Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
  • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
  • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
  • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
  • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
  • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
  • C12N9/222—Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes
  • C12N9/226—Class 2 CAS enzyme complex, e.g. single CAS protein

Definitions

• the present invention generally relates to the production of doubled haploids through androgenesis.
• SEQUENCE LISTING This application is accompanied by a sequence listing entitled 82745_ST26.xml, created September 19, 2022 which is approximately 225 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).
• BACKGROUND Microspores are haploid cells containing the gametic number of chromosomes and can be induced to form embryoids (pseudoembryos). This process is commonly referred to as microspore embryogenesis or androgenesis. Haploid embryoids produced from androgenesis can then develop into haploid plants. Haploid plants are generally frail and infertile. Chromosome doubling resulting from spontaneous or chemical induction doubles the chromosomes, resulting in a stable, homozygous, and fertile diploid or amphidiploid plants. Thus, microspores can form homozygous doubled haploid plants (DH) in one generation by androgenesis.
• DH homozygous doubled haploid plants
• haploid induction haploid induction
• Haploid induction generally produces fewer than 100 haploid embryos per induced ear.
• the present disclosure targets selected genes to block or interfere with the starch accumulation pathway in microspores that can serve as the universal trigger for androgenesis.
• SUMMARY Hybrid breeding requires rapid development of pure inbreds.
• Current doubled haploid (DH) technology and methods are expensive. It takes great effort to prepare millions of immature haploid embryos through a standard haploid induction process.
• millions of microspores can be easily isolated from one tassel of corn (or a few spikes of wheat) with a blender in a few minutes.
• Androgenesis from haploid microspores to DH lines in one generation, is the most efficient DH production method.
• the methods herein reduced expression of at least one pollen starch biosynthesis pathway gene selected from ADP glucose pyrophosphorylase, Waxy1, Hexokinase5, Phosphoglucomutase1, Phosphoglucomutase2, and Invertase2.
• the at least one pollen starch pathway gene is reduced by knockout or knockdown utilizing gene editing or RNAi.
• the gene editing is through the use of a site directed nuclease.
• the gene editing is done using a CRISPR nuclease (e.g., Cas12a).
• a CRISPR nuclease e.g., Cas12a
• the androgenic response is increased at least 40% compared to the wildtype.
• Docket no.82745-US-L-ORG-NAT-1 BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING
• SEQ ID NO: 1 is the nucleotide sequence for construct 25639.
• SEQ ID NO: 2 is the nucleotide sequence for construct 25635.
• SEQ ID NO: 3 is the nucleotide sequence for construct 25637.
• SEQ ID NO: 4 is the nucleotide sequence for construct 25619.
• SEQ ID NO: 5 is the nucleotide sequence for construct 25634.
• SEQ ID NO: 6 is the nucleotide sequence for construct 25636.
• SEQ ID NO: 7 is the nucleotide sequence for construct 25662.
• SEQ ID NO: 8 is the nucleotide sequence for construct 25638.
• SEQ ID NO: 9 is the nucleotide sequence for target gene phosphoglucomutase1.
• SEQ ID NO: 10 is the nucleotide sequence for target gene phosphoglucomutase2.
• SEQ ID NO: 11 is the nucleotide sequence for target gene ADP glucose pyrophosphorylaseII.
• SEQ ID NO: 12 is the nucleotide sequence for target gene Invertase2.
• SEQ ID NO: 13 is the nucleotide sequence for target gene Hexokinase5.
• SEQ ID NO: 14 is the nucleotide sequence for target gene Waxy1.
• SEQ ID NO: 15 is the nucleotide sequence for the gRNA target 1 of ADP glucose pyrophosphorylaseII.
• SEQ ID NO: 16 is the nucleotide sequence for the gRNA target 2 of ADP glucose pyrophosphorylaseII.
• SEQ ID NO: 17 is the nucleotide sequence for the RNAi target of ADP glucose pyrophosphorylaseII.
• SEQ ID NO: 18 is the nucleotide sequence for the gRNA target 1 of Hexokinase5.
• SEQ ID NO: 19 is the nucleotide sequence for the gRNA target 2 of Hexokinase5.
• SEQ ID NO: 20 is the nucleotide sequence for the RNAi target of Hexokinase5.
• SEQ ID NO: 21 is the nucleotide sequence for the gRNA target 1 of Waxy1. Docket no.82745-US-L-ORG-NAT-1
• SEQ ID NO: 22 is the nucleotide sequence for the gRNA target 2 of Waxy1.
• SEQ ID NO: 23 is the nucleotide sequence for the RNAi target of Waxy1.
• SEQ ID NO: 24 is the nucleotide sequence for the gRNA target 1 of Invertase2.
• SEQ ID NO: 25 is the nucleotide sequence for the gRNA target 2 of Invertase2.
• SEQ ID NO: 26 is the nucleotide sequence for the RNAi target of Invertase2.
• SEQ ID NO: 27 is the nucleotide sequence for the gRNA target 1 of phosphoglucomutase1.
• SEQ ID NO: 28 is the nucleotide sequence for the gRNA target 1 of phosphoglucomutase2.
• SEQ ID NO: 29 is a primer sequence for TaqMan Assay 3682.
• SEQ ID NO: 30 is a primer sequence for TaqMan Assay 3682.
• SEQ ID NO: 31 is the probe sequence for TaqMan Assay 3682.
• SEQ ID NO: 32 is a primer sequence for TaqMan Assay 3683.
• SEQ ID NO: 33 is a primer sequence for TaqMan Assay 3683.
• SEQ ID NO: 34 is the probe sequence for TaqMan Assay 3683.
• SEQ ID NO: 35 is a primer sequence for TaqMan Assay 3684.
• SEQ ID NO: 36 is a primer sequence for TaqMan Assay 3684.
• SEQ ID NO: 37 is the probe sequence for TaqMan Assay 3684.
• SEQ ID NO: 38 is a primer sequence for TaqMan Assay 3685.
• SEQ ID NO: 39 is a primer sequence for TaqMan Assay 3685.
• SEQ ID NO: 40 is the probe sequence for TaqMan Assay 3685.
• SEQ ID NO: 41 is a primer sequence for TaqMan Assay 3686.
• SEQ ID NO: 42 is a primer sequence for TaqMan Assay 3686.
• SEQ ID NO: 43 is the probe sequence for TaqMan Assay 3686.
• SEQ ID NO: 44 is a primer sequence for TaqMan Assay 3687.
• SEQ ID NO: 45 is a primer sequence for TaqMan Assay 3687. Docket no.82745-US-L-ORG-NAT-1
• SEQ ID NO: 46 is the probe sequence for TaqMan Assay 3687.
• SEQ ID NO: 47 is a primer sequence for TaqMan Assay 3688.
• SEQ ID NO: 48 is a primer sequence for TaqMan Assay 3688.
• SEQ ID NO: 49 is the probe sequence for TaqMan Assay 3688.
• SEQ ID NO: 50 is a primer sequence for TaqMan Assay 3689.
• SEQ ID NO: 51 is a primer sequence for TaqMan Assay 3689.
• SEQ ID NO: 52 is the probe sequence for TaqMan Assay 3689.
• SEQ ID NO: 53 is a primer sequence for TaqMan Assay 3690.
• SEQ ID NO: 54 is a primer sequence for TaqMan Assay 3690.
• SEQ ID NO: 55 is the probe sequence for TaqMan Assay 3690.
• SEQ ID NO: 56 is a primer sequence for TaqMan Assay 3691.
• SEQ ID NO: 57 is a primer sequence for TaqMan Assay 3691.
• SEQ ID NO: 58 is the probe sequence for TaqMan Assay 3691.
• the terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims.
• a cell refers to one or more cells, and in some embodiments can refer to a tissue and/or an organ.
• the phrase “at least one”, when employed herein to refer to an entity refers Docket no.82745-US-L-ORG-NAT-1 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 that entity, including but not limited to all whole number values between 1 and 100 as well as whole numbers greater than 100.
• allele refers to a variant or an alternative sequence form at a genetic locus.
• diploids a single allele is inherited by a progeny individual separately from each parent at each locus.
• the two alleles of a given locus present in a diploid organism occupy corresponding places on a pair of homologous chromosomes, although one of ordinary skill in the art understands that the alleles in any particular individual do not necessarily represent all of the alleles that are present in the species.
• the term “amplified” or “amplify” means the construction of multiple copies of a nucleic acid molecule or multiple copies complementary to the nucleic acid molecule using at least one of the nucleic acid molecules as a template.
• Amplification systems include the polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicase systems, transcription-based amplification system (TAS), and strand displacement amplification (SDA). See, e.g., Diagnostic Molecular Microbiology: Principles and Applications, PERSING et al., Ed., American Society for Microbiology, Washington, D.C.
• amplicon The product of amplification is termed an “amplicon.”
• the phrase “A, B, C, and/or D” includes A, B, C, and D individually, but also includes any and all combinations Docket no.82745-US-L-ORG-NAT-1 and subcombinations of A, B, C, and D (e.g., AB, AC, AD, BC, BD, CD, ABC, ABD, and BCD).
• one of 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).
• 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. As used herein, the phrase “consisting of” excludes any element, step, or ingredient not specifically recited.
• the disclosed subject matter thus also encompasses nucleic acids that encode polypeptides that in some embodiments consist essentially of amino acid sequences that are at least 95% identical to that SEQ ID NO: 2 or 3 as well as nucleic acids that encode polypeptides that in some embodiments consist of amino acid sequences that are at least 95% identical to that SEQ ID NO: 2 or 3.
• the methods for the disclosed subject matter comprise the steps that are disclosed herein, in some embodiments the methods for the presently disclosed subject matter consist essentially of the steps that are disclosed, and in some embodiments the methods for the presently disclosed subject matter consist of the steps that are disclosed herein.
• the term “de novo haploid induction” refers to the triggering 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. Docket no.82745-US-L-ORG-NAT-1
• the term “elite line” or “inbred line” refers to any line that has resulted from breeding and selection for superior agronomic performance.
• embryoids or “pseudoembryos” refers to clusters of embryo-like structures formed in vitro. These embryoids can form into fully grown plants.
• RNA e.g., mRNA, rRNA, tRNA, or snRNA
• Gene expression can be regulated at many stages in the process.
• expression may refer to the transcription of the antisense RNA only or the dsRNA only.
• expression refers to the transcription and stable accumulation of sense (mRNA) or functional RNA.
• “Expression” may also refer to the production of protein.
• the term “gene” refers to a hereditary unit including a sequence of DNA that occupies a specific location on a chromosome and that contains the genetic instruction for a particular characteristic or trait in an organism.
• the term “genotype” refers to the genetic constitution of a cell or organism.
• An individual's “genotype for a set of genetic markers” includes the specific alleles, for one or more genetic marker loci, present in the individual.
• a genotype can relate to a single locus or to multiple loci, whether the loci are related or unrelated and/or are linked or unlinked.
• an individual’s genotype relates to one or more genes that are related in that the one or more of the genes are involved in the expression of a phenotype of interest (e.g., a quantitative trait as defined herein).
• a genotype comprises a sum of one or more alleles present within an individual at one or more genetic loci of a quantitative trait.
• a genotype is expressed in terms of a haplotype (defined herein below).
• a haplotype defined herein below.
• the term “germplasm” refers to the totality of the genotypes of a population or another group of individuals (e.g., a species).
• the term “germplasm” can also refer to plant material; e.g., a group of plants that act as a repository for various alleles.
• adapted germplasm refers to plant materials of proven genetic superiority; e.g., for a Docket no.82745-US-L-ORG-NAT-1 given environment or geo-graphical area
• non-adapted germplasm refers to plant materials of unknown or unproven genetic value; e.g., for a given environment or geographical area; as such, the phrase “non- adapted germplasm” refers in some embodiments to plant materials that are not part of an established breeding population and that do not have a known relationship to a member of the established breeding population.
• a plant referred to as “haploid” has a single set (genome) of chromosomes and the reduced number of chromosomes (1n) in the haploid plant is equal to that of the gamete.
• a plant referred to as “doubled haploid” is developed by doubling the haploid set of chromosomes (from 1n to 2n). A plant or seed that is obtained from a doubled haploid plant that is selfed to any number of generations may still be identified as a doubled haploid plant. A doubled haploid plant is considered a homozygous plant.
• a plant is considered to be doubled haploid if it is fertile, even if the entire vegetative part of the plant does not consist of the cells with the doubled set of chromosomes; that is, a plant will be considered doubled haploid if it contains viable gametes, even if it is chimeric.
• heterologous when used in reference to a gene or nucleic acid refers to a gene encoding a factor that is not in its natural environment (i.e., has been altered by the hand of man).
• a heterologous gene may include a gene from one species introduced into another species.
• a heterologous gene may also include a gene native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to a non-native promoter or enhancer polynucleotide, etc.).
• Heterologous genes further may comprise plant gene polynucleotides that comprise cDNA forms of a plant gene; the cDNAs may be expressed in either a sense (to produce mRNA) or anti-sense orientation (to produce an anti-sense RNA transcript that is complementary to the mRNA transcript).
• heterologous genes are distinguished from endogenous plant genes in that the heterologous gene polynucleotide are typically joined to polynucleotides comprising regulatory elements such as promoters that are not found naturally associated with the gene for the protein encoded by the heterologous gene or with plant gene polynucleotide in the chromosome, or are associated with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed).
• a “heterologous” polynucleotide is a polynucleotide not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring polynucleotide.
• the term “heterozygous” means a genetic condition existing when different alleles reside at corresponding loci on homologous chromosomes.
• the term “homozygous” means a genetic condition existing when identical alleles reside at corresponding loci on homologous chromosomes.
• 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.
• nucleic acid or polynucleotide is not a nucleic acid as it occurs in its natural context, if it indeed has a naturally occurring counterpart.
• non-isolated nucleic acids are nucleic acids such as DNA and RNA, which are found in the state they exist in nature.
• a given polynucleotide e.g., a gene
• the isolated nucleic acid molecule may be present in single-stranded or double-stranded form. Alternatively, it may contain both the sense and antisense strands (i.e., the nucleic acid molecule may be double- stranded).
• the nucleic acid molecules of the present invention are understood to be isolated.
• locus refers to a position (e.g., of a gene, a genetic marker, or the like) on a chromosome of a given species.
• human-induced mutation refers to any mutation that occurs as a result of either direct or indirect human action. This term includes, but is not limited to, mutations obtained by any method of targeted mutagenesis.
• hybrid refers to offspring produced by crossing two genetically dissimilar parent plants. The resulting progeny of this cross are a “bi-parental” population.
• 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 marker or marker locus, through nucleic acid hybridization. Marker probes comprising about 8, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more contiguous nucleotides can be used for nucleic acid hybridization.
• the term “molecular marker” can be used to refer to a genetic marker, as defined above, or an encoded product thereof (e.g., a protein) used as a point of reference Docket no.82745-US-L-ORG-NAT-1 when identifying the presence/absence of a SCD or other locus of interest.
• a molecular marker can be derived from genomic nucleotide sequences or from expressed nucleotide sequences (e.g., from an RNA, a cDNA, etc.). The term also refers to nucleotide sequences complementary to or flanking the marker sequences, such as nucleotide sequences used as probes and/or primers capable of amplifying the marker sequence.
• Nucleotide sequences are “complementary” when they specifically hybridize in solution (e.g., according to Watson- Crick base pairing rules). This term also refers to the genetic markers that indicate a trait by the absence of the nucleotide sequences complementary to or flanking the marker sequences, such as nucleotide sequences used as probes and/or primers capable of amplifying the marker sequence.
• nucleotide sequence As used herein, the terms “nucleotide sequence,” “polynucleotide,” “nucleic acid sequence,” “nucleic acid molecule,” and “nucleic acid fragment” refer to a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural, and/or altered nucleotide bases.
• a “nucleotide” is a monomeric unit from which DNA or RNA polymers are constructed and consists of a purine or pyrimidine base, a pentose, and a phosphoric acid group.
• Nucleotides are referred to by their single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleotide.
• sequence identity 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.
• sequence identity refers to the value obtained using the Needleman and Wunsch algorithm ((1970) J. Mol.
• 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 Docket no.82745-US-L-ORG-NAT-1 EMBL-EBI search and sequence analysis tools APIs in 2019.” Madeira et al. Nucleic Acids Research, June 2019, 47(W1):W636-W641.
• equivalent program refers to any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by EMBOSS Needle.
• substantially identical nucleic acid or amino acid sequences may perform substantially the same function.
• introduction refers to both a natural and artificial process whereby genomic regions of one species, variety or cultivar are moved into the genome of another species, variety or cultivar, by crossing those species. The process may optionally be completed by backcrossing to the recurrent parent.
• ORF open reading frame
• an ORF refers to a nucleic acid sequence that encodes a polypeptide.
• an ORF comprises a translation initiation codon, a translation termination (i.e., stop) codon, and the nucleic acid sequence there between that encodes the amino acids present in the polypeptide.
• initiation codon and “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).
• 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.
• a phenotype is directly controlled by a single gene or genetic locus (i.e., corresponds to a “single gene trait”).
• a haploid plant evaluated for SCD phenotype can refer to fertile pollen shed and/or a seed obtained via pollination with that pollen.
• a phenotype is the result of interactions among several genes, which in some embodiments also results from an interaction of the plant and/or plant cell with its environment.
• the term “plant” can refer to a whole plant, any part thereof, or a cell or tissue culture derived from a plant.
• the term “plant” can refer to any of: whole plants, plant components or organs (e.g., leaves, stems, roots, etc.), plant tissues, seeds and/or plant cells, unless otherwise specified.
• a plant cell is a cell of a plant, taken from a plant, or derived through culture from a cell taken from a plant.
• plant cell includes without limitation cells within seeds, Docket no.82745-US-L-ORG-NAT-1 suspension cultures, embryos, meristematic regions, callus tissue, leaves, shoots, gametophytes, sporophytes, pollen, and microspores.
• plant part refers to a part of a plant, including single cells and cell tissues such as plant cells that are intact in plants, cell clumps, and tissue cultures from which plants can be regenerated.
• plant parts include, but are not limited to, single cells and tissues from pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems, shoots, and seeds; as well as scions, rootstocks, protoplasts, calli, and the like.
• population means a genetically heterogeneous collection of plants sharing a common genetic derivation.
• primer refers to an oligonucleotide which is capable of annealing to a nucleic acid target (in some embodiments, annealing specifically to a nucleic acid target) allowing a DNA polymerase and/or reverse transcriptase to attach thereto, thereby serving as a point of initiation of DNA synthesis when placed under conditions in which synthesis of a primer extension product is induced (e.g., in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH).
• one or more pluralities of primers are employed to amplify plant nucleic acids (e.g., using the polymerase chain reaction; PCR).
• 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.
• 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.
• the terms “progeny” and “progeny plant” refer to a plant generated from a vegetative or sexual reproduction from one or more parent plants.
• haploid induction the seed on the female parent is haploid, thus not a progeny of the inducing haploid line.
• the progeny of the haploid seed is not the only desired progeny.
• a progeny plant can be obtained by cloning or selfing a single parent plant, or by crossing two or more parental plants.
• 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 F1 or F2 or still further generations.
• An F1 is a first-generation progeny produced from parents at least one of which is used for the first time as donor of a Docket no.82745-US-L-ORG-NAT-1 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 F1s, F2s, and the like.
• An F1 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 F1 hybrids.
• the term “regenerate,” and grammatical variants thereof refers to the production of a plant from tissue culture.
• spontaneous haploid plant refers to a plant whose florets have undergone spontaneous doubling.
• tissue of a spontaneously doubled haploid plant may retain their haploid state (e.g., root, leaf, stem).
• 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.
• 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.
• haploid induction rate means the number of surviving haploid kernels over the total number of kernels after an ear is pollinated with haploid inducer pollen.
• HIR haploid induction rate
• the pollen starch biosynthesis pathway gene is selected from the group consisting of ADP glucose pyrophosphorylase, Waxy1, Hexokinase5, Phosphoglucomutase1 and Phosphoglucomutase2, and Invertase2.
• the ADP glucose pyrophosphorylase, Waxy1, Hexokinase5, Phosphoglucomutase1 and Phosphoglucomutase2, and Invertase2 genes comprise SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 13, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 12, respectively.
• the pollen starch biosynthesis pathway gene knockout may be achieved through gene editing while the knockdown may be achieved through RNAi.
• the gene editing is done using a site directed nuclease selected from the group consisting of a CRISPR nuclease, a meganuclease, a zinc-finger nuclease, and a transcription-activator like effector nuclease.
• the site-directed nuclease is a CRISPR nuclease
• the CRISPR nuclease is selected from the group consisting of Cas5, Cas6, Cas7, Cas8, Cas9, Cas12a, Cas12b, Cas12i, Cas12j, Cas12L, Cas12e, Cas12c, Cas12d, Cas12g, Cas12h, TnpB, Cas13a, Cas13b, Cas14, and nickase or deactivated versions thereof.
• the CRISPR nuclease is Cas12a.
• the knockout is a single gene knockout comprising use of two gRNAs and the two gRNAs comprise SEQ ID NOs: 15 and 16. In one embodiment, the knockout is a single gene knockout comprising use of two gRNAs and the two gRNAs comprise SEQ ID NOs: 18 and 19. In one embodiment, the knockout is a single gene knockout comprising use of two gRNAs and the two gRNAs comprise SEQ ID NOs: 21 and 22. In one embodiment, the knockout is a single gene knockout comprising use of two gRNAs and the two gRNAs comprise SEQ ID NOs: 24 and 25.
• the knockout comprises use of multiplexed gRNAs, wherein the gRNAs comprise SEQ ID NOs: 15, 16, 18, 19, 21, 22, 27, and 28.
• the increased androgenic response is increased at least 40% compared to the wild type.
• obtained microspores are selected from the group consisting of maize, rice, and wheat. The obtained microspores may be from maize. Also described herein are methods of generating a haploid plant. The methods comprise of first obtaining androgenic microspores, wherein the androgenic microspores have an increased androgenic response compared to the wild type. The obtained microspores are cultured, and a haploid plant is regenerated from said microspores.
• the increased androgenic response is achieved through knocking out or knocking down at least one pollen starch biosynthesis pathway gene.
• the pollen starch biosynthesis pathway gene is selected from the group consisting of ADP glucose pyrophosphorylase, Waxy1, Hexokinase5, Phosphoglucomutase1 and Phosphoglucomutase2, and Invertase2 and may comprise SEQ ID NOs: 11, SEQ ID NO: 14, SEQ ID NO: 13, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 12, respectively.
• the knockout may be achieved through gene editing while the knockdown may be achieved through RNAi.
• the gene editing may be done using a site directed nuclease selected from the group consisting of a CRISPR nuclease, a meganuclease, a zinc-finger nuclease, and a transcription-activator like effector nuclease.
• a site directed nuclease selected from the group consisting of a CRISPR nuclease, a meganuclease, a zinc-finger nuclease, and a transcription-activator like effector nuclease.
• the site directed nuclease is a CRISPR nuclease and CRISPR nuclease may be selected from the group consisting of Cas5, Cas6, Cas7, Cas8, Cas9, Cas12a, Cas12b, Cas12i, Cas12j, Cas12L, Cas12e, Cas12c, Cas12d, Cas12g, Cas12h, TnpB, Cas13a, Cas13b, Cas14, and nickase or deactivated versions thereof.
• the CRISPR nuclease may be Cas12a.
• the knockout is a single gene knockout comprising use of two gRNAs and the two gRNAs comprise SEQ ID NOs: 15 and 16. In one embodiment, the knockout is a single gene knockout comprising use of two gRNAs and the two gRNAs comprise SEQ ID NOs: 18 and 19. In one embodiment, the knockout is a single gene knockout comprising use of two gRNAs and the two gRNAs comprise SEQ ID NOs: 21 and 22. In one embodiment, the knockout is a single gene knockout comprising use of two gRNAs and the two gRNAs comprise SEQ ID NOs: 24 and 25.
• the knockout comprises use of multiplexed gRNAs, wherein the gRNAs comprise SEQ ID NOs: 15, 16, 18, 19, 21, 22, 27, and 28.
• the increased androgenic response is increased at least 40% compared to the wild type.
• obtained microspores are selected from the group consisting of maize, rice, and wheat. The obtained microspores may be from maize.
• the methods comprise doubling the chromosomes of the haploid plant to produce a doubled haploid plant. The chromosome doubling may be achieved via chemical induction or spontaneous doubling.
• chromosome doubling is achieved via chemical induction and achieved by a chemical selected from the group consisting of colchicine, trifluralin, pronamide, dithipyr, nitrous oxide, and oryzalin.
• the method of generating doubled haploid plants from microspores occurs in one generation. Also provided herein are plants produced from the methods described. EXAMPLES 1. Androgenesis protocol for testing present hypothesis A modified androgenesis protocol (See Zheng et al., 2003) was used to compare GE- modified microspores with wild type microspores for potential androgenesis (i.e., number of calli per microspores from 100 florets).
• Tassels with microspores at mid-to late-uninucleate stage were harvested, wrapped in aluminum foil, and stored in the dark at 6 – 8°C for 14 days. Then, the microspores were isolated with a blender and purified with maltose gradient centrifugation. Isolated microspores were cultured at 28°C in the dark with two fresh wheat ovaries per mL of culture media from wheat variety AC Nanda to generate calli or Docket no.82745-US-L-ORG-NAT-1 embryoids. This protocol was used as a baseline for comparing GE-modified microspores to non-modified microspores for androgenesis potential. 2.
• Transformable genotypes candidate gene selection, vector construction, and event selection Genotypes There were four genotypes (3 inbreds and 1 hybrid (F1)) tested to select options for later transformation to produce transgenic microspores.
• Hybrid 1 and Inbred 1 were selected for transformation to host the designed constructs based on their transformability data and preferences (Table 1). The inbred was selected solely for the FIREWORKS editing approach while the hybrid was selected for all eight constructs in Table 2 below.
• Table 1 List of genotypes tested for transformability to host constructs.
• T0 plants were generated through agrobacterium-mediated transformation following established procedures (insert a reference Zhonget al., 2018.). Genomic DNA and total RNA was isolated from the T0 plants and subjected to molecular analysis. A TaqMan copy number assay combined with targeted Next Generation Sequencing (NGS) or a TaqMan copy number assay combined with qRT-PCR was used to detect the knockout (KO) and knockdown (KD) events, respectively. T0 transgenic plants carrying monoallelic or biallelic mutations as well as a single copy of the transgene without vector backbone were selected as T0 KO plants. Transgenic plants showing reduced gene expression by qRT-PCR were selected as T0 KD events.
• NGS Next Generation Sequencing
• KD knockdown
• T0 events were grown in a greenhouse and crossed with the DH inbred line 1 as the female to generate T1 plants (T0 ⁇ inbred line 1). Androgenic response was evaluated on both T0 and T1 plants. Non-transgenic plants were used as a control. 3. Androgenesis evaluation on microspores In our evaluation, we observed three instances of increased androgenic response of GE- microspores of KO or KD genes at T0 generation as well as the subsequent T1 generation (produced by a first back cross). First, the most responsive KD T0 event was in the hybrid line 1 genotype from RNAi construct 25639.
• T1 generation microspores had a 300 to 600% increase of androgenic response.
• This increase in the T1 generation, compared to the T0 generation, may have been due to less stressed and short plants as was the case in T0.
• a FIREWORK construct (25619) simultaneously targeting several genes (ADP glucose pyrophosphorylase, Waxy1, Hexokinase 5, and Phosphoglucomutase) in inbred line 1 resulted in over a 400% increase of androgenic response in terms of embryoid-like structure (ELS) from cultured T0 microspores from 100 spikelets compared to non-edited microspores (wild type).
• ELS embryoid-like structure

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Genetics & Genomics (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Organic Chemistry (AREA)
• Molecular Biology (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Biotechnology (AREA)
• General Health & Medical Sciences (AREA)
• Biomedical Technology (AREA)
• General Engineering & Computer Science (AREA)
• Biochemistry (AREA)
• Microbiology (AREA)
• Medicinal Chemistry (AREA)
• Biophysics (AREA)
• Botany (AREA)
• Cell Biology (AREA)
• Physics & Mathematics (AREA)
• Plant Pathology (AREA)
• Nutrition Science (AREA)
• Virology (AREA)
• Developmental Biology & Embryology (AREA)
• Environmental Sciences (AREA)
• Gastroenterology & Hepatology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
• Medicines Containing Plant Substances (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=90455281

Family Applications (1)

Country Status (7)

Family Cites Families (2)

• 2023
  • 2023-09-18 CA CA3266969A patent/CA3266969A1/en active Pending
  • 2023-09-18 EP EP23869061.4A patent/EP4590833A2/de active Pending
  • 2023-09-18 WO PCT/US2023/074445 patent/WO2024064610A2/en not_active Ceased
  • 2023-09-18 CN CN202380067786.6A patent/CN119907859A/zh active Pending
  • 2023-09-18 AU AU2023347818A patent/AU2023347818A1/en active Pending
  • 2023-09-19 AR ARP230102487A patent/AR130520A1/es unknown
• 2025
  • 2025-03-19 CL CL2025000808A patent/CL2025000808A1/es unknown

Also Published As

Similar Documents

Legal Events