means that the interval or the segment is localized on the side of the reference point close to the chromosome centromere.
  • stringency refers to the hybridization conditions. High stringency is when base pairing is more difficult, low stringency is when base pairing is easier.
  • the stringency of the hybridization conditions depends, for example, on the salt concentration or ionic strength and the temperature. In general, the stringency can be increased by raising the temperature and/or by reducing the salt content.
  • stringent hybridization conditions should be understood to mean those conditions under which a hybridization takes place primarily only between homologous nucleic acid molecules.
  • hybridization conditions in this respect refers not only to the actual conditions prevailing during actual agglomeration of the nucleic acids, but also to the conditions prevailing during the subsequent washing steps.
  • stringent hybridization conditions may also include hybridization at 68°C in 0.25 M sodium phosphate, pH 7.2, 7 % SDS, 1 mM EDTA and 1 % BSA for 16 hours and subsequently washing twice with 2 x SSC and 0.1 % SDS at 68°C.
  • hybridization takes place under stringent conditions.
  • interval or “chromosomal interval” means a continuous linear segment on a genomic DNA, which is present in an individual chromosome in a plant or on a chromosome fragment and which is usually defined through two markers which represent the end points of the interval on the distal and proximal side.
  • Example 1 Fine mapping and cloning of the resistance gene locus on chromosome 5
  • the donor line TropicalD2 was crossed and backcrossed with the susceptible line RP7601 to create an introgression line population.
  • Individual lines of the introgression line population contain different segments of the donor line in the background of the recurrent parent, so that the whole donor genome is covered.
  • 100 introgression lines were planted in the field at two different locations. Of these lines, 9 lines contained a 4.5 cM donor segment on chromosome 5 (89.0 - 93.5 cM). These lines displayed NCLB scores up to 3.3 scoring notes better than the recurrent parent.
  • the effect was -0.47, describing the effect that the region of the introgression lines had on the NCLB resistance trait. The value is descriptive and used when selecting regions of interest and is not a statistical value.
  • Illumina whole genome sequencing the whole genome sequencing of the selected donor lines allows the analysis and characterization of the QTL region.
  • the genes that confer resistance may not be present in existing maize reference sequences. Therefore, it was important to sequence the resistant donor lines and identify any genes that may be missing from the existing reference sequences or the susceptible parent.
  • short read sequencing is very accurate and allows the identification of SNPs and other smaller polymorphisms between the resistant and susceptible parents. This can help with the identification of candidate genes, as a gene underlying the resistance should have a polymorphism between the resistant and susceptible parent lines. These polymorphisms can then also be used for marker development. This has been completed for lines: 5WV2002 and RP7601.
  • Illumina sequencing and genome assembly relies on short reads. These short reads create problems during genome assembly, as complex loci or repetitive regions may not be assembled properly. Large structural variations may also be missed when a reference genome would be used for scaffolding. To address this, the inventors performed long-read ONT sequencing of the parents and created high-quality genome assemblies. This has been completed for 5WV2002 and RP7601.
  • RNAseq time course serves two purposes. On the one hand, it allows identification of candidate genes. For example, if a gene not previously considered to be involved in resistance has an interesting expression pattern after infection, which is unique to the resistant parent, then said gene would be a very strong candidate gene. On the other hand, it allows validation of candidate genes, including the ability to exclude genes. For example, if an NLR (Nucleotide-binding site leucine-rich repeat gene) candidate gene would be present within the locus but not expressed after infection, the inventors can conclude that it is unlikely to be involved in the resistance response.
  • NLR Nucleotide-binding site leucine-rich repeat gene
  • 170 genes are annotated in the B73 AGPvO4 reference genome and 175 genes are annotated in the RP7601 Oxford Nanopore Technology de novo assembly. These were investigated for putative candidate genes. Seven candidate genes were selected for further in-depth analysis: Zm00001d014649, Zm00001d014650, Zm00001d014654, Zm00001d014687, Zm00001d014641 , Zm00001d014657, and
  • Validation of candidate genes will be done via (a) transformation of the susceptible maize line A188, (b) knock out with genome editing of the recombinant line RP7601nHTD5a, and (c) analysis of differential gene expression in infected and control plants of the recombinant line 5WV2002 and the recurrent parent line RP7601.
  • RNAseq experiments gene expression will be assessed in the recombinant line 5WV2002 and the recurrent parent line RP7601 at different time points after infection. Obtained RNAseq reads will be mapped on the 5WV2002 Oxford Nanopore Technology de novo assembly, so that candidate genes within the target QTL region can be confirmed or new ones identified.

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Abstract

La présente invention concerne des plants de maïs présentant une résistance aux maladies améliorée. En particulier, l'invention concerne des procédés d'identification ou de génération de plants de maïs et de parties ou de cellules de ceux-ci, présentant une résistance aux maladies. La présente invention concerne en outre des plants de maïs présentant une résistance aux maladies améliorée, produits au moyen des procédés de l'invention.
PCT/EP2025/050969 2024-01-17 2025-01-15 Plants de maïs présentant une résistance aux maladies améliorée Pending WO2025153582A1 (fr)

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