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 increased haploid production, and thereby the maize plant has increased haploid production. Also provided are various methods. Methods include obtaining a plant with increased haploid production and a method of selecting a first maize plant or germplasm that displays increased haploid production.

Description

[0001] INCREASING HAPLOID INDUCTION

[0002] FIELD OF THE INVENTION

[0003] The presently disclosed subject matter relates generally to breeding and haploid induction.

[0004] SEQUENCE LISTING

[0005] This application is accompanied by a sequence listing entitled 83041_ST26.mxl, created September 7, 2023, which is approximately 19 kilobytes in size. This sequence listing is incorporated herein by reference in its entirety. This sequence listing is submitted herewith via EFS-Web, and is in compliance with 37 C.F.R. § 1.824(a)(2)- (6) and (b).

[0006] BACKGROUND

[0007] Mapping QTL associated with increased haploid induction in com is an ongoing area of interest due to the growing importance of double haploids for accelerated breeding in com and other species. In com, most haploid induction is initiated by making a cross with a line that is known to contain the inducer genotype. However, for many decades, the genes contributing to induction of haploids has not been understood by the maize breeding community. Initial studies to identify the 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 Doubled 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.” Genetics 202 (4): 1267-76. https: / / doi.org / 10.1534 / genetics.115.184234.) as well as several secondary QTL that could further enhance haploid induction rates, including a QTL on chromosome 9. 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

[0008] 1

[0009] SUBSTITUTE SHEET (RULE 26) unexplored beyond these initial mapping studies. Therefore, there exists a need for a more precise genomic location and testing in a wider set of germplasm for these secondary QTL to enable selective breeding for an improved haploid inducer line.

[0010] SUMMARY

[0011] The desire for creating or obtaining haploid inducers continues to expand due to the importance of doubled haploid plants for accelerated breeding. With that, interest in and work to map QTL associated with haploid induction also continues to expand. To address this interest, 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. Also provided herein is 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. Additionally provided herein is 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.

[0012] BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

[0013] SEQ ID NO: 1 is a nucleotide sequence of a forward primer used to amplify marker SM10934.

[0014] SEQ ID NO: 2 is a nucleotide sequence of a forward primer used to amplify marker SM10934.

[0015] SEQ ID NO: 3 is a nucleotide sequence of a forward primer used to amplify marker SM10934.

[0016] 2

[0017] SUBSTITUTE SHEET (RULE 26) SEQ ID NO: 4 is a nucleotide sequence of a reverse primer used to amplify marker SM10934.

[0018] SEQ ID NO: 5 is a nucleotide sequence of the SM10934 marker target sequence.

[0019] SEQ ID NO: 6 is a nucleotide sequence of a forward primer used to amplify marker SM10932.

[0020] SEQ ID NO: 7 is a nucleotide sequence of a forw ard primer used to amplify marker SM10932.

[0021] SEQ ID NO: 8 is a nucleotide sequence of a forw ard primer used to amplify marker SM10932.

[0022] SEQ ID NO: 9 is a nucleotide sequence of a reverse primer used to amplify marker SM10932.

[0023] SEQ ID NO: 10 is a nucleotide sequence of the SMI 0932 marker target sequence.

[0024] DEFINITIONS

[0025] While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0026] All technical and scientific terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art.

[0027] References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques and / or substitutions of equivalent techniques that would be apparent to one of skill in the art. While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0028] Following long-standing patent law convention, the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. For example, the phrase “a cell” refers to one or more cells, and in some embodiments can refer to a tissue and / or an organ. Similarly, 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

[0029] 3

[0030] 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.

[0031] Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” The term “about,” as used herein when referring to a measurable value such as an amount of mass, weight, time, volume, concentration or percentage is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1 % from the specified amount, as such variations are appropriate to perform the disclosed methods and / or employ the discloses compositions, nucleic acids, polypeptides, etc. Accordingly, unless indicated to the contrary', the numerical parameters set forth in this specification and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently disclosed subject matter.

[0032] As used herein, the term “allele” refers to a variant or an alternative sequence form at a genetic locus. In 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.

[0033] As used herein, 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.”

[0034] As used herein, the term “and / or” when used in the context of a list of entities, refers to the entities being present singly or in combination. Thus, for example, 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

[0035] 4

[0036] SUBSTITUTE SHEET (RULE 26) BCD). In some embodiments, 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).

[0037] As used herein, the phrase “associated with” refers to a recognizable and / or assay able relationship between two entities. For example, 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. Similarly, 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.

[0038] As used herein, the term “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.

[0039] As used herein, the term “cDNA” refers to a single-stranded or a double-stranded DNA that is complementary to and derived from mRNA.

[0040] As used herein, the term “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.

[0041] The term “comprising,” which is synonymous with “including,” “containing,” and “characterized by,” is inclusive or open-ended and does not exclude additional, unrecited elements and / or method steps. “Comprising” is a term of art that means that the named elements and / or steps are present, but that other elements and / or steps can be added and still fall within the scope of the relevant subject matter.

[0042] As used herein, the phrase “consisting of’ excludes any element, step, or ingredient not specifically recited. When 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.

[0043] As used herein, the phrase “consisting essentially of’ limits the scope of the related disclosure or claim to the specified materials and / or steps, plus those that do not materially affect the basic and novel characteristic(s) of the disclosed and / or claimed subject matter.

[0044] With respect to the terms “comprising,” “consisting essentially of,” and “consisting of,” where one of these three terms is used herein, the presently disclosed and claimed subject

[0045] 5

[0046] SUBSTITUTE SHEET (RULE 26) mater can include in some embodiments the use of either of the other two terms. For example, if 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. Similarly, it is also understood that in some embodiments the methods for the disclosed subject mater comprise the steps that are disclosed herein, in some embodiments the methods for the presently disclosed subject mater consist essentially of the steps that are disclosed, and in some embodiments the methods for the presently disclosed subject mater consist of the steps that are disclosed herein.

[0047] As used herein, 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.

[0048] As used herein, the term “elite line” or “inbred 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.

[0049] As used herein, the term “elite inducer” refers to plants bred to be high-performing haploid inducers. For example, herein, 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.

[0050] As used herein, the term “expression” when used with reference to a polynucleotide, such as a gene, ORF or portion thereof, or a transgene in plants, refers to the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through “transcription” of the gene (i.e., via the enzymatic action of an RNA polymerase), and into protein where applicable (e.g. if a gene encodes a protein), through “translation” of mRNA. Gene expression can be regulated at many stages in the process. For example, in the case of

[0051] 6

[0052] SUBSTITUTE SHEET (RULE 26) antisense or dsRNA constructs, respectively, expression may refer to the transcription of the antisense RNA only or the dsRNA only. In embodiments, “expression” refers to the transcription and stable accumulation of sense (mRNA) or functional RNA. “Expression” may also refer to the production of protein.

[0053] As used herein, 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.

[0054] As used herein, 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. As is known in the art, 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. In some embodiments, 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). Thus, in some embodiments a genotype comprises a sum of one or more alleles present within an individual at one or more genetic loci of a quantitative trait. In some embodiments, a genotype is expressed in terms of a haplotype (defined herein below).

[0055] 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.

[0056] As used herein, 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. The phrase “adapted germplasm” refers to plant materials of proven genetic superiority; e.g., for a given environment or geo-graphical area, while the phrases “non-adapted germplasm”, “raw germplasm”, and “exotic germplasm” refer 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.

[0057] As used herein, a plant referred to as “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. As used herein, a plant referred to as “doubled haploid” is developed by doubling

[0058] 7

[0059] 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.

[0060] As used herein, the term “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). For example, 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). In one aspect of the invention, 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). Further, in embodiments, 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.

[0061] As used herein, the term “heterozygous” means a genetic condition existing when different alleles reside at corresponding loci on homologous chromosomes.

[0062] As used herein, the term “homozygous” means a genetic condition existing when identical alleles reside at corresponding loci on homologous chromosomes.

[0063] As used herein, “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

[0064] 8

[0065] 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.

[0066] As used herein, the term “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. In contrast, non-isolated nucleic acids are nucleic acids such as DNA and RNA, which are found in the state they exist in nature. For example, a given polynucleotide (e.g., a gene) is found on the host cell chromosome in proximity to neighboring genes. 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). In a preferred embodiment, the nucleic acid molecules of the present invention are understood to be isolated.

[0067] As used herein, the term “locus” refers to a position (e.g., of a gene, a genetic marker, or the like) on a chromosome of a given species.

[0068] As used herein, “maternal 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.

[0069] As used herein, the term “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.

[0070] As used herein, the term “hybrid” refers to offspring produced by crossing two genetically dissimilar parent plants. The resulting progeny of this cross are a “bi-parental” population.

[0071] As used herein, “logarithm of the odds” or “LOD score” refers to the statistical likelihood that two genes are in proximity on a chromosome.

[0072] As used herein, 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

[0073] 9

[0074] 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.

[0075] As used herein, 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.

[0076] 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 (usually found in their 5 '-monophosphate form) 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.

[0077] The term “identify” or “identical” in the context of two nucleic acid or amino acid sequences, 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. Unless otherwise stated, sequence identify as used herein refers to the value obtained using the Needleman and Wunsch algorithm ((1970) J. Mol. Biol. 48:443-453) implemented in the EMBOSS Needle alignment tool using default matrix files EBLOSUM62 for protein with default parameters (Gap Open = 10, Gap Extend =0.5, End Gap Penalty = False, End Gap

[0078] 10

[0079] SUBSTITUTE SHEET (RULE 26) Open = 10, End Gap Extend = 0.5) or DNAfull for nucleic acids with default parameters (Gap Open = 10, Gap Extend =0.5, End Gap Penalty = False, End Gap Open = 10, End Gap Extend = 0.5); or any equivalent program thereof. EMBOSS Needle is available, e.g., from EMBL-EBI such as at the following website: ebi.ac.uk / Tools / psa / emboss_needle / and as described in the following publication: “The EMBL-EBI search and sequence analysis tools APIs in 2019.” Madeira et al. Nucleic Acids Research, June 2019, 47(W1):W636-W641. The term “equivalent program” as used herein 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. In some embodiments, substantially identical nucleic acid or amino acid sequences may perform substantially the same function.

[0080] As used herein, the terms “introgression”, “introgressed” and “introgressing” refer 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.

[0081] The term “open reading frame” (ORF) refers to a nucleic acid sequence that encodes a polypeptide. In some embodiments, 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. The terms “initiation codon” and “termination 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).

[0082] As used herein, the terms “phenotype,” “phenotypic trait” or “trait” refer 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. In some cases, a phenotype is directly controlled by a single gene or genetic locus (i.e., corresponds to a “single gene trait”). For example, in the case of a haploid plant evaluated for SCD, phenotype can refer to fertile pollen shed and / or a seed obtained via pollination with that pollen. In other cases, 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.

[0083] As used herein, the term “plant” can refer to a whole plant, any part thereof, or a cell or tissue culture derived from a plant. Thus, the term “plant” can refer to any of the following: whole

[0084] 11

[0085] SUBSTITUTE SHEET (RULE 26) plants, plant components or organs (e.g., leaves, stems, roots, etc.), plant tissues, seeds and / or plant cells, unless otherwise specified.

[0086] A plant cell is a cell of a plant, taken from a plant, or derived through culture from a cell taken from a plant. Thus, the term ‘‘plant cell” includes without limitation cells within seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, shoots, gametophytes, sporophytes, pollen, and microspores. The phrase “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. Examples of 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.

[0087] As used herein, the term “population” means a genetically heterogeneous collection of plants sharing a common genetic derivation.

[0088] As used herein, the term “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). In some embodiments, one or more pluralities of primers are employed to amplify plant nucleic acids (e.g., using the polymerase chain reaction; PCR).

[0089] 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, a probe is of sufficient length to form a stable and sequence-specific duplex molecule with its complement, and as such can be employed in some embodiments to detect a sequence of interest present in a plurality of nucleic acids.

[0090] As used herein, the terms “progeny” and “progeny plant” refer to a plant generated from a vegetative or sexual reproduction from one or more parent plants. In 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. There is also the HI seed and subsequent plant and seed progeny of the haploid inducing plant. Both the haploid seed and

[0091] 12

[0092] SUBSTITUTE SHEET (RULE 26) the HI seed can be progeny. A progeny plant can be obtained by cloning or selfing a single parent plant, or by crossing two or more parental plants. For instance, a progeny plant can be obtained by cloning or selfing of a parent plant or by crossing two parental plants and include selfings as well as the Fl or F2 or still further generations. 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.

[0093] As used herein, “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. As used herein, “F2-H generation” refers to progeny produced by said Fl plants above selfing to produce F2, which was then crossed by a haploid inducer.

[0094] As used herein, “QTL penetrance” refers to the presence of an associated phenotype with a QTL locus.

[0095] As used herein, “quantitative trait locus” or “quantitative trait loci” or “QTL” refers to a locus associated with a particular trait.

[0096] As used herein, “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).

[0097] As used herein, the phrase “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.

[0098] As used herein, the term “reference sequence” refers to a defined nucleotide sequence used as a basis for nucleotide sequence comparison.

[0099] As used herein, the term “regenerate,” and grammatical variants thereof, refers to the production of a plant from tissue culture.

[0100] 13

[0101] SUBSTITUTE SHEET (RULE 26) As used herein, “spontaneous chromosome doubling” (“SCD”) or “spontaneous haploid genome doubling” or “haploid male fertility” or “spontaneous genome doubling” are used interchangeably to describe the doubling of haploid genomes without any intervention. SCD allows for the correct meiotic reduction of chromosomes and subsequent formation of mature pollen. In the present disclosure, SCD was calculated by dividing the number of fertile haploid plants / by the total number of plants.

[0102] As used herein, “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).

[0103] As used herein, the phrase “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.

[0104] Longer sequences typically hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Sambrook & Russell, 2001. Generally, stringent conditions are selected to be about 5-10°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium).

[0105] 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).

[0106] 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. For PCR, 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).

[0107] 14

[0108] SUBSTITUTE SHEET (RULE 26) As used herein, 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.

[0109] As used herein, the term “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.

[0110] As used herein, the term “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.

[0111] As used herein, haploid induction rate (“HIR”) 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).

[0112] DETAILED DESCRIPTION

[0113] Provided herein is 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. In one embodiment, the maize plant is a haploid inducer maize plant. In another embodiment, the haploid inducer maize plant may further comprise inducer allele ZmMATL. In another embodiment, 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, 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

[0114] 15

[0115] 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.

[0116] Also provided herein is a method of obtaining a plant with increased haploid production. 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. In one embodiment, the haploid inducer maize plant may further comprise inducer allele ZmMATL. In another embodiment, 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.

[0117] In another embodiment of the invention, provided herein is 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. In another embodiment, 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. In an embodiment, the SYN Chr5 QTL is detected using a composition comprising a detectable label. In one embodiment, 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. In another embodiment, the haploid inducer maize plant may further comprise inducer alleles ZmMATL and ZmDMP. In an embodiment, the

[0118] 16

[0119] SUBSTITUTE SHEET (RULE 26) 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. In an emdobiment, the color marker gene is a R1-SCM2 color marker gene. In an embodiment, the first maize plant displaying increased haploid production an elite line. In another embodiment, 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. In a final embodiment, provided herein is 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.

[0120] EXAMPLES

[0121] 1. Identification of QTL on chromosome 5 associated with increased haploid induction rate in multiple inducer populations.

[0122] Qhirl (QTL for Haploid Induction Ratel), qhir8, as well as the R1 [Panavas T., Weir J., Walker E.L. (1999). The structure and para-mutagenicity of the R-marbled haplotype of Zea mays. Genetics 153 979-991] and COLORED ALEURONE1 (Cl) [Paz-Ares, J., Ghosal, D., Saedler, H. Molecular analysis of the Cl -I allele from Zea mays: a dominant mutant of the regulatory Cl locus. (1990) EMBO J. 9 315-321] anthocyanin pathway genes useful for identifying haploid offspring are penetrant across germplasm.

[0123] Because haploid induction has become a routine step of the breeding process for maize at many large agricultural companies, the breeding of elite haploid inducers with increased rates of haploid induction and other favorable traits has become critical for doubled haploid production. Elite maize haploid inducer lines are homozygous for mat'. a loss of function allele caused by a 4bp insertion in the fourth exon of a patatin-like phospholipase A2a (called MATRILINEAL [MATL] [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], NOT LIKE DAD [NLD] [Liu, C., Li, X., Meng, D., Zhong, Y., Chen, C., Dong, D. et al. A 4-bp insertion at ZmPLAl encoding a putative phospholipase A generates haploid induction in maize. Mol. Plant 10, 520-522 (2017)], or PHOSPHOLIPASE Al [PLA1] [Liu, C„ Li, X., Meng, D„ Zhong, Yu., Chen, C„

[0124] 17

[0125] SUBSTITUTE SHEET (RULE 26) Dong, X. et al. (2017) A 4-bp Insertion at ZmPLAl Encoding a Putative Phospholipase A Generates Haploid Induction in Maize Molecular Plant 10, 520-522], The allele underlies the qhirl QTL and confers a maternal haploid induction rate (HIR) of 1% to 7% in isolation (i.e., the other 93 to 99% of offspring are diploid); the exact HIR depends on environmental conditions as well as male and female genetic background. Elite inducers should also be homozygous for the HI enhancer allele at qhir8, which combines with qhirl to give 10 to 20% HIR. According to internal mapping data, qhir8 is located on chromosome 9 between position 3.444,422 and 11,360,090 in the B73-REFERENCE-GRAMENE-4.0 (https: / / maizegdb.com) reference genome, which aligns with what was discovered by an academic group [Liu, C., Li, X., Meng, D., Zhong, Yu., Chen, C., Dong, X. et al. (2017) A 4- bp Insertion at ZmPLAl Encoding a Putative Phospholipase A Generates Haploid Induction in Maize Molecular Plant 10, 520-522], The HI allele in qhir8 likely comprises 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 haploid induction in maize. Nature Plants 5 575-580], In both monocots and dicots, 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. Nat Plants 6, 466-472; 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],

[0126] Elite inducers also have a dominant allele at the R1 locus if color selection is being used in the DH process [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] to facilitate sorting of haploid from diploid seed. 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. Some maize germplasms may also contain anthocyanin inhibitor alleles

[0127] 18

[0128] 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.

[0129] To improve doubled-haploid production rates, we created new maternal haploid inducer lines that would produce an increased number of haploids per cross, the foundation of an elite inducer line. Existing haploid induction lines (SYN-INB75Z and SYN-INB86Z) were crossed by an acquired inducer Procera Haploid Inducer-1 (PHI-1) (acquired from Expert Agro Farming SRL in Romania), which has been characterized to have an 11-12% haploid induction rate [Rotarenco, V., G. Dicu, State, D, and S. Fuia. 2010. “New Inducers of Maternal Haploids in Maize. ” Maize Genetics Cooperation Newsletter, no. 84: 21- -22.] In both cases, the resulting Fl progeny were self-pollinated to generate a biparental F2 population for mapping.

[0130] In the F2 generation, SNP markers were used to select for the inducer alleles for 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], 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. Theor Appl Genet 128, 2507-2515.], and the R1-SCM2 color marker genes.

[0131] In the F2 and subsequent generations, SNP marker data was used in combination with phenotypic data to select plants with the highest 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. In each generation where 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. Genotyping of qhir8, R1-SCM2, and the inhibitor locus in SYN-INBC34 led to the selection of hundreds of individuals, including controls, for test crossing, which followed pre-screening for agronomic, pollen production, and anthesis-silking interval traits. 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. The 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

[0132] 19

[0133] 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. In multiple F3 breeding populations, 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).

[0134] Table 1. Examples of 3 F3 inducer breeding populations with QTLs detected on chromosome

[0135] 5. 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.

[0136] 2. Fine-Mapping of chromosome 5 QTL

[0137] The initial placement of the chromosome 5 QTL by the F2 and F3 generation of the SYN- INB75Z x PHI-1 and SYN-INB86Z x PHI-1 studies (Table 1) was not reliable for selection

[0138] 20

[0139] SUBSTITUTE SHEET (RULE 26) in breeding populations because there was an insufficient number of SNP markers in the identified region on chromosome 5. In the F4 generation, 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 resulting progeny of these selfed heterozygotes were expected to have strong (>10%) HIRs because of the presence of the mutations in mail and dmp but segregated for the chromosome 5 QTL alleles. This population structure allowed us to directly measure the impact on the HIR of any minor QTL, such as the one we identified on chromosome 5, across the whole population by comparing plants with and without the favorable QTL allele using SNP markers and correlating with their HIR by independent plant phenotyping. Each plant in this fine-mapping population was treated as an individual male inducer and was test crossed to 2 different hybrid females of different genetic backgrounds. In total, 39 males were test crossed by two different female lines at 6 ears per tester. After discarding ears with fewer than 50 kernels, 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%.

[0140] Table 2. Physical locations of markers used for mapping chromosome 5 QTL

[0141] 21

[0142] SUBSTITUTE SHEET (RULE 26)

[0143]

[0144] 22

[0145] SUBSTITUTE SHEET (RULE 26)

[0146] 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).

[0147] SUBSTITUTE SHEET (RULE 26)

[0148]

[0149] SUBSTITUTE SHEET (RULE 26)

[0150] 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).

[0151] 25

[0152] SUBSTITUTE SHEET (RULE 26)

[0153] SUBSTITUTE SHEET (RULE 26)

[0154] 3. Comparing chromosome 5 QTL to published QTL

[0155] Prigge et al. (2012) and Hu et al. (2016) previously published a total of 4 QTL on chromosome 5 identified in haploid inducer lines independent of Syngenta’s germplasm. The markers defining each of the locations 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 QTL (SM10934 SEQ ID NO: 5 and SM10932 SEQ ID 10) are similarly mapped to B73-RefGen-v3, the 4 QTL regions identified by these two publications do not overlap with the QTL identified in the present study (Table 4).

[0156] 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 maize genome.

[0157] 27

[0158] SUBSTITUTE SHEET (RULE 26)

[0159] Table 5. DNA sequences for markers defining the SYN Chr5 QTL.

[0160] 28

[0161] SUBSTITUTE SHEET (RULE 26)

[0162] REFERENCES

[0163] 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]

[0164] 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],

[0165] Liu, C., Li, X., Meng, D., Zhong, Yu., Chen, C., Dong, X. et al. (2017) A 4-bp Insertion at ZmPLAl Encoding a Putative Phospholipase A Generates Haploid Induction in Maize Molecular Plant 10, 520-522],

[0166] 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. Theor Appl Genet 128, 2507-2515 ],

[0167] Panavas T., Weir J., Walker E.L. (1999). The structure and paramutagenicity of the R- marbled haplotype of Zea mays. Genetics 153 979-991]

[0168] Paz-Ares, J., Ghosal, D., Saedler, H. Molecular analysis of the Cl-I allele from Zea mays: a dominant mutant of the regulatory Cl locus. (1990) EMBO J. 9 315-321]

[0169] Pngge, 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],

[0170] 29

[0171] 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

[0172] 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

[0173] [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],

[0174] SUBSTITUTE SHEET (RULE 26)

Claims

What is claimed is:

1. A maize 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, Thereby the maize plant has increased haploid production.

2. The maize plant of claim 1, wherein the maize plant is a haploid inducer maize plant.

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

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

5. The haploid inducer maize plant of claim 2, wherein the haploid inducer maize plant further comprises inducer alleles ZmMATL and ZmDMP6. The haploid inducer maize plant of claim 3, wherein the haploid inducer maize plant further comprises a marker gene.

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

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

9. The haploid inducer maize plant of claim 8, wherein the anthocyanin marker gene is selected from the group consisting of R1-SCM2 and Rl-nj.

10. The haploid inducer maize plant of claim 9, wherein the haploid inducer maize plant further comprises a R1-SCM2 color marker gene.

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

12. The maize plant of claim 1, wherein the QTL is located on chromosome 5 between positions 129,909,257 and 155,088,562 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 that is 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. thereby obtaining a plant with increased haploid production.31SUBSTITUTE SHEET (RULE 26)14. The plant of claim 13, wherein the plant with increased haploid production is a maize plant.

15. The maize plant of claim 14, wherein the maize plant is a haploid inducer maize plant.

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

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

18. The haploid inducer maize plant of claim 15, wherein the haploid inducer maize plant further comprises inducer alleles ZmMATL and ZmDMP19. The haploid inducer maize plant of claim 16, wherein the haploid inducer maize plant further comprises a marker gene.

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

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

22. The haploid inducer maize plant of claim 21, wherein the anthocyanin marker gene is selected from the group consisting of R1-SCM2 and Rl-nj.

23. The haploid inducer maize plant of claim 22, wherein the haploid inducer maize plant further comprises a R1-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 displays increased haploid production, the method comprising: a. isolating nucleic acids 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 said first maize plant or germplasm, or selecting a progeny of said first maize plant or germplasm, comprising the Syn Chr5 QTL that is associated with increased haploid production.32SUBSTITUTE SHEET (RULE 26)27. The method of claim 26, further comprising 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.

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

29. The maize plant of claim 26, wherein the first maize plant is a haploid inducer maize plant.

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

31. The haploid inducer maize plant of claim 29, wherein the haploid inducer maize plant further comprises inducer allele ZmDMP.

32. The haploid inducer maize plant of claim 29, wherein the haploid inducer maize plant further comprises inducer alleles ZmMATL and ZmDMP33. The haploid inducer maize plant of claim 30, wherein the haploid inducer maize plant further comprises a marker gene.

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

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

36. The haploid inducer maize plant of claim 35, wherein the anthocyanin marker gene is selected from the group consisting of R1-SCM2 and Rl-nj.

37. The haploid inducer maize plant of claim 36, wherein the haploid inducer maize plant further comprises a R1-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 on chromosome 5 between positions 129,909,257 and 155,088,562 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: 9.33SUBSTITUTE SHEET (RULE 26)

Images