Markers associated with spontaneous chromosome doubling

Abstract

Provided herein is a method for selecting and generating germplasm that exhibits spontaneous chromosome doubling when in a haploid state. The method includes isolating nucleic acid from a first corn plant or germplasm and detecting SCD-QTL1 associated with chromosome doubling. The method further includes selecting the first corn plant or germplasm or a progeny of the first corn plant or germplasm that includes SCD-QTL1 associated with spontaneous chromosome doubling.

Description

[Technical field]

[0001] The subject matter disclosed herein generally relates to plant breeding and chromosome doubling. More specifically, the subject matter relates to methods for obtaining and producing plants that exhibit spontaneous chromosome doubling when in a haploid state. Furthermore, the methods herein relate to transferring a locus of interest and a QTL associated with spontaneous chromosome doubling.

[0002] Sequence Listing This application is accompanied by a sequence listing entitled 82610_ST26.xml, created on December 15, 2022, which is approximately 24 kilobytes in size. This sequence listing is incorporated herein by reference in its entirety. This sequence listing is submitted herewith and complies with 37 C.F.R. § 1.824(a)(2)-(6) and (b). [Background technology]

[0003] Spontaneous chromosome doubling ("SCD") is a rare trait in maize. Understanding its mechanism of action and the genetic loci associated with this trait have become an area of ​​academic research interest in recent years due to the potential value of doubled haploid breeding. Polyploid plants cannot produce fertile gametes during meiosis and must double to obtain fertile pollen and egg cells. Thus, a haploid maize plant without any intervention is expected to have completely sterile pollen unless an SCD is present that allows for correct meiosis of the chromosomes and the subsequent formation of mature pollen. Various studies have identified 35 QTLs and SNPs associated with this trait in biallelic maize populations (reviewed in Boerman, 2020). Several studies focused on mapping QTLs associated with SCD in maize (Zea mays) have identified loci on all maize chromosomes (Chaikam, 2019; Chaikam, 2020; Fuente, 2019; Ma, 2018; Molenaar, 2019; Ren, 2019; Vanous, 2019; Ren, 2017; Trampe, 2020; Wu, 2017; Yang, 2019). However, very few follow-up studies have been published. An exception is a strong QTL on chromosome 5, including the candidate gene BUBR1, identified in several studies and in U.S. Patent Publication No. 20190106703. However, the QTL on chromosome 5 has not shown predictable penetrance across the various germplasms we work with. Therefore, there is a need to identify SCD QTLs that are more predictable and penetrant across germplasms.

[0004] Doubled haploid plants are important, useful and advantageous for plant breeders. Breeders cross inbred parent lines (also called elite lines), one as male and one as female, to form hybrid seeds. The process of developing substantially homozygous inbred parent lines usually requires plants to be selected and self-pollinated (self-fertilized) for many generations to become nearly homozygous. This process is time-consuming and expensive. To shorten the time to develop homozygous inbred lines in corn, rice, wheat, barley and other crops, breeders may choose to use haploid derivative lines to induce haploid seed production in hybrid parents. Typically, obtaining doubled haploid plants requires obtaining haploid plants as the product of crosses between normal plants and haploid derivative plants, or culturing haploid gametophytes into haploid sporophytes via microspore culture, anther culture, ovule culture or ovary culture. Haploid plant sporophytes are sterile when developed without artificial intervention, unless there is some form of spontaneous chromosome doubling, and contain only half the normal number of chromosomes for that plant species. See ST Chalyk, Properties of maternal haploid maize plants and potential application to maize breeding, EUPHYTICA 79:13-18 (1994), at 14, col. 2. For example, maize is considered a diploid organism containing 20 chromosomes (i.e., two copies of each set of 10 different chromosomes). See, for example, MP Maguire, Chromosome behavior at premeiotic mitosis in maize, J. HEREDITY 74:93-96 (1983), at Table 1. In contrast, haploid maize plants have only 10 chromosomes (i.e., one copy of each of the 10 chromosomes). For a haploid plant to be fertile, the chromosomal complement must be doubled in at least the male and female germ lines, if not the entire plant.Chromosome doubling in plants is typically accomplished using liquid mitotic spindle poisons, which are toxic chemicals that can be used to treat human cancer, or the chemicals can also be microtubule-blocking herbicides. See Y. Wan, et al. Wan, et al., The use of antimicrotubule herbicides for the production of doubled haploid plants from anther-derived maize callus, THEOR.APPL.GENET.81(2):205-211(1991). Application of such chromosome doubling agents (e.g., colchicine) doubles the chromosomes of haploid plants to form doubled haploid homozygous inbred lines. Summary of the Invention [Means for solving the problem]

[0005] Without this doubling step, the maize haploid plant is male sterile, i.e. lacks the ability to produce pollen. Here, the inventors have identified an SCD QTL on maize chromosome 7, which indicates that the presence of the SCD QTL allows the maize plant to spontaneously double its haploid genome, at least in the floret, without the need to apply a chromosome doubling agent. When this spontaneous doubling occurs, the plant regains male fertility, i.e. the ability to produce pollen. The inventors provide a method for selecting a first maize plant or germplasm that exhibits SCD when in the haploid state. Additionally, a method for obtaining a spontaneously doubled haploid plant with male fertility is provided. The inventors also provide a method for crossing the first maize plant or germplasm with a second maize plant or germplasm to transfer the locus of interest, the transferred plant exhibiting SCD in the haploid state. Additionally, the inventors encompass a method of transferring a locus of interest between a first corn plant and a second corn plant, resulting in a corn plant that exhibits the locus of interest and an SCD QTL, The locus of interest comprises a quantitative trait locus or allele associated with a trait selected from the group consisting of increased yield, increased water optimization, increased disease resistance, increased drought tolerance and increased herbicide resistance. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] Brief description of the sequences in the sequence listing SEQ ID NO:1 is the forward primer used to amplify marker SM0077AQ in the TaqMan assay.

[0007] SEQ ID NO:2 is the reverse primer used to amplify marker SM0077AQ in the TaqMan assay.

[0008] SEQ ID NO:3 is the forward primer used to amplify marker SM11142 in the TaqMan assay.

[0009] SEQ ID NO:4 is the forward primer used to amplify marker SM11142 in the TaqMan assay.

[0010] SEQ ID NO:5 is the reverse primer used to amplify marker SM11142 in a TaqMan assay.

[0011] SEQ ID NO:6 is the SM0077AQ marker target sequence.

[0012] SEQ ID NO:7 is the SM11142 marker target sequence.

[0013] definition While the following terms are believed to be well understood by those of ordinary skill in the art, the following definitions are set forth to facilitate the description of the subject matter disclosed herein.

[0014] All technical and scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art, unless otherwise defined below. References to technology used herein are intended to refer to technology commonly understood in the art, including variations in technology and / or equivalent technical substitutions that would be apparent to those skilled in the art. While the following terms are believed to be fully understood by those skilled in the art, the following definitions are set forth to facilitate the description of the subject matter disclosed herein.

[0015] In accordance with 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 certain embodiments may refer to a tissue and / or an organ. Similarly, the phrase "at least one," when used 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 the applicable entity, including, but not limited to, all integer values ​​from 1 to 100, as well as integer values ​​greater than 100.

[0016] Unless otherwise indicated, all numerical values ​​expressing quantities of ingredients, reaction conditions, and the like used in the specification and claims are to be understood in each instance to be modified 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 the particular amount, 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%, such variations being appropriate for carrying out the disclosed methods and / or using the disclosed compositions, nucleic acids, polypeptides, and the like. Thus, unless indicated to the contrary, the numerical parameters set forth in the specification and appended claims are approximations that may vary depending upon the desired properties sought to be obtained by the subject matter disclosed herein.

[0017] As used herein, "0% pollen fertility" or "male sterility" or "complete pollen sterility" refers to 0% spontaneous chromosome doubling (SCD) in the context of the present disclosure. More specifically, in this context, 0% pollen fertility refers to a haploid plant that cannot produce fertile pollen. In the present disclosure, a haploid plant (1n genome) cannot reduce to a 0.5n genome, so it cannot complete meiosis, and the haploid plant also cannot produce fertile pollen. Therefore, when referring to 0% pollen fertility in the present disclosure, the present disclosure refers to 0% SCD. To produce fertile pollen from a haploid plant, the genome must be doubled chemically or by SCD.

[0018] As used herein, the term "allele" refers to a variant or alternative sequence form at a locus. In diploids, a single allele is inherited separately from each parent at each locus to a progeny individual. Although the two alleles at a given locus present in a diploid organism occupy corresponding positions in a pair of homologous chromosomes, those skilled in the art will understand that the alleles in any particular individual do not necessarily represent all of the alleles present in a species.

[0019] As used herein, the term "amplification" or "amplifying" refers to constructing multiple copies of a nucleic acid molecule or multiple copies complementary to a nucleic acid molecule using at least one of the nucleic acid molecules as a template. Amplification systems include the polymerase chain reaction (PCR) system, the ligase chain reaction (LCR) system, nucleic acid sequence-based amplification (NASBA, Cangene, Mississauga, Ontario), the Q-beta replicase system, the transcription-based amplification system (TAS) and strand displacement amplification (SDA). See, for example, Diagnostic Molecular Microbiology: Principles and Applications, PERSING et al., Ed., American Society for Microbiology, Washington, DC (1993). The product of amplification is called an "amplicon."

[0020] As used herein, when used in connection with a list of entities, the term "and / or" refers to the entities present singly or in combination. Thus, for example, the phrase "A, B, C and / or D" includes not only A, B, C and D individually, but also all combinations and subcombinations of A, B, C and D (e.g., AB, AC, AD, BC, BD, CD, ABC, ABD and BCD). In some embodiments, one or more of the elements referred to by "and / or" may also be present individually in single or multiple occurrences in combinations and / or subcombinations.

[0021] As used herein, the phrase "associated with" refers to a recognizable and / or testable relationship between two entities. For example, the phrase "associated with spontaneous chromosome doubling (SCD)" refers to a trait, locus, gene, allele, marker, phenotype, etc., or expression thereof, the presence or absence of which may affect the extent and / or degree to which a plant has the capacity for SCD. Thus, a marker is "associated with" a trait when it is associated with the trait and the presence of the marker is an indication of whether and / or to what extent a desired trait or trait morphology occurs in a plant / germplasm containing the marker. Similarly, a marker is "associated with" an allele when it is associated with an allele and the presence of the marker is an indication of whether the allele is present in a plant / germplasm containing the marker. For example, a "marker associated with SCD" refers to a marker whose presence or absence can be used to predict whether and / or to what extent a plant will exhibit SCD.

[0022] As used herein, the term "backcrossing" is understood within the present invention to refer to the process of repeatedly backcrossing hybrid progeny to one of the parents.

[0023] As used herein, the term "cDNA" refers to a single- or double-stranded DNA that is complementary to and derived from mRNA.

[0024] As used herein, the term "chromosome" is used herein as recognized in the art to mean a self-replicating genetic structure within the cell nucleus that contains the cellular DNA and carries the linear arrangement of genes.

[0025] The term "comprising," which is synonymous with "comprising," "containing," and "characterized by," is inclusive or open-ended and does not exclude additional, unrecited elements and / or method steps. "Comprising" is a terminology that means that the specified elements and / or steps are present, but that other elements and / or steps can be added and still be included within the scope of the relevant subject matter.

[0026] As used herein, the phrase "consisting of" excludes any element, step, or ingredient not specifically recited. When the phrase "consisting of" appears in a clause in the body of a claim rather than immediately following the preamble, it limits only the elements recited in that clause and does not exclude other elements from the claim as a whole.

[0027] As used herein, the phrase "consisting essentially of" limits the scope of the associated disclosure or claims to the particular materials and / or steps and methods that do not materially affect the basic and novel characteristics of the disclosed and / or claimed subject matter.

[0028] With respect to the terms "comprising," "consisting essentially of," and "consisting of," when one of these three terms is used herein, the subject matter disclosed and claimed herein may, in certain embodiments, include the use of either of the other two terms. For example, when the subject matter relates to a nucleic acid encoding a polypeptide comprising an amino acid sequence that, in certain embodiments, is at least 95% identical to SEQ ID NO: 2 or 3. It is understood that the disclosed subject matter herein also encompasses, in certain embodiments, a nucleic acid encoding a polypeptide consisting essentially of an amino acid sequence that is at least 95% identical to SEQ ID NO: 2 or 3, as well as a nucleic acid encoding a polypeptide consisting of an amino acid sequence that, in certain embodiments, is at least 95% identical to SEQ ID NO: 2 or 3. Similarly, it is also understood that, in certain embodiments, a method for the disclosed subject matter comprises the steps disclosed herein, in certain embodiments, a method for the subject matter disclosed herein consists essentially of the steps disclosed, and in certain embodiments, a method for the subject matter disclosed herein consists of the steps disclosed herein.

[0029] As used herein, the term "de novo haploid induction" refers to the triggering of haploid induction by the introduction of a spontaneous haploid inducer. Such introduction may be accomplished by topical spray, artificial 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.

[0030] As used herein, the term "elite line" or "inbred line" refers to any line resulting from breeding and selection for superior agronomic performance. An elite line has stable genetics, i.e., is reasonably or nearly isogenic across its entire genome. Stated differently, an elite line is reasonably or nearly homozygous for all alleles in its genome.

[0031] As used herein, the term "expression", when used with respect to a polynucleotide such as a gene, an ORF or a portion thereof, or a transgene in a plant, refers to the process of converting the genetic information encoded in the gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) by "transcription" of the gene (i.e., by the enzymatic action of RNA polymerase), and, where applicable (e.g., if the gene codes for a protein), into a protein by "translation" of the mRNA. Gene expression can be regulated at many stages in the process. For example, in the case of an antisense or dsRNA construct, respectively, expression can refer to the transcription of only the antisense RNA or only the dsRNA. In embodiments, "expression" refers to the transcription and stable accumulation of sense (mRNA) or functional RNA. "Expression" can also refer to the production of a protein.

[0032] As used herein, the term "gene" refers to a hereditary unit that occupies a specific location on a chromosome and contains a sequence of DNA that carries the genetic instructions for a particular characteristic or trait in an organism.

[0033] 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 particular alleles for one or more genetic marker loci present in the individual. As is known in the art, genotypes can be associated with single or multiple loci, regardless of whether the loci are related or unrelated and / or linked or unlinked. In some embodiments, an individual's genotype is associated with one or more genes that are related in that the one or more genes are involved in the expression of a phenotype of interest (e.g., a quantitative trait as defined herein). Thus, in some embodiments, the genotype includes the sum of one or more alleles present at one or more loci of a quantitative trait within the individual. In some embodiments, the genotype is expressed in terms of haplotypes (defined herein below).

[0034] A "genetic map" is a description, typically depicted in graphical or tabular form, of the genetic linkage relationships among genetic loci on one or more chromosomes within a given species.

[0035] As used herein, the term "germplasm" refers to the totality of genotypes of a population or another population (e.g., a species). The term "germplasm" may also refer to plant material, e.g., a group of plants that serves as a repository for various alleles. The phrase "adapted germplasm" refers to plant material with proven genetic superiority for a given environment or geographic region, for example, whereas the phrases "non-adapted germplasm," "unprocessed germplasm," and "exotic germplasm" refer to plant material with unknown or unproven genetic value for a given environment or geographic region, for example, and thus the phrase "non-adapted germplasm" in some embodiments refers to plant material that is not part of an established breeding population and whose relationship to members of the established breeding population is unknown.

[0036] As used herein, a plant referred to as "haploid" has a single set of chromosomes (genome), and the reduced number of chromosomes (1n) in a haploid plant is equal to that of a gamete. As used herein, a plant referred to as "doubled haploid" is generated by doubling the haploid set of chromosomes (1n to 2n). A plant or seed obtained from a doubled haploid plant that has been selfed to any number of generations can 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 growing part of the plant does not consist of cells with two sets of chromosomes, i.e., a plant will be considered doubled haploid if it contains viable gametes, even if it is a chimera.

[0037] As used herein, the term "heterologous" when used in reference to a gene or nucleic acid refers to a gene that encodes an element that is not present in its natural environment (i.e., altered by the hand of man). For example, a heterologous gene can include a gene from one species that has been introduced into another species. A heterologous gene can also include a gene native to an organism that has been modified in some way (e.g., mutated, added in multiple copies, linked to a non-native promoter or enhancer polynucleotide, etc.). A heterologous gene can further include plant gene polynucleotides, including cDNA forms of plant genes, where the cDNA can be expressed in either sense (to produce mRNA) or antisense orientation (to produce an antisense RNA transcript complementary to the mRNA transcript). In one aspect of the invention, a heterologous gene is distinguished from an endogenous plant gene in that a heterologous gene polynucleotide is typically associated with a polynucleotide that contains regulatory elements, such as a promoter, that is not naturally associated with the gene for the protein encoded by the heterologous gene or the plant gene polynucleotide in a chromosome, or is associated with a portion of the chromosome not found in nature (e.g., a gene expressed at a locus where the gene is not normally expressed). Furthermore, in embodiments, a "heterologous" polynucleotide is a polynucleotide that is not naturally associated with the host cell into which it is introduced, including multiple non-naturally occurring copies of a naturally occurring polynucleotide.

[0038] As used herein, the term "heterozygosity" refers to the genetic situation in which different alleles are present at corresponding loci on homologous chromosomes.

[0039] As used herein, the term "homozygous" refers to the genetic situation in which identical alleles are present at corresponding loci on homologous chromosomes.

[0040] 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. The plants are checked for fertile pollen every day during the pollen shedding period (7-14 days in the field), and each plant is assigned a number equal to the number of days the plant shed pollen (e.g., a plant assigned a number of 5 is a plant that shed pollen for 5 days). Sterile plants are scored as 0, which means that the plant shed pollen for 0 days. In the absence of other interventions, haploid plants with a score greater than zero are haploid plants with some level of SCD.

[0041] As used herein, the term "isolated" when used in relation to the nucleic acid molecule or polynucleotide of the present invention refers to a polynucleotide that has been identified in the respective source organism and isolated / separated from the context of its chromosomal polynucleotide. An isolated nucleic acid or polynucleotide is not a nucleic acid as it exists in a natural context, even if it has a naturally occurring counterpart. In contrast, a non-isolated nucleic acid is a nucleic acid found in a naturally occurring state, such as DNA and RNA. For example, a given polynucleotide (e.g., a gene) is present on a host cell chromosome in the vicinity of adjacent genes. An isolated nucleic acid molecule can exist in single-stranded or double-stranded form. Alternatively, it can include both the sense and antisense strands (i.e., the nucleic acid molecule can be double-stranded). In a preferred embodiment, the nucleic acid molecule of the present invention is understood to be isolated.

[0042] As used herein, the term "locus" refers to a location (e.g., a gene, a genetic marker, etc.) on a chromosome of a given species.

[0043] As used herein, "maternal haploid induction line" refers to a line that produces pollen and, when crossed as male, results in female development of haploid seeds. "Paternal haploid induction line" refers to a line that, when used in crosses as female, results in androgenic development of haploid seeds. Haploid induction line plants can use either of these maternal or paternal mechanisms to derive haploids.

[0044] As used herein, the term "artificial mutation" refers to any mutation that occurs as a result of direct or indirect human action. This term includes, but is not limited to, mutations obtained by any method of targeted mutagenesis.

[0045] As used herein, the term "hybrid" refers to the progeny produced by crossing two genetically distinct parent plants. The progeny resulting from this cross is a "biparental" population.

[0046] As used herein, "log of the odds" or "LOD score" refers to the statistical likelihood that two genes are close to each other on a chromosome.

[0047] 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 by nucleic acid hybridization, e.g., a nucleic acid probe complementary to all or a portion of a marker or marker locus. Marker probes containing about 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more consecutive nucleotides can be used for nucleic acid hybridization.

[0048] As used herein, the term "molecular marker" may be used to refer to a genetic marker as defined above or its encoded product (e.g., protein) that is used as a reference point when identifying the presence / absence of SCD or other loci of interest. Molecular markers may be derived from genomic or expressed nucleotide sequences (e.g., RNA, cDNA, etc.). The term also refers to nucleotide sequences that are complementary to or adjacent to a marker sequence, such as nucleotide sequences used as probes and / or primers that can amplify the marker sequence. Nucleotide sequences are "complementary" if they specifically hybridize in solution (e.g., according to Watson-Crick base pairing rules). The term also refers to genetic markers that exhibit a trait by the absence of nucleotide sequences that are complementary to or adjacent to a marker sequence, such as nucleotide sequences used as probes and / or primers that can amplify the marker sequence.

[0049] 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, and optionally contains synthetic, non-natural and / or altered nucleotide bases. "Nucleotides" are the monomeric units from which DNA or RNA polymers are built, and consist of a purine or pyrimidine base, a pentose, and a phosphate group. Nucleotides (usually found in their 5'-monophosphate form) are designated by their one-letter code as follows: "A" stands for adenylate or deoxyadenylate (for RNA or DNA, respectively), "C" stands for cytidylate or deoxycytidylate, "G" stands for guanylate or deoxyguanylate, "U" stands for uridylate, "T" stands for deoxythymidylate, "R" stands for purine (A or G), "Y" stands for pyrimidine (C or T), "K" stands for G or T, "H" stands for A or C or T, "I" stands for inosine, and "N" stands for any nucleotide.

[0050] In the context of two nucleic acid or amino acid sequences, the term "identity" or "same" 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) compared to a test ("subject") sequence when the two sequences are aligned as a whole. Unless otherwise indicated, sequence identity as used herein refers to the value obtained using the Needleman and Wunsch algorithm ((1970) J. Mol. Biol. 48:443-453) as implemented in the EMBOSS needle alignment tool, or any equivalent program, using the default matrix file EBLOSUM62 for proteins with default parameters (gap open=10, gap extension=0.5, end gap penalty=false, end gap open=10, end gap extension=0.5) or DNAfull for nucleic acids with default parameters (gap open=10, gap extension=0.5, end gap penalty=false, end gap open=10, end gap extension=0.5). EMBOSS Needle is available, for example, from EMBL-EBI, for example at the following website: ebi.ac.uk / Tools / psa / emboss_needle / , and described in the following publication: "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 program" refers to any sequence comparison program that generates an alignment having identical nucleotide or amino acid residue matches and identical percent sequence identity for any two sequences of interest 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.

[0051] As used herein, the terms "introgression," "introgressed," and "introgressing" refer to both the natural and artificial process in which a genomic region of one species, variety, or cultivar is moved into the genome of another species, variety, or cultivar by crossing the species. This process can optionally be completed by backcrossing to the recurrent parent.

[0052] The term "open reading frame" (ORF) refers to a nucleic acid sequence that encodes a polypeptide. In certain embodiments, an ORF includes a translation initiation codon, a translation termination (i.e., stop) codon, and the nucleic acid sequence therebetween that encodes the amino acids present in the polypeptide. The terms "initiation codon" and "termination codon" refer to three adjacent units of nucleotides (i.e., codons) in a coding sequence that specify the initiation and chain termination, respectively, of protein synthesis (mRNA translation).

[0053] As used herein, the term "phenotype", "phenotypic trait" or "trait" refers to one or more traits of a plant or plant cell. A phenotype may be observable by the naked eye or any other means of evaluation known in the art, such as microscopy, biochemical analysis or electromechanical assay. In some cases, a phenotype is directly controlled by a single gene or locus (i.e., corresponds to a "single gene trait"). For example, in the case of a haploid plant evaluated for SCD, the phenotype may refer to the fertile pollen that is dispersed and / or the seeds obtained by pollination with that pollen. In other cases, a phenotype is the result of the interaction between several genes, and in some embodiments, the interaction between the plant and / or plant cell and its environment.

[0054] As used herein, the term "plant" may refer to a whole plant, any part thereof, or a cell or tissue culture derived from a plant. Thus, unless otherwise specified, the term "plant" may refer to either a whole plant, a plant component or organ (e.g., leaves, stems, roots, etc.), a plant tissue, a seed, and / or a plant cell.

[0055] A plant cell is a plant cell obtained by culture from a cell taken from a plant. Thus, the term "plant cell" includes, but is not limited to, cells in seeds, suspension cultures, embryos, meristematic tissue parts, callus tissue, leaves, shoots, gametophytes, sporophytes, pollen, and microspores. The phrase "plant part" refers to a part of a plant from which a plant can be regenerated, including single cells and cell tissues such as intact plant cells in a plant, cell mass, and tissue culture. Examples of plant parts include, but are not limited to, pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems, shoots, and seeds, as well as single cells and tissues derived from shoots, rhizomes, protoplasts, callus, etc.

[0056] As used herein, the term "population" means a genetically heterogeneous collection of plants that share a common genetic origin.

[0057] As used herein, the term "primer" refers to an oligonucleotide that can anneal (and in some embodiments specifically anneal) to a nucleic acid target and serve as a point of initiation of DNA synthesis when placed under conditions that induce the synthesis of a primer extension product (e.g., in the presence of nucleotides and agents for polymerization such as DNA polymerase and at a suitable temperature and pH). In some embodiments, one or more primers are used to amplify plant nucleic acids (e.g., using the polymerase chain reaction, PCR).

[0058] As used herein, the term "probe" refers to a nucleic acid (e.g., a single-stranded nucleic acid or a strand or subsequence of a double-stranded or higher order nucleic acid) that can form a hydrogen-bonded duplex with a complementary sequence in a target nucleic acid sequence. Typically, a probe has a length sufficient to form a stable and sequence-specific double-stranded molecule with its complement, and thus can be used in certain embodiments to detect a sequence of interest present in a plurality of nucleic acids.

[0059] As used herein, the terms "progeny" and "progeny plant" refer to plants produced from vegetative or sexual reproduction from one or more parent plants. In haploid induction, the seeds of the female parent are haploid, so they are not the progeny of the derived haploid line. The progeny of haploid seeds are not the only desirable progeny. There are also HI seeds and subsequent plant and seed progeny of haploid-derived plants. Both haploid seeds and HI seeds can be progeny. Progeny plants are obtained by cloning or selfing a single parent plant or by crossing two or more parent plants. For example, progeny plants are obtained by cloning or selfing a parent plant or by crossing two parent plants, including selfing and F1 or F2 or even other generations. F1 is the first generation progeny produced from a parent, at least one of which is used for the first time as a trait donor, while the progeny of the second generation (F2) and subsequent generations (F3, F4, etc.) are samples produced from selfing, outcrossing, backcrossing and / or other crosses such as F1, F2, etc. Thus, the F1 may be (and in some embodiments is) a hybrid resulting from a cross between two pure breeding parents (i.e., the pure breeding parents are each homozygous for the trait or allele thereof of interest), while the F2 may be (and in some embodiments is) the progeny resulting from self-pollination of the F1 hybrid.

[0060] As used herein, "F1-H generation" refers to the progeny produced by first crossing an inbred line containing the SCD trait with an inbred line (F1) lacking the SCD trait. The progeny from the first cross was then crossed with a maternal haploid derivative to produce the F1-H generation. As used herein, "F2-H generation" refers to the progeny produced when the F1 plant is selfed to produce an F2, which is then crossed with a haploid derivative.

[0061] As used herein, "QTL penetrance" refers to the presence of a phenotype associated with a QTL locus.

[0062] As used herein, "quantitative trait locus" or "quantitative trait loci" or "QTL" refers to a genetic locus associated with a particular trait.

[0063] As used herein, "R1-nj" refers to the R1-Navajo anthocyanin marker, which is a marker known in the art to be used as a visual marker to distinguish haploids from diploids (or aneuploids).

[0064] As used herein, the term "recombination" refers to the exchange of DNA fragments between two DNA molecules or chromatids of paired chromosomes across regions of similar or identical nucleotide sequences ("crossing over"). It is understood that "recombination event" as used herein refers, in one embodiment, to meiotic crossing over.

[0065] As used herein, the term "reference sequence" refers to a defined nucleotide sequence used as a basis for a nucleotide sequence comparison.

[0066] As used herein, the term "regenerate" and grammatical variations thereof refer to the production of plants from tissue culture.

[0067] 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 the haploid genome without any intervention. SCD allows for correct meiosis of chromosomes and the subsequent formation of mature pollen. In this disclosure, SCD was calculated by dividing the number of fertile haploid plants by the total number of plants.

[0068] As used herein, a "spontaneously doubled haploid plant" refers to a plant in which the floret has undergone spontaneous doubling. Other tissues of a spontaneously doubled haploid plant may retain their haploid state (e.g., roots, leaves, stems).

[0069] As used herein, "SCD-QTL1" refers to a currently identified QTL on chromosome 7. This QTL is defined by a region of chromosome 7 flanked by two markers, SM0077AQ and SM11142. These markers correspond to physical locations in the B73_v4 reference genome.

[0070] As used herein, the phrase "stringent hybridization conditions" refers to conditions under which a polynucleotide will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but will not hybridize to substantially other sequences. Stringent conditions are sequence-dependent and may be different under different circumstances.

[0071] Longer sequences typically hybridize specifically at higher temperatures. Extensive guidelines for nucleic acid hybridization can be found in Sambrook & Russell, 2001. In general, 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 (when the target sequence is present in excess, 50% of the probes are occupied at equilibrium at Tm). 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 salt) 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., more than 50 nucleotides).

[0072] Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide. Further exemplary stringent hybridization conditions include 50% formamide, 5xSSC and 1% SDS or SSC incubated at 42°C, 1% SDS incubated at 65°C, with one or more washes in 0.2xSSC 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 from about 32°C to 48°C (or higher), depending on primer length. Further guidance for determining hybridization parameters is provided in many references (see, for example, Ausubel et al., 1999).

[0073] As used herein, the term "trait" refers to a phenotype of interest, a gene that contributes to the phenotype of interest, as well as a nucleic acid sequence associated with a gene that contributes to the phenotype of interest.

[0074] As used herein, the term "transgene" refers to a nucleic acid molecule that is introduced into an organism or one or more of its ancestors by some form of artificial transfer technique. Artificial transfer techniques thus produce "transgenic organisms" or "transgenic cells." It is understood that while artificial transfer techniques may be performed on (or may develop into) cells within and / or into ancestors, any progeny individual that has the artificially transferred nucleic acid molecule or a fragment thereof will still be 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.

[0075] As used herein, the term "targeted mutagenesis" or "mutagenesis technique" refers to any method of mutagenesis that results in the deliberate mutation of a selected gene. Targeted mutagenesis includes the methods CRISPR, TILLING, TALEN, and other methods that have not yet been disclosed but can be used to achieve the same result.

[0076] As used herein, haploid induction rate ("HIR") means the number of viable haploid kernels relative to the total number of kernels after pollinating an ear with haploid-inducing pollen.

[0077] The present disclosure relates inter alia to spontaneously doubled haploid plants. In one embodiment, the present disclosure is a method of selecting a first corn plant or germplasm that exhibits spontaneous chromosome doubling ("SCD") when in a haploid state. The method includes isolating nucleic acid from the first corn plant or germplasm and detecting SCD-QTL1, which is associated with spontaneous chromosome doubling, comprising SEQ ID NOs: 6 and 7. The method further includes selecting the first corn plant or germplasm, or selecting a progeny of the first corn plant or germplasm that includes SCD-QTL1 associated with spontaneous chromosome doubling. In another embodiment, the method further includes crossing the selected first corn plant or germplasm with a second corn plant or germplasm. The introgressed corn plant or germplasm exhibits increased spontaneous chromosome doubling ("SCD") when in a haploid state. SCD-QTL1 is detected using a composition comprising a detectable label.

[0078] In another embodiment, the present disclosure includes a method for obtaining a spontaneously doubled haploid plant. The method includes obtaining a first corn plant comprising SCD-QTL1 comprising SEQ ID NO: 6 and SEQ ID NO: 7, which is associated with spontaneous chromosome doubling, crossing the first corn plant with a second corn plant, obtaining progeny from the cross and selecting haploid progeny therefrom, and allowing the haploid progeny to undergo spontaneous chromosome doubling. The spontaneously doubled haploid plant can include reproductive tissue capable of producing fertile gametes. The spontaneously doubled haploid plant is male fertile.

[0079] In another embodiment, the first corn plant is a haploid derived corn plant. The first corn plant comprises a mutation in CENH3 or IG1 (see, for example, U.S. Patent No. 8,618,354 and WO 2011 / 044132, which are incorporated herein by reference in their entirety). In another embodiment, the second corn plant is a haploid derived corn plant. The second corn plant comprises a mutation in ZmMATL (see, for example, U.S. Patent No. 9,677,082, U.S. Patent No. 10,448,588 and WO 2017 / 087682, which are incorporated herein by reference in their entirety).

[0080] In another embodiment, the disclosure includes a method of transferring a locus of interest. The method includes first obtaining a first corn plant comprising SCD-QTL1, which comprises SEQ ID NO: 6 and SEQ ID NO: 7, associated with spontaneous chromosome doubling. The method then includes crossing the first corn plant with a second corn plant comprising the locus of interest, and obtaining progeny from the cross between the first corn plant and the second corn plant. In addition, the method optionally includes backcrossing the progeny obtained by crossing the first and second corn plants from above with the first corn plant or the second corn plant. The method further includes screening the progeny of the cross for the presence of the locus of interest and the SCD QTL, and selecting progeny that are at least heterozygous for the locus of interest and at least heterozygous for the SCD QTL. In one embodiment, the screening of the progeny for the presence of the locus of interest and the SCD-QTL is screened by genotype. In another embodiment, the screening of the progeny for the presence of the locus of interest and the SCD-QTL is by phenotypic screening.Furthermore, a corn plant is produced by the method, the corn plant belonging to a non-rigid stem heterosis group, a rigid stem heterosis group, or a tropical heterosis group.A corn progeny plant is produced from the corn plant.The corn plant is a hybrid corn plant or an elite corn plant.

[0081] In another embodiment, the locus of interest of the method of introgressing a locus of interest comprises a quantitative trait locus associated with a trait selected from the group consisting of increased yield, increased water optimization, increased disease resistance, increased drought tolerance and increased herbicide resistance. Further, the locus of interest comprises a transgene. EXAMPLES

[0082] Example 1. Screening for SCD in Syngenta maize germplasm To better understand the SCD trait, we screened 20 lines reported by maize breeders to have SCD. This was done by crossing our own inbred lines with haploid inducers, isolating haploids using genetic color markers, and growing the regenerated haploid plants under normal greenhouse conditions in the absence of chemical doubling agents. Under these conditions, a typical maize haploid is 100% male sterile and does not shed any pollen (0% pollen fertility is defined as 0% SCD). Plants were evaluated for pollen fertility daily during the pollen shedding period, and all pollen shed from the ear (i.e., released into the air) was presumed to be fertile. Male-sterile maize plants usually do not have anthers emerge from the ear, and in such cases, no pollen shedding is observed. However, four of these lines (SYN004, SYN007, SYN013, and K22) showed SCD at rates greater than 15%. For the remaining 16 lines, the average SCD was 5% (see Table 1). To confirm the greenhouse observations, a subset of lines, including some of the top performing lines, was also grown under field conditions (see Table 1). Four of the top performing lines were selected to create a QTL mapping population for the SCD trait.

[0083] [Table 1]

[0084] Example 2. QTL identification and validation Identification of QTLs in a biparental F1-H population To map the SCD trait, biparental populations were generated by crossing inbred lines with the SCD trait (e.g., SYN004 and SYN007) with inbred lines that did not show a high rate of the SCD phenotype (e.g., NP2222 and SYN006). The F1 progeny of the cross (presumed to be heterozygous for any SCD-related loci) was crossed with the maternal haploid derivative line, RWK, and seeds were selected for haploidy using selectable markers (see, e.g., Iowa State University website: www.doubledhaploid.biotech.iastate.edu / haploid-selection) ("Hohenheim method") to generate the F1-H generation. The doubled haploid seeds were sown in a greenhouse in full sun. Individual plants were phenotyped by counting the number of days of continuous pollen shedding (HMF score) and by evaluating the kernels produced from self-pollinations to ensure that the observed pollen was viable. Individual plants were genotyped using Affymetrix Axiom chip arrays (see, e.g., thermofisher.com / us / en / home / life-science / microarray-analysis / agrigenomics-solutions-microarrays-gbs / axiom-genotyping-solution-agrigenomics) to correlate array results with phenotypic results and determine regions of the genome associated with the doubling trait. For two of the SCD inbred lines, association mapping did not identify any meaningful QTLs. For the remaining lines (SYN004 and SYN007), a total of four QTLs with LOD scores >5 were considered significant, especially when similar loci appeared in multiple F1-H populations (Table 2).

[0085] [Table 2]

[0086] Validation of QTLs in a biparental F2-H population Validation of these four QTLs was performed in the next generation. F1 plants from the biparental population were selfed to generate F2, which were then crossed by a haploid induction system to generate the F2-H haploid population. These F2-H populations were grown in three locations. Individual haploid plants were screened by daily pollen checks (HMF score) and grain number measurements as described above. The observed phenotypes (see Table 3) were then compared with the Axiom genotype results for the population of 475 plants, and QTL association mapping was performed in the same way as was performed for the F1-H generation. The previously identified QTLs on chromosomes 2 and 10 (Table 2) failed to obtain significant scores in this second generation. However, the QTL on chromosome 7 was similarly strong (LOD score >5) in the second generation in both populations where it was expected. This QTL on chromosome 7 was named SCD-QTL1.

[0087] [Table 3]

[0088] Example 3. Fine mapping of QTL boundaries Fine mapping and SNP genotyping in the F2-H population In the same F2-H population grown for QTL validation, plants were also genotyped within the expected QTL interval on chromosome 7 using SNP-based TaqMan assay markers with predicted polymorphisms between parental alleles in the F2-H population (see Table 4). These markers cover the 113.9-154.6 Mb region of chromosome 7 (position from the B73_v4 reference genome) and were selected and / or designed based on the genome sequences of the parental lines. Comparing these results, the genotype from the parent with the SCD trait (B line in Table 5) is associated with a higher than average haploid pollen fertility rate between markers SM0077AQ and SM11142. These two markers flanked a 2.54 Mb region (B73_v4 coordinates) containing 46 annotated gene models and showed 74% pollen fertility in this study. To confirm the statistical significance of the intervals defined by recombination mapping, 466 haploids that were either AA or BB genotypes at markers SM0077AQ and SM11142 from the F2-H population were selected and subjected to single-factor ANOVA analysis. Comparison of the HMF scores of the A and B pedigrees (mean 1.94 and 6.27 HMF score units, respectively) revealed a P value of 8.44*10 -32 Based on all analyses, we defined SCD-QTL1 by a region of chromosome 7 flanked by markers SM0077AQ and SM11142, which corresponds to its physical location in B73 as determined by searching the DNA sequences associated with each of the TaqMan assays in its reference genome. The DNA sequences of these TaqMan markers are shown in Table 6.

[0089] [Table 4-1] [Table 4-2] [Table 4-3]

[0090] [Table 5]

[0091] [Table 6]

[0092] Example 4. Comparative mapping of previously published QTLs and / or SNPs with SCD-QTL1 In two published genome-wide association mapping studies, Ma (2018) and Yang (2019) have previously identified a QTL associated with SCD on maize chromosome 7. However, the QTLs defined in these studies do not overlap with the SCD-QTL1 defined herein using the B73_v4 reference genome. Ma (2018) identified two SNPs associated with SCD in his mapping population, one at 113,165,535 bp and one at 170,671,328 bp in the B73_v2 genome (available at maizegdb.org). A BLAST search of the target sequence of marker SM0077AQ (SEQ ID NO: 6) in maizegdb.org indicates that the relative position of the sequence between B73_v2 and B73_v4 is located at the left border of SCD-QTl1 starting at 120,202,757 bp in B73_v2. This region does not overlap with the region identified by Ma (2018). Yang (2019) identified three QTLs associated with chromosome 7 in two different maize lines and gave the approximate genomic location of the QTLs in cM (map distance) in the linkage map obtained for that study, which is consistent with the map obtained for B73_v2. Yang et al. also provide chromosome bin numbers for the QTLs, but these numbers are imprecise because the bins can be very large (e.g., bin 7.02 spans 13.8-128.1 Mb, occupying 62% of chromosome 7 in the B73_v control). Using the test linkage map, the three identified loci (qHMF7a, qHMF7b and qHMF7c) are located at 26.51-28.51 cM, 41.51-43.51 cM and 74.41 cM. SCD-QTL1 is located more than 97.2 cM away from the start of the chromosome according to the results of target sequence BLAST in the maizeGDB genome browser, and therefore does not overlap with any of the QTLs identified in Yang (2019). Considering these relative coordinates, we are confident that SCD-QTL1 is a novel QTL that has not been previously identified in the academic literature.

[0093] Example 5. Phenotype Analysis To compare the spontaneous chromosome doubling (SCD) phenotype observed in the high doubling accelerated lines observed in the initial screen, two lines, namely SYN007 and SYN004, were selected for more detailed phenotypic analysis, including pollen viability tests and ploidy analysis of various tissue types. Ploidy analysis can be easily performed on various plant tissues by determining the average DNA content of a population of nuclei extracted from fresh tissues using flow cytometry (Dolezel, 2007). No SCD was observed in diploids from these lines when seedling leaves, adult leaves, pollen, and periclinal tissues were analyzed by flow cytometry. However, in haploid plants, doubling was easily detected in male reproductive tissues (anthers, pollen, spikelets) using flow cytometry. However, no SCD was observed in vegetative tissues (leaves, roots, stems), indicating that developmentally, the SCD observed in these lines occurs only after the initiation of floral organs.

[0094] The metabolic activity of pollen can be measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay (Firmage and Dafni, 2001). In the presence of active oxidoreductase enzymes within the pollen grain, MTT is reduced from a yellow solution to an insoluble purple formazan. Pollen grains that turn purple in the presence of MTT are considered metabolically active and therefore viable. In the absence of chromosome doubling, haploid pollen is always sterile, so the presence of viable pollen in haploid plants is a direct result of SCD. In this experiment, anthers were collected from each of the two lines at the mature pollen shedding stage. Pollen from individual anthers was then released into the MTT solution by gently crushing the tissue with a plastic pestle. Pollen collected from individual anthers of line SYN007 typically showed a mixture of viable and non-viable pollen. However, pollen from individual anthers of SYN004 was either 100% viable or 0% viable. Furthermore, when comparing the lengths of individual anthers, only in SYN004 were anthers with viable (i.e., doubled) pollen twice as long as anthers with non-viable (i.e., non-doubled) pollen. Anthers within a floret were typically all the same length. This data suggests that SCD in SYN007 occurs quite late, after the pollen precursors have already been specified and become distinct from one another. However, SYN004 SCD must occur at an earlier stage of flower development, roughly when the individual florets are being specified but before the anthers begin to form. These conclusions are based on principles of maize flower development, where initially a small group of dividing cells undergo a series of differentiation events to become founder cells of the entire tassel. If SCD occurs very early in development, a large portion of the tassel, and perhaps the entire tassel, would be expected to show SCD. The phenotype of enlarged anthers (larger cells indicate higher ploidy levels) and the overall population of viable pollen in SYN004 suggest that SCD in this lineage occurs in cells that precede the complete anther during developmental progression.The mixed viability of pollen within individual anthers and the absence of fully doubled (larger) anthers suggests that SGD in SYN007 occurs later, after most of the floral organs are already present and before the pollen is mature.

[0095] Example 6. QTL penetrance in the absence of additional doubling treatments in diverse germplasm Penetrance of QTLs is generally germplasm dependent, based on other, often unidentified, genetic factors segregating in diverse germplasm backgrounds. To examine how widely penetrant SCD-QTL1 is in more diverse lines, a panel of 17 elite inbred lines was selected from Syngenta's North America breeding population. These lines represented several heterosis groups (rigid-stem, nonrigid-stem, and iodent varieties) as well as a highly diverse maturity group. To generate diverse germplasm populations for phenotypic screening, the QTL donor SYN004 was crossed with each of the 17 elite lines. These F1 populations were backcrossed to the 17 recurrent parents to generate the BC1 generation and backcrossed once more to generate the BC2 generation. In the BC2 population, lines spanning the entire QTL interval were genotyped using SNP assay markers that define the QTL (Table 5) to identify plants that were heterozygous for the advantageous (SYN004, doubled) allele. These heterozygotes were then crossed with haploid derivatives and the resulting haploids were identified in mature seeds based on the R-nj colormaker. From the resulting haploids, 50% of them were positive for the advantageous allele and 50% were negative, creating an ideal control group for comparing field performance.

[0096] Mature haploid seeds were directly sown in the field and seedlings were again genotyped with SNP markers (Table 5) to confirm the QTL allele status and to remove all potential polyploids from the population. Plants were grown under standard nursery conditions, but with additional shade cloth cover to reduce light intensity until pollen shedding. Individual plant phenotypes were collected for each haploid, measuring the number of days to pollen shedding and the number of kernels per ear after self-pollination. In the absence of colchicine or other chemical doubling treatments, the haploid phenotype would be expected to be zero days to pollen shedding and zero kernels. Indeed, this expectation was observed in the recurrent parental control, where the favorable allele of SCD-QTL1 was not present. However, in the BC2 haploid population, it was observed that many lines had more fertile pollen in individuals with favorable alleles of SCD-QTL1 (Table 7). This was the case for 7 of the 17 elite lines, showing moderate levels of QTL penetrance. These same lines also had less abundant seed set (5 of 17 lines had more kernels per ear when the favorable allele of the SCD1-QTL was present), possibly indicating that additional factors contributing to female doubling (haploid female fertility) may not be present in some lines.

[0097] [Table 7]

[0098] Example 7. QTL penetrance in diverse germplasm in combination with colchicine doubling treatment To test whether SCD-QTL1 has an additive effect on haploid male fertility using a combination of a chemical doubling treatment such as colchicine, 17 elite BC2 lines from Example 6 that had undergone one additional backcross generation were used to create BC3. Heterozygotes at SCD-QTL1 were identified by genotyping, and the material was crossed with a haploid inducer, embryos were isolated from immature ears, and haploids were regenerated for transplantation into the field. Immediately after isolation, embryos from each of the 17 BC3 populations were divided into two treatment groups. One group received a doubling treatment with colchicine, and the other group did not receive the chemical doubling agent. Thus, there were four treatment groups to compare within each elite BC3 population: (1) SCD-QTL with colchicine (haploid male fertility + colchicine), (2) unfavorable QTL allele with colchicine (positive control), (3) SCD-QTL1 without colchicine (haploid male fertility alone), and (4) unfavorable QTL allele without colchicine (negative control). All lines were transplanted in triplicate over a period of approximately six months, and data were pooled for analysis.

[0099] In this study, the sample size was large enough (minimum 50 haploids per treatment group) to allow statistical analysis to be performed to determine whether the observed differences in grain set between groups with and without the favorable allele of SCD-QTL1 were statistically significant using a Student's T-test (Table 8). Three of the 17 lines showed a significant increase in grain set (p-value < 0.05) in the presence of SCD-QTL1, whereas three of the 17 lines showed a significant decrease (p-value < 0.05). Seven of the lines did not show any significant effect of the QTL, and four were refractory to all doubled haploid treatments (these were excluded from the analysis). Of the seven lines that did not show statistically significant differences, some showed a positive trend, but it was not significant. The three lines negatively correlated with SCD-QTL1 are still a relatively large region (~2 Mb) and may represent other factors co-segregating with SCD-QTL1 that contains 62 potential gene candidates. In doubled haploid production, the number of individual haploids producing more than 5 grains per spike with colchicine treatment is an important indicator of success. Comparing only the colchicine treatment group (which showed an overall better performance in both pollen and grain production than SCD-QTL1 alone), five lines showed an increased percentage of haploids that produced selfed spikes with more than 5 grains, indicating that the presence of favorable alleles of SCD-QTL1 increases the efficiency of doubled haploid production in some lines (Table 8).

[0100] [Table 8-1] [Table 8-2] [Table 8-3]

Claims

1. 1. A method for selecting a first corn plant or germplasm that exhibits spontaneous chromosome doubling ("SCD") when in a haploid state, comprising: a. isolating nucleic acid from the first corn plant or germplasm; b. detecting in said first maize plant or germplasm an SCD-QTL1 associated with spontaneous chromosome doubling, said SCD-QTL1 comprising SEQ ID NOs: 6 and 7; c. selecting the first corn plant or germplasm, or a progeny of the first corn plant or germplasm, that includes the SCD-QTL1 associated with spontaneous chromosome doubling; The method includes:

2. 2. The method of claim 1, further comprising crossing the selected first corn plant or germplasm with a second corn plant or germplasm to generate a progeny corn plant, wherein the progeny corn plant or germplasm exhibits increased spontaneous chromosome doubling ("SCD") when in a haploid state.

3. The method of claim 1, wherein the SCD-QTL1 is detected using a composition comprising a detectable label.

4. 1. A method for obtaining a spontaneously doubled haploid plant, comprising the steps of: a. Obtaining a first maize plant comprising a SCD-QTL1 comprising SEQ ID NOs: 6 and 7, said SCD-QTL1 being associated with spontaneous chromosome doubling; b. crossing the first corn plant with a second corn plant; c. Obtaining progeny from the cross of step b and selecting haploid progeny therefrom; d. allowing the haploid progeny to undergo spontaneous chromosome doubling; thereby obtaining a spontaneously doubled haploid plant.

5. The method of claim 3 , wherein the spontaneously doubled haploid plant comprises reproductive tissue capable of producing fertile gametes.

6. 5. The method of claim 4, wherein the spontaneously doubled haploid plant is male fertile.

7. 4. The method of claim 3, wherein the first corn plant is a haploid derived corn plant.

8. The method of claim 6 , wherein the first corn plant comprises a mutation in CENH3 or IG1.

9. The method of claim 3 , wherein the second corn plant is a haploid derived corn plant.

10. The method of claim 8 , wherein the second corn plant comprises a mutation in ZmMATL.

11. 1. A method for transferring a locus of interest, comprising the steps of: a. Obtaining a first maize plant comprising a SCD-QTL1 comprising SEQ ID NOs: 6 and 7, said SCD-QTL1 being associated with spontaneous chromosome doubling; b. crossing the first corn plant with a second corn plant that contains the locus of interest; c. obtaining progeny from the mating of step b.; d. optionally, backcrossing the progeny of step c. to the first corn plant or the second corn plant; e. screening the progeny of step c. or step d. for the presence of the locus of interest and the SCD QTL; f. Selecting progeny that are at least heterozygous for the locus of interest and at least heterozygous for the SCD QTL. thereby obtaining a maize plant containing said locus of interest and said SCD QTL.

12. 11. The method of claim 10, wherein screening of the offspring for the presence of a locus of interest and the SCD-QTL is by genotypic screening.

13. The method of claim 10, wherein screening of the progeny for the presence of the locus of interest and the SCD-QTL is by phenotypic screening.

14. 11. The method of claim 10, wherein the locus of interest comprises a quantitative trait locus associated with a trait selected from the group consisting of increased yield, increased water optimization, increased disease resistance, increased drought tolerance and increased herbicide resistance.

15. 14. The method of claim 13, wherein the locus of interest comprises a transgene.

16. A corn plant produced by the method of claim 10.

17. 16. The corn plant of claim 15, which belongs to the non-rigid stem heterosis group, the rigid stem heterosis group, or the tropical heterosis group.

18. 16. The corn plant of claim 15, which is a hybrid corn plant or an elite corn plant.

19. 16. A progeny corn plant produced from the corn plant of claim 15.

20. A detection kit comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:

5.

21. 1. A method for producing spontaneously doubled haploid maize plants, comprising: a. identifying a first maize plant or germplasm comprising a SCD-QTL1 comprising SEQ ID NO:6 and SEQ ID NO:7, which is associated with spontaneous chromosome doubling; b. crossing the first corn plant with a second corn plant; c. Obtaining progeny from the cross of step b and selecting haploid progeny therefrom; d. allowing the haploid progeny to undergo spontaneous chromosome doubling; thereby producing a spontaneously doubled haploid plant.

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