WO2025064264A1 - Augmentation de l'induction haploïde - Google Patents

Augmentation de l'induction haploïde Download PDF

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Publication number
WO2025064264A1
WO2025064264A1 PCT/US2024/045960 US2024045960W WO2025064264A1 WO 2025064264 A1 WO2025064264 A1 WO 2025064264A1 US 2024045960 W US2024045960 W US 2024045960W WO 2025064264 A1 WO2025064264 A1 WO 2025064264A1
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Prior art keywords
maize plant
haploid
plant
inducer
haploid inducer
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PCT/US2024/045960
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English (en)
Inventor
Becky Breitinger
Brent Delzer
Azhaguvel PERUMAL
Rachel EGGER
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Syngenta Crop Protection AG Switzerland
Syngenta Crop Protection LLC
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Syngenta Crop Protection AG Switzerland
Syngenta Crop Protection LLC
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Priority to CN202480058273.3A priority Critical patent/CN121843585A/zh
Publication of WO2025064264A1 publication Critical patent/WO2025064264A1/fr
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    • 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
    • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
    • A01H1/08—Methods for producing changes in chromosome number
    • 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
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • 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
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/46—Gramineae or Poaceae, e.g. ryegrass, rice, wheat or maize
    • A01H6/4684—Zea mays [maize]

Definitions

  • the presently disclosed subject matter relates generally to breeding and haploid induction.
  • the chromosome 1 and chromosome 9 QTL were later determined to be the genes MATRILINEAL (MATL) and ZmDMP, respectively (Kelliher et al. 2017, Zhong et al. 2019).
  • the other secondary QTL that may contribute to further improvement in haploid induction rates when used in combination with MATL and DMP remain relatively
  • the disclosure herein provides a maize plant comprising a QTL on chromosome 5 (SYN Chr5 QTL).
  • the QTL on chromosome 5 is associated with increased haploid induction.
  • a method of obtaining a plant with increased haploid production by obtaining a first plant comprising a SYN Chr5 QTL and crossing it with a second plant followed by obtaining progeny from said cross containing the SYN Chr5 QTL, thereby obtaining a plant with increased haploid production.
  • a method of selecting a first maize plant or germplasm that displays increased haploid production wherein nucleic acids are isolated from the first maize plant or germplasm followed by detecting in the first maize plant or germplasm a SYN Chr5 QTL associated with increased haploid production and selecting said first maize plant or germplasm, or selecting a progeny of said first maize plant or germplasm, comprising SYN Chr5 QTL that is associated with increased haploid production. Further, the method comprises crossing said selected first maize plant or germplasm with a second maize plant or germplasm, wherein the introgressed maize plant or germplasm displays increased haploid production.
  • SEQ ID NO: 1 is a nucleotide sequence of a forward primer used to amplify marker SM10934.
  • SEQ ID NO: 2 is a nucleotide sequence of a forward primer used to amplify marker SM10934.
  • SEQ ID NO: 3 is a nucleotide sequence of a forward primer used to amplify marker SM10934.
  • SUBSTITUTE SHEET (RULE 26) SEQ ID NO: 4 is a nucleotide sequence of a reverse primer used to amplify marker SM10934.
  • SEQ ID NO: 5 is a nucleotide sequence of the SM10934 marker target sequence.
  • SEQ ID NO: 6 is a nucleotide sequence of a forward primer used to amplify marker SM10932.
  • SEQ ID NO: 7 is a nucleotide sequence of a forw ard primer used to amplify marker SM10932.
  • SEQ ID NO: 8 is a nucleotide sequence of a forw ard primer used to amplify marker SM10932.
  • SEQ ID NO: 9 is a nucleotide sequence of a reverse primer used to amplify marker SM10932.
  • SEQ ID NO: 10 is a nucleotide sequence of the SMI 0932 marker target sequence.
  • the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims.
  • a cell refers to one or more cells, and in some embodiments can refer to a tissue and/or an organ.
  • the phrase “at least one”, when employed herein to refer to an entity refers to, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, or more of
  • SUBSTITUTE SHEET (RULE 26) that entity, including but not limited to all whole number values between 1 and 100 as well as whole numbers greater than 100.
  • allele refers to a variant or an alternative sequence form at a genetic locus.
  • diploids a single allele is inherited by a progeny individual separately from each parent at each locus.
  • the two alleles of a given locus present in a diploid organism occupy corresponding places on a pair of homologous chromosomes, although one of ordinary skill in the art understands that the alleles in any particular individual do not necessarily represent all of the alleles that are present in the species.
  • the term “amplified” or “amplify” means the construction of multiple copies of a nucleic acid molecule or multiple copies complementary' to the nucleic acid molecule using at least one of the nucleic acid molecules as a template.
  • Amplification systems include the polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicase systems, transcription-based amplification system (TAS), and strand displacement amplification (SDA). See, e.g., Diagnostic Molecular Microbiology: Principles and Applications, PERSING et al., Ed., American Society for Microbiology, Washington, D.C. (1993). The product of amplification is termed an “amplicon.”
  • the phrase “A, B, C, and/or D” includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D (e.g., AB, AC, AD, BC, BD, CD, ABC, ABD, and
  • one or more of the elements to which the “and/or” refers can also individually be present in single or multiple occurrences in the combinations(s) and/or subcombination(s).
  • a marker is “associated with” a trait when it is linked to it and when the presence of the marker is an indicator of whether and/or to what extent the desired trait or trait form will occur in a plant/germplasm comprising the marker.
  • a marker is “associated with” an allele when it is linked to it and when the presence of the marker is an indicator of whether the allele is present in a plant/germplasm comprising the marker.
  • backcrossing is understood within the scope of the invention to refer to a process in which a hybrid progeny is repeatedly crossed back to one of the parents.
  • cDNA refers to a single-stranded or a double-stranded DNA that is complementary to and derived from mRNA.
  • chromosome is used herein as recognized in the art as meaning the self-replicating genetic structure in the cellular nucleus containing the cellular DNA and bearing the linear array of genes.
  • the phrase “consisting of’ excludes any element, step, or ingredient not specifically recited.
  • the phrase “consists of’ appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.
  • SUBSTITUTE SHEET (RULE 26) mater can include in some embodiments the use of either of the other two terms.
  • a subject mater relates in some embodiments to nucleic acids that encode polypeptides comprising amino acid sequences that are at least 95% identical to a SEQ ID NO: 2 or 3. It is understood that the disclosed subject mater thus also encompasses nucleic acids that encode polypeptides that in some embodiments consist essentially of amino acid sequences that are at least 95% identical to that SEQ ID NO: 2 or 3 as well as nucleic acids that encode polypeptides that in some embodiments consist of amino acid sequences that are at least 95% identical to that SEQ ID NO: 2 or 3.
  • the term “de novo haploid induction” refers to the tnggering of haploid induction by the introduction of a spontaneous-haploid inducing agent. Such introduction can be achieved by topical spray, hand-pollination, mutagenesis, or transgenic methods.
  • the terms “de novo haploid induction,” “de novo HI,” and “haploid induction de novo” are used interchangeably throughout this specification.
  • elite line refers to any line that has resulted from breeding and selection for superior agronomic performance.
  • An elite line has stable genetics, i.e., it is reasonably or nearly isogenic across its genome. Said another way, an elite line is reasonably or nearly homozygous for all alleles in its genome.
  • an elite inducer refers to plants bred to be high-performing haploid inducers.
  • an elite inducer may contain a haploid induction rate (HIR) greater than 15% and mutations in the MATL gene, mutations in the ZmDMP gene, a dominant locus at the R1 gene for color selection or other marker for haploid selection, and/or additional QTL associated with increased rates of haploid induction or other traits favorable for a haploid inducer line.
  • HIR haploid induction rate
  • RNA e.g., mRNA, rRNA, tRNA, or snRNA
  • Gene expression can be regulated at many stages in the process. For example, in the case of
  • expression may refer to the transcription of the antisense RNA only or the dsRNA only.
  • expression refers to the transcription and stable accumulation of sense (mRNA) or functional RNA. “Expression” may also refer to the production of protein.
  • the term “gene” refers to a hereditary' unit including a sequence of DNA that occupies a specific location on a chromosome and that contains the genetic instruction for a particular characteristic or trait in an organism.
  • the term “genotype” refers to the genetic constitution of a cell or organism.
  • An individual's “genotype for a set of genetic markers” includes the specific alleles, for one or more genetic marker loci, present in the individual.
  • a genotype can relate to a single locus or to multiple loci, whether the loci are related or unrelated and/or are linked or unlinked.
  • an individual’s genotype relates to one or more genes that are related in that the one or more of the genes are involved in the expression of a phenotype of interest (e.g., a quantitative trait as defined herein).
  • a genotype comprises a sum of one or more alleles present within an individual at one or more genetic loci of a quantitative trait.
  • a genotype is expressed in terms of a haplotype (defined herein below).
  • a “genetic map” is a description of genetic linkage relationships among loci on one or more chromosomes within a given species, generally depicted in a diagrammatic or tabular form.
  • the term “germplasm” refers to the totality of the genotypes of a population or another group of individuals (e.g., a species).
  • the term “germplasm” can also refer to plant material, e.g., a group of plants that act as a repository for various alleles.
  • adapted germplasm refers to plant materials of proven genetic superiority; e.g., for a given environment or geo-graphical area
  • non-adapted germplasm refers to plant materials of unknown or unproven genetic value; e g., for a given environment or geographical area; as such, the phrase “non-adapted germplasm” refers in some embodiments to plant materials that are not part of an established breeding population and that do not have a known relationship to a member of the established breeding population.
  • haploid has a single set (genome) of chromosomes and the reduced number of chromosomes (In) in the haploid plant is equal to that of the gamete.
  • doubled haploid is developed by doubling
  • SUBSTITUTE SHEET (RULE 26) the haploid set of chromosomes (from In to 2n).
  • a plant or seed that is obtained from a doubled haploid plant that is crossed to itself (selfed) to any number of generations may still be identified as a doubled haploid plant.
  • a doubled haploid plant is considered a homozygous plant.
  • a plant is considered to be doubled haploid if it is fertile, even if the entire vegetative part of the plant does not consist of the cells with the doubled set of chromosomes; that is, a plant will be considered doubled haploid if it contains viable gametes, even if it is chimeric.
  • heterologous when used in reference to a gene or nucleic acid refers to a gene encoding a factor that is not in its natural environment (i.e., has been altered by the hand of man).
  • a heterologous gene may include a gene from one species introduced into another species.
  • a heterologous gene may also include a gene native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to a non-native promoter or enhancer polynucleotide, etc.).
  • Heterologous genes further may comprise plant gene polynucleotides that comprise cDNA forms of a plant gene; the cDNAs may be expressed in either a sense (to produce mRNA) or anti-sense orientation (to produce an anti -sense RNA transcript that is complementary to the mRNA transcript).
  • heterologous genes are distinguished from endogenous plant genes in that the heterologous gene polynucleotide are typically joined to polynucleotides comprising regulatory elements such as promoters that are not found naturally associated with the gene for the protein encoded by the heterologous gene or with plant gene polynucleotide in the chromosome, or are associated with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed).
  • a “heterologous” polynucleotide is a polynucleotide not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring polynucleotide.
  • heterozygous means a genetic condition existing when different alleles reside at corresponding loci on homologous chromosomes.
  • homozygous means a genetic condition existing when identical alleles reside at corresponding loci on homologous chromosomes.
  • HMF score or “Haploid Male Fertility score” refers to the number of days a plant sheds pollen. The method used herein measures each individual plant. Plants are checked for fertile pollen shed every day over the course of the pollen shedding period (7 - 14 days in the field), and each plant is assigned a number equal to the number of days it shed
  • SUBSTITUTE SHEET (RULE 26) pollen (e.g., a plant assigned number 5 equals a plant shedding pollen for 5 days). A sterile plant is scored as a zero, meaning there were zero days it shed pollen.
  • isolated when used in the context of the nucleic acid molecules or polynucleotides of the present invention, refers to a polynucleotide that is identified within and isolated/separated from its chromosomal polynucleotide context within the respective source organism.
  • An isolated nucleic acid or polynucleotide is not a nucleic acid as it occurs in its natural context, if it indeed has a naturally occurring counterpart.
  • non-isolated nucleic acids are nucleic acids such as DNA and RNA, which are found in the state they exist in nature.
  • a given polynucleotide e.g., a gene
  • the isolated nucleic acid molecule may be present in single-stranded or double-stranded form. Alternatively, it may contain both the sense and antisense strands (i.e., the nucleic acid molecule may be double-stranded).
  • the nucleic acid molecules of the present invention are understood to be isolated.
  • locus refers to a position (e.g., of a gene, a genetic marker, or the like) on a chromosome of a given species.
  • haploid inducer refers to a line that produces pollen and, when crossed as a male, results in the gynogenic development of haploid seeds.
  • a “paternal haploid inducer” refers to a line that when used as a female in a cross, results in androgenic development of haploid seeds.
  • a haploid inducer plant can use either of these maternal or paternal mechanisms to derive haploids and can be generically referred to as an “inducer” without specifying the mechanism of a particular line.
  • human-induced mutation refers to any mutation that occurs because of either direct or indirect human action. This term includes, but is not limited to, mutations obtained by any method of targeted mutagenesis.
  • hybrid refers to offspring produced by crossing two genetically dissimilar parent plants. The resulting progeny of this cross are a “bi-parental” population.
  • logarithm of the odds or “LOD score” refers to the statistical likelihood that two genes are in proximity on a chromosome.
  • the terms “marker probe” and “probe” refer to a nucleotide sequence or nucleic acid molecule that can be used to detect the presence or absence of a sequence within a larger sequence, e.g., a nucleic acid probe that is complementary to all of or a portion of the
  • SUBSTITUTE SHEET (RULE 26) marker or marker locus, through nucleic acid hybridization. Marker probes comprising about 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more contiguous nucleotides can be used for nucleic acid hybridization.
  • the term “molecular marker” can be used to refer to a genetic marker, as defined above, or an encoded product thereof (e.g., a protein) used as a point of reference when identifying the presence/absence of a SCD or other locus of interest.
  • a molecular marker can be derived from genomic nucleotide sequences or from expressed nucleotide sequences (e.g., from an RNA, a cDNA, etc.). The term also refers to nucleotide sequences complementary to or flanking the marker sequences, such as nucleotide sequences used as probes and/or primers capable of amplifying the marker sequence.
  • Nucleotide sequences are “complementary” when they specifically hybridize in solution (e.g., according to Watson- Crick base pairing rules). This term also refers to the genetic markers that indicate a trait by the absence of the nucleotide sequences complementary to or flanking the marker sequences, such as nucleotide sequences used as probes and/or primers capable of amplifying the marker sequence.
  • nucleotide sequence As used herein, the terms “nucleotide sequence,” “polynucleotide,” “nucleic acid sequence,” “nucleic acid molecule,” and “nucleic acid fragment” refer to a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural, and/or altered nucleotide bases.
  • 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 are referred to by their single letter designation as follows: “A” for adenylate or deoxy adenylate (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.
  • sequence identify refers to the percentage of identical nucleotides or amino acids in a linear polynucleotide or amino acid sequence of a reference (“query”) sequence (or its complementary strand) as compared to a test (“subject”) sequence when the two sequences are globally aligned.
  • equivalent program refers to any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by EMBOSS Needle.
  • substantially identical nucleic acid or amino acid sequences may perform substantially the same function.
  • introduction refers to both a natural and artificial process whereby genomic regions of one species, variety or cultivar are moved into the genome of another species, variety or cultivar, by crossing those species. The process may optionally be completed by backcrossing to the recunent parent.
  • ORF open reading frame
  • an ORF refers to a nucleic acid sequence that encodes a polypeptide.
  • an ORF comprises a translation initiation codon, a translation termination (i.e., stop) codon, and the nucleic acid sequence there between that encodes the amino acids present in the polypeptide.
  • initiation codon and terminal codon refer to a unit of three adjacent nucleotides (i.e., a codon) in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation).
  • phenotype refers to one or more traits of a plant or plant cell.
  • the phenotype can be observable to the naked eye, or by any other means of evaluation known in the art, e.g., microscopy, biochemical analysis, or an electromechanical assay.
  • a phenotype is directly controlled by a single gene or genetic locus (i.e., corresponds to a “single gene trait”).
  • phenotype can refer to fertile pollen shed and/or a seed obtained via pollination with that pollen.
  • a phenotype is the result of interactions among several genes, which in some embodiments also results from an interaction of the plant and/or plant cell with its environment.
  • plant can refer to a whole plant, any part thereof, or a cell or tissue culture derived from a plant.
  • plant can refer to any of the following: whole
  • SUBSTITUTE SHEET plants, plant components or organs (e.g., leaves, stems, roots, etc.), plant tissues, seeds and/or plant cells, unless otherwise specified.
  • a plant cell is a cell of a plant, taken from a plant, or derived through culture from a cell taken from a plant.
  • plant cell includes without limitation cells within seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, shoots, gametophytes, sporophytes, pollen, and microspores.
  • plant part refers to a part of a plant, including single cells and cell tissues such as plant cells that are intact in plants, cell clumps, and tissue cultures from which plants can be regenerated.
  • plant parts include, but are not limited to, single cells and tissues from pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems, shoots, and seeds; as well as scions, rootstocks, protoplasts, calli, and the like.
  • population means a genetically heterogeneous collection of plants sharing a common genetic derivation.
  • primer refers to an oligonucleotide which is capable of annealing to a nucleic acid target (in some embodiments, annealing specifically to a nucleic acid target) allowing a DNA polymerase and/or reverse transcriptase to attach thereto, thereby serving as a point of initiation of DNA synthesis when placed under conditions in which synthesis of a primer extension product is induced (e.g., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH).
  • one or more pluralities of primers are employed to amplify plant nucleic acids (e.g., using the polymerase chain reaction; PCR).
  • progeny and “progeny plant” refer to a plant generated from a vegetative or sexual reproduction from one or more parent plants.
  • haploid induction the seed on the female parent is haploid, thus not a progeny of the inducing haploid line.
  • the progeny of the haploid seed is not the only desired progeny.
  • An Fl is a first-generation progeny produced from parents at least one of which is used for the first time as donor of a trait, while progeny of second generation (F2) or subsequent generations (F3, F4, and the like) are specimens produced from selfings, intercrosses, backcrosses, and/or other crosses of FIs, F2s, and the like.
  • An Fl can thus be (and in some embodiments is) a hybrid resulting from a cross between two true breeding parents (i.e., parents that are true-breeding are each homozygous for a trait of interest or an allele thereof), while an F2 can be (and in some embodiments is) a progeny resulting from self-pollination of the Fl hybrids.
  • Fl-H generation refers to progeny produced by first crossing an inbred containing the SCD trait with an inbred line absent the SCD trait (Fl). The progeny from said first cross was then crossed by a maternal haploid inducer line, producing the Fl-H generation.
  • F2-H generation refers to progeny produced by said Fl plants above selfing to produce F2, which was then crossed by a haploid inducer.
  • QTL penetrance refers to the presence of an associated phenotype with a QTL locus.
  • quantitative trait locus or “quantitative trait loci” or “QTL” refers to a locus associated with a particular trait.
  • Rl-nj refers to the R1 -Navajo anthocyanin marker. It is a marker used as a visual marker for distinguishing haploids from diploids (or aneuploids).
  • recombination refers to an exchange of DNA fragments between two DNA molecules or chromatids of paired chromosomes (a “crossover”) over in a region of similar or identical nucleotide sequences.
  • a “recombination event” is herein understood to refer in some embodiments to a meiotic crossover.
  • reference sequence refers to a defined nucleotide sequence used as a basis for nucleotide sequence comparison.
  • the term “regenerate,” and grammatical variants thereof, refers to the production of a plant from tissue culture.
  • SCD SUBSTITUTE SHEET
  • spontaneously doubled haploid plant refers to a plant whose florets have undergone spontaneous doubling. Other tissues of a spontaneously doubled haploid plant may retain their haploid state (e.g., root, leaf, stem).
  • stringent hybridization conditions refers to conditions under which a polynucleotide hybridizes to its target subsequence, typically in a complex mixture of nucleic acids, but to essentially no other sequences. Stringent conditions are sequencedependent and can be different under different circumstances.
  • Tm thermal melting point
  • Exemplary stringent conditions are 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 can also be achieved with the addition of destabilizing agents such as formamide.
  • Additional exemplary stringent hybridization conditions include 50% formamide, 5x SSC, and 1 % SDS incubating at 42°C; or SSC, 1 % SDS, incubating at 65°C; with one or more washes 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 can vary between about 32°C and 48°C (or higher) depending on primer length. Additional guidelines for determining hybridization parameters are provided in numerous references (see e.g., Ausubel et al., 1999).
  • the term “trait” refers to a phenotype of interest, a gene that contributes to a phenotype of interest, as well as a nucleic acid sequence associated with a gene that contributes to a phenotype of interest.
  • transgene refers to a nucleic acid molecule introduced into an organism or one or more of its ancestors by some form of artificial transfer technique.
  • the artificial transfer technique thus creates a “transgenic organism” or a “transgenic cell.” It is understood that the artificial transfer technique can occur in an ancestor organism (or a cell therein and/or that can develop into the ancestor organism) and yet any progeny individual that has the artificially transferred nucleic acid molecule or a fragment thereof is still considered transgenic even if one or more natural and/or assisted breedings result in the artificially transferred nucleic acid molecule being present in the progeny individual.
  • targeted mutagenesis or “mutagenesis strategy” refers to any method of mutagenesis that results in the intentional mutagenesis of a chosen gene.
  • Targeted mutagenesis includes the methods CRISPR, TILLING, TALEN, and other methods not yet discovered but which may be used to achieve the same outcome.
  • haploid induction rate means the number of surviving haploid kernels over the total number of kernels after an ear is pollinated with haploid inducer pollen (a percent of total progeny per good pollination).
  • a maize plant comprising a QTL.
  • the QTL comprises SEQ ID NO: 5 and SEQ ID NO: 10 and may be associated with increased haploid production.
  • the maize plant is a haploid inducer maize plant.
  • the haploid inducer maize plant may further comprise inducer allele ZmMATL.
  • the haploid inducer maize plant may further comprise inducer allele ZmDMP.
  • the haploid inducer maize plant may further comprise inducer alleles ZmMATL and ZmDMP.
  • the haploid inducer maize plant may further comprise a marker gene, the marker gene may be a color marker, and the color marker gene is an anthocyanin marker gene.
  • the anthocyanin marker gene is selected from the group consisting of R1-SCM2 and Rl-nj.
  • the haploid inducer maize plant may further comprise a R1-SCM2 color marker gene.
  • the maize plant comprising the QTL may be an elite line. The QTL of the
  • SUBSTITUTE SHEET (RULE 26) maize plant is located on chromosome 5 between positions 129,909,257 and 155,088,562 using coordinates from B73-REFERENCE-GRAMENE-4.0.
  • the method comprises obtaining a first plant comprising a SYN Chr5 QTL comprising SEQ ID NO: 5 and SEQ ID NO: 10 that is associated with increased haploid production.
  • the first plant is crossed with a second plant and progeny from said cross is obtained follow by selection of progeny containing the SYN Chr5 QTL, thereby obtaining a plant with increased haploid production.
  • the plant with increased haploid production may be a maize plant and may also be a haploid inducer maize plant.
  • the haploid inducer maize plant may further comprise inducer allele ZmMATL.
  • the haploid inducer maize plant may further comprise inducer allele ZmDMP. In yet another embodiment, the haploid inducer maize plant may further comprise inducer alleles ZmMATL and ZmDMP.
  • the haploid inducer maize plant may further comprise a marker gene and said marker gene may is a color marker gene.
  • the color marker gene is an anthocyanin marker gene and is selected from the group consisting of R1-SCM2 and Rl-nj. In one embodiment, the haploid inducer maize plant further comprises a R1-SCM2 color marker gene. In an embodiment, the maize plant with increased haploid production is an elite line.
  • the maize plant with increased haploid induction comprises the SYN Chr5 QTL that is located on chromosome 5 between positions 129,909,257 and 155,088,562 using coordinates from B73- REFERENCE-GRAMENE-4.0.
  • a method of selecting a first maize plant or germplasm that displays increased haploid production wherein nucleic acids are isolated from the first maize plant or germplasm followed by detecting in the first maize plant or germplasm a SYN Chr5 QTL comprising SEQ ID NOs: 5 and 10 that is associated with increased haploid production and selecting said first maize plant or germplasm, or selecting a progeny of said first maize plant or germplasm, comprising SYN Chr5 QTL that is associated with increased haploid production.
  • the method further comprises crossing said selected first maize plant or germplasm with a second maize plant or germplasm, wherein the introgressed maize plant or germplasm displays increased haploid production.
  • the SYN Chr5 QTL is detected using a composition comprising a detectable label.
  • the first is a haploid inducer maize plant.
  • the haploid inducer maize plant may further comprise inducer allele ZmMATL or it may further comprise inducer allele ZmDMP.
  • the haploid inducer maize plant may further comprise inducer alleles ZmMATL and ZmDMP.
  • the haploid inducer maize plant may further comprise inducer alleles ZmMATL and ZmDMP.
  • haploid inducer maize plant of the method further comprises a marker gene and may be a color marker gene.
  • the color marker gene may be an anthocyanin marker gene and selected from the group consisting of R1-SCM2 and Rl-nj.
  • the color marker gene is a R1-SCM2 color marker gene.
  • the first maize plant displaying increased haploid production an elite line.
  • the SYN Chr5 QTL of the first maize plant displaying increased haploid production is located on chromosome 5 between positions 129,909,257 and 155,088,562 using coordinates from B73-REFERENCE- GRAMENE-4.0.
  • a detection kit comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.
  • DMP knockout leads to a low rate of HI [Zhong, Y., Liu, C., Qi, X , Jiao, Y., Wang, D., Wang, Y. et al. (2019) Mutation of ZmDMP enhances haploid induction in maize. Nature Plants 5 575-580; Zhong, Y., Chen, B., Li, M., Wang, D., Jiao, Y., Qi, X., et al. (2020) A DMP-triggered in vivo maternal haploid induction system in the dicotyledonous Arabidopsis.
  • the R-navajo [Chaikam, V., Nair, S.K., Babu, R., Martinez, L., Tejomurtula, J., and Boddupalli, P.M. (2015) Analysis of effectiveness of Rl-nj anthocyanin marker for in vivo haploid identification in maize and molecular markers for predicting the inhibition of Rl-nj expression.
  • Theor Appl Genet 128, 159-171] allele is associated with strong anthocyanin accumulation in the aleurone layer and weak accumulation in embryos.
  • the R1 -scutellum (R1-SCM2) allele is associated with strong anthocyanin accumulation in the embryo and aleurone.
  • R1 is between -139 Mb to -140 Mb on chromosome 10 in B73-REFERENCE- GRAMENE-4.0.
  • SUBSTITUTE SHEET (RULE 26) including the dominant Cl-I (inhibitor) allele of colored aleuronel (Zm00001eb373660) on chromosome 9 starting at position 11,117,066 in B73-REFERENCE-GRAMENE-4.0.
  • HIR HIR is typically measured as a percent of total progeny per good pollination, where a good pollination is an ear with greater than 80 kernels.
  • haploid induction phenotyping was performed, the segregating inducer plants were test crossed (i.e. , used as male pollen donors and crossed onto female tester ears) to assess their relative HIRs. Lines to be tested for HIR were planted in quadruplicates, with intervening tester rows.
  • the HIR was determined by isolating embryos from test crossed ears at 15 to 20 days after pollination into embryo rescue media (4.43 grams of Murashige and Skoog basal media with vitamins, 30 grams of sucrose, and 70 milligrams of salicylic acid). Petri dishes were exposed to light, and the purple (diploid) and cream-colored (haploid) embryos were counted.
  • HIR of lines fixed for all HI tested alleles ranged from 10-19% HIR, like the control (RWKS/Z21S//RWKS). Lines without matl and qhir8 fixed show a lower HIR.
  • the phenotypic data was then aligned with SNP markers genotyped at
  • SUBSTITUTE SHEET (RULE 26) thousands of informative SNPs in the genome using an Axiom SNP array for the purpose of identifying the candidate QTL that may be associated with increased HIR.
  • this associated analysis identified a QTL on chromosome 5 (between 60 - 144 cM) that conferred between a two to four percent increase in the number of haploids per ear (Table 1).
  • the phenotypic traits tracked as proxies for haploid induction were the percent haploids per ear (%Hap), number of haploids per ear (#Hap/Ear), and total seeds per ear (#Seeds/Ear) all of which can increase the total haploid induction rate.
  • SUBSTITUTE SHEET (RULE 26) in breeding populations because there was an insufficient number of SNP markers in the identified region on chromosome 5.
  • additional markers were added to the genotyping panel to increase the density around the chromosome 5 QTL and increase the resolution by which its boundaries could be defined (Table 2).
  • a mapping population was selected by selfing the parents of the F3 generation that were fixed for all other known haploid-induction-related QTL and were heterozygous for the chromosome 5 QTL according to flanking markers from the F3 generation.
  • the haploid induction rate was determined from a total of 209 well-pollinated ears. With this data, the QTL was narrowed to between 129,909,257 and 155,088,562 on chromosome 5 (coordinates using B73- REFERENCE-GRAMENE-4.0, which is publicly available at MaizeGDB.org). Individuals fixed for the favorable inducer allele (A Line in Tables 3a and 3b) had an average HIR of 21.6% compared to those with the unfavorable allele (B Line in Tables 3a and 3b) where the average HIR was 17.9%, for an average difference of 3.7%.
  • Table 3a Fine-mapping data from all inducer genotypes represented in the F4 studies of lines derived from SYN-INB75Z x PHI-1 and SYN-INB86Z x PHI-1 crosses. At each marker, the SNP is called as A Line (favorable HIR parent), B Line (unfavorable HIR parent), heterozygous (Het), or no data (ND).
  • Table 3b Fine-mapping data from all inducer genotypes represented in the F4 studies of lines derived from SYN-INB75Z x PHI-1 and SYN-INB86Z x PHI-1 crosses. At each marker, the SNP is called as A Line (favorable HIR parent), B Line (unfavorable HIR parent), heterozygous (Het), or no data (ND).
  • SUBSTITUTE SHEET (RULE 26) Zhong, Y., Chen, B., Li, M., Wang, D., Jiao, Y., Qi, X., et al. (2020) A DMP-triggered in vivo maternal haploid induction system in the dicotyledonous Arabidopsis. Nat Plants 6, 466-472

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Abstract

L'invention concerne une plante de maïs comprenant un QTL, le QTL comprenant SEQ ID No : 5 et SEQ ID No : 10, et le QTL étant associé à une production d'haploïdes accrue, et ainsi la plante de maïs ayant une production d'haploïdes accrue. L'invention concerne également divers procédés. Les procédés consistent à obtenir une plante ayant une production d'haploïdes accrue et un procédé de sélection d'une première plante de maïs ou d'un germoplasme qui affiche une production d'haploïdes accrue.
PCT/US2024/045960 2023-09-21 2024-09-10 Augmentation de l'induction haploïde Pending WO2025064264A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070192889A1 (en) * 1999-05-06 2007-08-16 La Rosa Thomas J Nucleic acid molecules and other molecules associated with transcription in plants and uses thereof for plant improvement
WO2023164393A2 (fr) * 2022-02-23 2023-08-31 Syngenta Crop Protection Ag Marqueurs associés à un doublement de chromosome spontané

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20070192889A1 (en) * 1999-05-06 2007-08-16 La Rosa Thomas J Nucleic acid molecules and other molecules associated with transcription in plants and uses thereof for plant improvement
WO2023164393A2 (fr) * 2022-02-23 2023-08-31 Syngenta Crop Protection Ag Marqueurs associés à un doublement de chromosome spontané

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