Increasing haploid induction

Abstract

Provided herein is a maize plant comprising a QTL wherein the QTL comprises SEQ ID NO: 5 and SEQ ID NO: 10, and wherein the QTL is associated with an increased haploid production, and whereby the maize plant has an increased haploid production. Various methods are also provided. Methods include obtaining a plant with increased haploid production, and selecting a first maize plant or germplasm exhibiting increased haploid production.

Description

Technical Field

[0001] The subject matter disclosed in this application generally relates to breeding and haploid induction. sequence list

[0002] This application is accompanied by a sequence list named 83041_ST26.mxl, created on 7 September 2023, which is approximately 19 kilobytes in size. This sequence list is incorporated herein by reference in its entirety. The sequence list is filed with this application via EFS-Web and is compliant with 37 CFR § 1.824(a)(2)-(6) and (b). Background Technology

[0003] Due to the increasing importance of double haploids for accelerated breeding in maize and other species, QTL mapping associated with enhanced haploid induction in maize is a region of ongoing interest. In maize, most haploid induction is initiated by crossing with lines known to contain the inducing line genotype. However, for decades, the maize breeding community has not identified genes that contribute to haploid induction. Preliminary studies identifying factors associated with haploid induction identified a QTL on chromosome 1 required for haploid induction in maize (Prigge, Vanessa, Xiaowei Xu, Liang Li, Raman Babu, Shaojiang Chen, Gary N. Atlin, and Albrecht E. Melchinger. 2012. “New Insights into the Genetics of in Vivo Induction of Maternal Haploids, the Backbone of Double Haploid Technology in Maize.” Genetics 190 (2): 781–93; Hu, Haixiao, Tobias A. Schrag, Regina Peis, Sandra Unterseer, Wolfgang Schipprack, Shaojiang Chen, Jinsheng Lai, et al. 2016. “The Genetic Basis of Haploid Induction in Maize Identified with a Novel Genome-Wide Association Method.” [Genetic Basis of Haploid Induction in Maize Identified Using Novel Genome-Wide Association Approach]"Genetics [Genetics] 202 (4): 1267–76. https: / / doi.org / 10.1534 / genetics.115.184234.) and several secondary QTLs that could further enhance haploid induction rates, including a QTL on chromosome 9. The chromosome 1 and chromosome 9 QTLs were subsequently identified as the genes MATRILINEAL (MATL) and ZmDMP, respectively (Kelliher et al. 2017, Zhong et al. 2019). Other secondary QTLs that may contribute to further improvements in haploid induction rates when used in combination with MATL and DMP remain relatively unexplored beyond these preliminary mapping studies.Therefore, more precise genomic mapping and testing of these secondary QTLs are needed in a wider range of germplasms to enable selective breeding of improved haploid inducible lines. Summary of the Invention

[0004] Due to the importance of double haploid plants for accelerated breeding, the demand for generating or obtaining haploid inducible lines continues to grow. Consequently, interest and work on locating QTLs associated with haploid induction are also increasing. To address this interest, this paper discloses a maize plant containing a QTL on chromosome 5 (SYN Chr5 QTL). The QTL on chromosome 5 is associated with increased haploid induction. This paper also provides a method for obtaining plants with increased haploid production by obtaining a first plant containing the SYN Chr5 QTL and crossing it with a second plant, subsequently obtaining progeny from the cross containing the SYN Chr5 QTL, thereby obtaining plants with increased haploid production. Furthermore, this paper provides a method for selecting a first corn plant or germplasm exhibiting increased haploid production, wherein nucleic acids are isolated from the first corn plant or germplasm, followed by detection of SYNChr5 QTLs associated with increased haploid production in the first corn plant or germplasm, and selection of the first corn plant or germplasm, or selection of its progeny, wherein the first corn plant or germplasm or its progeny contains SYNChr5 QTLs associated with increased haploid production. Additionally, the method includes crossing the selected first corn plant or germplasm with a second corn plant or germplasm, wherein the introgressed corn plant or germplasm exhibits increased haploid production. A brief description of sequences in a sequence list.

[0005] SEQ ID NO: 1 is the nucleotide sequence of the forward primer used to amplify the marker SM10934.

[0006] SEQ ID NO: 2 is the nucleotide sequence of the forward primer used to amplify the marker SM10934.

[0007] SEQ ID NO: 3 is the nucleotide sequence of the forward primer used to amplify the marker SM10934.

[0008] SEQ ID NO: 4 is the nucleotide sequence of the reverse primer used to amplify the marker SM10934.

[0009] SEQ ID NO: 5 is the nucleotide sequence of the SM10934-tagged target sequence.

[0010] SEQ ID NO: 6 is the nucleotide sequence of the forward primer used to amplify the marker SM10932.

[0011] SEQ ID NO: 7 is the nucleotide sequence of the forward primer used to amplify the marker SM10932.

[0012] SEQ ID NO: 8 is the nucleotide sequence of the forward primer used to amplify the marker SM10932.

[0013] SEQ ID NO: 9 is the nucleotide sequence of the reverse primer used to amplify the marker SM10932.

[0014] SEQ ID NO: 10 is the nucleotide sequence of the SM10932-tagged target sequence.

[0015] definition

[0016] While it is believed that the following terms will be well understood by those skilled in the art, the following definitions are set forth in order to explain the subject matter disclosed in this application.

[0017] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques used herein are intended to refer to techniques commonly understood in the art, including variations and / or equivalents of those techniques that are readily apparent to one of ordinary skill in the art. While it is believed that the following terms will be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate the interpretation of the subject matter disclosed in this application.

[0018] Under the long-standing Patent Law Convention, the terms “a,” “an,” and “the” used in this application (including the claims) mean “one or more.” For example, the phrase “a cell” refers to one or more cells, and in some embodiments may refer to tissues and / or organs. Similarly, the phrase “at least one” when used herein to refer to an entity means, 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 that entity, including but not limited to all integer values ​​between 1 and 100 and integers greater than 100.

[0019] Unless otherwise indicated, all figures representing amounts of components, reaction conditions, etc., used in this specification and claims should be understood to be modified in all cases by the term "about". As used herein, the term "about", when referring to a measurable value such as mass, weight, time, volume, concentration, or percentage, means to cover variations of ±20% from a specified amount in some embodiments, ±10% in some embodiments, ±5% in some embodiments, ±1% in some embodiments, ±0.5% in some embodiments, and ±0.1% in some embodiments, because such variations are suitable for performing the disclosed methods and / or using the disclosed compositions, nucleic acids, peptides, etc. Therefore, unless indicated to the contrary, the numerical parameters listed in this specification and the appended claims are approximate values ​​that may vary depending on the desired characteristics sought to be obtained from the subject matter of this disclosure.

[0020] As used herein, the term "allelic gene" refers to a variant or alternative sequence form at a genetic locus. In diploid organisms, a single allele at each locus is inherited by offspring from each parent, respectively. While those skilled in the art will understand that an allele in any particular individual does not necessarily represent all alleles present in that species, the two alleles at a given locus in a diploid organism occupy corresponding positions on a pair of homologous chromosomes.

[0021] As used herein, the terms “amplified” or “amplified” refer to the construction of multiple copies of a nucleic acid molecule or multiple copies complementary to that nucleic acid molecule using at least one nucleic acid molecule as a template. Amplification systems include polymerase chain reaction (PCR) systems, ligase chain reaction (LCR) systems, nucleic acid sequence-based amplification (NASBA, Cangene, Mississauga, Ontario), Q-β replicase systems, transcription-based amplification (TAS) systems, and strand displacement amplification (SDA). See, for example, Diagnostic Molecular Microbiology: Principles and Applications, eds. PERSING et al., American Society for Microbiology, Washington, DC, (1993). The product of amplification is called an “amplifier”.

[0022] As used herein, the term “and / or” when used in the context of enumerating entities refers to entities existing individually or in combination. Thus, for example, the phrase “A, B, C and / or D” includes A, B, C, and D individually, but also any and all combinations and sub-combinations of A, B, C, and D (e.g., AB, AC, AD, BC, BD, CD, ABC, ABD, and BCD). In some embodiments, one or more elements referred to by “and / or” may also exist individually in one or more occurrences in one or more combinations and / or one or more sub-combinations.

[0023] As used herein, the phrase “associated with” refers to an identifiable and / or measurable relationship between two entities. For example, a marker is “associated with” a trait when it is linked to the trait and when the presence of the marker indicates whether and / or to what extent the desired trait or trait form will occur in the plant / germstone containing the marker. Similarly, a marker is “associated with” an allele when it is linked to the allele and when the presence of the marker indicates the presence of the allele in the plant / germstone containing the marker.

[0024] As used herein, the term "backcross" within the scope of this invention should be understood as a method of repeatedly crossing a hybrid offspring with one of its parents.

[0025] As used in this article, the term "cDNA" refers to single-stranded or double-stranded DNA that is complementary to and derived from mRNA.

[0026] As used herein, the term "chromosome" as is generally accepted in the art refers to a self-replicating gene structure in the cell nucleus containing cellular DNA and having a linear gene array.

[0027] The term "comprising" is synonymous with "including," "containing," and "characterized by," and is inclusive or open-ended, and does not exclude additional unlisted elements and / or method steps. "Comprising" means that the specified elements and / or steps exist, but other elements and / or steps may be added and still fall within the scope of the relevant subject matter.

[0028] As used herein, the phrase “composes of” excludes any element, step, or component not specifically listed. When the phrase “composes of” appears in a clause of the body of a claim, rather than directly following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.

[0029] As used herein, the phrase “consistently of…” limits the scope of the relevant disclosure or claim to the specified materials and / or steps, plus those that do not substantially affect one or more basic and novel features of the disclosed and / or claimed subject matter.

[0030] Regarding the terms “comprising,” “substantially consisting of,” and “consisting of,” when one of these three terms is used herein, the subject matter disclosed and claimed in this application may include the use of any of the other two terms in some embodiments. For example, if the subject matter in some embodiments relates to a nucleic acid encoding a polypeptide comprising at least 95% of the same amino acid sequence as SEQ ID NO: 2 or 3. It should be understood that the disclosed subject matter therefore also covers nucleic acids encoding a polypeptide comprising at least 95% of the same amino acid sequence as SEQ ID NO: 2 or 3 in some embodiments, and nucleic acids encoding a polypeptide comprising at least 95% of the same amino acid sequence as SEQ ID NO: 2 or 3 in some embodiments. Similarly, it should also be understood that in some embodiments, the method for the disclosed subject matter includes the steps disclosed herein, in some embodiments, the method for the subject matter disclosed herein consists substantially of the disclosed steps, and in some embodiments, the method for the subject matter disclosed herein consists of the steps disclosed herein.

[0031] As used herein, the term "de novo haploid induction" refers to the induction of haploidy by introducing a spontaneous haploid inducer. Such introduction can be achieved by local spraying, manual pollination, mutagenesis, or transgenic methods. Throughout this specification, the terms "de novo haploid induction," "de novo HI," and "haploid induction de novo" are used interchangeably.

[0032] As used herein, the term "superior line" or "inbred line" refers to any line resulting from breeding and selection aimed at achieving superior agronomic performance. A superior line has stable genetics, meaning it is rationally or nearly isogenetic in its genome. In other words, a superior line is rationally or nearly homozygous for all alleles in its genome.

[0033] As used herein, the term “superior inducible line” refers to a plant bred as a high-performance haploid inducible line. For example, in this article, a superior inducible line may contain a haploid induction rate (HIR) greater than 15% and mutations in the MATL gene, mutations in the ZmDMP gene, a dominant locus on the R1 gene for color selection or other markers for haploid selection, and / or additional QTLs associated with increased haploid induction rate or other traits favorable to haploid inducible line varieties.

[0034] As used herein, when used in conjunction with a reference polynucleotide (such as a plant gene, an ORF or a portion thereof, or a transgene), the term "expression" refers to a method of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) via the "transcription" of the gene (i.e., via the enzymatic action of an RNA polymerase) and, where applicable (e.g., if the gene encodes a protein), into a protein via the "translation" of the mRNA. Gene expression can be regulated at many stages of this method. For example, in the case of an antisense construct or a dsRNA construct, expression may refer only to the transcription of antisense RNA or only to the transcription of dsRNA. In the embodiments, "expression" refers to the transcription and stable accumulation of sense (mRNA) or functional RNA. "Expression" can also refer to the production of proteins.

[0035] As used herein, the term "gene" refers to a genetic unit consisting of a DNA sequence that occupies a specific location on a chromosome and contains the genetic instructions for a specific characteristic or trait in an organism.

[0036] As used herein, the term "genotype" refers to the genetic makeup of a cell or organism. An individual's "genotype of a set of genetic markers" includes specific alleles at one or more genetic marker loci present in the individual. As is known in the art, a genotype can involve a single locus or multiple loci, whether these loci are related or unrelated, and / or linked or non-linked. In some embodiments, an individual's genotype involves one or more related genes because one or more of these genes are involved in the expression of a desired phenotype (e.g., a quantitative trait as defined herein). Thus, in some embodiments, a genotype includes the sum of one or more alleles present at one or more genetic loci of an individual's intrinsic quantitative trait. In some embodiments, the genotype is represented by a haplotype (defined below).

[0037] "Genetic mapping" is a description of the genetic linkage between loci on one or more chromosomes within a given species, usually depicted in diagram or tabular form.

[0038] As used herein, the term "germstone" refers to the totality of genotypes of a population or another group of individuals (e.g., a species). The term "germstone" can also refer to plant material; for example, a group of plants that serve as repositories of different alleles. The phrase "adapted germplasm" refers to plant material that has been shown to possess genetic advantages; for example, for a given environment or geographic region. The phrases "unadapted germplasm," "primitive germplasm," and "exotic germplasm" refer to plant material whose genetic value is unknown or unproven; for example, for a given environment or geographic region. Thus, the phrase "unadapted germplasm" in some embodiments refers to plant material that is not part of an established breeding population and has no known relationship to members of an established breeding population.

[0039] As used herein, a plant referred to as a “haploid” has a single set of chromosomes (genome), and the reduced number of chromosomes (1n) in a haploid plant is equal to the number of chromosomes in its gametes. As used herein, a plant referred to as a “double haploid” is developed by doubling the haploid chromosome set (from 1n to 2n). Plants or seeds obtained from double haploid plants that have been crossed with themselves (self-pollinated) to any number of generations can still be identified as double haploid plants. Double haploid plants are considered homozygous plants. If a plant is fertile, it is considered double haploid even if the entire vegetative part of the plant is not composed of cells with doubled chromosome sets; that is, if a plant contains viable gametes, it will be considered double haploid even if it is chimeric.

[0040] As used herein, when referring to the use of genes or nucleic acids, the term "heterologous" means that the gene encoding a factor is not in its natural environment (i.e., has been artificially altered). For example, heterologous genes can include genes introduced from one species into another. Heterologous genes can also include genes that are natural to an organism but have been altered in some way (e.g., through mutation, addition of multiple copies, ligation to a non-natural promoter or enhancer polynucleotide, etc.). Heterologous genes can further include plant gene polynucleotides comprising the cDNA form of a plant gene; the cDNA can be expressed in a sense direction (to produce mRNA) or an antisense direction (to produce an antisense RNA transcript complementary to the mRNA transcript). In one aspect of the invention, the difference between a heterologous gene and an endogenous plant gene is that the heterologous gene polynucleotide typically binds to a polynucleotide containing regulatory elements such as a promoter, and these polynucleotides are not found to be naturally associated with the gene encoding the protein of the heterologous gene or with plant gene polynucleotides in chromosomes, or these polynucleotides are associated with a portion of chromosome not found in nature (e.g., a gene expressed at a locus that does not normally express the gene). Furthermore, in embodiments, "heterologous" polynucleotides are polynucleotides not naturally associated with the host cell in which they are introduced, including multiple non-natural copies of naturally occurring polynucleotides.

[0041] As used in this article, the term "heterozygous" refers to the genetic condition that exists when different alleles are located at corresponding loci on homologous chromosomes.

[0042] As used in this article, the term "homozygous" refers to the genetic condition that exists when the same alleles are located at corresponding loci on homologous chromosomes.

[0043] As used in this article, the “HMF score” or “haploid male fertility score” refers to the number of days a plant sheds pollen. The method used in this article measures each individual plant. During the pollen shedding period (7–14 days in the field), the shedding of fertile pollen from the plant was checked daily, and each plant was assigned a number equal to the number of days it shed pollen (e.g., assigning plant number 5 equals 5 days of pollen shedding). Sterile plants were scored zero, meaning they shed pollen for zero days.

[0044] As used herein, when used in the context of nucleic acid molecules or polynucleotides of the present invention, the term "isolated" refers to a polynucleotide that is identified and isolated / separated in the context of a chromosomal polynucleotide in the corresponding source organism. If an isolated nucleic acid or polynucleotide does indeed have a naturally occurring counterpart, then the isolated nucleic acid or polynucleotide is not the nucleic acid it is present in in its natural environment. In contrast, non-isolated nucleic acids are nucleic acids (such as DNA and RNA) that are found in their naturally occurring state. For example, a given polynucleotide (e.g., a gene) is found on a host cell chromosome near an adjacent gene. Isolated nucleic acid molecules can exist in single-stranded or double-stranded form. Alternatively, they can contain a sense strand and an antisense strand (i.e., the nucleic acid molecule can be double-stranded). In preferred embodiments, the nucleic acid molecules of the present invention are understood to be isolated.

[0045] As used in this article, the term “locus” refers to a position on a chromosome in a given species (e.g., the position of a gene, genetic marker, etc.).

[0046] As used herein, a “maternal haploid inducing line” refers to a line that produces pollen and, when used as a male in hybridization, causes the development of the gynogenetic nucleus in haploid seeds. A “paternal haploid inducing line” refers to a line that, when used as a female in hybridization, causes the development of the androgenetic nucleus in haploid seeds. Haploid inducing line plants can derive haploids using either of these maternal or paternal mechanisms, and when no specific mechanism for a particular line is specified, they can be collectively referred to as “inducing lines”.

[0047] As used herein, the term "human-induced mutation" refers to any mutation resulting from direct or indirect human intervention. This term includes, but is not limited to, mutations obtained through any targeted mutagenesis method.

[0048] As used in this article, the term "hybrid" refers to the offspring produced by crossing two genetically different parent plants. The offspring obtained from this hybridization are a "biparental" population.

[0049] As used in this article, "log of dominance" or "LOD score" refers to the statistical probability that two genes are close on a chromosome.

[0050] As used herein, the terms "labeled 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, such as a nucleic acid probe that is fully or partially complementary to a label or labeled locus through nucleic acid hybridization. Labeled probes containing approximately 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more consecutive nucleotides can be used for nucleic acid hybridization.

[0051] As used herein, when identifying the presence / absence of an SCD or other target locus, the term "molecular marker" can be used to refer to a genetic marker as defined above, or its coding product (e.g., a protein) used as a reference point. Molecular markers can be derived from genomic nucleotide sequences or expressed nucleotide sequences (e.g., derived from RNA, cDNA, etc.). The term also refers to nucleotide sequences complementary to or flanking the marker sequence, such as nucleotide sequences used as probes and / or primers capable of amplifying the marker sequence. These nucleotide sequences are "complementary" (e.g., according to the Watson-Crick base pairing principle) when they specifically hybridize in solution. The term also refers to genetic markers indicating traits by the absence of nucleotide sequences complementary to or flanking the marker sequence (such as nucleotide sequences used as probes and / or primers capable of amplifying the marker sequence).

[0052] As used herein, the terms “nucleotide sequence,” “polynucleotide,” “nucleic acid sequence,” “nucleic acid molecule,” and “nucleic acid fragment” refer to a single-stranded or double-stranded polymer of RNA or DNA, optionally containing synthetic, non-natural, and / or modified nucleotide bases. A “nucleotide” is a monomeric unit from which a DNA or RNA polymer is constructed and consists of a purine or pyrimidine base, a pentose sugar, and a phosphate group. Nucleotides (usually found in their 5'-monophosphate form) are designated by their single-letter names as follows: “A” for adenosine or deoxyadenosine (for RNA or DNA, respectively), “C” for cytidine or deoxycytidine, “G” for guanylic acid or deoxyguanylic acid, “U” for uridine, “T” for deoxythymidine, “R” for purine (A or G), “Y” for pyrimidine (C or T), “K” for G or T, “H” for A, C, or T, “I” for inosine, and “N” for any nucleotide.

[0053] In the case of two nucleic acid or amino acid sequences, the term "identity" or "identical" refers to the percentage of identical nucleotides or amino acids in the linear polynucleotide or amino acid sequence of the reference ("query") sequence (or its complementary strand) when the two sequences are fully compared, such as when compared with the test ("test") sequence. Unless otherwise stated, sequence identity as used herein refers to values ​​obtained using the Needleman and Wunsch algorithm ((1970) J. Mol. Biol. [Molecular Biology Journal] 48:443-453) implemented in the EMBOSS Needle alignment tool, using the default matrix file EBLOSUM62 (for proteins) and default parameters (vacancy open = 10, vacancy extension = 0.5, terminal vacancy penalty = false, terminal vacancy open = 10, terminal vacancy extension = 0.5) or DNAfull (for nucleic acids) and default parameters (vacancy open = 10, vacancy extension = 0.5, terminal vacancy penalty = false, terminal vacancy open = 10, terminal vacancy extension = 0.5); or any equivalent procedure thereof. The EMBOSS Needle is available, for example, from EMBL-EBI, at ebi.ac.uk / Tools / psa / emboss_needle / and as described in the publication: “The EMBL-EBI search and sequence analysis tools APIs in 2019.” Madeira et al., Nucleic Acids Research, June 2019, 47(W1):W636-W641. As used herein, the term “equivalent procedure” refers to any sequence comparison procedure that, for any two sequences in question, produces alignments with the same nucleotide or amino acid residue matches and the same percentage of sequence identity when compared to the corresponding alignments generated by the EMBOSS Needle. In some embodiments, substantially identical nucleic acid or amino acid sequences may function substantially identically.

[0054] As used herein, the terms “introgression,” “introgressed,” and “introgressing” refer to both natural and artificial processes in which a genomic region of one species, variety, or cultivar is transferred to the genome of another species, variety, or cultivar by hybridization. This process may optionally be accomplished through backcrossing with a recurrent parent.

[0055] The term "open reading frame" (ORF) refers to a nucleic acid sequence encoding a polypeptide. In some embodiments, the ORF comprises a translation start codon, a translation termination (i.e., stop) codon, and the nucleic acid sequence encoding amino acids present in the polypeptide therein. The terms "start codon" and "stop codon" refer to a unit of three adjacent nucleotides (i.e., codons) in the coding sequence, which correspondingly indicate the initiation and termination of protein synthesis (mRNA translation).

[0056] As used herein, the terms “phenotype,” “phenotypic trait,” or “trait” refer to one or more characteristics of a plant or plant cell. A phenotype can be observed with the naked eye or by any other assessment method known in the art (e.g., microscopy, biochemical analysis, or electromechanical assay). In some cases, a phenotype is directly controlled by a single gene or genetic locus (i.e., corresponding to a “monogenous trait”). For example, in the case of haploid plants evaluating SCD, the phenotype may refer to shed fertile pollen and / or seeds obtained via pollination with that pollen. In other cases, a phenotype is the result of interactions between several genes, and in some embodiments, it is also caused by interactions between the plant and / or plant cells and their environment.

[0057] As used herein, the term "plant" can refer to the whole plant, any part thereof, or a cell or tissue culture derived from a plant. Therefore, unless otherwise stated, the term "plant" can refer to any of the following: the whole plant, plant components or organs (e.g., leaves, stems, roots, etc.), plant tissues, seeds, and / or plant cells.

[0058] Plant cells are plant cells obtained from plants or derived from cells taken from plants through culture. Therefore, the term "plant cell" includes, but is not limited to, cells within seeds, suspension cultures, embryos, meristematic zones, callus, leaves, buds, gametophytes, sporophytes, pollen, and microspores. The phrase "plant part" refers to a portion of a plant, including single cells and cellular tissues (such as intact plant cells), cell masses, and tissue cultures that can regenerate plants. Examples of plant parts include, but are not limited to, single cells and tissues derived from: pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems, buds, and seeds; as well as scions, rhizomes, protoplasts, callus, etc.

[0059] As used in this article, the term "population" refers to a genetically heterogeneous collection of plants that share a common genetic derivation.

[0060] As used herein, the term "primer" refers to an oligonucleotide that, when placed under conditions inducing primer extension synthesis (e.g., in the presence of nucleotides and reagents for polymerization, such as DNA polymerase, and at suitable temperature and pH), is capable of annealing to a nucleic acid target (specifically annealing to a nucleic acid target in some embodiments) to allow DNA polymerase and / or reverse transcriptase to attach thereto, thereby serving as a starting point for DNA synthesis. In some embodiments, one or more primers are used to amplify plant nucleic acids (e.g., using polymerase chain reaction; PCR).

[0061] As used herein, the term "probe" refers to a nucleic acid (e.g., a single-stranded nucleic acid or a strand of a double-stranded or higher-order nucleic acid, or a subsequence thereof) that can form a hydrogen-bonded duplex with a complementary sequence in a target nucleic acid sequence. Typically, probes are of sufficient length to form stable and sequence-specific duplex molecules with their complementary sequences, and this can be used in some embodiments to detect a target sequence present in multiple nucleic acids.

[0062] As used herein, the terms “offspring” and “offspring plants” refer to plants produced from one or more parent plants through asexual or sexual reproduction. In haploid induction, the seeds on the maternal parent are haploid and therefore not offspring of the induced haploid line. Offspring from haploid seeds are not the only desired offspring. There are also HI seeds and subsequent plants and seed offspring from haploid-induced plants. Both haploid seeds and HI seeds can be offspring. Offspring plants can be obtained by cloning a single parent plant, self-pollinating a single parent plant, or by crossing two or more parents. For example, offspring plants can be obtained by cloning or self-pollinating a single parent plant or by crossing two parents, and include self-pollinators and F1 or F2 generations or even further back. F1 is the first generation of offspring generated from two parents (at least one of the two parents is used as a donor for the trait for the first time), while the second generation (F2) or subsequent generations (F3, F4, etc.) are samples generated from self-crossing, intercrossing, backcrossing, and / or other hybridizations of F1, F2, etc. Therefore, F1 can be (and in some embodiments is) a hybrid produced from crossing two purebred parents (i.e., purebred parents are homozygous for the target trait or their alleles), while F2 can be (and in some embodiments is) an offspring produced from self-pollination of the F1 hybrid.

[0063] As used herein, "F1-H generation" refers to the offspring produced by first crossing an inbred line containing the SCD trait with an inbred line lacking the SCD trait (F1). The offspring from this first cross are then crossed with a haploid inducing line from the maternal parent to produce the F1-H generation. As used herein, "F2-H generation" refers to the offspring produced by self-pollinating the aforementioned F1 plants to produce F2, and then crossing them with a haploid inducing line.

[0064] As used in this article, "QTL penetrance" refers to the presence of a phenotype associated with a QTL locus.

[0065] As used in this article, “quantitative trait locus” or “quantitative trait loci” or “QTL” refers to a locus associated with a specific trait.

[0066] As used in this article, "R1-nj" refers to the R1-Navajo anthocyanin marker. It is a visual marker used to distinguish between haploids and diploids (or aneuploids).

[0067] As used herein, the phrase “recombination” refers to the exchange (“crossing-over”) of DNA fragments between two DNA molecules or chromatids on a paired chromosome in regions of similar or identical nucleotide sequences. “Recombination event” is understood herein to refer, in some embodiments, to meiotic crossing-over.

[0068] As used in this article, the term "reference sequence" refers to a defined nucleotide sequence used as the basis for nucleotide sequence comparison.

[0069] As used in this article, the term “regeneration” and its grammatical variations refer to the generation of plants from tissue cultures.

[0070] As used herein, “spontaneous chromosome doubling” (“SCD”), “spontaneous haploid genome doubling”, “haploid male fertility”, or “spontaneous genome doubling” are used interchangeably to describe the doubling of a haploid genome without any intervention. SCD allows for the proper reduction of chromosome meiosis and subsequent formation of mature pollen. In this disclosure, SCD is calculated by dividing the number of fertile haploid plants by the total number of plants.

[0071] As used in this article, a “spontaneous double haploid plant” is a plant whose florets have undergone spontaneous doubling. Other tissues of spontaneous double haploid plants may retain their haploid state (e.g., roots, leaves, stems).

[0072] As used in this article, the phrase "strict hybridization conditions" refers to the conditions under which polynucleotides typically hybridize with their target sequences (but essentially not with other sequences) in a complex mixture of nucleic acids. Strict conditions are sequence-dependent and can vary in different environments.

[0073] Typically, longer sequences hybridize specifically at higher temperatures. A comprehensive guide to nucleic acid hybridization can be found in Sambrook and Russell, 2001. Generally, for specific sequences at a defined ionic strength pH, ​​stringent conditions are chosen to be approximately 5°C to 10°C below the specific melting point (Tm). Tm is the temperature at which 50% of the probe complementary to the target hybridizes to the target sequence at equilibrium (in the presence of an excess of the target sequence, 50% of the probe is occupied at Tm at a given ionic strength, pH, and nucleic acid concentration). Exemplary stringent conditions include: a salt concentration of less than approximately 1.0 M sodium ions, typically approximately 0.01 to 1.0 M sodium ion concentration (or other salt) at pH 7.0 to 8.3, and a temperature of at least approximately 30°C for short probes (e.g., 10 to 50 nucleotides) and at least approximately 60°C for long probes (e.g., greater than 50 nucleotides).

[0074] Tight conditions can also be achieved by adding a destabilizing agent, such as formamide. Other exemplary tight hybridization conditions include incubation at 42°C with 50% formamide, 5x SSC, and 1% SDS; or incubation at 65°C with SSC and 1% SDS; followed by one or more washes at 65°C in 0.2x SSC and 0.1% SDS. For PCR, a temperature of approximately 36°C is typically used for low-tight amplification, but the annealing temperature can vary between approximately 32°C and 48°C (or higher) depending on the primer length. Further guidelines for determining hybridization parameters are provided in numerous references (see, for example, Ausubel et al., 1999).

[0075] As used in this article, the term "trait" refers to the desired phenotype, the gene that contributes to the desired phenotype, and the nucleic acid sequence associated with the gene that contributes to the desired phenotype.

[0076] As used herein, the term "transgenic" refers to a nucleic acid molecule introduced into an organism or one or more of its ancestors through some form of artificial transfer technique. Thus, artificial transfer techniques establish "transgenic organisms" or "transgenic cells." It should be understood that artificial transfer techniques can occur in ancestral organisms (or cells therein and / or cells that can develop into ancestral organisms), and any offspring individual possessing an artificially transferred nucleic acid molecule or fragment thereof is still considered transgenic, even if one or more natural and / or assisted breeding results in the presence of the artificially transferred nucleic acid molecule in the offspring individual.

[0077] As used herein, the term “targeted mutagenesis” or “mutation strategy” refers to any mutagenesis method that intentionally induces mutagenesis in a selected gene. Targeted mutagenesis includes CRISPR, TILLING, TALEN methods, and other methods that have not yet been discovered but can be used to achieve the same results.

[0078] As used in this article, haploid induction rate (“HIR”) means the number of haploid grains that survive after pollination of the ear with pollen from a haploid inducing line, divided by the total number of grains (the percentage of the total offspring that were successfully pollinated each time). Detailed Implementation

[0079] This document provides a corn plant containing a QTL. The QTL includes SEQ ID NO: 5 and SEQ ID NO: 10 and may be associated with increased haploid production. In one embodiment, the corn plant is a haploid induction line corn plant. In another embodiment, the haploid induction line corn plant may further include the induction line allele ZmMATL. In another embodiment, the haploid induction line corn plant may further include the induction line allele ZmDMP. In yet another embodiment, the haploid induction line corn plant may further include both the induction line alleles ZmMATL and ZmDMP. The haploid induction line corn plant may further include a marker gene, which 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 R1-nj. The haploid induction line corn plant may further include the R1-SCM2 color marker gene. Maize plants containing QTLs can be considered superior strains. Using coordinates from B73-REFERENCE-GRAMENE-4.0, the QTLs of maize plants are located on chromosome 5 between positions 129,909,257 and 155,088,562.

[0080] This document also provides a method for obtaining plants with increased haploid production. The method includes obtaining a first plant containing the SYN Chr5 QTL, which is associated with increased haploid production, comprising SEQ ID NO: 5 and SEQ ID NO: 10. The first plant is crossed with a second plant, and progeny from the cross are obtained. Progeny containing the SYN Chr5 QTL are then selected to obtain plants with increased haploid production. The plants with increased haploid production may be maize plants or haploid-inducing maize lines. In one embodiment, the haploid-inducing maize line may further contain the inducing line allele ZmMATL. In another embodiment, the haploid-inducing maize line may further contain the inducing line allele ZmDMP. In yet another embodiment, the haploid-inducing maize line may further contain both the inducing line alleles ZmMATL and ZmDMP. The haploid-inducing maize line may further contain a marker gene, and the marker gene may be a color marker gene. The color marker gene is an anthocyanin marker gene selected from the group consisting of R1-SCM2 and R1-nj. In one embodiment, the haploid-induced maize line further includes the R1-SCM2 color marker gene. In one embodiment, the haploid-inducible maize line is a superior line. The haploid-inducible maize line contains the SYN Chr5 QTL, which, using coordinates from B73-REFERENCE-GRAMENE-4.0, is located on chromosome 5 at positions 129,909,257 and 155,088,562.

[0081] In another embodiment of the invention, a method is provided for selecting a first maize plant or germplasm exhibiting increased haploid production, wherein nucleic acids are isolated from the first maize plant or germplasm, followed by detection of a SYN Chr5 QTL comprising SEQ ID NO: 5 and 10 in the first maize plant or germplasm associated with increased haploid production, and selection of the first maize plant or germplasm, or selection of progeny of the first maize plant or germplasm, wherein the first maize plant or germplasm or its progeny comprises a SYNChr5 QTL associated with increased haploid production. In another embodiment, the method further comprises crossing the selected first maize plant or germplasm with a second maize plant or germplasm, wherein the introgressed maize plant or germplasm exhibits increased haploid production. In one embodiment, a composition comprising a detectable marker is used to detect the SYN Chr5 QTL. In one embodiment, the first is a haploid-inducing maize line. The haploid inducible maize line may further include the inducible line allele ZmMATL, or it may further include the inducible line allele ZmDMP. In another embodiment, the haploid inducible maize line may further include both the inducible line alleles ZmMATL and ZmDMP. In one embodiment, the haploid inducible maize line of this method further includes a marker gene, which may be a color marker gene. The color marker gene may be anthocyanin marker genes selected from the group consisting of R1-SCM2 and R1-nj. In one embodiment, the color marker gene is the R1-SCM2 color marker gene. In one embodiment, the first maize plant exhibiting increased haploid production is a superior line. In another embodiment, using coordinates from B73-REFERENCE-GRAMENE-4.0, the SYN Chr5 QTL of the first maize plant exhibiting increased haploid production is located between positions 129,909,257 and 155,088,562 on chromosome 5. In a final embodiment, this document provides a test 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. Example

[0082] 1. Identify QTLs on chromosome 5 associated with increased haploid induction rates in multiple induction line populations.

[0083] Qhir1 (a QTL for haploid induction rate 1), qhir8, and R1 were used to identify haploid progeny [Panavas T., Weir J., Walker EL (1999). The structure and para-mutagenicity of the R-marbled haplotype of Zea mays. Genetics 153 979-991] and colored aleurone 1 (C1) [Paz-Ares, J., Ghosal, D., Saedler, H. Molecular analysis of the C1-I allele from Zea mays: a dominant mutant of the regulatory C1 locus. (1990) EMBO J. 9 315–321] Anthocyanin pathway genes exhibit penetrance across germplasm.

[0084] Because haploid induction has become a routine step in the corn breeding process for many large agricultural companies, the breeding of superior haploid induction lines with increased haploid induction rates and other advantageous traits has become crucial for the production of double haploids. Superior maize haploid induction lines are homozygous for matl, which is caused by the loss of a functional allele due to a 4 bp insertion in the fourth exon of patatin-like phospholipase A2α (referred to as MATRILINEAL [MATL][Kelliher, T., Starr, D., Richbourg, L., Chintamanani, S., Delzer, B., Nuccio, ML et al. (2017) MATRILINEAL, a sperm-specific phospholipase, triggers maize haploid induction [MATRILINEAL, a sperm-specific phospholipase, triggers maize haploid induction in maize] Nature [Nature] 542 105–109], NOT LIKE DAD [NLD] [Liu, C., Li, X., Meng, D., Zhong, Y., Chen, C., Dong, D. et al. A 4-bp insertion at ZmPLA1 encoding a putative phospholipase A generates Haploid induction in maize. [A 4-bp insertion at ZmPLA1 encoding a putative phospholipase A generates haploid induction in maize] Mol. Plant [Molecular Plant] 10, 520–522 (2017)] or phospholipase A1 [PLA1] [Liu, C., Li, X., Meng, D., Zhong, Yu., Chen, C., Dong, X. et al. (2017) A 4-bp Insertion at ZmPLA1 Encoding a Putative Phospholipase A Generates Haploid Induction in Maize [A 4-bp insertion at ZmPLA1 encoding a putative phospholipase A generates haploid induction in maize] Molecular Plant [Molecular Plant] 10, 520–522]). This allele forms the basis of the qhir1 QTL and alone confers 1% to 7% maternal haploid induction rate (HIR) (i.e., the other 93% to 99% of offspring are diploid); the exact HIR depends on environmental conditions and the male and female genetic background.The superior inducible line should also be homozygous for the HI enhancer allele at qhir8, which combines with qhir1 to produce 10% to 20% HIR. According to internal mapping data, qhir8 is located at positions 3,444,422 and 11,360,090 on chromosome 9 in the B73-REFERENCE-GRAMENE-4.0 (https: / / maizegdb.com) reference genome, which is consistent with the findings of the research group [Liu, C., Li, X., Meng, D., Zhong, Yu., Chen, C., Dong, X. et al. (2017) A 4-bp Insertion at ZmPLA1 Encoding a Putative Phospholipase A Generates Haploid Induction in Maize [Molecular Plant] 10, 520-522]. The HI allele in qhir8 may contain a mutation in the DUF679 domain membrane protein 7 (DMP) gene [Zhong, Y., Liu, C., Qi, X., Jiao, Y., Wang, D., Wang, Y. et al. (2019) Mutation of ZmDMP enhances haploidinduction in maize. Nature Plants 5 575-580].In both monocots and dicots, DMP knockout leads to low HI rates. [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. Nat Plants 6 466-472; Zhong, Y., Chen, B., Wang, D., Zhu, et al. (2020) A DMP-triggered in vivo maternal haploid induction system in the dicotyledonous Arabidopsis. Nat Plants 6 466-472; Zhong, Y., Chen, B., Wang, D., Zhu, et al. (2020) A DMP-triggered in vivo maternal haploid induction system in the dicotyledonous Arabidopsis. X., Li, M., Zhang, J. et al. In vivo maternal haploidinduction in tomato. Plant Biotechnol J. 20, 250–252.

[0085] If color selection is used during the DH process, superior inducible lines also possess a dominant allele at the R1 locus [Chaikam, V., Nair, SK, Babu, R., Martinez, L., Tejomurtula, J., and Boddupalli, PM (2015) Analysis of effectiveness of R1-nj anthocyanin marker for in vivo haploid identification in maize and molecular markers for predicting the inhibition of R1-nj expression. Theor ApplGenet [Theoretical and Applied Genetics] 128, 159-171] to facilitate the sorting of haploids from diploid seeds. R-navajo [Chaikam, V., Nair, SK, Babu, R., Martinez, L., Tejomurtula, J., and Boddupalli, PM (2015) Analysis of effectiveness of R1-nj anthocyanin marker for in vivo haploid identification in maize and molecular markers for predicting the inhibition of R1-nj expression. Theor ApplGenet [Theoretical and Applied Genetics] 128, 159-171] The R1-navajo allele was associated with strong anthocyanin accumulation in the aleurone layer and weak accumulation in the embryo. The R1-scutellum (R1-SCM2) allele was associated with strong anthocyanin accumulation in both the embryo and aleurone layer. In B73-REFERENCE-GRAMENE-4.0, R1 is located between approximately 139 Mb and approximately 140 Mb on chromosome 10.Some corn germplasm may also contain anthocyanin inhibitor alleles, including the dominant C1-I (inhibitor) allele on chromosome 9 in B73-REFERENCE-GRAMENE-4.0, starting at position 11,117,066, in colored aleurone layer 1 (Zm00001eb373660).

[0086] To improve the production rate of double haploids, we created new maternal haploid inducing lines that produce an increased number of haploids with each hybridization, forming the basis for superior inducing lines. Existing haploid inducing lines (SYN-INB75Z and SYN-INB86Z) were crossed with the acquired inducing line Procera haploid inducing line-1 (PHI-1) (obtained from Expert Agro Farming in Romania), which has been characterized as having a haploid induction rate of 11%–12% [Rotarenco, V., G. Dicu, State, D, and S. Fuia. 2010. “New Inducers of Maternal Haploids in Maize.” Maize Genetics Cooperation Newsletter, Vol. 84: 21–22.] In both cases, the resulting F1 offspring will self-pollinate to produce a parental F2 population for localization.

[0087] In the F2 generation, SNP markers were used to select matl [Prigge, V. Xiaowei, X., Liang, L., Babu, R., Chen, S., Atlin, G. et al. (2012) New insights into the genetics of in vivo induction of maternal haploids, the backbone of doubled haploid technology in maize. Genetics 190, 781–793] and dmp [Liu, C., Li, W., Zhong, Y., Dong, X., Hu, H., Tian, ​​X. et al. (2015) Fine mapping of qhir8 affecting in vivo haploid induction in maize. TheorAppl Genet 128, [2507-2515.] and the induced alleles of the R1-SCM2 color marker gene.

[0088] In F2 and subsequent generations, SNP marker data were combined with phenotypic data to select plants with the highest HIR. HIR is typically measured as the percentage of total progeny that were well pollinated each time, where well pollination is defined as an ear with more than 80 grains. In each generation for haploid-induced phenotypic typing, isolated inducing lines were subjected to test hybridization (i.e., used as male pollen donors and crossed onto female test ears) to assess their relative HIR. Lines with the HIR to be tested were planted in quadruplicate, with test rows inserted. Genotyping of the qhir8, R1-SCM2, and inhibitor loci in SYN-INBC34 resulted in the selection of hundreds of individuals (including controls) for test hybridization, following pre-screening for agronomic, pollen production, and flowering-silking interval traits. HIR was determined by isolating embryos from test hybrids 15–20 days post-pollination into embryo rescue medium (4.43 g Murashige and Skoog basal medium containing vitamins, 30 g sucrose, and 70 mg salicylic acid). The culture dishes were exposed to light, and purple (diploid) and cream-colored (haploid) embryos were counted. As with the control (RWKS / Z21S / / RWKS), the HIR range for all allele-fixed lines against all HI tests was 10%–19%. Unfixed matl and qhir8 lines showed lower HIR. Phenotypic data were then compared with SNP markers used to genotype the genome with thousands of informative SNPs using the Axiom SNP array to identify candidate QTLs potentially associated with increased HIR. In multiple F3 breeding populations, this correlation analysis identified a QTL on chromosome 5 (between 60 and 144 cM) that increased the number of haploids per ear by 2% to 4% (Table 1).

[0089] Table 1. Examples of three F3 induction line breeding populations in which QTLs were detected on chromosome 5. The phenotypic traits monitored as alternative indicator traits for haploid induction were percentage of haploids per ear (%haploids), number of haploids per ear (#haploids / ear), and total number of seeds per ear (#seeds / ear), all of which can increase the total haploid induction rate.

[0090]

[0091] 2. Fine mapping of QTLs on chromosome 5

[0092] Initial localization of chromosome 5 QTLs using SYN-INB75Z x PHI-1 and SYN-INB86Z x PHI-1 in F2 and F3 generations (Table 1) was unreliable for selection in the breeding population due to insufficient number of SNP markers in the identified regions on chromosome 5. In F4, additional markers were added to the genotype set to increase the density around chromosome 5 QTLs and improve the resolution that could define their boundaries (Table 2). Localization populations were selected by self-crossing the parents in F3 generations, based on flanking markers from F3, which were fixed against all other known haploid-induced QTLs and heterozygous for chromosome 5 QTLs. Due to the presence of mutations in matl and dmp, the offspring of these self-crossed heterozygotes were expected to have a strong (>10%) HIR, but segregation would occur for the chromosome 5 QTL alleles. This population structure allowed us to directly measure the impact of any minor QTL (e.g., the QTL we identified on chromosome 5) on HIR across the entire population by comparing plants with and without favorable QTL alleles using SNP markers and by associating them with their HIRs through independent plant phenotypic typing. Each plant in this finely mapped population was treated as a separate male inducing line and hybridized with two different hybrid females with different genetic backgrounds. A total of 39 males were hybridized with the two different female lines, with 6 ears per tester. After discarding ears with fewer than 50 grains, haploid induction rates were determined from a total of 209 well-pollinated ears. Using this data, the QTLs on chromosome 5 were narrowed down to between 129,909,257 and 155,088,562 (coordinates used using B73-REFERENCE-GRAMENE-4.0, which is publicly available at MaizeGDB.org). The mean Hierarchical Influence (HIR) of individuals with favorable inducible alleles (strain A in Tables 3a and 3b) was 21.6%, while the mean HIR of individuals with unfavorable alleles (strain B in Tables 3a and 3b) was 17.9%, with a mean difference of 3.7%.

[0093] Table 2. Physical locations of markers used to locate QTLs on chromosome 5

[0094]

[0095] Table 3a. Fine mapping data of all induced line genotypes represented in the F4 study of lines derived from the crosses of SYN-INB75Z x PHI-1 and SYN-INB86Z x PHI-1. At each marker, the SNP is referred to as line A (favorable HIR parent), line B (unfavorable HIR parent), heterozygous (Het), or no data (ND).

[0096]

[0097] Table 3b. Fine mapping data of all induced line genotypes represented in the F4 study of lines derived from the crosses of SYN-INB75Z x PHI-1 and SYN-INB86Z x PHI-1. At each marker, the SNP is referred to as line A (favorable HIR parent), line B (unfavorable HIR parent), heterozygous (Het), or no data (ND).

[0098]

[0099] 3. Compare the QTLs on chromosome 5 with publicly available QTLs.

[0100] Prigge et al. (2012) and Hu et al. (2016) previously published a total of four QTLs on chromosome 5 identified in haploid inducible lines independent of Syngenta germplasm. The markers defining each location in those publications were mapped to the B73-RefGen-v3 genome (database available at https: / / maizegdb.org / genome / assembly / B73%20RefGen_v3). When the markers defining the SYN-chromosome 5 QTLs (SM10934 SEQ ID NO: 5 and SM10932 SEQ ID 10) were similarly mapped to B73-RefGen-v3, the four QTL regions identified by these two publications did not overlap with the QTLs identified in this study (Table 4).

[0101] Table 4. Physical locations of markers defining the left and right boundaries of HIR QTLs identified on chromosome 5 by Prigge et al., Hu et al., and Syngenta in the B73-RefGen-v3 corn genome.

[0102]

[0103] Table 5. DNA sequences that define the markers for SYN Chr5 QTL.

[0104] References

[0105] Chaikam, V., Nair, S.K., Babu, R., Martinez, L., Tejomurtula, J., andBoddupalli, P.M. (2015) Analysis of effectiveness of R1-nj anthocyanin markerfor in vivo haploid identification in maize and molecular markers forpredicting the inhibition of R1-nj expression. Theor Appl Genet 128, 159-171]

[0106] Kelliher, T., Starr, D., Richbourg, L., Chintamanani, S., Delzer, B.,Nuccio, M. L. et al. (2017) MATRILINEAL, a sperm-specific phospholipase,triggers maize haploid induction Nature 542 105–109],

[0107] Liu, C., Li, X., Meng, D., Zhong, Yu., Chen, C., Dong, X. et al.(2017) A 4-bp Insertion at ZmPLA1 Encoding a Putative Phospholipase AGenerates Haploid Induction in Maize Molecular Plant 10, 520-522].

[0108] Liu, C., Li, W., Zhong, Y., Dong, X., Hu, H., Tian, X. et al. (2015)Fine mapping of qhir8 affecting in vivo haploid induction in maize. TheorAppl Genet 128, 2507-2515.],

[0109] Panavas T., Weir J., Walker EL. Structure and paramutagenicity of the R-marbled haplotype of Zea mays. Genetics 153 979–991].

[0110] Paz-Ares , J. , Ghosal , D. , Saedler , H. Molecular analysis of the C1-Iallele from Zea mays: a dominant mutant of the regulatory C1 locus. (1990)EDUCATION J. 9 315–321]

[0111] Prigge , V. Xiaowei , X. , Liang , L. , Babu , R. , Chen , S. , Atlin , G. etal . (2012) New insights into the genetics of in vivo induction of maternalhaploids, the backbone of doubled haploid technology in maize. Genetics 190,781–793].

[0112] 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 thedicotyledonous Arabidopsis. Nat Plants 6, 466–472

[0113] Zhong , Y. , Chen , B. , Wang , D. , Zhu , X. , Li , M. , Zhang , J. et al. In vivo maternal haploid induction in tomato. Plant Biotechnol J. 20, 250–252

[0114] [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 5575-580].

Claims

1. A Zea mays plant comprising a QTL, a. wherein the QTL comprises SEQ ID NO: 5 and SEQ ID NO: 10, and b. wherein the QTL is associated with increased haploid production, whereby the Zea mays plant has increased haploid production.

2. The Zea mays plant of claim 1, wherein the Zea mays plant is a haploid inducer line Zea mays plant.

3. The haploid inducer line Zea mays plant of claim 2, wherein the haploid inducer line Zea mays plant further comprises the inducer line allele ZmMATL.

4. The haploid inducer line Zea mays plant of claim 2, wherein the haploid inducer line Zea mays plant further comprises the inducer line allele ZmDMP.

5. The haploid inducer line Zea mays plant of claim 2, wherein the haploid inducer line Zea mays plant further comprises the inducer line alleles ZmMATL and ZmDMP.

6. The haploid inducer line Zea mays plant of claim 3, wherein the haploid inducer line Zea mays plant further comprises a marker gene.

7. The haploid inducer line Zea mays plant of claim 6, wherein the marker gene is a color marker gene.

8. The haploid inducer line Zea mays plant of claim 7, wherein the marker gene is an anthocyanin marker gene.

9. The haploid inducer line Zea mays plant of claim 8, wherein the anthocyanin marker gene is selected from the group consisting of Rl-SCM2 and Rl-nj.

10. The haploid inducer line Zea mays plant of claim 9, wherein the haploid inducer line Zea mays plant further comprises the Rl-SCM2 color marker gene.

11. The Zea mays plant of claim 1, wherein the Zea mays plant is an elite line.

12. The Zea mays plant of claim 1, wherein the QTL is located between position 129,909,257 and 155,088,562 on chromosome 5 using coordinates from B73-REFERENCE-GRAMENE-4.

0.

13. A method of obtaining a plant with increased haploid production, the method comprising a. obtaining a first plant comprising a SYN Chr5 QTL comprising SEQ ID NO: 5 and SEQ ID NO: 10 associated with increased haploid production; b. crossing the first plant with a second plant; c. obtaining progeny from the cross of step b and selecting progeny containing the SYN Chr5 QTL therefrom, d. whereby a plant with increased haploid production is obtained.

14. The plant of claim 13, wherein the plant with increased haploid production is a Zea mays plant.

15. The Zea mays plant of claim 14, wherein the Zea mays plant is a haploid inducer line Zea mays plant.

16. The haploid inducer line Zea mays plant of claim 15, wherein the haploid inducer line Zea mays plant further comprises the inducer line allele ZmMATL.

17. The inducer line maize plant of claim 15, wherein the inducer line maize plant further comprises the inducer line allele ZmDMP.

18. The inducer line maize plant of claim 15, wherein the inducer line maize plant further comprises the inducer line alleles ZmMATL and ZmDMP.

19. The inducer line maize plant of claim 16, wherein the inducer line maize plant further comprises a marker gene.

20. The inducer line maize plant of claim 19, wherein the marker gene is a color marker gene.

21. The inducer line maize plant of claim 20, wherein the marker gene is an anthocyanin marker gene.

22. The inducer line maize plant of claim 21, wherein the anthocyanin marker gene is selected from the group consisting of Rl-SCM2 and Rl-nj.

23. The inducer line maize plant of claim 22, wherein the inducer line maize plant further comprises the Rl-SCM2 color marker gene.

24. The maize plant of claim 14, wherein the maize plant is an elite line.

25. The maize plant of claim 14, wherein the SYN Chr5 QTL is located on chromosome 5 between positions 129,909,257 and 155,088,562 using coordinates from B73-REFERENCE-GRAMENE-4.

0.

26. A method of selecting a first maize plant or germplasm that exhibits increased haploid production, the method comprising: a. isolating nucleic acid from the first maize plant or germplasm; b. 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 c. selecting the first maize plant or germplasm, or a progeny of the first maize plant or germplasm, that comprises the Syn Chr5 QTL that is associated with increased haploid production.

27. The method of claim 26, further comprising crossing the selected first maize plant or germplasm with a second maize plant or germplasm, wherein the introgressed maize plant or germplasm exhibits increased haploid production.

28. The method of claim 26, wherein the SYN Chr5 QTL is detected using a composition comprising a detectable marker.

29. The maize plant of claim 26, wherein the first maize plant is an inducer line maize plant.

30. The inducer line maize plant of claim 29, wherein the inducer line maize plant further comprises the inducer line allele ZmMATL.

31. The haploid-inducing line maize plant of claim 29, wherein the haploid-inducing line maize plant further comprises the inducible line allele ZmDMP.

32. The haploid-inducing line maize plant of claim 29, wherein the haploid-inducing line maize plant further comprises the inducible line alleles ZmMATL and ZmDMP.

33. The haploid-inducing line maize plant of claim 30, wherein the haploid-inducing line maize plant further comprises a marker gene.

34. The haploid-inducing line maize plant of claim 33, wherein the marker gene is a color marker gene.

35. The haploid-inducing line maize plant of claim 34, wherein the marker gene is an anthocyanin marker gene.

36. The haploid-inducing line maize plant of claim 35, wherein the anthocyanin marker gene is selected from the group consisting of Rl-SCM2 and Rl-nj.

37. The haploid-inducing line maize plant of claim 36, wherein the haploid-inducing line maize plant further comprises the Rl-SCM2 color marker gene.

38. The maize plant of claim 26, wherein the maize plant is an elite line.

39. The maize plant of claim 26, wherein the QTL is located between positions 129,909,257 and 155,088,562 on chromosome 5 using coordinates from B73-REFERENCE-GRAMENE-4.

0.

40. 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.

Images