WO2020239680A2 - Stimulateur d'induction d'haploïdes - Google Patents

Stimulateur d'induction d'haploïdes Download PDF

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WO2020239680A2
WO2020239680A2 PCT/EP2020/064420 EP2020064420W WO2020239680A2 WO 2020239680 A2 WO2020239680 A2 WO 2020239680A2 EP 2020064420 W EP2020064420 W EP 2020064420W WO 2020239680 A2 WO2020239680 A2 WO 2020239680A2
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plant
sequence
seq
nucleic acid
haploid
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WO2020239680A3 (fr
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Monika KLOIBER-MAITZ
Milena OUZUNOVA
Christof BOLDUAN
Silke WIECKHORST
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KWS SAAT SE & Co. KGaA
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    • 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
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
    • A01H1/08—Methods for producing changes in chromosome number
    • 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

Definitions

  • the invention relates to the field of plant biotechnology, more particularly genetic engineering and gene editing, as well as plant breeding.
  • the invention provides nucleic acids which, after transcription or after expression in a plant, are suitable for enhancing the haploid induction capability of a haplotype inducer plant, such as caused by a mutated patatin-like phospholipase (PLA1).
  • PLA1 patatin-like phospholipase
  • both parent lines used in the induction cross are both diploids, so their gametes (egg cells and sperm cells) are haploids.
  • Haploid induction is frequently a medium to low penetrance trait of the inducer line, so the resulting progeny, depending on the species or situation, may be either diploid (if no genome loss takes place) or haploids (if genome loss does indeed take place).
  • haploid induction is something of a misnomer, because the haploid progeny produced will have a gametic chromosome number, and thus would not really be haploids, but rather diploids (if the parent is tetraploid) or triploids (if the parent is hexaploid) and so on.
  • haploids possess half the number of chromosomes of either parent; thus haploids of diploid organisms (e.g., maize) exhibit monoploidy; haploids of tetraploid organisms (e.g., ryegrasses) exhibit diploidy; haploids of hexaploid organisms (e.g., wheat) exhibit triploidy.
  • diploid organisms e.g., maize
  • haploids of tetraploid organisms e.g., ryegrasses
  • haploids of hexaploid organisms e.g., wheat
  • Haploid induction can occur during self-pollination or intercrossing of two lines within the same species, or it can occur during wide crosses, where it can be viewed as a hybridization barrier, preventing the formation of interspecific hybrids.
  • the most commonly employed method of inducing haploids is through the use of an intraspecific haploid inducer male line, which is primarily triggered by rearrangements of, mutations in, and/or recombinations, insertion, or deletions within a region of chromosome 1, specifically the PHOSPHOLIPASE Al (PLA1) gene, also known as NOT LIKE DAD1 (NLDI) and MATRILINEAL (MATL) (WO 2016/075255 Al; Kelliher et al. (2017).
  • PPA1 PHOSPHOLIPASE Al
  • MATRILINEAL MATRILINEAL
  • Haploid inducer (HI) maize lines contain a quantitative trait locus (QTL) on Chromosome 1 responsible for at least 66% of the variation in haploid induction. The QTL causes haploid induction at different rates when it is introgressed into various backgrounds. All maize haploid inducer lines used in the seed industry are derivatives of the founding HI line, known as Stock6, and all have the haploid inducer chromosome 1 QTL mutation.
  • haploid seed or embryos are specifically produced by making crosses between a haploid inducer male (i.e., haploid inducer pollen) and virtually any ear that one chooses - the ear could be of any inbred, hybrid, or other germplasm.
  • Haploids are produced when the haploid inducer pollen DNA is not fully transmitted and/or maintained through the first cell divisions of the embryos. The resulting phenotype is not fully penetrant, with some ovules containing haploid embryos, and others containing diploid embryos, aneuploid embryos, chimeric embryos, or aborted embryos.
  • the haploid kernels have embryos that contain only the maternal DNA plus normal triploid endosperm.
  • haploid embryos or seed are typically segregated from diploid and aneuploid siblings using a phenotypic or genetic marker screen and grown or cultured into haploid plants. These plants are then converted either naturally or via chemical manipulation (e.g., using an anti-microtubule agent such as colchicine) into doubled haploid (DH) plants which then produce inbred seed.
  • DH plants Plant breeding is facilitated by the use of doubled haploid (DH) plants.
  • the production of DH plants enables plant breeders to obtain inbred lines without multi- generational inbreeding, thus decreasing the time required to produce homozygous plants.
  • DH plants provide an invaluable tool to plant breeders, particularly for generating inbred lines, QTL mapping, cytoplasmic conversions, trait introgression, and F2 screening for high throughput trait improvement. A great deal of time is spared as homozygous lines are essentially generated in one generation, negating the need for multigenerational single-seed decent (conventional inbreeding).
  • DH plants are entirely homozygous, they are very amenable to quantitative genetics studies.
  • the production of haploid seed is critical for the doubled haploid breeding process. Haploid seed are produced on maternal germplasm when fertilized with pollen from a gynogenetic inducer, such as Stock 6 and Stock 6-derivative lines.
  • HIR8 locus had been narrowed down to a 789 kb region flanked by markers 4292232 and umcl867. Within the 789 kb intervals, there were 35 genes according to the B73 reference sequence annotated. However, the region is non-conserved and marker development in this region was not possible and candidate genes had not been selected based on expression and regulatory sequences or any other analyses.
  • the present invention is based on the identification of genes for the haploid inducer locus qhir8.
  • genes could be identified that, individually or in any combination, are responsible for and/or involved in haploid induction capability, in particular in enhancing the haploid induction capability of an inducer plant caused by a mutated patatin-like phospholipase (PLA1).
  • PHA1 mutated patatin-like phospholipase
  • HIR8 genes helps to improve the maize inducer and to develop an effective inducer in Sorghum or other species, in particular where the phospholipase system is functional for maternal induction.
  • Sorghum is of interest.
  • PLA1 gene of Sorghum are able to confer the capability of a haploid inducer to a sorghum plant. Due to the knowledge of the genetic nature of the HIR8 locus, the haploid inducer rates in Sorghum based on mutated PLA1 can be further increased.
  • Another application of the knowledge of the joint effect of HIR1 and HIR8 is the use haploid induction simultaneously with gene editing.
  • the disclosure of WO 2018/102816 relates to this concept and is herein incorporated by reference in its entirety.
  • the present invention is of particular relevance, given the lack of markers in the non- conserved regions.
  • the original markers used for haplotyping and annotation were based on B73.
  • the most interesting regulatory regions were missed working only with B73.
  • the previous failure to develop a TILLING population of RWS, necessitated identification of functional mutants instead of knockout mutants in a non-inducing line.
  • the invention in an aspect relates to means and methods for enhancing the induction capability of a haploid inducer plant, in particular caused by a mutated patatin-like phospholipase (PLA1). More particularly, nucleic acids are provided, which, after transcription or after expression in a plant, are suitable for enhancing the induction capability of an inducer plant caused by a mutated patatin-like phospholipase (PLA1) through manipulation of the identified gene(s).
  • the invention also relates to methods for producing a plant which is a carrier of an enhancer for the induction capability of an inducer plant, in particular caused by a mutated patatin-like phospholipase (PLA1) by mutagenizing an endogenous DNA sequence that is identical to a nucleic acid described herein or by transforming a plant with the nucleic acid(s) described herein, as well as methods and molecular markers for identifying such carriers.
  • the invention further relates to methods for producing plants that are suitable for use as haploid inductor, as well as the haploid inducer plants and parts thereof.
  • the invention further relates to methods for producing haploid plants or double haploid plants, in particular by crossing a plant with a haploid inducer plant according to the invention.
  • the invention also relates to haploid plants and double haploid plants.
  • the invention also relates to methods of editing plant genomic DNA using a haploid inducer plant according to the invention and transforming it so that it contains DNA coding for
  • a nucleic acid molecule comprising a nucleotide sequence, which:
  • (i) is a sequence selected from the group consisting of SEQ ID NOs: 1, 4, 5, 8, 10, 12, 15,
  • (ii) has a coding sequence selected from the group consisting of SEQ ID NOs: 2, 6, 13, 16,
  • (iii) is complementary to the sequence from (i) or (ii); or
  • (iv) is at least 80% identical to the sequence from (i) or (ii); or
  • (v) encodes a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7, 9, 11, 14, 17, 20, 23 and 37, or a functional part of the protein, preferably SEQ ID NOs: 3, 11, 17, and 37, or a functional part of the protein; or
  • (vi) encodes a protein comprising an amino acid sequence which is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 3, 7, 9, 11, 14, 17, 20, 23 and 37, or a functional part of the protein, preferably SEQ ID NOs: 3, 11, 17, and 37, or a functional part of the protein; or
  • a nucleic acid molecule comprising a nucleotide sequence, which:
  • (i) is a sequence selected from the group consisting of SEQ ID NOs: 24, 27, and 32, or a functional fragment thereof, preferably SEQ ID NO: 24, or a functional fragment thereof; or
  • (ii) has a coding sequence selected from the group consisting of SEQ ID NOs: 25, 28 and 33; preferably SEQ ID NO: 25; or
  • (iii) is complementary to the sequence from (i) or (ii); or
  • (iv) is at least 80% identical to the sequence from (i) or (ii); or
  • (v) encodes a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 29 and 34, or a functional part of the protein, preferably SEQ ID NO: 26, 29 and 34 or a functional part of the protein; or
  • (vi) encodes a protein comprising an amino acid sequence which is at least 80% identical to a sequence selected from the group consisting of SEQ ID NOs: 26, 29 and 34, or a functional part of the protein, preferably SEQ ID NO: 26, or a functional part of the protein; or hybridizes with a sequence from (iii) under stringent conditions.
  • nucleic acid according to statement 3 which is an RNAi molecule.
  • nucleic acid molecule according to any of statements 1 or 3-4, wherein said nucleic acid, after transcription or expression in a plant, or the nucleic acid molecule according to statement 2, wherein said nucleotide, after silencing in a plant, is suitable for enhancing the haploid induction capability of a haploid inducer plant.
  • nucleic acid molecule according to statement 5 wherein said haploid inducer plant comprises the HIR1 QTL.
  • haploid inducer plant comprises a mutated patatin-like phospholipase (PLA1).
  • nucleic acid molecule according to statement 8, wherein said PLA1 has a sequence identical to the PLA1 sequence of Stock 6, RWK, RWS, UH400, AX5707RS, or NP2222-mai/.
  • (iii) encoding the amino acid sequence of SEQ ID NO: 43, 49 or 50 or a sequence which is at least 80% identical to SEQ ID NO: 43, 49 or 50 comprises one or more mutation, preferably producing a change in the transcription or expression rate of the PLA1 sequence in a plant, in comparison to a non-mutagenized wild-type plant, or a change in the activity or stability of the encoded amino acid sequence in a plant, in comparison to a non-mutagenized wild-type plant, preferably wherein the mutation causes the property of a haploid inductor plant or increases the induction capability of a haploid inductor plant,
  • (A) a deletion or mutation in SEQ ID NO: 41, 42, 47 or 48, preferably which leads to an early stop codon causing a shortening of the encoded amino acid sequence, wherein preferably the amino acid sequence at the C-terminal end is shortened by 5 or more amino acids, and/or
  • (D) causes an amino acid exchange in the amino acid sequence of SEQ ID NO: 43, 49 or 50 at position 58, wherein arginine is replaced by glutamine (R58Q);
  • (E) causes an amino acid exchange in the amino acid sequence of SEQ ID NO: 43, 49 or 50 at position 74, wherein aspartate is replaced by asparagine (D74N);
  • (F) causes an amino acid exchange in the amino acid sequence of SEQ ID NO: 43, 49 or 50 at position 78, wherein glycine is replaced by arginine (G78R);
  • (G) causes an amino acid exchange in the amino acid sequence of SEQ ID NO: 43, 49 or 50 at position 160, wherein valine is replaced by isoleucine (V160I);
  • (I) is a knock-out mutation.
  • a vector comprising the nucleic acid according to any of statements 1 to 11.
  • a plant cell comprising the nucleic acid or vector according to any of statements 1 to 17.
  • a plant or a part thereof comprising the plant cell according to statement 18.
  • a method for producing a (transgenic) plant preferably a (transgenic) plant which is carrier of an enhancer for the haploid induction capability of an inducer plant, preferably a haploid inducer plant as defined in any of statements 5 to 11 comprising: al) transforming at least one plant cell with a nucleic acid molecule according to statement 1 or 3 or a vector according to any of statements 11 to 17 for overexpression of myosin heavy chain,
  • a method for producing a (mutated) plant preferably a (mutated) plant which is carrier of an enhancer for the haploid induction capability of an inducer plant, preferably a haploid inducer plant as defined in any of statements 5 to 11
  • said mutation has at least one mutation in a regulatory sequence of said endogenous DNA sequence, wherein said mutation produces an increase in the transcription or expression rate of the endogenous DNA sequence that is identical to a nucleic acid according to statement 1 in the identified plant or a decrease in the transcription or expression rate of the endogenous DNA sequence that is identical to a nucleic acid according to statement 2 in the identified plant, in comparison to a non-mutagenized wild-type plant, or wherein said mutation produces an increased activity or stability of a protein or polypeptide encoded by the endogenous DNA sequence that is identical to a nucleic acid according to statement 1 in the identified plant or an decreased activity or stability of a protein or polypeptide encoded by the endogenous DNA sequence that is identical to a nucleic acid according to statement 2 in the identified plant, in comparison to a non-mutagenized wild-type plant, preferably wherein said at least one mutation causes the property of an enhancer of the haploid induction capability of a haploid inducer plant, preferably a hap
  • a method for producing a plant preferably a plant which comprises or which is carrier of an enhancer for the haploid induction capability of an inducer plant, preferably a haploid inducer plant as defined in any of statements 5 to 11 comprising overexpressing the myosin heavy chain gene or protein, or increasing the myosin heavy chain mRNA or protein stability or functionality.
  • a method for producing a plant preferably a plant which comprises or which is carrier of an enhancer for the haploid induction capability of an inducer plant, preferably a haploid inducer plant as defined in any of statements 5 to 11 comprising repressing the aquaporin gene or protein, or decreasing the aquaporin mRNA or protein stability or functionality.
  • a method for producing a plant preferably a plant which comprises or which is carrier of an enhancer for the haploid induction capability of an inducer plant, preferably a haploid inducer plant as defined in any of statements 5 to 11 comprising mutating the PPR gene, preferably resulting in a PPR protein having the sequence as set forth in SEQ ID NO: 17.
  • step a) is a cell comprising a mutated patatin-like phospholipase (PLA1), preferably as defined in any one of the statements 9-11 .
  • PHA1 patatin-like phospholipase
  • a plant preferably a Zea mays or Sorghum bicolor plant, comprising
  • nucleotide sequence encoding the amino acid according to SEQ ID NO: 11, or
  • a plant preferably a Zea mays or Sorghum bicolor plant, comprising
  • nucleotide sequence encoding the amino acid according to SEQ ID NO: 40.
  • Method for identification/selection of a an enhancer for the haploid induction capability of an inducer plant preferably a haploid inducer plant as defined in any of statements 5 to 11, comprising steps of
  • step b)(iii) or step b)(iv) comprises comparing the expression level and/or activity of said one or more gene with a predetermined threshold.
  • a method for the production of a haploid plant or doubled haploid plant comprising the following steps:
  • step b) generating a haploid plant from the seed or embryo selected in step b),
  • d) optionally, doubling the set of chromosomes via a chromosome doubling agent or via spontaneous doubling, optionally wherein the doubling agent is colchicine, pronamide, dithipyr, or trifluralin.
  • An isolated polynucleic acid specifically hybridising with a nucleotide sequence molecule of SEQ ID NO: 1, 2, 4, 10, 15, 16, 17, 24, 25, 30, 35, 36, 38, or 39; or the complement or the reverse complement thereof.
  • a method of editing or modifying plant genomic DNA or RNA comprising:
  • first plant is a haploid inducer plant as obtained by a method according to any of statements 21 to 27 or 36, or a plant according to any of statements 19, 28 to 33, or 37, and wherein said first plant is capable of expressing a DNA or RNA modification enzyme and optionally at least one guide nucleic acid;
  • the second plant comprises the plant genomic DNA or RNA which is to be edited or modified
  • step (c) selecting at least one haploid progeny produced by the pollination of step (c) wherein the haploid progeny comprises the genome of the second plant but not the first plant, and the genome of the haploid progeny has been edited or modified by the DNA or RNA editing or modification enzyme and optional at least one guide nucleic acid delivered by the first plant. 41.
  • the DNA or RNA editing or modification enzyme is a site-directed nuclease selected from the group consisting of meganucleases (MNs), zinc-finger nucleases (ZFNs), transcription-activator like effector nucleases (TALENs), Cas nucleases, such as Cas9 nuclease, Cfpl nuclease, Casl3 nuclease, dCas9-Fokl, dCpfl-Fokl, chimeric Cas9- cytidine deaminase, chimeric Cas9-adenine deaminase, chimeric FENI-Fokl, and Mega-TALs, a nickase Cas9 (nCas9), chimeric dCas9 non-Fokl nuclease and dCpfl non-Fokl nuclease;
  • MNs meganucleases
  • ZFNs zinc-finger nucleases
  • DNA or RNA modification enzyme is as defined in any of statements 50 to 62.
  • chromosome doubling agent is colchicine, pronamide, dithipyr, trifluralin, or another known anti-microtubule agent.
  • polymorphism is a SNP, deletion or insertion
  • exemplary polymorphisms are given in table 2.
  • a plant or plant part obtainable by the method according to any of statements 21 to 27, 36, or 40, or a plant according to any of statements 19, 28 to 33, 37, or 45 comprising or expressing a DNA or RNA modification enzyme, or a nucleic acid encoding a DNA or RNA modification enzyme, and optionally a guide nucleic acid.
  • DNA or RNA modification enzyme is a ZFN, TALEN, meganuclease, or a Cas effector protein.
  • Cas effector protein is a type II, type V (such as type Va or type Vb), or type VI (such as type Via or type Vlb) effector protein.
  • Cas effector protein is Cas9, Casl2 (such as Casl2a or Casl2b), or Casl3 (such as Casl3a or Casl3b).
  • heterologous domain comprises base editing activity, nucleotide deaminase activity, methylase activity, demethylase activity, translation activation activity, translation repression activity, transcription activation activity, transcription repression activity, transcription release factor activity, chromatin modifying or remodeling activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-strand RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity, and nucleic acid binding activity.
  • Figure 1 Protein/sequence map of a fragment of the myosin (Zea mays) protein indicating the mutation V325A caused by a mutation in the coding sequence of the myosin gene. Protein sequences of wildtype (SEQ ID NO: 9) and mutated (SEQ ID NO: 11) myosin fragments are shown.
  • the terms "one or more” or “at least one”, such as one or more or at least one member(s) of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g., any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members.
  • haploid inducer and "haploid inductor” are used as synonyms herein and refer to a plant that is capable of producing fertilized seeds or embryos which have a single (haploid) chromosome set from a crossing with a plant of the same genus, preferably a plant of the same species, which is not a haploid inducer.
  • Haploid induction is frequently a medium to low penetrance trait of the inducer line, so the resulting progeny, depending on the species or situation, may be either diploid (if no genome loss takes place) or haploids (if genome loss does indeed take place).
  • haploid induction is something of a misnomer, because the haploid progeny produced will have a gametic chromosome number, and thus would not really be haploids, but rather diploids (if the parent is tetraploid) or triploids (if the parent is hexaploid) and so on.
  • haploids possess half the number of chromosomes of either parent; thus haploids of diploid organisms (e.g., maize) exhibit monoploidy; haploids of tetraploid organisms (e.g., ryegrasses) exhibit diploidy; haploids of hexaploid organisms (e.g., wheat) exhibit triploidy.
  • diploid organisms e.g., maize
  • haploids of tetraploid organisms e.g., ryegrasses
  • haploids of hexaploid organisms e.g., wheat
  • any haploid inducer can be used.
  • a haploid inducer as referred to herein can be a plant selected and/or derived from the lines Stock 6, RWK, RWS, UH400, AX5707RS, NP2222-mai/, or any other haploid inducer line, in particular a line comprising the HIR1 QTL.
  • a haploid inducer as referred to herein can be a mutant plant selected from the plants as disclosed in US 2017/327832 A1 (or a plant of a different species having the corresponding mutation(s)), incorporated herein by reference in its entirety, in particular the tilling mutants having a D74N (exchange of aspartate at Position 74 for asparagin) or G78R (exchange of glycine at Position 78 for arginine) in the PLA1 gene of maize. It will be understood that corresponding amino acids can be mutated in other plants.
  • a haploid inducer as referred to herein can be a mutant plant selected from the plants as described in WO 2018/158301 (or a plant of a different species having the corresponding mutation(s)), incorporated herein by reference in its entirety.
  • a haploid inducer as referred to herein can be a plant with mutated PLA1 according to SEQ ID NOs: 38-40 or 44-46, or as defined herein elsewhere.
  • a haploid inducer as referred to herein can be a mutant plant selected from the plants as described in Kalinowska et al. (2016) "State-of-the-art and novel developments of in vivo haploid technologies"; Theor. Appl. Genetics; 132(3):593-605; incorporated herein by reference in its entirely.
  • PLA1 refers to patatin-like phospholipase, also known as NOT LIKE DAD1 (NLDI) and MATRILINEAL (MATL), as for instance defined in Kelliher et al. (2017).
  • MATRILINEAL a sperm-specific phospholipase, triggers maize haploid induction. Nature, 542(7639), 105.
  • PLA1 may originate from maize but equally the PLA1 orthologues may be used herein, such as Sorghum PLA1.
  • wild type PLA1 or PLA1 originating from a line which is not a haploid inducer (or a haploid inducer which is not caused by PLA1 mutations) is PLA1 from PH207 or B73, i.e. PLA1 having a genomic, coding, or protein sequence (substantially) identical to the PLA1 sequence from PH207 or B73, or an orthologue thereof.
  • wild type PLA1 or PLA1 originating from a line which is not a haploid inducer is PLA1 having a genomic, coding, or protein sequence which is at least 80% identical (preferably over the entire length) to the PLA1 sequence from PH207 or B73 (or an orthologue thereof), preferably at least 85%, such as at least 86%, 87%, 88%, 89%, more preferably at least 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • wild type PLA1 or PLA1 originating from a line which is not a haploid inducer is PLA1 having a genomic sequence which is at least 80% identical (preferably over the entire length) to the PLA1 sequence as set forth in SEQ ID NO: 41 or 47 (or an orthologue thereof), preferably at least 85%, such as at least 86%, 87%, 88%, 89%, more preferably at least 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • wild type PLA1 or PLA1 originating from a line which is not a haploid inducer is PLA1 having a coding sequence which is at least 80% identical (preferably over the entire length) to the PLA1 sequence as set forth in SEQ ID NO: 42 or 48 (or an orthologue thereof), preferably at least 85%, such as at least 86%, 87%, 88%, 89%, more preferably at least 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • wild type PLA1 or PLA1 originating from a line which is not a haploid inducer is PLA1 having a protein sequence which is at least 80% identical (preferably over the entire length) to the PLA1 sequence as set forth in SEQ ID NO: 43, 49, or 50 (or an orthologue thereof), preferably at least 85%, such as at least 86%, 87%, 88%, 89%, more preferably at least 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • wild type PLA1 variants such as orthologues
  • wild type PLA1 preferably excludes any of the PLA1 mutations described below.
  • such variants have comparable expression levels, stability, and/or activity as PLA1 from PH207 or B73, i.e. expression, stability, and/or activity does not differ by more than 100%, preferably does not differ by more than 50%.
  • mutated PLA1 as referred to herein causes the property of a haploid inducer plant or increases the induction capability of a haploid inducer plant.
  • PLA1 having the wild type sequence as set forth in SEQ ID NOs: 41-43 or SEQ ID NOs: 47-50 is mutated.
  • mutated PLA1 as referred to herein has an altered (mRNA or protein) expression level compared to (unmutated) wild type PLA1 (such as PH207 of B73 PLA1). In certain embodiments, mutated PLA1 as referred to herein has an altered activity compared to (unmutated) wild type PLA1 (such as PH207 of B73 PLA1). In certain embodiments, mutated PLA1 as referred to herein has an altered stability compared to (unmutated) wild type PLA1 (such as PH207 of B73 PLA1). In certain embodiments, mutated PLA1 as referred to herein has a reduced (mRNA or protein) expression level compared to (unmutated) wild type PLA1 (such as PH207 of B73 PLA1).
  • mutated PLA1 as referred to herein has a reduced activity compared to (unmutated) wild type PLA1 (such as PH207 of B73 PLA1). In certain embodiments, mutated PLA1 as referred to herein has a reduced stability compared to (unmutated) wild type PLA1 (such as PH207 of B73 PLA1). Altered or decreased expression, activity, and stability are to be interpreted as defined herein elsewhere. In certain embodiments PLA1 having the wild type sequence as set forth in SEQ ID NOs: 41-43 or SEQ ID NOs: 47-50 is mutated to have an altered (decreased) expression, activity, and/or stability.
  • mutated PLA1 as referred to herein has an altered or reduced expression level, activity, and/or stability as described above, and causes the property of a haploid inducer plant or increases the induction capability of a haploid inducer plant.
  • PLA1 having the wild type sequence as set forth in SEQ ID NOs: 41-43 or SEQ ID NOs: 47-50 is mutated to have an altered (decreased) expression, activity, and/or stability, and causes the property of a haploid inducer plant or increases the induction capability of a haploid inducer plant.
  • mutated PLA1 (genomic sequence, coding sequence, or protein sequence) as referred to herein is a truncated PLA1.
  • mutated PLA1 (genomic sequence, coding sequence, or protein sequence) as referred to herein comprises one or more deletion such as an internal deletion of one or more nucleotides or amino acids, or a 3' and/or 5' or N-terminal and/or C-terminal deletion of one or more nucleotides or amino acids.
  • mutated PLA1 (genomic sequence or coding sequence) as referred to herein comprises a premature stop codon.
  • mutated PLA1 (genomic sequence or coding sequence) as referred to herein comprises a frame shift. In certain embodiments, mutated PLA1 (genomic sequence or coding sequence) as referred to herein comprises a frame shift resulting in a premature stop codon. In certain embodiments, mutated PLA1 (genomic sequence or coding sequence) as referred to herein comprises a (point) mutation resulting in a premature stop codon. In certain embodiments, mutated PLA1 (genomic sequence or coding sequence) as referred to herein comprises a deletion resulting in a premature stop codon. In certain embodiments PLA1 having the wild type sequence as set forth in SEQ ID NOs: 41-43 or SEQ ID NOs: 47-50 is truncated, has a deletion, frame shift, point mutation, premature stop codon, as defined above.
  • mutated PLA1 (genomic sequence, coding sequence, or protein sequence) as referred to herein comprises a mutation (such as deletion, truncation, or frame shift) wherein the amino acid sequence at the C-terminal end is shortened by 5 or more amino acids, preferably 10 or more amino acids, such as 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, or 100 or more amino acids.
  • mutated PLA1 (genomic sequence, coding sequence, or protein sequence) as referred to herein comprises a mutation (such as deletion, truncation, or frame shift) wherein the amino acid sequence at the C-terminal end is shortened by 5 to 100 amino acids, preferably 10 to 100 amino acids, such as 15 to 100 amino acids, 10 to 90 amino acids, 15 to 90 amino acids, 10 to 80 amino acids, 15 to 80 amino acids, 10 to 70 amino acids, 15 to 70 amino acids, 10 to 60 amino acids, 15 to 60 amino acids.
  • PLA1 having the wild type sequence as set forth in SEQ ID NOs: 41-43 or SEQ ID NOs: 47-50 is C-terminally shortened as defined above.
  • mutated PLA1 (genomic sequence, coding sequence, or protein sequence) as referred to herein comprises a destabilizing mutation, i.e. a mutation affecting protein stability.
  • PLA1 having the wild type sequence as set forth in SEQ ID NOs: 41-43 or SEQ ID NOs: 47-50 comprises a destabilizing mutation.
  • mutated PLA1 (genomic sequence, coding sequence, or protein sequence) as referred to herein comprises a point mutation. In certain embodiments, mutated PLA1 (genomic sequence, coding sequence, or protein sequence) as referred to herein comprises a mutation in which one or more amino acids are exchanged. In certain embodiments, mutated PLA1 (genomic sequence, coding sequence, or protein sequence) as referred to herein comprises a mutation which causes an amino acid exchange between amino acid positions 57 and 289, referenced to SEQ ID NO: 43, 49, or 50, i.e.
  • mutated PLA1 comprises a mutation which causes an amino acid exchange at position 58, referenced to SEQ ID NO: 43, 49, or 50, preferably wherein the amino acid is replaced by glutamine (58Q), preferably wherein arginine is replaced by glutamine (R58Q).
  • mutated PLA1 (genomic sequence, coding sequence, or protein sequence) as referred to herein comprises a mutation which causes an amino acid exchange at position 74, referenced to SEQ ID NO: 43, 49, or 50, preferably wherein the amino acid is replaced by asparagine (74N), preferably wherein aspartate is replaced by asparagine (D74N).
  • mutated PLA1 (genomic sequence, coding sequence, or protein sequence) as referred to herein comprises a mutation which causes an amino acid exchange at position 78, referenced to SEQ ID NO: 43, 49, or 50, preferably wherein the amino acid is replaced by arginine (78R), preferably wherein glycine is replaced by arginine (G78R).
  • mutated PLA1 (genomic sequence, coding sequence, or protein sequence) as referred to herein comprises a mutation which causes an amino acid exchange at position 160, referenced to SEQ ID NO: 43, 49, or 50, preferably wherein the amino acid is replaced by isoleucine (1601), preferably wherein valine is replaced by isoleucine (V160I).
  • mutated PLA1 (genomic sequence, coding sequence, or protein sequence) as referred to herein comprises a mutation which causes an amino acid exchange at position 288, referenced to SEQ ID NO: 43, 49, or 50, preferably wherein the amino acid is replaced by leucine (288L), preferably wherein serine is replaced by leucine (S288L).
  • PLA1 having the wild type sequence as set forth in SEQ ID NOs: 41-43 or SEQ ID NOs: 47-50 comprises a point mutation/amino acid exchange as described above.
  • mutated PLA1 as referred to herein has a protein sequence as set forth in SEQ ID NO: 40 or 46, or a sequence at least 80%, preferably at least 85%, such as at least 90% or 95% identical (preferably over the entire length) to SEQ ID NO: 40 or 46 (with the proviso that such variant is a haploid inducer), or an orthologue comprising the corresponding mutation.
  • mutated PLA1 as referred to herein comprises a mutation in one or more of its regulatory sequences (in particular the promoter). In certain embodiments, mutated PLA1 (genomic sequence) as referred to herein, comprises a mutation in one or more of its regulatory sequences (in particular the promoter) which affect transcription. In certain embodiments, mutated PLA1 (genomic sequence) as referred to herein, comprises a mutation in one or more of its regulatory sequences (in particular the promoter) which reduce or (substantially) eliminate transcription. In certain embodiments PLA1 having the wild type sequence as set forth in SEQ ID NOs: 41-43 or SEQ ID NOs: 47-50 comprises a mutation in one or more of its regulatory sequences as defined above.
  • mutated PLA1 as referred to herein is knocked out or knocked down.
  • haploid induction rate or “induction rate” of a haploid inducer refers to the number or percentage of fertilized seeds or embryos that have a haploid chromosome set and which have arisen from a crossing of the haploid inducer with a plant of the same genus (preferably, a plant of the same species) which is not a haploid inducer.
  • the haploid induction rate of a haploid inducer in particular a haploid inducer comprising a mutated patatin-like phospholipase (PLA1), can be increased with at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8% or 0.9%, preferably with at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%, more preferably with at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 50%.
  • the number of fertilized seeds or embryos which have a haploid chromosome set and which have arisen from a crossing of the haploid inducer with a plant of the same genus (preferably, a plant of the same species) which is not a haploid inducer may thus be higher by at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8% or 0.9%, preferably at least 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%, more preferably, at least 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30% or 50%, than the number of haploid fertilized seeds or embryos which is achieved without the use of the nucleic acid as described herein.
  • plant includes whole plants, including descendants or progeny thereof.
  • plant part includes any part or derivative of the plant, including particular plant tissues or structures, plant cells, plant protoplast, plant cell or tissue culture from which plants can be regenerated, plant calli, plant clumps and plant cells that are intact in plants or parts of plants.
  • plant parts include fertilized or unfertilized seeds, embryos, pollen, ovules, leaves, roots, root tips, anthers, flowers, fruits, stems, shoots, and the like.
  • Plant parts may include processed plant parts or derivatives, including flower, oils, extracts etc.
  • the plant part or derivative comprises, consists of, or consists essentially of one or more, preferably all of stalks, leaves, and cobs. In certain embodiments, the plant part or derivative is leaves. In certain embodiments, the plant part or derivative is stalks. In certain embodiments, the plant part or derivative is cobs. In certain embodiments, the plant part or derivative comprises, consists of, or consists essentially of one or more, preferably all of stalks and leaves. In certain embodiments, the plant part or derivative comprises, consists of, or consists essentially of one or more, preferably all of stalks, and cobs. In certain embodiments, the plant part or derivative comprises, consists of, or consists essentially of one or more, preferably all of leaves and cobs.
  • the plant part or derivative is not (functional) propagation material, such as germplasm, a seed, or plant embryo or other material from which a plant can be regenerated. In certain embodiments, the plant part or derivative does not comprise (functional) male and female reproductive organs. In certain embodiments, the plant part or derivative is or comprises propagation material, but propagation material which does not or cannot be used (anymore) to produce or generate new plants, such as propagation material which have been chemically, mechanically or otherwise rendered non-functional, for instance by heat treatment, acid treatment, compaction, crushing, chopping, etc. in certain preferred embodiments, the plant part is corn cobs or stover.
  • a "plant” may be of any species from the dicotyledon, monocotyledon, and gymnosperm plants.
  • Non-limiting examples include Hordeum vulgare, Sorghum bicolor, Secale cereale, Triticale, Saccharum officinarium, Zea mays, Setaria italic, Oryza sativa, Oryza minuta, Oryza australiensis, Oryza alta, Triticum aestivum, Triticum durum, Hordeum bulbosum, Brachypodiurn distachyon, Hordeum marinum, Aegilops tauschii, Beta vulgaris, Helianthus annuus, Daucus glochidiatus, Daucus pusillus, Daucus muricatus, Daucus carota, Eucalyptus grandis, Erythranthe guttata, Genlisea aurea, Gossypium sp., Musa sp
  • maize refers to a plant of the species Zea mays, preferably Zea mays ssp mays.
  • B73 is a maize breeding line that is used as a model genotype in maize genetics and was used to create the first maize reference sequence.
  • the B73 reference genome as defined herein is the B73 AGPv2 genome and refers to the assembly B73 RefGen_v2 (also known as AGPv2, B73 RefGen_v2) as provided on the Maize Genetics and Genomics Database
  • RWS is a haploid inducer line based on Stock 6, and has the haploid inducer chromosome 1 QTL mutation (Hll).
  • W22 is a non-haploid inducer line but carrier of the enhancer allele on HIR8 locus.
  • sorghum refers to a plant of the genus Sorghum, and includes without limitation Sorghum bicolor, Sorghum sudanense, Sorghum bicolor c Sorghum sudanense, Sorghum c almum (Sorghum bicolor c Sorghum halepense), Sorghum arundinaceum, Sorghum drummondii, Sorghum halepense and/or Sorghum propinguum.
  • a "functional fragment" of a nucleotide sequence as used herein means a segment of a nucleotide sequence which has the functionality identical or comparable to the complete nucleotide sequence from which the functional fragment originates.
  • the functional fragment may possess a nucleotide sequence which is identical or homologous to the complete nucleotide sequence over a length of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94% 96%, 97%, 98% or 99%.
  • a "functional fragment" of a nucleotide sequence may also mean a segment of a nucleotide sequence which alters the functionality of the total nucleotide sequence, e.g., in the course of post-transcriptional gene silencing.
  • the functional fragment of a nucleotide sequence may include at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25, preferably at least 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120 or 140, more preferably at least 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or 1000 successive nucleotides of the complete nucleotide sequence.
  • a “functional part” of a protein means a segment of a protein, or a section of the amino acid sequence, that encodes for the protein, wherein the segment may exert functionality identical or comparable to the entire protein in a plant cell.
  • a functional part of a protein has, over a length of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96%, 97%, 98%, or 99%, an identical or— under conservative and semi-conservative amino acid exchanges— similar amino acid sequence to that of the protein from which the functional part originates.
  • sequence identity refers to the degree of identity between any given nucleic acid sequence and a target nucleic acid sequence. Percent sequence identity is calculated by determining the number of matched positions in aligned nucleic acid sequences, dividing the number of matched positions by the total number of aligned nucleotides, and multiplying by 100. A matched position refers to a position in which identical nucleotides occur at the same position in aligned nucleic acid sequences. Percent sequence identity can also be determined for any amino acid sequence.
  • a target nucleic acid or amino acid sequence is compared to the identified nucleic acid or amino acid sequence using the BLAST 2 Sequences (BI2seq) program from the stand-alone version of BLASTZ containing BLASTN and BLASTP.
  • This stand-alone version of BLASTZ can be obtained from Fish & Richardson's web site (World Wide Web at fr.com/blast) or the U.S. government's National Center for Biotechnology Information web site (World Wide Web at ncbi.nlm.nih.gov). Instructions explaining how to use the BI2seq program can be found in the readme file accompanying BLASTZ.
  • BI2seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm.
  • BLASTN is used to compare nucleic acid sequences
  • BLASTP is used to compare amino acid sequences.
  • the options are set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (e.g. , C: ⁇ seq I .txt); -j is set to a file containing the second nucleic acid sequence to be compared (e.g. , C: ⁇ seq2.txt); -p is set to blastn; -o is set to any desired file name (e.g.
  • C ⁇ output.txt
  • -q is set to - 1
  • -r is set to 2; and all other options are left at their default setting.
  • the following command will generate an output file containing a comparison between two sequences: C: ⁇ B12seq -i c: ⁇ seql .txt -j c: ⁇ seq2.txt -p blastn -o c: ⁇ output.txt -q - 1 -r 2. If the target sequence shares homology with any portion of the identified sequence, then the designated output file will present those regions of homology as aligned sequences. If the target sequence does not share homology with any portion of the identified sequence, then the designated output file will not present aligned sequences.
  • a length is determined by counting the number of consecutive nucleotides from the target sequence presented in alignment with the sequence from the identified sequence starting with any matched position and ending with any other matched position.
  • a matched position is any position where an identical nucleotide is presented in both the target and identified sequences. Gaps presented in the target sequence are not counted since gaps are not nucleotides. Likewise, gaps presented in the identified sequence are not counted since target sequence nucleotides are counted, not nucleotides from the identified sequence.
  • the percent identity over a particular length is determined by counting the number of matched positions over that length and dividing that number by the length followed by multiplying the resulting value by 100.
  • 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2. It also is noted that the length value will always be an integer.
  • isolated nucleic acid sequence refers to a nucleic acid sequence which is no longer in the natural environment from which it was isolated, e.g. the nucleic acid sequence in a bacterial host cell or in the plant nuclear or plastid genome.
  • sequence When referring to a “sequence” herein, it is understood that the molecule having such a sequence is referred to, e.g. the nucleic acid molecule.
  • a "host cell” or a “recombinant host cell” or “transformed cell” are terms referring to a new individual cell (or organism) arising as a result of at least one nucleic acid molecule, having been introduced into said cell.
  • the host cell is preferably a plant cell or a bacterial cell.
  • the host cell may contain the nucleic acid as an extra-chromosomally (episomal) replicating molecule, or comprises the nucleic acid integrated in the nuclear or plastid genome of the host cell, or as introduced chromosome, e.g. minichromosome.
  • the nucleic acid molecule as described herein comprises less than 50000 nucleotides. In certain embodiments, the nucleic acid molecule as described herein comprises less than 40000 nucleotides. In certain embodiments, the nucleic acid molecule as described herein comprises less than 30000 nucleotides. In certain embodiments, the nucleic acid molecule as described herein comprises less than 25000 nucleotides. In certain embodiments, the nucleic acid molecule as described herein comprises less than 20000 nucleotides. In certain embodiments, the nucleic acid molecule as described herein comprises less than 15000 nucleotides. In certain embodiments, the nucleic acid molecule as described herein comprises less than 10000 nucleotides.
  • the nucleic acid molecule as described herein comprises less than 5000 nucleotides. In certain embodiments, the nucleotide molecule as described herein comprises at least 100 nucleotides. In certain embodiments, the nucleic acid molecule as described herein comprises at least 100 nucleotides and less than 50000 nucleotides. In certain embodiments, the nucleic acid molecule as described herein comprises at least 100 nucleotides and less than 40000 nucleotides. In certain embodiments, the nucleic acid molecule as described herein comprises at least 100 nucleotides and less than 30000 nucleotides.
  • the nucleic acid molecule as described herein comprises at least 100 nucleotides and less than 25000 nucleotides. In certain embodiments, the nucleic acid molecule as described herein comprises at least 100 nucleotides and less than 20000 nucleotides. In certain embodiments, the nucleic acid molecule as described herein comprises at least 100 nucleotides and less than 15000 nucleotides. In certain embodiments, the nucleic acid molecule as described herein comprises at least 100 nucleotides and less than 10000 nucleotides. In certain embodiments, the nucleic acid molecule as described herein comprises at least 100 nucleotides and less than 5000 nucleotides.
  • nucleic acid sequence e.g. DNA or genomic DNA
  • nucleic acid sequence identity to a reference sequence or having a sequence identity of at least 80%>, e.g. at least 85%, 90%, 95%, 98%> or 99%> nucleic acid sequence identity to a reference sequence
  • said nucleotide sequence is considered substantially identical to the given nucleotide sequence and can be identified using stringent hybridisation conditions.
  • nucleic acid sequence comprises one or more mutations compared to the given nucleotide sequence but still can be identified using stringent hybridisation conditions.
  • hybridizing and “hybridization” refer to a process in which a single-stranded nucleic acid molecule is added to a nucleic acid strand that is complementary to the greatest possible extent, i.e., enters into base pairing.
  • Standard methods for hybridization are described in Sambrook et al. 2001, for example.
  • stringency relates to the hybridization conditions. High stringency is present when a base pairing is made more difficult; low stringency is present if a base pairing is made easier.
  • the stringency of the hybridization conditions depends upon the salt concentration, or ion strength, and the temperature. In general, the stringency may be increased by increasing the temperature and/or decreasing the salt content.
  • stringent hybridization conditions is meant herein those conditions under which a hybridization predominantly occurs only between homologous nucleic acid molecules.
  • hybridization conditions thereby relates not only to the conditions prevailing in the actual addition of the nucleic acids, but also to the conditions prevailing in the following washing steps.
  • Stringent hybridization conditions are, for example, conditions under which, predominantly, only those nucleic acid molecules are hybridized that have at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity.
  • Stringent hybridization conditions are, for example, hybridization in 4xSSC at 65° C, and subsequent repeated washing in O.lxSSC at 65° C. for approximately 1 hour in total.
  • the term "stringent hybridization conditions” that is used here may also mean hybridization at 68° C. in 0.25 M sodium phosphate, pH 7.2, 7% SDS, 1 mM EDTA and 1% BSA for 16 hours, and subsequent washing twice with 2xSSC and 0.1% SDS at 68° C.
  • a hybridization preferably occurs under stringent conditions.
  • “increase in the expression rate” or “increased expression rate” or “activation of the expression” or “overexpression” or a comparable phrase in certain embodiments means an increase in the expression rate of a nucleotide sequence by more than 10%, 15%, 20%, 25% or 30%, preferably by more than 40%, 50%, 60%, 70%, 80%, 90% or 100%, more preferably by more than 150%, 200%, 250%, 300%, 500%, or 1000%, in comparison to the specified reference, such as a plant not comprising the genetic or otherwise modifications according to the invention as described herein elsewhere (e.g. overexpression of the myosin heavy chain), or a reference plant, such as BL73.
  • the specified reference such as a plant not comprising the genetic or otherwise modifications according to the invention as described herein elsewhere (e.g. overexpression of the myosin heavy chain), or a reference plant, such as BL73.
  • an altered phenotype may be the enhanced induction capability of a haploid inductor caused by a mutated palatin-like phospholipase (PLA1).
  • “Increase in the transcription rate” or “increased transcription rate” or a comparable phrase in certain embodiments means an increase in the transcription rate of a nucleotide sequence by more than 10%, 15%, 20%, 25% or 30%, preferably by more than 40%, 50%, 60%, 70%, 80%, 90% or 100%, more preferably by more than 150%, 200%, 250%, 300%, 500%, or 1000%, in comparison to the specified reference, such as a plant not comprising the genetic or otherwise modifications according to the invention as described herein elsewhere (e.g. overexpression of the myosin heavy chain), or a reference plant, such as BL73.
  • the increase in the transcription rate preferably leads to a change of the phenotype of a plant in which the transcription rate is increased.
  • an altered phenotype may be the enhanced induction capability of a haploid inductor caused by a mutated palatin-like phospholipase (PLA1).
  • reducing the expression rate or “reduction in the expression rate” or “suppression of the expression,” “reduced expression rate,” or “repression” or a comparable phrase in certain embodiments means a reduction in the expression rate of a nucleotide sequence by more than 10%, 15%, 20%, 25% or 30%, preferably by more than 40%, 45%, 50%, 55%, 60% or 65%, more preferably by more than 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96% or 98% in comparison to the specified reference, such as a plant not comprising the genetic or otherwise modifications according to the invention as described herein elsewhere (e.g. repression of the aquaporin protein or gene), or a reference plant, such as BL73.
  • an altered phenotype may be the enhanced induction capability of a haploid inductor caused by a mutated palatin-like phospholipase (PLA1).
  • Reduction in the transcription rate or “reduced transcription rate” or a comparable phrase in certain embodiments means a reduction in the transcription rate of a nucleotide sequence by more than 10%, 15%, 20%, 25% or 30%, preferably by more than 40%, 45%, 50%, 55%, 60% or 65%, more preferably by more than 70%, 75%, 80%, 85%, 90%, 92%, 94%, 96% or 98% in comparison to the specified reference, such as a plant not comprising the genetic or otherwise modifications according to the invention as described herein elsewhere (e.g. repression of the aquaporin protein or gene), or a reference plant, such as BL73. However, it may also mean that the transcription rate of a nucleotide sequence is reduced by 100%.
  • the reduction in the transcription rate preferably leads to a change of the phenotype of a plant in which the transcription rate is reduced.
  • an altered phenotype may be the enhanced induction capability of a haploid inductor caused by a mutated palatin- like phospholipase (PLA1).
  • increased (protein) activity refers to increased activity of about at least 10%, preferably at least 30%, more preferably at least 50%, such as at least 20%, 40%, 60%, 80% or more, such as at least 85%, at least 90%, at least 95%, or more.
  • reduced (protein) activity refers to decreased activity of about at least 10%, preferably at least 30%, more preferably at least 50%, such as at least 20%, 40%, 60%, 80% or more, such as at least 85%, at least 90%, at least 95%, or more.
  • Activity is (substantially) absent or eliminated if activity is reduced at least 80%, preferably at least 90%, more preferably at least 95%.
  • activity is (substantially) absent, if no activity, in particular the wild type or native protein activity, can be detected.
  • (Protein) activity levels can be determined by any means known in the art, depending on the type of protein, such as by standard detection methods, including for instance enzymatic assays (for enzymes), transcription assays (for transcription factors), assays to analyse a phenotypic output, etc. Activity may be compared to a reference as defined above.
  • increased stability may refer to increased protein stability or increased RNA, such as mRNA stability. Stability of proteins or RNA can be determined by means known in the art, such as determination of protein/RNA half-life. Increased protein or RNA stability in certain embodiments means an increase of stability of about at least 10%, preferably at least 30%, more preferably at least 50%, such as at least 20%, 40%, 60%, 80% or more, such as at least 85%, at least 90%, at least 95%, or more, such as 100%, 200%, 300%, 400%, 500% or more.
  • Increased protein or RNA stability in certain embodiments means an increase in half-life of about at least 10%, preferably at least 30%, more preferably at least 50%, such as at least 20%, 40%, 60%, 80% or more, such as at least 85%, at least 90%, at least 95%, or more, such as a twp-fold, three-fold, four-fold, 5-fold or more increase in half-life. Stability may be compared to a reference as defined above.
  • RNA interference or "RNAi” is a biological process in which RNA molecules inhibit gene expression or translation, by neutralizing targeted mRNA molecules.
  • RNAs are the direct products of genes, and these small RNAs can bind to other specific messenger RNA (mRNA) molecules and either increase or decrease their activity, for example by preventing an mRNA from being translated into a protein.
  • mRNA messenger RNA
  • the RNAi pathway is found in many eukaryotes, including animals, and is initiated by the enzyme Dicer, which cleaves long double-stranded RNA (dsRNA) molecules into short double-stranded fragments of about 21 nucleotide siRNAs (small interfering RNAs).
  • siRNAs are unwound into two single-stranded RNAs (ssRNAs), the passenger strand and the guide strand.
  • the passenger strand is degraded and the guide strand is incorporated into the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • Mature miRNAs are structurally similar to siRNAs produced from exogenous dsRNA, but before reaching maturity, miRNAs must first undergo extensive post- transcriptional modification.
  • a miRNA is expressed from a much longer RNA-coding gene as a primary transcript known as a pri-miRNA which is processed, in the cell nucleus, to a 70- nucleotide stem-loop structure called a pre-miRNA by the microprocessor complex.
  • This complex consists of an RNase III enzyme called Drosha and a dsRNA-binding protein DGCR8.
  • the dsRNA portion of this pre-miRNA is bound and cleaved by Dicer to produce the mature miRNA molecule that can be integrated into the RISC complex; thus, miRNA and siRNA share the same downstream cellular machinery.
  • a short hairpin RNA or small hairpin RNA is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference.
  • RNAi molecules can be applied as such to/in the plant, or can be encoded by appropriate vectors, from which the RNAi molecule is expressed. Delivery and expression systems of RNAi molecules, such as siRNAs, shRNAs or miRNAs are well known in the art.
  • regulatory sequence as used herein relates to a nucleotide sequence which affects the specificity and/or the expression strength, e.g., in that the regulatory sequence mediates a defined tissue specificity.
  • a regulatory sequence may be located upstream of the transcription initiation point of a minimal promoter, but also downstream of it, e.g., as in a transcribed, but untranslated, leader sequence or within an intron.
  • a "vector” has its ordinary meaning in the art, and may for instance be a plasmid, a cosmid, a phage or an expression vector, a transformation vector, shuttle vector, or cloning vector; it may be double- or single-stranded, linear or circular; or it may transform a prokaryotic or eukaryotic host, either via integration into its genome or extrachromosomally.
  • the nucleic acid according to the invention is preferably operatively linked in a vector with one or more regulatory sequences which allow the transcription, and, optionally, the expression, in a prokaryotic or eukaryotic host cell.
  • a regulatory sequence— preferably, DNA— may be homologous or heterologous to the nucleic acid according to the invention.
  • the nucleic acid is under the control of a suitable promoter or terminator.
  • Suitable promoters may be promoters which are constitutively induced (example: 35S promoter from the "Cauliflower mosaic virus" (Odell et al., 1985); those promoters which are tissue-specific are especially suitable (example: Pollen-specific promoters, Chen et al. (2010), Zhao et al. (2006), or Twell et al. (1991)), or are development-specific (example: blossom-specific promoters).
  • Suitable promoters may also be synthetic or chimeric promoters which do not occur in nature, are composed of multiple elements, and contain a minimal promoter, as well as— upstream of the minimum promoter— at least one cis-regulatory element which serves as a binding location for special transcription factors. Chimeric promoters may be designed according to the desired specifics and are induced or repressed via different factors. Examples of such promoters are found in Gurr & Rushton (2005) or Venter (2007). For example, a suitable terminator is the nos-terminator (Depicker et al., 1982).
  • the vector may be introduced via conjugation, mobilization, biolistic transformation, agrobacteria-mediated transformation, transfection, transduction, vacuum infiltration, or electroporation.
  • operatively linked or "operably linked” means connected in a common nucleic acid molecule in such a manner that the connected elements are positioned and oriented relative to one another such that a transcription of the nucleic acid molecule may occur.
  • a DNA which is operatively linked with a promoter is under the transcriptional control of this promoter.
  • transformation refers to the transfer of isolated and cloned genes into the DNA, usually the chromosomal DNA or genome, of another organism.
  • Transgene refers to a genetic locus comprising a DNA sequence, such as a recombinant gene, which has been introduced into the genome of a plant cell or plant by transformation, such as Agrobacterium mediated transformation.
  • a plant cell or plant comprising a transgene stably integrated into its genome is referred to as “transgenic plant cell” or “transgenic plant”.
  • locus means a specific place or places or a site on a chromosome where for example a QTL, a gene or genetic marker is found.
  • QTL quantitative trait locus
  • a QTL may refer to a region of DNA that is associated with the differential expression of a quantitative phenotypic trait in at least one genetic background, e.g., in at least one breeding population.
  • the region of the QTL encompasses or is closely linked to the gene or genes that affect the trait in question.
  • An "allele of a QTL" can comprise multiple genes or other genetic factors within a contiguous genomic region or linkage group, such as a haplotype.
  • An allele of a QTL can denote a haplotype within a specified window wherein said window is a contiguous genomic region that can be defined, and tracked, with a set of one or more polymorphic markers.
  • a haplotype can be defined by the unique fingerprint of alleles at each marker within the specified window.
  • a QTL may encode for one or more alleles that affect the expressivity of a continuously distributed (quantitative) phenotype.
  • the QTL, marker, allele, or gene as described herein may be homozygous.
  • the QTL, marker, allele, or gene as described herein may be heterozygous.
  • the term “homozygote” refers to an individual cell or plant having the same alleles at one or more or all loci. When the term is used with reference to a specific locus or gene, it means at least that locus or gene has the same alleles. As used herein, the term “homozygous” means a genetic condition existing when identical alleles reside at corresponding loci on homologous chromosomes. As used herein, the term “heterozygote” refers to an individual cell or plant having different alleles at one or more or all loci. When the term is used with reference to a specific locus or gene, it means at least that locus or gene has different alleles.
  • the term "heterozygous” means a genetic condition existing when different alleles reside at corresponding loci on homologous chromosomes.
  • the QTL, marker(s), or gene(s) as described herein is/are homozygous.
  • the QTL, marker(s), or gene(s) as described herein are heterozygous.
  • the QTL, marker(s), or gene(s) allele(s) as described herein is/are homozygous.
  • the QTL, marker(s), or gene(s) allele(s) as described herein are heterozygous.
  • allele or “alleles” refers to one or more alternative forms, i.e. different nucleotide sequences, of a locus.
  • mutant alleles or “mutation” of alleles include alleles having one or more mutations, such as insertions, deletions, stop codons, base changes (e.g. , transitions or transversions), or alterations in splice junctions, which may or may not give rise to altered gene products. Modifications in alleles may arise in coding or non-coding regions (e.g. promoter regions, exons, introns or splice junctions).
  • a “marker” is a (means of finding a position on a) genetic or physical map, or else linkages among markers and trait loci (loci affecting traits).
  • the position that the marker detects may be known via detection of polymorphic alleles and their genetic mapping, or else by hybridization, sequence match or amplification of a sequence that has been physically mapped.
  • a marker can be a DNA marker (detects DNA polymorphisms), a protein (detects variation at an encoded polypeptide), or a simply inherited phenotype (such as the 'waxy' phenotype).
  • a DNA marker can be developed from genomic nucleotide sequence or from expressed nucleotide sequences (e.g., from a spliced RNA or a cDNA). Depending on the DNA marker technology, the marker may consist of complementary primers flanking the locus and/or complementary probes that hybridize to polymorphic alleles at the locus.
  • the term marker locus is the locus (gene, sequence or nucleotide) that the marker detects.
  • Marker or “molecular marker” or “marker locus” may also be used to denote a nucleic acid or amino acid sequence that is sufficiently unique to characterize a specific locus on the genome. Any detectable polymorphic trait can be used as a marker so long as it is inherited differentially and exhibits linkage disequilibrium with a phenotypic trait of interest.
  • Markers that detect genetic polymorphisms between members of a population are well- established in the art. Markers can be defined by the type of polymorphism that they detect and also the marker technology used to detect the polymorphism. Marker types include but are not limited to, e.g., detection of restriction fragment length polymorphisms (RFLP), detection of isozyme markers, randomly amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLPs), detection of simple sequence repeats (SSRs), detection of amplified variable sequences of the plant genome, detection of self-sustained sequence replication, or detection of single nucleotide polymorphisms (SNPs). SNPs can be detected e.g.
  • RFLP restriction fragment length polymorphisms
  • RAPD randomly amplified polymorphic DNA
  • AFLPs amplified fragment length polymorphisms
  • SSRs simple sequence repeats
  • SNPs single nucleotide polymorphisms
  • DNA sequencing via DNA sequencing, PCR-based sequence specific amplification methods, detection of polynucleotide polymorphisms by allele specific hybridization (ASFI), dynamic allele-specific hybridization (DASFI), molecular beacons, microarray hybridization, oligonucleotide ligase assays, Flap endonucleases, 5' endonucleases, primer extension, single strand conformation polymorphism (SSCP) or temperature gradient gel electrophoresis (TGGE).
  • DNA sequencing such as the pyrosequencing technology has the advantage of being able to detect a series of linked SNP alleles that constitute a haplotype. Flaplotypes tend to be more informative (detect a higher level of polymorphism) than SNPs.
  • a “marker allele”, alternatively an “allele of a marker locus”, can refer to one of a plurality of polymorphic nucleotide sequences found at a marker locus in a population.
  • allele refers to the specific nucleotide base present at that SNP locus in that individual plant.
  • “Fine-mapping” refers to methods by which the position of a QTL can be determined more accurately (narrowed down) and by which the size of the introgression fragment comprising the QTL is reduced.
  • Near Isogenic Lines for the QTL QTL-NILs
  • Such lines can then be used to map on which fragment the QTL is located and to identify a line having a shorter introgression fragment comprising the QTL.
  • Marker assisted selection (of MAS) is a process by which individual plants are selected based on marker genotypes.
  • Marker assisted counter-selection is a process by which marker genotypes are used to identify plants that will not be selected, allowing them to be removed from a breeding program or planting. Marker assisted selection uses the presence of molecular markers, which are genetically linked to a particular locus or to a particular chromosome region (e.g. introgression fragment, transgene, polymorphism, mutation, etc), to select plants for the presence of the specific locus or region (introgression fragment, transgene, polymorphism, mutation, etc).
  • a molecular marker genetically (closely) linked to a digestibility QTL as defined herein can be used to detect and/or select plants comprising the QTL on chromosome 7.
  • the closer the genetic linkage of the molecular marker to the locus e.g. about 7cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.5 cM or less), the less likely it is that the marker is dissociated from the locus through meiotic recombination.
  • the closer two markers are linked to each other e.g.
  • a marker "within 7 cM or within 5 cM, 3 cM, 2 cM, or 1 cM" of another marker refers to a marker which genetically maps to within the 7 cM or 5 cM, 3 cM, 2 cM, or 1 cM region flanking the marker (i.e. either side of the marker).
  • a marker within 5 Mb, 3 Mb, 2.5 Mb, 2 Mb, 1 Mb, 0.5 Mb, 0.4 Mb, 0.3 Mb, 0.2 Mb, 0.1 Mb, 50 kb, 20 kb, 10 kb, 5 kb, 2 kb, 1 kb or less of another marker refers to a marker which is physically located within the 5 Mb, 3 Mb, 2.5 Mb, 2 Mb, 1 Mb, 0.5 Mb, 0.4 Mb, 0.3 Mb, 0.2 Mb, 0.1 Mb, 50 kb, 20 kb, 10 kb, 5 kb, 2 kb, 1 kb or less, of the genomic DNA region flanking the marker (i.e.
  • LOD-score logarithm (base 10) of odds refers to a statistical test often used for linkage analysis in animal and plant populations. The LOD score compares the likelihood of obtaining the test data if the two loci (molecular marker loci and/or a phenotypic trait locus) are indeed linked, to the likelihood of observing the same data purely by chance. Positive LOD scores favor the presence of linkage and a LOD score greater than 3.0 is considered evidence for linkage. A LOD score of +3 indicates 1000 to 1 odds that the linkage being observed did not occur by chance.
  • a "marker haplotype” refers to a combination of alleles at a marker locus.
  • a "marker locus” is a specific chromosome location in the genome of a species where a specific marker can be found.
  • a marker locus can be used to track the presence of a second linked locus, e.g., one that affects the expression of a phenotypic trait.
  • a marker locus can be used to monitor segregation of alleles at a genetically or physically linked locus.
  • a “marker probe” is a nucleic acid sequence or molecule that can be used to identify the presence of a marker locus, e.g., a nucleic acid probe that is complementary to a marker locus sequence, through nucleic acid hybridization. Marker probes comprising 30 or more contiguous nucleotides of the marker locus ("all or a portion" of the marker locus sequence) may be used for nucleic acid hybridization. Alternatively, in some aspects, a marker probe refers to a probe of any type that is able to distinguish (i.e., genotype) the particular allele that is present at a marker locus.
  • molecular marker may be used to refer to a genetic marker or an encoded product thereof (e.g., a protein) used as a point of reference when identifying a linked locus.
  • a marker can be derived from genomic nucleotide sequences or from expressed nucleotide sequences (e.g., from a spliced RNA, a cDNA, etc.), or from an encoded polypeptide.
  • the term also refers to nucleic acid sequences complementary to or flanking the marker sequences, such as nucleic acids used as probes or primer pairs capable of amplifying the marker sequence.
  • a “molecular marker probe” is a nucleic acid sequence or molecule that can be used to identify the presence of a marker locus, e.g., a nucleic acid probe that is complementary to a marker locus sequence.
  • a marker probe refers to a probe of any type that is able to distinguish (i.e., genotype) the particular allele that is present at a marker locus.
  • Nucleic acids are "complementary" when they specifically hybridize in solution, e.g., according to Watson- Crick base pairing rules. Some of the markers described herein are also referred to as hybridization markers when located on an indel region, such as the non- collinear region described herein.
  • the insertion region is, by definition, a polymorphism vis a vis a plant without the insertion.
  • the marker need only indicate whether the indel region is present or absent. Any suitable marker detection technology may be used to identify such a hybridization marker, e.g. SNP technology is used in the examples provided herein.
  • Genetic markers are nucleic acids that are polymorphic in a population and where the alleles of which can be detected and distinguished by one or more analytic methods, e.g., RFLP, AFLP, isozyme, SNP, SSR, and the like.
  • the terms "molecular marker” and “genetic marker” are used interchangeably herein.
  • the term also refers to nucleic acid sequences complementary to the genomic sequences, such as nucleic acids used as probes.
  • Markers corresponding to genetic polymorphisms between members of a population can be detected by methods well- established in the art. These include, e.g., PCR-based sequence specific amplification methods, detection of restriction fragment length polymorphisms (RFLP), detection of isozyme markers, detection of polynucleotide polymorphisms by allele specific hybridization (ASFI), detection of amplified variable sequences of the plant genome, detection of self-sustained sequence replication, detection of simple sequence repeats (SSRs), detection of single nucleotide polymorphisms (SNPs), or detection of amplified fragment length polymorphisms (AFLPs).
  • ESTs expressed sequence tags
  • SSR markers derived from EST sequences and randomly amplified polymorphic DNA
  • a "polymorphism” is a variation in the DNA between two or more individuals within a population.
  • a polymorphism preferably has a frequency of at least 1 % in a population.
  • a useful polymorphism can include a single nucleotide polymorphism (SNP), a simple sequence repeat (SSR), or an insertion/deletion polymorphism, also referred to herein as an "indel".
  • SNP single nucleotide polymorphism
  • SSR simple sequence repeat
  • an insertion/deletion polymorphism also referred to herein as an "indel”.
  • the term “indel” refers to an insertion or deletion, wherein one line may be referred to as having an inserted nucleotide or piece of DNA relative to a second line, or the second line may be referred to as having a deleted nucleotide or piece of DNA relative to the first line.
  • “Physical distance” between loci (e.g. between molecular markers and/or between phenotypic markers) on the same chromosome is the actually physical distance expressed in bases or base pairs (bp), kilo bases or kilo base pairs (kb) or megabases or mega base pairs (Mb).
  • Genetic distance between loci is measured by frequency of crossing-over, or recombination frequency (RF) and is indicated in centimorgans (cM).
  • RF recombination frequency
  • cM centimorgans
  • One cM corresponds to a recombination frequency of 1%. If no recombinants can be found, the RF is zero and the loci are either extremely close together physically or they are identical. The further apart two loci are, the higher the RF.
  • a "physical map" of the genome is a map showing the linear order of identifiable landmarks (including genes, markers, etc.) on chromosome DNA. Flowever, in contrast to genetic maps, the distances between landmarks are absolute (for example, measured in base pairs or isolated and overlapping contiguous genetic fragments) and not based on genetic recombination (that can vary in different populations).
  • An allele "negatively” correlates with a trait when it is linked to it and when presence of the allele is an indicator that a desired trait or trait form will not occur in a plant comprising the allele.
  • An allele "positively” correlates with a trait when it is linked to it and when presence of the allele is an indicator that the desired trait or trait form will occur in a plant comprising the allele.
  • centimorgan is a unit of measure of recombination frequency.
  • One cM is equal to a 1 % chance that a marker at one genetic locus will be separated from a marker at a second locus due to crossing over in a single generation.
  • chromosomal interval designates a contiguous linear span of genomic DNA that resides in planta on a single chromosome.
  • the genetic elements or genes located on a single chromosomal interval are physically linked.
  • the size of a chromosomal interval is not particularly limited.
  • the genetic elements located within a single chromosomal interval are genetically linked, typically with a genetic recombination distance of, for example, less than or equal to 20 cM, or alternatively, less than or equal to 10 cM. That is, two genetic elements within a single chromosomal interval undergo recombination at a frequency of less than or equal to 20% or 10%.
  • closely linked in the present application, means that recombination between two linked loci occurs with a frequency of equal to or less than about 10% (i.e., are separated on a genetic map by not more than 10 cM). Put another way, the closely linked loci co-segregate at least 90% of the time. Marker loci are especially useful with respect to the subject matter of the current disclosure when they demonstrate a significant probability of co-segregation (linkage) with a desired trait (e.g., resistance to gray leaf spot).
  • Closely linked loci such as a marker locus and a second locus can display an inter-locus recombination frequency of 10% or less, preferably about 9% or less, still more preferably about 8% or less, yet more preferably about 7% or less, still more preferably about 6% or less, yet more preferably about 5% or less, still more preferably about 4% or less, yet more preferably about 3% or less, and still more preferably about 2% or less.
  • the relevant loci display a recombination a frequency of about 1 % or less, e.g., about 0.75% or less, more preferably about 0.5% or less, or yet more preferably about 0.25% or less.
  • Two loci that are localized to the same chromosome, and at such a distance that recombination between the two loci occurs at a frequency of less than 10% (e.g., about 9 %, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, 0.75%, 0.5%, 0.25%, or less) are also said to be "proximal to" each other.
  • two different markers can have the same genetic map coordinates. In that case, the two markers are in such close proximity to each other that recombination occurs between them with such low frequency that it is undetectable.
  • Linkage refers to the tendency for alleles to segregate together more often than expected by chance if their transmission was independent. Typically, linkage refers to alleles on the same chromosome. Genetic recombination occurs with an assumed random frequency over the entire genome. Genetic maps are constructed by measuring the frequency of recombination between pairs of traits or markers. The closer the traits or markers are to each other on the chromosome, the lower the frequency of recombination, and the greater the degree of linkage. Traits or markers are considered herein to be linked if they generally co- segregate. A 1/100 probability of recombination per generation is defined as a genetic map distance of 1.0 centiMorgan (1.0 cM).
  • linkage disequilibrium refers to a non-random segregation of genetic loci or traits (or both). In either case, linkage disequilibrium implies that the relevant loci are within sufficient physical proximity along a length of a chromosome so that they segregate together with greater than random (i.e., non-random) frequency. Markers that show linkage disequilibrium are considered linked. Linked loci co-segregate more than 50% of the time, e.g., from about 51 % to about 100% of the time.
  • linkage can be between two markers, or alternatively between a marker and a locus affecting a phenotype.
  • a marker locus can be "associated with” (linked to) a trait. The degree of linkage of a marker locus and a locus affecting a phenotypic trait is measured, e.g., as a statistical probability of co-segregation of that molecular marker with the phenotype (e.g., an F statistic or LOD score).
  • the genetic elements or genes located on a single chromosome segment are physically linked.
  • the two loci are located in close proximity such that recombination between homologous chromosome pairs does not occur between the two loci during meiosis with high frequency, e.g., such that linked loci co-segregate at least about 90% of the time, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.75%, or more of the time.
  • the genetic elements located within a chromosomal segment are also "genetically linked", typically within a genetic recombination distance of less than or equal to 50cM, e.g., about 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5, 0.25 cM or less.
  • two genetic elements within a single chromosomal segment undergo recombination during meiosis with each other at a frequency of less than or equal to about 50%, e.g., about 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25% or less.
  • “Closely linked” markers display a cross over frequency with a given marker of about 10% or less, e.g., 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25% or less (the given marker locus is within about 10 cM of a closely linked marker locus, e.g., 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5, 0.25 cM or less of a closely linked marker locus).
  • closely linked marker loci co- segregate at least about 90% the time, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.75%, or more of the time.
  • introduction refers to both a natural and artificial process whereby chromosomal fragments or genes 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.
  • introgression of a desired allele at a specified locus can be transmitted to at least one progeny via a sexual cross between two parents of the same species, where at least one of the parents has the desired allele in its genome.
  • transmission of an allele can occur by recombination between two donor genomes, e.g., in a fused protoplast, where at least one of the donor protoplasts has the desired allele in its genome.
  • the desired allele can be, e.g., detected by a marker that is associated with a phenotype, at a QTL, a transgene, or the like.
  • offspring comprising the desired allele can be repeatedly backcrossed to a line having a desired genetic background and selected for the desired allele, to result in the allele becoming fixed in a selected genetic background.
  • the process of "introgressing” is often referred to as “backcrossing” when the process is repeated two or more times.
  • “Introgression fragment” or “introgression segment” or “introgression region” refers to a chromosome fragment (or chromosome part or region) which has been introduced into another plant of the same or related species either artificially or naturally such as by crossing or traditional breeding techniques, such as backcrossing, i.e. the introgressed fragment is the result of breeding methods referred to by the verb "to introgress" (such as backcrossing).
  • the introgression fragment never includes a whole chromosome, but only a part of a chromosome.
  • the introgression fragment can be large, e.g. even three quarter or half of a chromosome, but is preferably smaller, such as about 15 Mb or less, such as about 10 Mb or less, about 9 Mb or less, about 8 Mb or less, about 7 Mb or less, about 6 Mb or less, about 5 Mb or less, about 4 Mb or less, about 3 Mb or less, about 2.5 Mb or 2 Mb or less, about 1 Mb (equals 1,000,000 base pairs) or less, or about 0.5 Mb (equals 500,000 base pairs) or less, such as about 200,000 bp (equals 200 kilo base pairs) or less, about 100,000 bp (100 kb) or less, about 50,000 bp (50 kb) or less, about 25,000 bp (25 kb) or less.
  • the introgression fragment can be large, e.g
  • a genetic element, an introgression fragment, or a gene or allele conferring a trait (such as improved digestibility) is said to be “obtainable from” or can be “obtained from” or “derivable from” or can be “derived from” or “as present in” or “as found in” a plant or plant part as described herein elsewhere if it can be transferred from the plant in which it is present into another plant in which it is not present (such as a line or variety) using traditional breeding techniques without resulting in a phenotypic change of the recipient plant apart from the addition of the trait conferred by the genetic element, locus, introgression fragment, gene or allele.
  • the genetic element, locus, introgression fragment, gene or allele can thus be transferred into any other genetic background lacking the trait.
  • pants comprising the genetic element, locus, introgression fragment, gene or allele can be used, but also progeny/descendants from such plants which have been selected to retain the genetic element, locus, introgression fragment, gene or allele, can be used and are encompassed herein.
  • a plant or genomic DNA, cell or tissue of a plant
  • comprises the same genetic element, locus, introgression fragment, gene or allele as obtainable from such plant can be determined by the skilled person using one or more techniques known in the art, such as phenotypic assays, whole genome sequencing, molecular marker analysis, trait mapping, chromosome painting, allelism tests and the like, or combinations of techniques. It will be understood that transgenic plants may also be encompassed.
  • genetic engineering As used herein the terms “genetic engineering”, “transformation” and “genetic modification” are all used herein as synonyms for the transfer of isolated and cloned genes into the DNA, usually the chromosomal DNA or genome, of another organism.
  • the present invention in an aspect relates to means and methods suitable for enhancing the haploid induction capability of an inducer plant.
  • the haploid inducer plant in certain embodiments may be a plant selected and/or derived from the lines Stock 6, RWK, RWS, UH400, AX5707RS, NP2222-mai/, or any other haploid inducer line.
  • a haploid inducer as referred to herein can be a mutant plant selected from the plants as disclosed in US 2017/327832 A1 (or a plant of a different species having the corresponding mutation(s)), incorporated herein by reference in its entirety, in particular the tilling mutants having a D74N (exchange of aspartate at Position 74 for asparagin) or G78R (exchange of glycine at Position 78 for arginine) in the PLA1 gene.
  • a haploid inducer as referred to herein can be a mutant plant selected from the plants as described in WO 2018/158301 (or a plant of a different species having the corresponding mutation(s)), incorporated herein by reference in its entirety.
  • a haploid inducer as referred to herein can be a plant with mutated PLA1 according to SEQ ID NOs: 38-40.
  • a haploid inducer as referred to herein can be a mutant plant selected from the plants as described in Kalinowska et al. (2016) "State-of-the-art and novel developments of in vivo haploid technologies"; Theor. Appl. Genetics; 132(3):593-605; incorporated herein by reference in its entirely; preferably wherein haploid induction is caused by a mutated patatin- like phospholipase (PLA1).
  • the invention provides a nucleic acid which, after transcription or expression in a plant or after silencing in a plant, is suitable for enhancing the haploid induction capability of an inducer plant, such as caused by a mutated patatin-like phospholipase (PLA1).
  • an endogenous DNA sequence in the genome of a plant, or in the genome of a plant haploid inductor, which is identical to one of the nucleic acids according to the invention may also be modified such that the property of a haploid inductor is mediated, or the induction capability of the haploid inductor is increased, after transcription or expression of the endogenous DNA sequence.
  • the nucleic acid of the present invention is preferably an isolated nucleic acid which is extracted from its natural or original environment (genetic context).
  • a nucleic acid may be double-stranded or single-stranded, and linear or circular. It may thereby be genomic DNA, synthetic DNA, cDNA, or an RNA type (for example, IncRNA, siRNA, or miRNA), wherein the nucleobase uracil occurs in RNA instead of the nucleobase thymine.
  • the nucleic acid according to the invention may be used as a transgene.
  • an endogenous DNA sequence in the genome of a plant, or in the genome of a haploid inductor, preferably a haploid inductor comprising a mutation in a patatin-like phospholipase (PLA1), which is identical to one of the nucleic acids according to the invention may also be modified such that the induction capability of an inducer plant, preferably caused by a mutated patatin- like phospholipase (PLA1) can be enhanced, after transcription or expression of the endogenous DNA sequence or after silencing of the endogenous DNA sequence, and optionally after crossing with a plant comprising a mutated patatin-like phospholipase (PLA1).
  • the nucleic acid of the present invention is preferably an isolated nucleic acid which is extracted or isolated from its natural or original environment (genetic context).
  • a nucleic acid may be double-stranded or single-stranded, and linear or circular. It may thereby be genomic DNA, synthetic DNA, cDNA, or an RNA type (for example, IncRNA, siRNA, or miRNA), wherein the nucleobase uracil occurs in RNA instead of the nucleobase thymine.
  • the nucleic acid which, after transcription or expression in a plant, is preferably suitable for enhancing the induction capability of an inducer plant, such as caused by a mutated patatin-like phospholipase (PLA1) may be a nucleic acid comprising a nucleotide sequence which:
  • (i) is a sequence selected from the group consisting of SEQ ID NOs: 1, 4, 5, 8, 10, 12,
  • (ii) has a coding sequence selected from the group consisting of SEQ ID NOs: 2, 6, 13,
  • (iii) is complementary to the sequence from (i) or (ii); or
  • (iv) is at least 80%, 82%, 84%, 86%, 88%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, or more preferably at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to the sequence from (i) or (ii); or
  • (v) encodes a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7, 9, 11, 14, 17, 20, 23 and 37, or a functional part of the protein, preferably SEQ ID NOs: 3, 11, 17, and 37, or a functional part of the protein; or
  • (vi) encodes a protein comprising an amino acid sequence which is at least 80%, 82%, 84%, 86%, 88%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, or more preferably at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7, 9, 11, 14, 17, 20, 23 and 37, or a functional part of the protein, preferably SEQ ID NOs: 3, 11, 17, and 37, or a functional part of the protein; or
  • nucleic acid hybridizes with a sequence from (iii) under stringent conditions.
  • This nucleic acid or an RNA encoded by the nucleic acid or a protein or polypeptide encoded by the nucleic acid, has a positive effect on the haploid induction capability of an inducer plant, preferably caused by a mutated patatin-like phospholipase (PLA1), and therefore, in the following, a nucleic acid which is defined here is designated as an induction-promoting nucleic acid.
  • Non-limiting examples of uses of the nucleic acid in order to enhance the induction capability of an inducer plant, preferably caused by a mutated patatin-like phospholipase (PLA1) may include transgenically or endogenously increasing the expression rate of the nucleic acid or the activity or stability of the encoded protein or of the encoded part of the protein. Additional methods and uses of the induction-promoting nucleic acid, as well as substances which comprise the induction-promoting nucleic acid, are disclosed further below.
  • the nucleic acid which, after silencing in a plant, is preferably suitable for enhancing the induction capability of an inducer plant, preferably caused by a mutated patatin-like phospholipase (PLA1) may be a nucleic acid comprising a nucleotide sequence which:
  • (i) is a sequence selected from the group consisting of SEQ ID NOs: 24, 27 and 32, or a functional fragment thereof, preferably SEQ ID NO: 24, or a functional fragment thereof ; or
  • (ii) has a coding sequence selected from the group consisting of SEQ ID NO: 25, 28 and 33, preferably SEQ ID NO: 25; or
  • (iii) is complementary to the sequence from (i) or (ii); or
  • (iv) is at least 80%, 82%, 84%, 86%, 88%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, or more preferably at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to the sequence from (i) or (ii); or
  • (v) encodes a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 29 and 34, or a functional part of the protein, preferably SEQ ID NO: 26, or a functional part of the protein; or
  • (vi) encodes a protein comprising an amino acid sequence which is at least 80%, 82%, 84%, 86%, 88%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, or more preferably at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 26, 29 and 34, or a functional part of the protein, preferably SEQ ID NO: 26, or a functional part of the protein; or
  • This nucleic acid or an RNA encoded by the nucleic acid or a protein or polypeptide encoded by the nucleic acid, has a negative effect on the haploid induction capability of an inducer plant, preferably caused by a mutated patatin-like phospholipase (PLA1), and therefore, in the following, a nucleic acid which is defined here is designated as an induction-inhibiting nucleic acid.
  • PHA1 patatin-like phospholipase
  • Non-limiting examples of uses of the nucleic acid in order to enhance the induction capability of an inducer plant, preferably caused by a mutated patatin-like phospholipase (PLA1) may include transgenically or endogenously reducing the expression rate of the nucleic acid or the activity or stability of the encoded protein or of the encoded part of the protein. Additional methods and uses of the induction-inhibiting nucleic acid, as well as substances which comprise the induction-inhibiting nucleic acid, are disclosed further below.
  • the nucleic acid which, after transcription or expression in a plant, is preferably suitable for enhancing the induction capability of an inducer plant, preferebly caused by a mutated patatin-like phospholipase (PLA1) may be a nucleic acid that encodes for an RNA that has a double-stranded portion, wherein at least one strand of the double- stranded portion has a nucleotide sequence which is homologous or identical to at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, preferably at least 30, 35, 40, 45, 50, 60, 70, 80, 90, 100,
  • nucleic acid 120, or 140, more preferably at least 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 successive nucleotides of a coding sequence of a nucleic acid that:
  • (i) has a sequence selected from the group consisting of SEQ ID Nos: 25, 28 and 33, or a fragment thereof, in a sense or anti-sense orientation; or
  • (iii) is at least 80%, 82%, 84%, 86%, or 88%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, or 96%, more preferably at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to the sequence from (i); or
  • nucleic acid hybridizes with the sequence from (ii) under stringent conditions.
  • such a nucleic acid may be used to suppress the expression of the induction-inhibiting nucleic acid described above.
  • Table 1 Sequence index and sequence association of the nucleotide and amino acid sequences.
  • the haploid induced plant comprises the HIR1 QTL ( qhirl ), as for instance described in Nair et al. (2017) "Dissection of a major QTL qhirl conferring maternal haploid induction ability in maize”; Theor Appl Genet; 130(6):1113-1122, incorporated herein by reference in its entirety.
  • the haploid induced plant comprises the HIR1 QTL sub-region qhirll. The skilled person will understand that a comparable or corresponding QTL in plants other than maize can also be used.
  • the haploid induced plant is selected from, or is derived from the following lines: Stock 6, RWK, RWS, UH400, AX5707RS, NP2222-mai/. It will be understood that "derived from” in this context means that the haploid induction phenotype is exhibited in the plant.
  • the haploid inducer plant comprises a mutated PLA1 gene, as defined herein elsewhere.
  • the mutated PLA1 gene may result in a gain-of-function, such as a dominant negative phenotype, (partial) loss-of-function, and may result in a knock-out or a truncated PLA1.
  • the PLA1 gene has a sequence, such as a genomic sequence, mRNA sequence, or preferably protein sequence identical to the corresponding sequence of the PLA1 gene in lines Stock 6, RWK, RWS, UH400, AX5707RS, NP2222-mai/.
  • the PLA1 gene has one or more of the following mutations: D74N, G78R, R58Q, V162I, S288L, or Q369stop.
  • the PLA1 gene has a genomic sequence as set forth in SEQ ID NO: 38. In certain embodiments, the PLA1 gene has a cDNA sequence as set forth in SEQ ID NO: 39. In certain embodiments, the PLA1 gene has a protein sequence as set forth in SEQ ID NO: 40.
  • the PLA1 gene has a genomic sequence as set forth in SEQ ID NO: 44. In certain embodiments, the PLA1 gene has a cDNA sequence as set forth in SEQ ID NO: 45. In certain embodiments, the PLA1 gene has a protein sequence as set forth in SEQ ID NO: 46.
  • the present invention relates to a vector which comprises a nucleic acid as described herein, preferably an induction-promoting nucleic acid or a nucleic acid encoding a double-stranded RNA as described herein.
  • the vector may be a plasmid, a cosmid, a phage or an expression vector, a transformation vector, shuttle vector, or cloning vector; it may be double- or single-stranded, linear or circular.
  • the vector may transform a prokaryotic or eukaryotic host, either via integration into its genome or extrachromosomally.
  • the vector is an expression vector.
  • the nucleic acid is preferably operatively linked in a vector with one or more regulatory sequences which allow the transcription, and optionally the expression, in a prokaryotic or eukaryotic host cell.
  • a regulatory sequence may be homologous or heterologous to the nucleic acid.
  • the nucleic acid is under the control of a suitable promoter or terminator.
  • Suitable promoters may be promoters which are constitutively induced, for example, the 35S promoter from the "Cauliflower mosaic virus" (Odell et al., 1985. Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter.) Tissue-specific promoters, e.g.
  • Suitable promoters may also be synthetic or chimeric promoters which do not occur in nature, and which are composed of multiple elements. Such synthetic or chimeric promoter may contain a minimal promoter, as well as at least one cis-regulatory element which serves as a binding location for special transcription factors.
  • Chimeric promoters may be designed according to the desired specifics and can be induced or repressed via different factors. Examples of such promoters are found in Gurr & Rushton (2005. Trends in Biotechnology 23(6): 275-282) or Venter (2007. Trends in Plant Science: 12(3): 118-124). For example, a suitable terminator is the nos-terminator (Depicker et al., 1982. Journal of Molecular and Applied Genetics 1(6): 561-573).
  • the vector is a conditional expression vector. In certain embodiments, the vector is a constitutive expression vector. In certain embodiments, the vector is a tissue-specific expression vector, such as a pollen-specific expression vector. In certain embodiments, the vector is an inducible expression vector. All such vectors are well-known in the art.
  • a host cell such as a plant cell, which comprises a nucleic acid as described herein, preferably an induction-promoting nucleic acid or a nucleic acid encoding a double-stranded RNA as described herein, or a vector as described herein.
  • the host cell may contain the nucleic acid as an extra-chromosomally (episomal) replicating molecule, or comprises the nucleic acid integrated in the nuclear or plastid genome of the host cell, or as introduced chromosome, e.g. minichromosome.
  • the host cell may be a prokaryotic (for example, bacterial) or eukaryotic cell (for example, a plant cell or a yeast cell).
  • the host cell may be an agrobacterium, such as Agrobacterium tumefaciens or Agrobacterium rhizogenes.
  • the host cell is a plant cell.
  • a nucleic acid described herein or a vector described herein may be introduced in a host cell via well-known methods, which may depend on the selected host cell, including, for example, conjugation, mobilization, biolistic transformation, agrobacteria-mediated transformation, transfection, transduction, vacuum infiltration, or electroporation.
  • methods for introducing a nucleic acid or a vector in an agrobacterium cell are well-known to the skilled person and may include conjugation or electroporation methods.
  • methods for introducing a nucleic acid or a vector into a plant cell are known (Sambrook et al., 2001) and may include diverse transformation methods such as biolistic transformation and agrobacterium-mediated transformation.
  • the present invention relates to a transgenic plant cell which comprises a nucleic acid as described herein, in particular an induction-promoting nucleic acid or a nucleic acid encoding a double-stranded RNA as described herein, as a transgene or a vector as described herein.
  • the present invention relates to a transgenic plant or a part thereof which comprises the transgenic plant cell.
  • such a transgenic plant cell or transgenic plant is a plant cell or plant which is, preferably stably, transformed with a nucleic acid as described herein, in particular an induction-promoting nucleic acid or a nucleic acid encoding a double-stranded RNA as described herein, or a vector as described herein.
  • the nucleic acid in the transgenic plant cell is operatively linked with one or more regulatory sequences which allow the transcription, and optionally the expression, in the plant cell.
  • a regulatory sequence may be homologous or heterologous to the nucleic acid.
  • the total structure made up of the nucleic acid according to the invention and the regulatory sequence(s) may then represent the transgene.
  • a part of a transgenic plant may be, for example, a fertilized or unfertilized seed, an embryo, a pollen, a tissue, an organ, or a plant cell, wherein the fertilized or unfertilized seed, the embryo, or the pollen are generated in the transgenic plant, and the nucleic acid as described herein, in particular an induction-promoting nucleic acid or a nucleic acid encoding a double- stranded RNA as described herein, is integrated into its genome as a transgene or the vector.
  • transgenic plant as used herein also includes a descendant of the transgenic plant described herein in whose genome the nucleic acid as described herein, in particular an induction-promoting nucleic acid or a nucleic acid encoding a double-stranded RNA as described herein, is integrated as a transgene or the vector.
  • plant cells that endogenously comprise a nucleic acid as described herein, in particular an induction-promoting nucleic acid or a nucleic acid encoding a double- stranded RNA as described herein, as well as a plant or a part thereof comprising such plants cells.
  • a protein or a polypeptide encoded by a nucleic acid as described herein may be suitable for enhancing the induction capability of a an inducer plant, preferably caused by caused by a mutated patatin-like phospholipase (PLA1).
  • the protein or polypeptide encoded by an induction-promoting nucleic acid described herein is preferred.
  • the protein or polypeptide may comprise an amino acid sequence selected from SEQ ID NOs: 3, 7, 9, 11, 14, 17, 20, 23, or 37, preferably SEQ ID NOs: 3, 11, 17, or 37, or from SEQ ID NOs: 26, 29 or 34.
  • the present invention relates to methods for the production of a plant comprising or which is carrier of an enhancer for the haploid induction capability of an inducer plant, such as caused by a mutated patatin-like phospholipase (PLA1).
  • PHA1 patatin-like phospholipase
  • a method for the production of a (transgenic) plant such as a plant which comprises or which is a carrier of an enhancer for the haploid induction capability of an inducer plant, preferably caused by a mutated patatin-like phospholipase (PLA1), said method comprising the following steps:
  • nucleic acid as described herein, such as an induction-promoting nucleic acid or a nucleic acid encoding a double-stranded RNA as described herein; or provision of a vector as described herein comprising said nucleic acid,
  • step b) regeneration of a transgenic plant from the at least one transgenic plant cell obtained in step b) or a transgenic progeny thereof
  • the introduced or transformed nucleic acid may be (over)expressed in a non-tissue-specific way, or in tissue-specific way, preferably in pollen.
  • the (over)expression of the introduced or transformed nucleic acid appears at least in pollen of the transgenic plant or in a tissue of pollen of the transgenic plant.
  • the method may further comprise the step e) of identification of a modified expression pattern in the transgenic plant obtained in step c) or in a plant part thereof, preferably in pollen of the transgenic plant or in a tissue of pollen of the transgenic plant.
  • the transcription or expression rate of the introduced induction-promoting nucleic acid is increased in the transgenic plant in comparison to a wild-type plant which, for example, was regenerated from an isogenic, untransformed plant cell, and/or
  • the expression rate of an endogenous DNA sequence having a nucleotide sequence that is identical to the induction-inhibiting nucleic acid as described herein is reduced, e.g. via a double-stranded RNA which is encoded by the introduced nucleic acid encoding a double-stranded RNA, in the transgenic plant in comparison to a wild-type plant which, for example, was regenerated from an isogenic, untransformed plant cell.
  • a verification of the transcription rate may take place via qRT-PCR, for example.
  • two or more of the induction-promoting nucleic acids and nucleic acids encoding a double-stranded RNA may be provided and the at least one plant cell may be transformed with said two or more nucleic acids.
  • These two or more nucleic acids may be comprised in one or more vectors.
  • one or more additional nucleic acids which are known to be suitable for the generation of a haploid inductor (for example, manipulated cenh3 gene (Ravi & Chan, 2010. Nature 464(7288): 615-618) or for enhancing the induction capability of a haploid inductor may be provided and transformed into the at least one plant cell, in addition to the induction- promoting nucleic acid and/or the nucleic acid encoding a double-stranded RNA as described herein.
  • a haploid inductor for example, manipulated cenh3 gene (Ravi & Chan, 2010. Nature 464(7288): 615-618) or for enhancing the induction capability of a haploid inductor
  • transgenic plant which can be or is produced with this method, including a descendant of the transgenic plant which comprises the introduced nucleic acid as a transgene, or a part of this plant.
  • a part of the plant may be a fertilized or unfertilized seed, an embryo, a pollen, a tissue, an organ, or a plant cell, wherein the fertilized or unfertilized seed, the embryo, or the pollen are generated at the transgenic plant, and the introduced nucleic acid is integrated into its genome as a transgene or the vector.
  • a method for the production of a (mutated) plant such as a (mutated) plant which is carrier of an enhancer for the haploid induction capability of an inducer plant, preferably caused by a mutated patatin-like phospholipase (PLA1), said method comprising the following steps: a) mutagenization of plant cells and subsequent regeneration of plants from the mutagenized plant cells or mutagenization of plants, and b) identification of a plant obtained in step a) which: has at least one mutation in an endogenous DNA sequence which is identical to an induction-promoting or an induction-inhibiting nucleic acid as described herein, or has at least one mutation in a regulatory sequence (for example, a promoter, enhancer, terminator, or intron) of said endogenous DNA sequence, wherein said mutation produces a change in the transcription or expression rate of the endogenous DNA sequence in the identified plant, in comparison to a non-mutagenized wild- type plant, or a change in the activity or stability
  • the change in the transcription rate or expression rate, or the change in the activity or stability appears at least in a pollen of the identified plant or in a tissue of a pollen of the identified plant.
  • step b) encompasses the identification of a plant obtained in step a) which (i): has at least one mutation in an endogenous DNA sequence that is identical to an induction-inducing nucleic acid as described herein, or has at least one mutation in in a regulatory sequence (for example, a promoter, enhancer, terminator, or intron) of said endogenous DNA sequence, wherein said mutation effects an increase in the transcription or expression rate of said endogenous DNA sequence or an increase in the activity or stability of a protein or polypeptide encoded by said endogenous DNA sequence; and/or which (ii): has at least one mutation in an endogenous DNA sequence that is identical to an induction-inhibiting nucleic acid as described herein, or has at least one mutation in a regulatory sequence (for example, a promoter, enhancer, terminator, or intron) of said endogenous DNA sequence, wherein said mutation effects a reduction in the transcription or expression rate of said endogenous DNA sequence or a reduction in the activity or stability
  • a "mutation" refers to a modification at the DNA level, and includes changes in the genetics and/or epigenetics.
  • An alteration in the genetics may include an insertion, a deletion, an introduction of a stop codon, a base change (e.g. , transition or transversion), or an alteration in splice junctions. These alterations may arise in coding or non-coding regions (e.g. promoter regions, exons, introns or splice junctions) of the endogenous DNA sequence.
  • an alteration in the genetics may be the exchange of at least one nucleobase in the endogenous DNA sequence or in a regulatory sequence of the endogenous DNA sequence.
  • nucleobase exchange takes place in a promoter, for example, this may lead to an altered activity of the promoter, since, for example, cis-regulator elements are modified such that the affinity of a transcription factor to the mutated cis-regulatory elements is altered in comparison to the wild-type promoter, so that the activity of the promoter with the mutated cis-regulatory elements is increased or reduced, depending upon whether the transcription factor is a repressor or inductor, or whether the affinity of the transcription factor to the mutated cis-regulatory elements is intensified or weakened.
  • nucleobase exchange occurs, e.g., in an encoding region of the endogenous DNA sequence, this may lead to an amino acid exchange in the encoded protein, which may produce an alteration in the activity or stability of the protein, in comparison to the wild-type protein.
  • An alteration in the epigenetics may take place via an altered methylation pattern of the DNA.
  • Mutagenesis may be performed in accordance with any of the techniques known in the art.
  • "mutagenization” or “mutagenesis” includes both conventional mutagenesis and location-specific mutagenesis or “genome editing” or “gene editing”.
  • modification at the DNA level is not produced in a targeted manner.
  • the plant cell or the plant is exposed to mutagenic conditions, such as TILLING, via UV light exposure or the use of chemical substances (Till et al., 2004).
  • An additional method of random mutagenesis is mutagenesis with the aid of a transposon.
  • Location-specific mutagenesis enables the introduction of modification at the DNA level in a target-oriented manner at predefined locations in the DNA. For example, TALENS, meganucleases, homing endonucleases, zinc finger nucleases, or a CRISPR/Cas System as further described herein may be used for this.
  • the identification of a plant in step b) takes place with the aid of molecular markers or probes.
  • markers may be based upon an SNP and may be specific to the mutation and include, for example, KASP and TaqMan markers.
  • DNA probes are primers or primer pairs which may be used in a PCR reaction.
  • TILLING mutants may be verified or identified by sequencing the target gene in a TILLING population, or via additional methods that verify the mispairings in the DNA, e.g., melting point analyses or use of mispairing-specific nucleases.
  • Mutants generated by means of transposons may also be verified by use of transposon-specific primers and target gene-specific primers in PCR, across the entire population and subsequent sequencing of PCR products.
  • a change in the expression rate in pollen may be determined with RT-PCR; the change in the stability may be determined by examining ubiquitin binding locations and prediction of changes to the tertiary structure, for example.
  • recombinant expression of the wild-type proteins, and the corresponding mutant proteins and subsequent biochemical activity tests are also suitable.
  • the invention relates to a molecular marker which is able to hybridize to nucleic acid molecule as described above, or a molecular marker which is able to detect a polymorphism genetically associated with or closely linked to nucleic acid molecule as defined above.
  • polymorphism is a SNP, deletion or insertion
  • exemplary polymorphisms are given in table 2.
  • Table 2 Polymorphic traits/polymorphisms for detection of gene enhancing haploid induction capability.
  • a plant which can be or is produced with the preceding method, including descendants of the plant which have the at least one mutation, or a part of this plant.
  • a part of the plant may be a fertilized or unfertilized seed, an embryo, a pollen, a tissue, an organ, or a plant cell, wherein the fertilized or unfertilized seed, the embryo, or the pollen are generated at the plant obtainable by the preceding method, and the at least one mutation is present in its genome.
  • a non-limiting example of a plant which can be produced with the preceding method is a plant, preferably Zea mays or Sorghum bicolor, which: has at least one mutation in an endogenous DNA sequence that is identical to a nucleic acid that (i) has a sequence selected from SEQ ID NOs: 1, 5, and 12, or a functional fragment thereof, preferably SEQ ID NO: 1; or (ii) has a coding sequence selected from SEQ ID NO: 2, 6 and 13, preferably SEQ ID NO: 2; or (ii) is complementary to the sequence from (i) or (ii); or (iii) is at least 80% identical to the sequence from (i) or (ii); or (iv) encodes for a protein having the amino acid sequence selected from SEQ ID NOs: 3, 7 and 14, preferably SEQ ID NO:
  • the mutation is preferably an alteration in the coding sequence of SEQ ID No: 1, 5 or 12 (for example, a point mutation), i.e. an alteration in SEQ ID NO: SEQ ID NO: 2, 6 or 13, which causes an amino acid exchange at the amino acid position 325 of SEQ ID No: 3, 7 or 14.
  • This may here involve a mutation according to SEQ ID NO: 10.
  • the mutation caused by TILLING in SEQ ID NO: 10 causes an amino acid exchange in the encoded amino acid at position 325, wherein the alanine is replaced by valine (A325V).
  • a plant preferably a Zea mays or Sorghum bicolor plant, comprising: i) the nucleotide sequence according to SEQ ID NO: 10, or
  • nucleotide sequence encoding the amino acid according to SEQ ID NO: 11, or iii) a nucleotide sequence encoding a myosin having at position 325 referenced to SEQ ID NO: 11 or 14 an amino acid different to alanine, preferably valine.
  • the methods for the production of a transgenic or mutated plant may further comprise the steps of:
  • the transgenic or mutated plant which is carrier of an enhancer for the haploid induction capability of a haploid inducer plant, preferably caused by a mutated patatin-like phospholipase (PLA1) obtained by the method with a haploid inducer plant preferably comprising a mutated patatin-like phospholipase (PLA1); and
  • a plant from the progeny which comprises A) the haploid inducer phenotype, preferably the mutated patatin-like phospholipase (PLA1) and B) the introduced nucleic acid in the method for producing a transgenic carrier plant or the at least one mutation created in the method for producing a mutated carrier plant.
  • the haploid inducer phenotype preferably the mutated patatin-like phospholipase (PLA1) and B) the introduced nucleic acid in the method for producing a transgenic carrier plant or the at least one mutation created in the method for producing a mutated carrier plant.
  • the selected plant may be suitable for use as a haploid inductor. Accordingly, the present invention also relates to a method for producing a plant suitable for use as a haploid inductor comprising the following steps:
  • a plant in particular a transgenic plant or a mutated plant, which is carrier of an enhancer for the haploid induction capability of an inducer plant, preferably caused by a mutated patatin-like phospholipase (PLA1) according to a method described herein;
  • PHA1 patatin-like phospholipase
  • PHA1 patatin-like phospholipase
  • a plant from the progeny which comprises A) the haploid inducer phenotype, preferably the mutated patatin-like phospholipase (PLA1) and B) the introduced nucleic acid in the method for producing a transgenic carrier plant described herein or the at least one mutation created in the method for producing a mutated carrier plant described herein.
  • the haploid inducer phenotype preferably the mutated patatin-like phospholipase (PLA1)
  • PUA1 mutated patatin-like phospholipase
  • the nucleic acid may be introduced in a plant cell preferably comprising the mutated patatin-like phospholipase (PLA1) or a plant cell comprising the haploid inducer phenotype, preferably the mutated patatin-like phospholipase (PLA1) may be mutagenized.
  • the plants obtained by such methods may also be suitable for use as haploid inductor.
  • the present invention relates to a method for the production of a haploid plant, said method comprising the following steps:
  • the plant which is suitable for use as a haploid inductor is preferably used as a pollen parent, and is crossed with a seed elder of the same genus, preferably of the same species.
  • the plant which is suitable for use as a haploid inductor may also be used as a seed parent and be crossed with a pollen elder of the same genus, preferably of the same species. Both cross partners in step a), thus, seed parent and pollen parent, may also be the same individual.
  • the crossing step then represents a selfing.
  • the selection of the haploid fertilized seed or embryo encompasses a step of the verification of the haploidy, and the separation of the haploid fertilized seed or embryo of polyploid fertilized seed or embryo.
  • the verification of the haploidy of a fertilized seed or embryo may take place phenotypically or genotypically, in that, for example, the inductor is provided with an embryo-specific dominant marker that is visible in all diploid descendants, but not in the induced haploid descendants.
  • the ploidy status may be determined via flow cytometry.
  • a complete, homozygotic pattern of molecular markers provides an indication of haploid plants.
  • the separation may take place automatically on the basis of data of the verification of the haploidy.
  • a haploid, fertilized seed, or embryo which is created upon crossing in step a) of the method for production of a haploid plant described herein, as well as a haploid plant which can be or is produced with this method, including its descendant, or a part of this plant.
  • the invention further relates to a doubled haploid (diploid) plant or a part thereof, wherein the doubled haploid (diploid) plant or a part thereof was generated by chromosome duplication of the haploid plant described herein or of the part thereof via e.g. use of a chromosome doubling agent or via spontaneous doubling.
  • a related aspect is directed to a method for the production of a double haploid plant, comprising the following steps:
  • the chromosome doubling agent may be colchicine, pronamide, dithipyr, trifluralin, or another known anti-microtubule agent.
  • Another aspect relates to the use of the induction-promoting nucleic acid and the nucleic acid encoding double-stranded RNA described herein, or a vector comprising said induction- promoting nucleic acid or said induction-inhibiting nucleic acid described herein, in a hapoid inducer plant, preferably a plant comprising a mutated patatin-like phospholipase (PLA1) to enhance the haploid induction capability of said plant.
  • a hapoid inducer plant preferably a plant comprising a mutated patatin-like phospholipase (PLA1) to enhance the haploid induction capability of said plant.
  • the present invention also relates to the use of a plant which is suitable for use as a haploid inductor as described herein, to produce a haploid, fertilized seed or embryo, a haploid plant, or a doubled haploid plant.
  • RNA that has a double-stranded portion, wherein at least one strand of the double-stranded portion has a nucleotide sequence which is homologous or identical to at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, preferably at least 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, or 140, more preferably at least 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 successive nucleotides of a coding sequence of a nucleic acid that:
  • (i) has a sequence selected from the group consisting of SEQ ID Nos: 25, 28 and 33, or a fragment thereof, in a sense or anti-sense orientation; or
  • (iii) is at least 80%, 82%, 84%, 86%, or 88%, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, or 96%, more preferably at least 97%, 97.5%, 98%, 98.5%, 99%, or 99.5% identical to the sequence from (i); or
  • RNA is suitable for external application to plants and is suitable for enhancing the induction capability of a haploid inductor plant caused by a mutated patatin-like phospholipase (PLA1).
  • the application preferably occurs at the point in time of the anther formation, pollen formation, or fertilization.
  • the RNA may be used by being sprayed in the form of a spray, or via additional ways of external application that are commonplace to the person skilled in the art, onto the plant tissue, or by spraying or mixing with additional additives before or after the flowering of the plant.
  • additives may be wetting agents, carrier substances, or RNA stabilizers, e.g., liposomes
  • Double-stranded RNA may be produced in vitro by means of methods known to the person skilled in the art. For example, the synthesis of double-stranded RNA may take place synthetically, wherein the RNA is formed directly in vitro. Starting from a double-stranded DNA, double-stranded RNA may also be synthesized via the formation of an mRNA transcript, which then forms a hairpin structure, for example.
  • the invention relates to a plant, preferably a plant of the genus Zea or Sorghum, preferably Zeaaria or Sorghum bicolor, comprising
  • the invention relates to a plant, preferably a plant of the genus Zea or Sorghum, preferably Zeaaria or Sorghum bicolor, comprising
  • nucleotide sequence according to SEQ ID NOs: 38 or 39, or ii) a nucleotide sequence encoding the protein according to SEQ ID NO: 40.
  • SEQ ID NOs: 38-40 are Zeaaria sequences, and that corresponding sequences, in particular comprising the corresponding mutations, may be used from different plant species, such as Sorghum.
  • the invention provides a method of editing a plant's genomic DNA or RNA (such as mRNA, microRNA, etc) or otherwise targeting or modifying such (for instance transcriptional or translational activation or repression, epigenomic modification, etc).
  • a plant's genomic DNA or RNA such as mRNA, microRNA, etc
  • targeting or modifying for instance transcriptional or translational activation or repression, epigenomic modification, etc.
  • This is done by taking a first plant— which is a haploid inducing plant and which also has encoded into its DNA the machinery necessary for accomplishing the editing, targeting, or modification (for example, a Cas9 enzyme and a guide RNA)— and using that first plant's pollen to pollinate a second plant.
  • the second plant is the plant to be edited. From that pollination event, progeny (e.g., embryos or seeds) are produced; at least one of which will be a haploid seed.
  • progeny e.g., embryos or
  • This haploid seed will only contain the chromosomes of the second plant; the first plant's chromosomes have vanished (having been eliminated, lost or degraded), but before doing so, the first plant's chromosomes permitted the gene-editing, targeting, or modification machinery to be expressed.
  • the first plant delivers the already- expressed editing, targeting or modification machinery upon pollination via the pollen tube.
  • the haploid inducer line is the female in the cross
  • the haploid inducing plant's egg cell contains the editing machinery that is present and perhaps already being expressed, upon fertilization with the "wild type" or non-haploid inducing pollen grain.
  • the haploid progeny obtained by the cross will also have had its genome or transcriptome edited, targeted, or otherwise modified.
  • Simultaneous editing plus haploid induction can be done in almost any crop via wide cross or de novo haploid induction for instance via CENH3 mutation (i.e., CENH3 -modified haploid inducer; see, e.g., WO 2017/004375, incorporated herein by reference in its entirety) or via lipid spray (see WO 2017/087682, incorporated herein by reference in its entirety).
  • CENH3 mutation i.e., CENH3 -modified haploid inducer
  • lipid spray see, e.g., WO 2017/087682, incorporated herein by reference in its entirety.
  • the editing machinery is any DNA or RNA modification enzyme, but is preferably a site-directed nuclease.
  • the site-directed nuclease is preferably CRISPR-based, but could also be a meganuclease, a transcription-activator like effector nuclease (TALEN), or a zinc finger nuclease.
  • the nuclease used in this invention could be Cas9, Cfpl, dCas9- Fokl, chimeric FENI- Fokl.
  • the DNA or RNA modification enzyme is a site-directed base editing enzyme such as Cas9-cytidine deaminase or Cas9-adeninie deaminase, wherein the Cas9 can have one or both of its nuclease activity inactivated, i.e. chimeric Cas9 nickase (nCas9) or deactivated Cas9 (dCas9) fused to cytidine deaminase or adenine deaminase.
  • the optional guide RNA targets the genome at the specific site intended to be edited.
  • the modification enzyme is mutated, such as catalytically inactive or a nickase.
  • the modification enzyme is fused to a heterologous domain, preferably a functional heterologous domain.
  • the 'functional) heterologous domain comprises base editing activity, nucleotide deaminase activity, methylase activity, demethylase activity, translation activation activity, translation repression activity, transcription activation activity, transcription repression activity, transcription release factor activity, chromatin modifying or remodeling activity, histone modification activity, nuclease activity, single-strand RNA cleavage activity, double-strand RNA cleavage activity, single-strand DNA cleavage activity, double-strand DNA cleavage activity, and nucleic acid binding activity.
  • the modification enzyme is modified.
  • the modification enzyme is modified, such as to increase or decrease target recognition specificity, increase or decrease nuclease activity, increase or decrease stability, and/or alter PAM recognition.
  • Gene editing refers to genetic engineering in which in which DNA or RNA is inserted, deleted, modified or replaced in the genome of a living organism. Gene editing may comprise targeted or non-targeted (random) mutagenesis. Targeted mutagenesis may be accomplished for instance with designer nucleases, such as for instance with meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system. These nucleases create site-specific double-strand breaks (DSBs) at desired locations in the genome.
  • ZFNs zinc finger nucleases
  • TALEN transcription activator-like effector-based nucleases
  • CRISPR/Cas9 clustered regularly interspaced short palindromic repeats
  • the induced double-strand breaks are repaired through nonhomologous end-joining (NHEJ) or homologous recombination (HR), resulting in targeted mutations or nucleic acid modifications.
  • NHEJ nonhomologous end-joining
  • HR homologous recombination
  • designer nucleases is particularly suitable for generating gene knockouts or knockdowns.
  • designer nucleases are developed which specifically induce a mutation in the gene, as described herein elsewhere, such as to generate a mutated gene or a knockout of the gene.
  • designer nucleases in particular RNA-specific CRISPR/Cas systems are developed which specifically target the mRNA, such as to cleave the mRNA and generate a knockdown of the gene/mRNA/protein. Delivery and expression systems of designer nuclease systems are well known in the art.
  • the nuclease or targeted/site-specific/homing nuclease is, comprises, consists essentially of, or consists of a (modified) CRISPR/Cas system or complex, a (modified) Cas protein, a (modified) zinc finger, a (modified) zinc finger nuclease (ZFN), a (modified) transcription factor-like effector (TALE), a (modified) transcription factor-like effector nuclease (TALEN), or a (modified) meganuclease.
  • said (modified) nuclease or targeted/site-specific/homing nuclease is, comprises, consists essentially of, or consists of a (modified) RNA-guided nuclease.
  • the nucleases may be codon optimized for expression in plants.
  • targeting of a selected nucleic acid sequence means that a nuclease or nuclease complex is acting in a nucleotide sequence specific manner.
  • the guide RNA is capable of hybridizing with a selected nucleic acid sequence.
  • hybridization or “hybridizing” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self-hybridizing strand, or any combination of these.
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of PGR, or the cleavage of a polynucleotide by an enzyme.
  • a sequence capable of hybridizing with a given sequence is referred to as the "complement" of the given sequence.
  • Gene editing may involve transient, inducible, or constitutive expression of the gene editing components or systems. Gene editing may involve genomic integration or episomal presence of the gene editing components or systems. Gene editing components or systems may be provided on vectors, such as plasmids, which may be delivered by appropriate delivery vehicles, as is known in the art. Preferred vectors are expression vectors.
  • Gene editing may comprise the provision of recombination templates, to effect homology directed repair (HDR).
  • HDR homology directed repair
  • a genetic element may be replaced by gene editing in which a recombination template is provided.
  • the DNA may be cut upstream and downstream of a sequence which needs to be replaced.
  • the sequence to be replaced is excised from the DNA.
  • FIDR the excised sequence is then replaced by the template.
  • the genes of the invention as described herein may be provided on/as a template.
  • the gene of the invention may be provided on/as a template. More advantageously however, the gene of the invention may be generated without the use of a recombination template, but solely through the endonuclease action leading to a double strand DNA break which is repaired by NHEJ, resulting in the generation of indels.
  • the nucleic acid modification or mutation is effected by a (modified) transcription activator-like effector nuclease (TALEN) system.
  • Transcription activator-like effectors can be engineered to bind practically any desired DNA sequence. Exemplary methods of genome editing using the TALEN system can be found for example in Cermak T. Doyle EL. Christian M. Wang L. Zhang Y. Schmidt C, et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 2011;39:e82; Zhang F. Cong L. Lodato S. Kosuri S. Church GM.
  • TALEs or wild type TALEs are nucleic acid binding proteins secreted by numerous species of proteobacteria.
  • TALE polypeptides contain a nucleic acid binding domain composed of tandem repeats of highly conserved monomer polypeptides that are predominantly 33, 34 or 35 amino acids in length and that differ from each other mainly in amino acid positions 12 and 13.
  • the nucleic acid is DNA.
  • polypeptide monomers or “TALE monomers” will be used to refer to the highly conserved repetitive polypeptide sequences within the TALE nucleic acid binding domain and the term “repeat variable di-residues” or “RVD” will be used to refer to the highly variable amino acids at positions 12 and 13 of the polypeptide monomers.
  • RVD repeat variable di-residues
  • the amino acid residues of the RVD are depicted using the lUPAC single letter code for amino acids.
  • a general representation of a TALE monomer which is comprised within the DNA binding domain is Xl-ll-(X12X13)-X14-33 or 34 or 35, where the subscript indicates the amino acid position and X represents any amino acid.
  • X12X13 indicate the RVDs.
  • the variable amino acid at position 13 is missing or absent and in such polypeptide monomers, the RVD consists of a single amino acid.
  • the RVD may be alternatively represented as X*, where X represents X12 and (*) indicates that X13 is absent.
  • the DNA binding domain comprises several repeats of TALE monomers and this may be represented as (Xl-ll-(X12X13)-X14-33 or 34 or 35)z, where in an advantageous embodiment, z is at least 5 to 40. In a further advantageous embodiment, z is at least 10 to 26.
  • the TALE monomers have a nucleotide binding affinity that is determined by the identity of the amino acids in its RVD.
  • polypeptide monomers with an RVD of Nl preferentially bind to adenine (A)
  • polypeptide monomers with an RVD of NG preferentially bind to thymine (T)
  • polypeptide monomers with an RVD of HD preferentially bind to cytosine (C)
  • polypeptide monomers with an RVD of NN preferentially bind to both adenine (A) and guanine (G).
  • polypeptide monomers with an RVD of IG preferentially bind to T.
  • the number and order of the polypeptide monomer repeats in the nucleic acid binding domain of a TALE determines its nucleic acid target specificity.
  • polypeptide monomers with an RVD of NS recognize all four base pairs and may bind to A, T, G or C.
  • TALEs The structure and function of TALEs is further described in, for example, Moscou et al., Science 326:1501 (2009); Boch et al., Science 326:1509-1512 (2009); and Zhang et al., Nature Biotechnology 29:149-153 (2011), each of which is incorporated by reference in its entirety.
  • the nucleic acid modification or mutation is effected by a (modified) zinc-finger nuclease (ZFN) system.
  • ZFN zinc-finger nuclease
  • the ZFN system uses artificial restriction enzymes generated by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain that can be engineered to target desired DNA sequences. Exemplary methods of genome editing using ZFNs can be found for example in U.S. Patent Nos.
  • ZF artificial zinc-finger
  • ZFP ZF protein
  • the first synthetic zinc finger nucleases were developed by fusing a ZF protein to the catalytic domain of the Type IIS restriction enzyme Fokl.
  • Fokl Type IIS restriction enzyme
  • Kim Y. G. et al., 1994, Chimeric restriction endonuclease, Proc. Natl. Acad. Sci. U.S. A. 91, 883-887; Kim, Y. G. et al., 1996, Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain. Proc. Natl. Acad. Sci. U.S. A. 93, 1156-1160).
  • ZFPs can also be designed as transcription activators and repressors and have been used to target many genes in a wide variety of organisms.
  • the nucleic acid modification is effected by a (modified) meganuclease, which are endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs).
  • a (modified) meganuclease which are endodeoxyribonucleases characterized by a large recognition site (double-stranded DNA sequences of 12 to 40 base pairs).
  • Exemplary method for using meganucleases can be found in US Patent Nos: 8,163,514; 8,133,697; 8,021,867; 8,119,361; 8,119,381; 8,124,369; and 8,129,134, which are specifically incorporated by reference.
  • the nucleic acid modification is effected by a (modified) CRISPR/Cas complex or system.
  • CRISPR/Cas Systems components thereof, and delivery of such components, including methods, materials, delivery vehicles, vectors, particles, and making and using thereof, including as to amounts and formulations, as well as Cas9CRISPR/Cas-expressing eukaryotic cells, Cas-9 CRISPR/Cas expressing eukaryotes, such as a mouse, reference is made to: US Patents Nos. 8,999,641, 8,993,233, 8,697,359,
  • the CRISPR/Cas system or complex is a class 2 CRISPR/Cas system. In certain embodiments, said CRISPR/Cas system or complex is a type II, type V, or type VI CRISPR/Cas system or complex.
  • the CRISPR/Cas system does not require the generation of customized proteins to target specific sequences but rather a single Cas protein can be programmed by an RNA guide (gRNA) to recognize a specific nucleic acid target, in other words the Cas enzyme protein can be recruited to a specific nucleic acid target locus (which may comprise or consist of RNA and/or DNA) of interest using said short RNA guide.
  • gRNA RNA guide
  • CRISPR/Cas or CRISPR system is as used herein foregoing documents refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas") genes, including sequences encoding a Cas gene and one or more of, a tracr (trans-activating CRISPR) sequence (e.g.
  • RNA(s) as that term is herein used (e.g., RNA(s) to guide Cas, such as Cas9, e.g. CRISPR RNA and, where applicable, transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from a CRISPR locus.
  • RNA(s) e.g., RNA(s) to guide Cas, such as Cas9, e.g. CRISPR RNA and, where applicable, transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from a CRISPR locus.
  • a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system).
  • target sequence refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex.
  • a target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides.
  • the gRNA is a chimeric guide RNA or single guide RNA (sgRNA).
  • the gRNA comprises a guide sequence and a tracr mate sequence (or direct repeat).
  • the gRNA comprises a guide sequence, a tracr mate sequence (or direct repeat), and a tracr sequence.
  • the CRISPR/Cas system or complex as described herein does not comprise and/or does not rely on the presence of a tracr sequence (e.g. if the Cas protein is Cpfl).
  • the term “crRNA” or “guide RNA” or “single guide RNA” or “sgRNA” or “one or more nucleic acid components" of a CRISPR/Cas locus effector protein comprises any polynucleotide sequence having sufficient complementarity with a target nucleic acid sequence to hybridize with the target nucleic acid sequence and direct sequence- specific binding of a nucleic acid-targeting complex to the target nucleic acid sequence.
  • the degree of complementarity when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.
  • Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (lllumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
  • the ability of a guide sequence (within a nucleic acid-targeting guide RNA) to direct sequence-specific binding of a nucleic acid -targeting complex to a target nucleic acid sequence may be assessed by any suitable assay.
  • a guide sequence, and hence a nucleic acid-targeting guide RNA may be selected to target any target nucleic acid sequence.
  • the target sequence may be DNA.
  • the target sequence may be genomic DNA.
  • the target sequence may be mitochondrial DNA.
  • the target sequence may be any RNA sequence.
  • the target sequence may be a sequence within a RNA molecule selected from the group consisting of messenger RNA (mRNA), pre-mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), micro-RNA (miRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), double stranded RNA (dsRNA), non coding RNA (ncRNA), long non-coding RNA (IncRNA), and small cytoplasmatic RNA (scRNA).
  • the target sequence may be a sequence within a RNA molecule selected from the group consisting of mRNA, pre-mRNA, and rRNA.
  • the target sequence may be a sequence within a RNA molecule selected from the group consisting of ncRNA, and IncRNA. In some more preferred embodiments, the target sequence may be a sequence within an mRNA molecule or a pre-mRNA molecule.
  • the gRNA comprises a stem loop, preferably a single stem loop.
  • the direct repeat sequence forms a stem loop, preferably a single stem loop.
  • the spacer length of the guide RNA is from 15 to 35 nt. In certain embodiments, the spacer length of the guide RNA is at least 15 nucleotides.
  • the spacer length is from 15 to 17 nt, e.g., 15, 16, or 17 nt, from 17 to 20 nt, e.g., 17, 18, 19, or 20 nt, from 20 to 24 nt, e.g., 20, 21, 22, 23, or 24 nt, from 23 to 25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt, e.g., 24, 25, 26, or 27 nt, from 27-30 nt, e.g., 27, 28, 29, or 30 nt, from 30-35 nt, e.g., 30, 31, 32, 33, 34, or 35 nt, or 35 nt or longer.
  • the CRISPR/Cas system requires a tracrRNA.
  • the "tracrRNA” sequence or analogous terms includes any polynucleotide sequence that has sufficient complementarity with a crRNA sequence to hybridize.
  • the degree of complementarity between the tracrRNA sequence and crRNA sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher.
  • the tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length.
  • the tracr sequence and gRNA sequence are contained within a single transcript, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin.
  • the transcript or transcribed polynucleotide sequence has at least two or more hairpins.
  • the transcript has two, three, four or five hairpins.
  • the transcript has at most five hairpins.
  • the portion of the sequence 5' of the final "N" and upstream of the loop may correspond to the tracr mate sequence, and the portion of the sequence 3' of the loop then corresponds to the tracr sequence.
  • the portion of the sequence 5' of the final "N" and upstream of the loop may alternatively correspond to the tracr sequence, and the portion of the sequence 3' of the loop corresponds to the tracr mate sequence.
  • the CRISPR/Cas system does not require a tracrRNA, as is known by the skilled person.
  • the guide RNA (capable of guiding Cas to a target locus) may comprise (1) a guide sequence capable of hybridizing to a target locus and (2) a tracr mate or direct repeat sequence (in 5' to 3' orientation, or alternatively in 3' to 5' orientation, depending on the type of Cas protein, as is known by the skilled person).
  • the CRISPR/Cas protein is characterized in that it makes use of a guide RNA comprising a guide sequence capable of hybridizing to a target locus and a direct repeat sequence, and does not require a tracrRNA.
  • the guide sequence, tracr mate, and tracr sequence may reside in a single RNA, i.e. an sgRNA (arranged in a 5' to 3' orientation or alternatively arranged in a 3' to 5' orientation), or the tracr RNA may be a different RNA than the RNA containing the guide and tracr mate sequence.
  • the tracr hybridizes to the tracr mate sequence and directs the CRISPR/Cas complex to the target sequence.
  • nucleic acid-targeting complex comprising a guide RNA hybridized to a target sequence and complexed with one or more nucleic acid-targeting effector proteins
  • modification results in modification (such as cleavage) of one or both DNA or RNA strands in or near (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence.
  • sequence(s) associated with a target locus of interest refers to sequences near the vicinity of the target sequence (e.g.
  • target sequence within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from the target sequence, wherein the target sequence is comprised within a target locus of interest).
  • the skilled person will be aware of specific cut sites for selected CRISPR/Cas systems, relative to the target sequence, which as is known in the art may be within the target sequence or alternatively 3' or 5' of the target sequence.
  • the unmodified nucleic acid-targeting effector protein may have nucleic acid cleavage activity.
  • the nuclease as described herein may direct cleavage of one or both nucleic acid (DNA, RNA, or hybrids, which may be single or double stranded) strands at the location of or near a target sequence, such as within the target sequence and/or within the complement of the target sequence or at sequences associated with the target sequence.
  • the nucleic acid-targeting effector protein may direct cleavage of one or both DNA or RNA strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence.
  • the cleavage may be blunt (e.g. for Cas9, such as SaCas9 or SpCas9).
  • the cleavage may be staggered (e.g. for Cpfl), i.e. generating sticky ends.
  • the cleavage is a staggered cut with a 5' overhang.
  • the cleavage is a staggered cut with a 5' overhang of 1 to 5 nucleotides, preferably of 4 or 5 nucleotides.
  • the cleavage site is upstream of the PAM.
  • the cleavage site is downstream of the PAM.
  • the nucleic acid-targeting effector protein that may be mutated with respect to a corresponding wild-type enzyme such that the mutated nucleic acid-targeting effector protein lacks the ability to cleave one or both DNA or RNA strands of a target polynucleotide containing a target sequence.
  • two or more catalytic domains of a Cas protein may be mutated to produce a mutated Cas protein substantially lacking all DNA cleavage activity.
  • a nucleic acid-targeting effector protein may be considered to substantially lack all DNA and/or RNA cleavage activity when the cleavage activity of the mutated enzyme is about no more than 25%, 10%, 5%, 1%, 0.1%, 0.01%, or less of the nucleic acid cleavage activity of the non-mutated form of the enzyme; an example can be when the nucleic acid cleavage activity of the mutated form is nil or negligible as compared with the non-mutated form.
  • modified Cas generally refers to a Cas protein having one or more modifications or mutations (including point mutations, truncations, insertions, deletions, chimeras, fusion proteins, etc.) compared to the wild type Cas protein from which it is derived.
  • derived is meant that the derived enzyme is largely based, in the sense of having a high degree of sequence homology with, a wildtype enzyme, but that it has been mutated (modified) in some way as known in the art or as described herein.
  • the target sequence should be associated with a PAM (protospacer adjacent motif) or PFS (protospacer flanking sequence or site); that is, a short sequence recognized by the CRISPR complex.
  • PAM protospacer adjacent motif
  • PFS protospacer flanking sequence or site
  • the precise sequence and length requirements for the PAM differ depending on the CRISPR enzyme used, but PAMs are typically 2-5 base pair sequences adjacent the protospacer (that is, the target sequence). Examples of PAM sequences are given in the examples section below, and the skilled person will be able to identify further PAM sequences for use with a given CRISPR enzyme.
  • engineering of the PAM Interacting (PI) domain may allow programing of PAM specificity, improve target site recognition fidelity, and increase the versatility of the Cas, e.g. Cas9, genome engineering platform.
  • Cas proteins such as Cas9 proteins may be engineered to alter their PAM specificity, for example as described in Kleinstiver BP et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature. 2015 Jul 23;523(7561):481-5. doi: 10.1038/naturel4592.
  • the method comprises allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence.
  • the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence.
  • the Cas protein as referred to herein may originate from any suitable source, and hence may include different orthologues, originating from a variety of (prokaryotic) organisms, as is well documented in the art.
  • the Cas protein is (modified) Cas9, preferably (modified) Staphylococcus aureus Cas9 (SaCas9) or (modified) Streptococcus pyogenes Cas9 (SpCas9).
  • the Cas protein is (modified) Cpfl, preferably Acidaminococcus sp., such as Acidaminococcus sp. BV3L6 Cpfl (AsCpfl) or Lachnospiraceae bacterium Cpfl, such as Lachnospiraceae bacterium MA2020 or Lachnospiraceae bacterium MD2006 (LbCpfl).
  • the Cas protein is (modified) C2c2, preferably Leptotrichia wadei C2c2 (LwC2c2) or Listeria newyorkensis FSL M6- 0635 C2c2 (LbFSLC2c2).
  • the (modified) Cas protein is C2cl.
  • the (modified) Cas protein is C2c3.
  • the (modified) Cas protein is Casl3b.
  • the nucleic acid modification is effected by random mutagenesis.
  • Cells or organisms may be exposed to mutagens such as UV radiation or mutagenic chemicals (such as for instance such as ethyl methanesulfonate (EMS)), and mutants with desired characteristics are then selected.
  • Mutants can for instance be identified by TILLING (Targeting Induced Local Lesions in Genomes).
  • TILLING Targeting Induced Local Lesions in Genomes.
  • the method combines mutagenesis, such as mutagenesis using a chemical mutagen such as ethyl methanesulfonate (EMS) with a sensitive DNA screening-technique that identifies single base mutations/point mutations in a target gene.
  • EMS ethyl methanesulfonate
  • the TILLING method relies on the formation of DNA heteroduplexes that are formed when multiple alleles are amplified by PCR and are then heated and slowly cooled. A "bubble” forms at the mismatch of the two DNA strands, which is then cleaved by a single stranded nucleases. The products are then separated by size, such as by HPLC. See also McCallum et al. "Targeted screening for induced mutations"; Nat Biotechnol. 2000 Apr;18(4):455-7 and McCallum et al. "Targeting induced local lesions IN genomes (TILLING) for plant functional genomics"; Plant Physiol. 2000 Jun;123(2):439-42.
  • RNA interference is a biological process in which RNA molecules inhibit gene expression or translation, by neutralizing targeted mRNA molecules.
  • RNA molecules Two types of small ribonucleic acid (RNA) molecules - microRNA (miRNA) and small interfering RNA (siRNA) - are central to RNA interference.
  • RNAs are the direct products of genes, and these small RNAs can bind to other specific messenger RNA (mRNA) molecules and either increase or decrease their activity, for example by preventing an mRNA from being translated into a protein.
  • RNAi pathway is found in many eukaryotes, including animals, and is initiated by the enzyme Dicer, which cleaves long double-stranded RNA (dsRNA) molecules into short double-stranded fragments of about 21 nucleotide siRNAs (small interfering RNAs). Each siRNA is unwound into two single- stranded RNAs (ssRNAs), the passenger strand and the guide strand. The passenger strand is degraded and the guide strand is incorporated into the RNA-induced silencing complex (RISC). Mature miRNAs are structurally similar to siRNAs produced from exogenous dsRNA, but before reaching maturity, miRNAs must first undergo extensive post-transcriptional modification.
  • RISC RNA-induced silencing complex
  • a miRNA is expressed from a much longer RNA-coding gene as a primary transcript known as a pri-miRNA which is processed, in the cell nucleus, to a 70-nucleotide stem-loop structure called a pre-miRNA by the microprocessor complex.
  • This complex consists of an RNase III enzyme called Drosha and a dsRNA-binding protein DGCR8.
  • the dsRNA portion of this pre-miRNA is bound and cleaved by Dicer to produce the mature miRNA molecule that can be integrated into the RISC complex; thus, miRNA and siRNA share the same downstream cellular machinery.
  • RNAi molecules may be an siRNA, shRNA, or a miRNA.
  • the RNAi molecules can be applied as such to/in the plant, or can be encoded by appropriate vectors, from which the RNAi molecule is expressed. Delivery and expression systems of RNAi molecules, such as siRNAs, shRNAs or miRNAs are well known in the art.
  • the invention relates to a polynucleic acid, preferably an isolated polynucleic acid, capable of specifically hybridizing with any of the nucleic acid molecules of the invention as described herein, or the complement or reverse complement thereof.
  • a polynucleic acid is capable of specifically hybridizing with a nucleotide sequence molecule of SEQ ID NO: 1, 2, 4, 10, 15, 16, 24, 25, 30, 35, 36, 38, or 39; or the complement or the reverse complement thereof. It will be understood that such polynucleic acid hybridizes specifically with the recited sequences if it does not hybridize with related sequences (e.g. mutated genes versus wild type genes).
  • the polynucleic acid comprises less than 500 nucleotides, such as less than 400 nucleotides, such as less than 300 nucleotides, such as less than 200 nucleotides, such as less than 100, nucleotides, such as preferably less than 80 nucleotides, more preferably less than 60 nucleotides, most preferably less than 40 nucleotides. In certain embodiments, such polynucleic acids comprise at least 5 nucleotides, preferably at least 10 nucleotides, more preferably at least 15 nucleotides.
  • polynucleic acids can therefore be used to discriminate between the haploid inducer enhancers according to the invention and other non-haploid inducer enhancer plants.
  • the polynucleic acid comprises less than 500 nucleotides, such as less than 400 nucleotides, such as less than 300 nucleotides, such as less than 200 nucleotides, such as less than 100, nucleotides, such as preferably less than 80 nucleotides, more preferably less than 60 nucleotides, most preferably less than 40 nucleotides; and at least 5 nucleotides, preferably at least 10 nucleotides, more preferably at least 15 nucleotides.
  • such polynucleic acids comprise from 10 to 60 nucleotides, preferably from 15 to 40 nucleotides.
  • such polynucleic acids are primers or probes, as described herein elsewhere.
  • the invention further relates to a kit comprising one or more of the above polynucleic acid.
  • Example 1 QTL analysis and identification of candidate genes
  • the functionality of the QTL HIR8 on chromosome 9 was shown by comparing the induction rate of recombinants carrying only the functional mutation (HIR1 carrying the PLA1 gene), only the QTL on chromosome 9 (HIR8) or both:
  • the haplotype of the region of interest is identical between RWS, KW5526 and the public line W22.
  • KW5526 was sequenced (40x coverage, lllumina) and the genic sequences were compared. No differences could be identified.
  • the functionality of the HIR8 allele in KW5526 was shown in an F2 line containing HIR1 of RWS and HIR8 of KW5526. The induction rate increased from 1.6% (control lines) to 4%.
  • upstream region SEQ ID NO: 4
  • a fragment of 31.7 kb length is inserted in W22.
  • Annotation showed two additional genes, one zinc ion binding gene with two exons and UTR, i.e. all characteristics of a functional gene.
  • the second gene seems to be a transposable element.
  • the RNASeq data of RWS pollen, which were mapped to W22, did not show any expression of these genes.
  • Myosin itself is expressed 2-3 fold higher than in control lines. The introgression in the promotor may be responsible for this effect.
  • the line PH207 is not carrier of the HIR8 QTL and has been used for generation of a TILLING population.
  • the TILLING population of PH207 was screened for myosin mutants. These were crossed to a mutant of HIR1, which has an induction rate of 1-2% on its own. Double homozygous plants were tested for induction using a glossy tester and subsequent flowcytometry.
  • this mutation in the myosin gene may disrupt a helix structure around amino acid position 325 (Fig. 1) leading to either an increase of the stability of the protein or its functionality, thus mimicking an effect of overexpression.
  • Any of these genes individually, or any combination of the genes, may be responsible for the effect of haploid induction.

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Abstract

La présente invention concerne des procédés d'induction d'haploïdes, basés sur le QTL HIR8 sur le chromosome 9 du maïs, ainsi que des gènes et des marqueurs associés. L'invention concerne en outre l'identification d'inducteurs d'haploïdes, ainsi que des procédés pour générer de manière transgénique des inducteurs d'haplotypes ainsi que la combinaison de l'induction d'haplotypes et de l'édition de gènes.
PCT/EP2020/064420 2019-05-25 2020-05-25 Stimulateur d'induction d'haploïdes WO2020239680A2 (fr)

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