In vivo haploid induction line of sunflower

Abstract

The present invention provides a Sunflower plant having haploid inducer activity. In particular, haploid inducer activity is obtained by targeting two CENH3 copies present in sunflower to reduce its expression and / or activity. The invention also provides polynucleotides and polypeptides carrying certain mutations, including knock-down or knock-out mutations, as well as methods of producing and identifying the plants according to the invention.

Description

Technical Field

[0001] This invention provides a *Helianthus* plant with haploid inducer activity. Specifically, haploid inducer activity is obtained by targeting two copies of CENH3 present in *Helianthus* plants to reduce their expression and / or activity. This invention also provides polynucleotides and polypeptides carrying specific knockdown or knockout mutations, as well as methods for preparing and identifying the plant described herein. Background of the Invention The generation and application of haploids is one of the most powerful biotechnological means for improving cultivated plants. The advantage of haploids for breeders is that, after doubling and haploidization, homozygosity can be achieved in the first generation, allowing for the cultivation of doubled haploid plants and obtaining high homozygosity without multiple self-crossing / backcrossing steps. Furthermore, the value of haploids in plant research and breeding lies in the fact that the starting cells for doubled haploids are products of meiosis, resulting in a diverse and genotype-fixed population. Therefore, the generation of doubled haploids not only provides ideal genetic variation for crop improvement but is also an important means of constructing mapping populations, recombinant inbred lines, and rapidly obtaining homozygous mutants and transgenic lines.

[0003] Haploid plants can be prepared either in vitro or in vivo. In vitro haploid preparation is achieved through cell culture, including androgenesis via anther or pollen culture, or gynogenesis via ovule or ovary culture. Appropriate techniques have been developed for different plant species.

[0004] Haploid plants can acquire genomic elimination in vivo through interspecific and intraspecific hybridization, in which one of the paternal genomes is eliminated after fertilization. Studies have shown that modifying a centriole protein, specifically the centriole-specific histone CENH3 in Arabidopsis, can induce genomic elimination after fertilization (Ravi and Chan, Haploid plants produced by centromere-mediated genome elimination). Nature (Vol. 464, 2010, 615-619). Using modified haploid inducible lines, haploidization occurred in the offspring when haploid inducer plants were crossed with wild-type plants. Interestingly, the haploid inducible lines were stable during self-pollination, suggesting that competition between modified and wild-type centrioles in the developing hybrid embryo leads to centriolein inactivation in the inducer parent, resulting in the elimination of the uniparental chromosome. However, Ravi and Chan only used the transgenic system and did not demonstrate any data on its transfer to crops. Despite the efforts of many scientists to achieve this transfer, it has proven to be more difficult than expected.

[0005] Wang et al. (Haploid induction by a maize cenh3 null mutant, Sci. Adv. ,2021, 7(4), eabe2299) demonstrated the generation of paternal haploids in maize using a heterozygous CenH3 knockout mutant.

[0006] For sunflower (Helianthus), there is currently no effective double haploid (DH) technology available, either in vitro or in vivo. Until recently, all in vivo systems based on mutations in known candidate genes (such as CenH3, patatin-like phospholipase, or KNL2) have failed.

[0007] Therefore, one objective of this invention is to provide an efficient method for producing haploid sunflower plants to improve crop production.

[0008] More specifically, one objective of this invention is to identify mutations in the sunflower genome that confer activity on haploid inducers.

[0009] Another objective of this invention is to provide sunflower plants with haploid induction rates to allow for the development of improved plants. Invention Overview In one aspect, the present invention relates to a sunflower plant having haploid inducer activity, comprising (i) The first nucleotide sequence encoding the first CENH3 protein, and (ii) The second nucleotide sequence encoding the second type of CENH3 protein. The combined expression and / or activity of the first and second CENH3 proteins is reduced by about 60% to about 95% compared to wild-type sunflowers, preferably by about 65% to about 90%, and particularly preferably by about 75% to about 80%.

[0011] In one embodiment of the plant described above, three of the four alleles encoding the first and second CENH3 proteins carry at least one mutation, including at least one knockdown or knockout mutation.

[0012] In another embodiment of the above-mentioned plant, the expression of the first and second CENH3 proteins is reduced due to the presence of a double-stranded RNA molecule or a group of molecules that target the first and second nucleotide sequences for gene silencing.

[0013] In one embodiment of the plant according to any of the above embodiments, the first nucleotide sequence is represented by the sequence of SEQ ID NO: 1 or 2, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the corresponding reference sequence of SEQ ID NO: 3 or 4, and / or the second nucleotide sequence is represented by the sequence of SEQ ID NO: 3 or 4, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the corresponding reference sequence of SEQ ID NO: 3 or 4.

[0014] In one embodiment of the plant according to any of the above embodiments, the first CENH3 protein is represented by the amino acid sequence of SEQ ID NO: 5, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 5 as the corresponding reference sequence, and / or wherein the second CENH3 protein is represented by the amino acid sequence of SEQ ID NO: 6, or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 6 as the corresponding reference sequence.

[0015] In one embodiment of the above-described plant, at least one mutation in the first CENH3 protein, preferably a mutation resulting in at least one knockdown or knockout mutation, is selected from: (a) The glutamine at position 94 of the sequence of SEQ ID NO: 5 is substituted, preferably resulting in a stop codon; (b) The tryptophan at position 93 in the sequence of SEQ ID NO: 5 is substituted, preferably resulting in a stop codon; (c) The glutamic acid at position 103 in the sequence of SEQ ID NO: 5 is substituted, preferably with lysine; (d) The histidine at position 47 of the sequence of SEQ ID NO: 5 is substituted, preferably with tyrosine; (e) The asparagine at position 43 of the sequence of SEQ ID NO: 5 is substituted, preferably with lysine; (f) The glycine at position 41 of the sequence of SEQ ID NO: 5 is substituted, preferably with glutamic acid; (g) The leucine at position 56 in the sequence of SEQ ID NO: 5 is substituted, preferably with phenylalanine; (h) The serine at position 87 in the sequence of SEQ ID NO: 5 is substituted, preferably with threonine; (i) The threonine at position 53 in the sequence of SEQ ID NO: 5 is substituted, preferably with methionine; (j) The isoleucine at position 70 of SEQ ID NO: 5 is substituted, preferably with threonine; (k) The lysine at position 9 of the sequence SEQ ID NO: 5 is substituted, preferably with arginine; and wherein At least one mutation in the second CENH3 protein, preferably a mutation leading to functional knockdown or elimination, is selected from: (l) At least one nucleotide in the ATG nucleic acid sequence encoding methionine at position 1 of the sequence SEQ ID NO: 6 is deleted, inserted, or substituted, resulting in a knockout mutation, preferably achieved by frameshift insertion or deletion or nonsense mutation, or resulting in an exchange of the encoded amino acid at position 1, for example, replacing methionine with isoleucine. (m) The glutamine at position 96 of the sequence SEQ ID NO: 6 is substituted, preferably resulting in a stop codon, and (n) In the sequence of SEQ ID NO: 6, isoleucine at position 118 is substituted, preferably with phenylalanine, and (o) According to public reference XRQ2, a mutation occurs at position 93963324 on chromosome 15.

[0016] In one embodiment, the plant carries at least mutation (a) and one mutation selected from mutations (l), (m), (n), and (o) (as defined above), or the plant carries at least mutation (b) and one mutation selected from mutations (l), (m), (n), and (o) (as defined above), or the plant carries at least mutation (c) and one mutation selected from mutations (l), (m), (n), and (o) (as defined above), or the plant carries at least mutation (d) and one mutation selected from mutations (l), (m), (n), and (o) A mutation (as defined above), or the plant carries at least one mutation (e) and one mutation selected from mutations (l), (m), (n), and (o) (as defined above), or the plant carries at least one mutation (f) and one mutation selected from mutations (l), (m), (n), and (o) (as defined above), or the plant carries at least one mutation (g) and one mutation selected from mutations (l), (m), (n), and (o) (as defined above), or the plant carries at least one mutation (h) and one mutation selected from mutations (l), (m), (n), and (o) The plant carries at least one mutation (as defined above), or the plant carries at least one mutation selected from mutations (l), (m), (n) and (o) (as defined above), or the plant carries at least one mutation (j) and one mutation selected from mutations (l), (m), (n) and (o) (as defined above), or the plant carries at least one mutation (k) and one mutation selected from mutations (l), (m), (n) and (o) (as defined above), preferably the plant carries at least one mutation (a) and (l) as defined above, or the plant carries at least one mutation (k) and one mutation selected from mutations (l), (m), (n) and (o) (as defined above), preferably the plant carries at least one mutation (a) and (l) as defined above, or the plant carries at least one mutation (j) and one mutation selected from mutations (l), (m), (n) and (o) (as defined above), preferably the plant carries at least one mutation (a) and one mutation (l) as defined above, or the plant carries at least one mutation (i ... The plant carries at least the mutations (a) and (m) as defined above, or the plant carries at least the mutations (a) and (n) as defined above, or the plant carries at least the mutations (a) and (o) as defined above, or the plant carries at least the mutations (b) and (l) as defined above, or the plant carries at least the mutations (b) and (m) as defined above, or the plant carries at least the mutations (b) and (o) as defined above, or the plant carries at least one of the mutations (f), (g), (h), (i) and (k) and the mutation (m) as defined above.

[0017] In another aspect, the present invention relates to a polynucleotide or a group of polynucleotides encoding a protein represented by the amino acid sequence of SEQ ID NO: 5 and / or 6 or a sequence representing a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5 or 6 (as respective reference sequences), and carrying at least one knockout mutation, preferably selected from the mutations defined above or a combination of at least two knockout combinations, preferably as defined above.

[0018] In another aspect, the present invention relates to polypeptides encoded by polynucleotides or a group of polynucleotides as defined above.

[0019] In another aspect, the present invention relates to a carrier comprising a polynucleotide or a group of polynucleotides as defined above.

[0020] In one aspect, the present invention also relates to cells comprising polynucleotides or groups of polynucleotides as defined above, polypeptides as defined above, or carriers as defined above.

[0021] In another aspect, the present invention relates to a method for producing plants or plant parts, particularly sunflowers, preferably plants or plant parts as defined in any of the above embodiments, comprising: (a) By means of gene editing technology or by using random or directed mutagenesis, at least one mutation, preferably a knockdown or knockout mutation, is introduced into the genome of a plant or part of a plant (preferably a sunflower) into the first nucleotide sequence encoding a first CENH3 protein and the second nucleotide sequence encoding a second CENH3 protein, such that three of the four alleles encoding the first and second CENH3 proteins are knocked down or knocked out; or (b) Using gene editing technology, RNAi molecules or a group of RNAi molecules are stably or transiently integrated into a plant or plant part by transformation or insertion, wherein these RNAi molecules target, bind to, or hybridize to the first nucleotide sequence encoding a first CENH3 protein and the second nucleotide sequence encoding a second CENH3 protein. Furthermore, optionally, a plant can be regenerated from the plant portion in (a) or (b). Preferably, the first CENH3 protein is represented by the amino acid sequence of SEQ ID NO: 5 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 5, and / or the second CENH3 protein is represented by the amino acid sequence of SEQ ID NO: 6 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 6.

[0022] In one embodiment of the above method, at least one mutation in the first CENH3 protein, preferably a mutation that functionally results in knockout or knockdown, is selected from: (a) The glutamine at position 94 of the sequence of SEQ ID NO: 5 is substituted, preferably resulting in a stop codon; (b) The tryptophan at position 93 in the sequence of SEQ ID NO: 5 is substituted, preferably resulting in a stop codon; (c) The glutamic acid at position 103 in the sequence of SEQ ID NO: 5 is substituted, preferably with lysine; (d) The histidine at position 47 of the sequence of SEQ ID NO: 5 is substituted, preferably with tyrosine; (e) The aspartic acid at position 43 of the sequence of SEQ ID NO: 5 is substituted, preferably with lysine; (f) The glycine at position 41 of the sequence of SEQ ID NO: 5 is substituted, preferably with glutamic acid; (g) The leucine at position 56 in the sequence of SEQ ID NO: 5 is substituted, preferably with phenylalanine; (h) The serine at position 87 in the sequence of SEQ ID NO: 5 is substituted, preferably with threonine; (i) The threonine at position 53 in the sequence of SEQ ID NO: 5 is substituted, preferably with methionine; (j) The isoleucine at position 70 of SEQ ID NO: 5 is substituted, preferably with threonine; (k) The lysine at position 9 of the sequence of SEQ ID NO: 5 is substituted, preferably with arginine; At least one mutation in the second CENH3 protein, preferably a knockout or knockdown mutation, is selected from: (l) At least one nucleotide in the ATG nucleic acid sequence encoding methionine at position 1 of the sequence in SEQ ID NO: 6 is deleted, inserted, or substituted, resulting in a knockout mutation, preferably by frameshift insertion or deletion or nonsense mutation, or resulting in an exchange of the encoded amino acid at position 1, for example, methionine is exchanged for isoleucine. (m) In the sequence of SEQ ID NO: 6, the glutamine at position 96 is substituted, preferably resulting in a stop codon. (n) In the sequence of SEQ ID NO: 6, isoleucine at position 118 is substituted, preferably with phenylalanine. (o) According to public reference XRQ2, a mutation occurs at position 93963324 on chromosome 15; Preferably, in step (a), at least one mutation (a) as defined above and one mutation selected from mutations (l), (m), (n), and (o) are introduced; or at least one mutation (b) as defined above and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation (c) as defined above and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation (d) as defined above and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation (e) as defined above and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation (f) as defined above and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation (g) as defined above and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation (h) as defined above and one mutation selected from mutations (l), (m), (n), and (o). Mutations of (l), (m), (n) and (o), or at least the above-defined mutation (i) and a mutation selected from mutations (l), (m), (n) and (o), or at least the above-defined mutation (j) and a mutation selected from mutations (l), (m), (n) and (o), or at least the above-defined mutation (k) and a mutation selected from mutations (l), (m), (n) and (o), preferably at least the above-defined mutations (a) and (l), or at least the above-defined mutations (a) and (m), or at least the above-defined mutations (a) and (n), or at least the above-defined mutations (a) and (o), or at least the above-defined mutations (b) and (l), or at least the above-defined mutations (b) and (m), or at least the above-defined mutations (b) and (o), or at least one of the above-defined mutations (f), (g), (h), (i) and (k) and mutation (m).

[0023] In another aspect, the present invention relates to the use of the above-defined mutations or combinations of the above-defined mutations or the above-defined polynucleotides or the above-defined polypeptides or the above-defined vectors for generating plants having haploid inducer activity, preferably sunflower plants.

[0024] In another aspect, the present invention relates to a method for identifying sunflower plants with haploid inducer activity, preferably as defined in any of the above embodiments, comprising: (a) Screening for mutations in the first nucleotide sequence encoding the first type of CENH3 protein and the second nucleotide sequence encoding the second type of CENH3 protein, preferably with knockdown or deletion mutations, or (b) Screening for reduced expression of the first and second nucleotide sequences encoding the first and second CENH3 proteins.

[0025] Preferably, the first nucleotide sequence is represented by the sequence of SEQ ID NO:1 or 2 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO:1 or 2, and / or the second nucleotide sequence is represented by the sequence of SEQ ID NO:3 or 4 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO:3 or 4. and / or The first CENH3 protein is represented by the amino acid sequence of SEQ ID NO:5 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO:5, and / or the second CENH3 protein is represented by the amino acid sequence of SEQ ID NO:6 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO:6. More preferably, at least one knockdown or knockout mutation in the first CENH3 protein is selected from: (a) The glutamine at position 94 of SEQ ID NO:5 is substituted, preferably resulting in a stop codon; (b) The tryptophan at position 93 of SEQ ID NO:5 is substituted, preferably resulting in a stop codon; (c) The glutamic acid at position 103 of SEQ ID NO:5 is substituted, preferably with lysine; (d) Histidine at position 47 of SEQ ID NO:5 is substituted, preferably with tyrosine; (e) Asparagine at position 43 of SEQ ID NO:5 is substituted, preferably with lysine; (f) Glycine at position 41 of SEQ ID NO:5 is substituted, preferably with glutamic acid; (g) Leucine at position 56 of SEQ ID NO:5 is substituted, preferably with phenylalanine; (h) The serine at position 87 of SEQ ID NO:5 is substituted, preferably with threonine; (i) Threonine at position 53 of SEQ ID NO:5 is substituted, preferably with methionine; (j) The isoleucine at position 70 of SEQ ID NO:5 is substituted, preferably with threonine; (k) The 9th lysine residue in SEQ ID NO:5 is substituted, preferably with arginine; and At least one mutation in the second CENH3 protein, preferably a knockdown or knockout mutation, is selected from: (l) At least one nucleotide of the ATG nucleic acid sequence encoding methionine at position 1 of the sequence SEQ ID NO:6 is deleted, inserted, or substituted, resulting in a knockout mutation (preferably by frameshift insertion or deletion or nonsense mutation) or resulting in the exchange of the amino acid encoded at position 1 (e.g., methionine is exchanged for isoleucine). (m) The glutamine at position 96 of SEQ ID NO:6 is substituted, preferably resulting in a stop codon. (n) The isoleucine at position 118 of SEQ ID NO:6 is substituted, preferably with phenylalanine. (o) A mutation occurs at position 93963324 on chromosome 15 according to the public reference XRQ2; More preferably, the plant contains at least one mutation as defined above (a) and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation as defined above (b) and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation as defined above (c) and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation as defined above (d) and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation as defined above (e) and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation as defined above (f) and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation as defined above (g) and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation as defined above (h) and one mutation selected from mutations (l), (m) The mutation is one of the above-defined mutations (i) and (l), (m), (n), (o), or at least the above-defined mutation (j) and the above-defined mutation (j), or the above-defined mutation (k) and the above-defined mutation (k), preferably at least the above-defined mutations (a) and (l), or at least the above-defined mutations (a) and (m), or at least the above-defined mutations (a) and (n), or at least the above-defined mutations (a) and (o), or at least the above-defined mutations (b) and (l), or at least the above-defined mutations (b) and (m), or at least the above-defined mutations (b) and (o), or at least the above-defined mutations (f), (g), (h), (i), (k) and mutation (m).

[0026] sequence list SEQ ID NO: 1 HanXRQr2_Chr14g0649211_genomic SEQ ID NO: 2 HanXRQr2_Chr14g0649211_cDNA SEQ ID NO: 3 HanXRQr2_Chr15g0703621-T1_genomic SEQ ID NO: 4 HanXRQr2_Chr15g0703621-T1_cDNA SEQ ID NO: 5 HanXRQ2_Chr14g0649211_Protein SEQ ID NO: 6 HanXRQr2_Chr15g0703621-T1_Protein SEQ ID NO: 7 Marker ha0000ab98 SEQ ID NO: 8, marked ha0000ac07 SEQ ID NO: 9, marked ha88508s02 SEQ ID NO: 10 Intentionally skipped sequence SEQ ID NO: 11 Marker ha88508s08 SEQ ID NO: 12, marked ha88508s09 SEQ ID NO: 13 Marker ha88508s10 SEQ ID NO: 14 Marker ha88508s12 SEQ ID NO: 15, marked ha88508s13 SEQ ID NO: 16 Marker ha88508s14 SEQ ID NO: 17 Marker ha88508s17 SEQ ID NO: 18 Marker ha88508s18 SEQ ID NO: 19 Marker ha88508s20 SEQ ID NO: 20 Marker ha88508s25 SEQ ID NO: 21 Marker ha88509s06 SEQ ID NO: 22, marked ha88509s15 definition In this article, "haploid plant" or "haploid plant cell" refers to a plant or plant cell with only one set of chromosomes, where each chromosome is unpaired. The number of chromosomes in one set is called the haploid number, denoted by the symbol n. A "gamete" is a haploid cell; two gametes combine during fertilization to form a "zygote," which has n pairs of chromosomes, meaning a total of 2n chromosomes. Each pair of chromosomes contains one chromosome from each gamete, called a homologous chromosome. Cells and organisms with paired homologous chromosomes are "diploid."

[0027] In the context of this disclosure, "doubling haploid" refers to the genotype formed when a haploid cell undergoes chromosome doubling. Doubling haploids can arise spontaneously or through chromosome doubling from haploid cells, organisms, or materials. Therefore, doubling haploids are homozygous. For polyploid cells (2n = 3x, 4x, 5, 6x, or more), the corresponding polyploid haploid may contain more than one copy of the same haploid genome.

[0028] In the context of this invention, "plant with haploid inducer activity," "haploid inducer," or "haploid inducer line" refers to a genetically modified plant or plant line that, upon fertilization with a wild-type plant, produces haploid offspring in at least 0.1%, at least 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, preferably at least 1.0%, preferably at least 2.0%, at least 3.0%, at least 4.0%, at least 5.0%, at least 6.0%, at least 7.0%, at least 8.0%, at least 9.0%, at least 10.0%, at least 11.0%, at least 12.0%, at least 13.0%, at least 14.0%, and at least 15.0% of instances. Because the chromosome of the haploid inducer is eliminated, the resulting haploid offspring contain only the genetic material of the wild-type parent.

[0029] As used herein, “reduced (protein) expression” refers to a certain percentage reduction in the expression rate of the nucleotide sequence encoding the protein compared to a specific reference (e.g., a plant that does not contain the genetic modifications or other modifications of the present invention described elsewhere herein, or a reference plant such as a wild-type plant).

[0030] The term "reduced (protein) activity" used in this article refers to a percentage reduction in activity compared to a reference (e.g., wild-type plant). The (protein) activity of CENH3 can be assessed by quantifying the centromere localization of CENH3 using an antibody against CENH3 and detecting it using confocal microscopy.

[0031] In the context of this disclosure, "knockout" or "knockdown" of a target gene refers to the complete or partial inactivation of the target gene, respectively. In the case of knockdown, the expression of the target gene is usually reduced, while in the case of "knockout," the (functional) gene is not expressed at all.

[0032] "Double-stranded RNA molecules or a group of molecules that target specific nucleic acid sequences" refers to RNAi, microRNA (miRNA), or small interfering RNA (siRNA) that participate in gene silencing (RNA interference) and post-gene expression transcriptional regulation.

[0033] The terms “RNA interference,” “RNAi,” “RNA silencing,” or “gene silencing,” used interchangeably in this article, refer to a gene downregulation (or knockdown) mechanism that has been proven to exist in all eukaryotes. This mechanism was first discovered in plants and was then called “post-transcriptional gene silencing” or “PTGS.” In RNAi, a small RNA guides a specific effector protein to a target nucleotide sequence via complementary base pairing, leading to the degradation of the target. A “gene silencing construct” or “RNAi construct” typically contains so-called “sense” and “antisense” sequences. Both the sense and antisense sequences are complementary sequences that exist in opposite orientations within the nucleic acid sequence. If the nucleic acid construct contains a sense sequence and a corresponding antisense sequence, these two complementary sequences form an RNA double strand post-transcriptionally, resulting in an “RNA hairpin.” In an RNA hairpin, the sense and antisense sequences together form a double strand and are separated by “intercalated intron loop sequences,” forming a loop within the hairpin structure. An “RNAi construct” can also contain more than one pair of sense and antisense sequences and form multiple loops. "RNAi constructs" can be provided in the form of expression constructs or vectors / plasmids.

[0034] A nucleic acid (construct) "targets" a genomic sequence or gene when it contains sequence information that allows recognition of that sequence or gene and can therefore interfere with that sequence (e.g., through site-specific cleavage or silencing). Targeting can be achieved through direct interaction with the genomic sequence itself or through interaction with the transcript of the genomic sequence. For example, if the nucleic acid construct of the present invention contains or encodes a sense and a corresponding antisense sequence targeting the genomic sequence, the construct activates an RNA silencing or RNA interference (RNAi) mechanism post-transcriptionally, leading to the destruction of the transcript of the genomic target sequence, thereby inhibiting target expression. In another case, the nucleic acid construct may encode a site-specific nuclease and guide RNA, thereby leading to the cleavage of the target sequence.

[0035] The "stop codon" is a nucleotide triplet that serves as the termination signal in the protein translation process. The "start codon" is the first nucleotide triplet in the messenger RNA transcript, translated by the ribosomes.

[0036] "Gene editing technology" refers to the use of site-specific nucleases or site-specific nicking enzymes, or their functionally active fragments or variants, along with homologous guide RNA (or pegRNA or crRNA), to guide a relevant CRISPR nuclease to its target site for cleavage. "Site-specific nucleases" are nucleases or their active fragments that can specifically recognize and cleave DNA at a specific target site. These nucleases typically produce double-strand breaks (DSBs), which are subsequently repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). These nucleases include zinc finger nucleases, transcription activator-like effector nucleases, engineered homing nucleases, recombinases, transposases, and macronucleases, as well as CRISPR nucleases and / or any combination, variant, or active fragment thereof.

[0037] The term "introduction" as used in this article, referring to the introduction of specific constructs or effectors, such as genome editing systems or gene silencing constructs, can be performed by any method known to those skilled in the art, such as transformation or transfection. "Transformation" or "transfection" of plant cells with a construct or a set of constructs or effector molecules refers to any well-established technique for introducing nucleic acid molecules into cells, such as bioballistic methods (e.g., particle bombardment), microinjection, permeabilization of the cell membrane through various treatments (e.g., electroporation or polyethylene glycol treatment), or Agrobacterium-mediated transformation. Typically, integration of nucleic acid constructs (e.g., through transformation) can be achieved using techniques generally known to those skilled in the art. For example, nucleic acid constructs can be integrated into plant cells by infecting plant tissues or plant cells with Agrobacterium containing the sequence of the nucleic acid to be transferred, located on a plasmid into which it can be integrated into the plant genome. Bioballistic transfer integration is another option, in which the nucleic acid construct to be integrated into the plant cell is attached to gold or tungsten particles, which are then injected into the cell at high speed. Another method known to technicians for integrating nucleic acid constructs into plant cells is protoplast transformation, in which polyvinyl glycol is added to the protoplast in the presence of the nucleic acid molecule to be integrated, or the protoplast is exposed to a short current pulse, making the protoplast membrane temporarily permeable to the nucleic acid construct.

[0038] Introducing modifications into the plant genome can also be done through mutagenesis. "Random mutagenesis" refers to techniques that introduce modifications or mutations into nucleic acid sequences in a random or non-site-specific (non-targeted) manner. For example, mutations can be induced by certain chemicals such as EMS (ethyl methanesulfonate) or ENU (N-ethylnitrosourea), or by physical methods such as irradiation with ultraviolet or gamma rays. On the other hand, "targeted" or "site-specific modification" relies on site-specific effectors such as nucleases, nickases, recombinases, transposases, and base editors. These tools recognize specific target sequences and allow the introduction of modifications at specific locations within the target sequence.

[0039] "TILLING" (targeted induction of localized lesions in the genome) is a process that identifies mutations in a specific gene after (non-specific) mutagenesis. Mutagenesis can be performed, for example, using chemical mutagens such as EMS. Single-base mutations are then identified using sensitive DNA screening techniques. Methods for performing TILLING are known to those skilled in the art.

[0040] The term "transient integration" herein refers to the transient introduction of at least one nucleic acid and / or amino acid sequence according to this disclosure, preferably integrated into a delivery vector and / or recombinant construct, into a target structure, such as a plant cell or cell system, with or without the aid of a delivery vector, wherein the at least one nucleic acid or nucleotide sequence is introduced under suitable reaction conditions such that the at least one nucleic acid sequence does not integrate into the endogenous nucleic acid material of the target structure, the whole genome, and therefore the at least one nucleic acid sequence does not integrate into the endogenous DNA of the target cell. Therefore, in the case of transient integration, the introduced genetic construct is not inherited by the offspring of the target structure (e.g., plant cells). The at least one nucleic acid and / or amino acid sequence or the product of its transcription, translation, processing, post-translational modification, or complex construction is only transient, i.e., present in a constitutive or inducible form, and is therefore only active in the target cell for a limited time to exert its effect. Therefore, at least one sequence introduced via transient integration is not inherited by the offspring of the cell. However, the effects mediated by at least one sequence or effector introduced transiently may potentially be inherited by the offspring of the target cell. "Stable integration" therefore refers to the integration of a nucleic acid or nucleotide sequence into the genome of a target cell or cellular system of interest, which includes the nuclear genome as well as genomes contained in other organelles.

[0041] The term "vector" refers to an element used to introduce a nucleic acid construct or a set of nucleic acid constructs into a cellular system. The vector may be a plasmid or plasmid vector, a granule, an artificial yeast chromosome (YAC), a bacterial artificial chromosome (BAC) or a P1 artificial chromosome (PAC), a phage particle, a bacterial phage-based vector, an isolated single-stranded or double-stranded nucleic acid sequence comprising a linear or circular DNA and RNA sequence, or a mixture thereof, for introduction or transformation into plants, plant cells, tissues, organs, or materials in accordance with this disclosure.

[0042] As used herein, the terms "plant," "plant cell," or "plant part" refer to plant organisms, plant organs, differentiated and undifferentiated plant tissues, plant cells, seeds and their derivatives and offspring, particularly including plant structures, plant protoplasts, plant cells or tissue cultures capable of regenerating plants, plant callus, plant clumps, and intact plant cells present in plants or plant parts, such as seeds, grains, rachis, fruits, flowers, cotyledons, leaves, stems, buds, roots, root tips, and straw. Plant cells include, but are not limited to, cells derived from seeds, mature and immature cells or organs, including embryos, meristems, seedlings, callus at different stages of differentiation, leaves, flowers, roots, shoots, male or female gametophytes, sporophytes, pollen, pollen tubes, microspores, and protoplasts.

[0043] Whenever this disclosure relates to the percentage identity between nucleic acid or amino acid sequences, these values ​​are defined as results obtained using the EMBOSS Water double sequence alignment tool (https: / / www.ebi.ac.uk / Tools / psa / emboss_water / ). The alignment or sequence comparison used herein refers to the comparison of two sequences over their entire length. The tool for local sequence alignment provided by the European Molecular Biology Laboratory (EMBL) and the European Bioinformatics Institute (EBI) employs a modified Smith-Waterman algorithm (see www.ebi.ac.uk / Tools / psa / and Smith, TF & Waterman, MS "Identification of common molecular subsequences" Journal of Molecular Biology, 1981 147 (1):195-197). Default parameters defined by EMBL-EBI are used during alignment. These parameters are (i) for amino acid sequences: matrix = BLOSUM62, vacancy opening penalty = 10, vacancy expansion penalty = 0.5; or (ii) for nucleic acid sequences: matrix = DNAfull, vacancy opening penalty = 10, vacancy expansion penalty = 0.5. Experts are well aware that, for example, a protein-coding sequence can be “codon-optimized” if it will be used in an organism different from its original source.

[0044] Detailed description The gene encoding CENH3 in sunflower (HanXRQr2_Chr15g0703621) was published in 2015 (Nagaki et al., 2015, Front Plant Sci. 2015; 6: 912.). In the context of this invention, a second gene encoding another copy of CENH3 (HanXRQr2_Chr14g0649211) was unexpectedly discovered in sunflower, which is significantly different from the previously published CENH3 at the N-terminus. Both copies are transiently expressed in different biological systems. The centromere localization of this protein was assessed using an anti-sunflower CENH3 antibody and detected by confocal microscopy.

[0045] Similar to other plants, the genome size of the genus *Helianthus* exhibits considerable variation, with most of this variation attributable to differences in ploidy levels. In fact, sunflowers used as agricultural plants typically comprise diploid (2n=2x=34) species. Furthermore, tetraploid (2n=4x=68) and hexaploid (2n=6x=102) species have been observed and described in neopolyploidization events that occurred millions of years after the genus diverged (Kantar et al., Briefings in Functional Genomics, Volume 13, Issue 4, July 2014, Pages 328–340, https: / / doi.org / 10.1093 / bfgp / elu004).

[0046] The inventors of this invention have now discovered that to achieve haploid inducer activity in sunflower plants, it is necessary to reduce the expression of two different CENH3 proteins observed in diploid sunflower germplasm. However, some residual activity must still be retained, otherwise the plant cannot survive.

[0047] Therefore, in a first aspect, the present invention relates to sunflower plants having haploid inducer activity and comprising the following: (i) The first nucleotide sequence encoding the first CENH3 protein, and (ii) The second nucleotide sequence encoding a second CENH3 protein, which differs from the naturally occurring first CENH3 protein. The combined expression and / or activity of the first and second CENH3 proteins was reduced by about 60% to about 95% compared to wild-type sunflower plants, preferably by about 65% to about 90%, and particularly preferably by about 75% to about 80%.

[0048] In diploid sunflower plants, there are several possibilities for reducing the expression and / or activity of the first and second CENH3 proteins. In polyploid sunflower species, even more possibilities exist. In diploid species, at least one allele encoding one of these two different CENH3 proteins should carry at least one mutation to reduce the total dose of CENH3 protein produced.

[0049] Therefore, in some implementations, the presence of at least one mutation in only one allele encoding one of the two different CENH3 proteins may have had a strong effect.

[0050] In a preferred embodiment, at least one mutation is present in at least one allele encoding the first and second CENH3 proteins to achieve a reduction in the expression and / or activity of the first and second CENH3 proteins, such that the total residual activity is reduced by about 60% to about 95% compared to wild-type sunflowers, preferably by about 65% to about 90%, and particularly preferably by about 75% to about 80%.

[0051] To achieve this reduction, mutations can be introduced, preferably knockdown or knockout mutations in two CENH3 genes or their alleles. However, in the final plant, one copy needs to be heterozygous to retain sufficient activity for the plant to survive. As used herein, "mutation" is defined as a change in at least one nucleotide position in a DNA molecule compared to a reference molecule without the mutation. This can include point mutations, deletion mutations, or insertion mutations, or in some embodiments, combinations thereof. As used herein, "knockout" and "knockdown" mutations represent the functional outcome of a mutation, including at least one point mutation, insertion mutation, or deletion mutation. In the case of knockout, this can be a complete or near-complete loss of function of the original gene product (RNA or protein), or in the case of "knockdown," it can be a reduction in the original function of the gene product.

[0052] In one embodiment of the above-mentioned plant, three of the four alleles encoding the first and second CENH3 proteins carry at least one mutation, preferably at least one knockdown or knockout mutation.

[0053] Preferably, the sunflower plant described in the first aspect is not a plant that is obtained or available through an inherently biological process.

[0054] Therefore, the present invention also relates to a plant belonging to the genus *Helianthus* that has haploid inducer activity, comprising... (i) The first nucleotide sequence encoding the first CENH3 protein, and (ii) The second nucleotide sequence encoding the second type of CENH3 protein. Among them, three of the four alleles encoding the first and second CENH3 proteins carry at least one mutation, preferably at least one knockdown or knockout mutation.

[0055] Alternatively, gene silencing can be used to reduce the expression of both CENH3 genes while maintaining sufficient residual activity.

[0056] In another embodiment of the above-described plant, the expression of the first and second CENH3 proteins is reduced due to the presence of double-stranded RNA molecules or a group of molecules that silence genes targeting the first and second nucleotide sequences.

[0057] The molecule or set of molecules that silences the gene targeting the first and second nucleotide sequences can be an RNAi construct, a microRNA (miRNA), or a small interfering RNA (siRNA) that recognizes the CENH3 sequence through base pairing. Two molecules can be used, each recognizing one CENH3 sequence. The transcribed CENH3 sequence that pairs with the molecular bases is then degraded, resulting in decreased expression of the CENH3 gene. Advantageously, this approach strongly knocks down CENH3 to provide haploid inducer activity while maintaining an activity level sufficient to avoid lethality.

[0058] As described above, in the context of this invention, a second copy of CENH3 was identified in sunflower. The genomic sequence of this copy is represented by the sequence SEQ ID NO: 1, and the cDNA sequence is represented by the sequence SEQ ID NO: 2. Previously disclosed copies of CENH3 are represented by the sequences SEQ ID NO: 3 (genomic sequence) and SEQ ID NO: 4 (cDNA).

[0059] In one embodiment of the plant according to any of the above embodiments, the first nucleotide sequence is represented by the sequence of SEQ ID NO: 1 or 2, or by a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 3 or 4, and / or wherein the second nucleotide sequence is represented by the sequence of SEQ ID NO: 3 or 4, or by a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 3 or 4, which are the respective reference sequences.

[0060] The amino acid sequence of the newly identified CENH3 copy is represented by the sequence in SEQ ID NO: 5, while the amino acid sequence of the previously published copy is represented by the sequence in SEQ ID NO: 6.

[0061] In one embodiment of the plant according to any of the above embodiments, the first CENH3 protein is represented by the amino acid sequence of SEQ ID NO:5, or by a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO:5, which serves as its respective reference sequence, and / or the second CENH3 protein is represented by the amino acid sequence of SEQ ID NO:6, or by a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO:6, which serves as its respective reference sequence.

[0062] In the context of this invention, haploid inducer activity was evaluated for knockdown and knockout mutations of two CENH3 copies.

[0063] TILLING reverse selection was performed on the two CENH3 copies. Twenty-three individual mutations were identified, 19 of which were tested for their performance in inducing haploid plants (HIR, haploid induction rate). HIR was tested by crossing mutants with heterozygous test organisms. Progeny were screened using 4–8 polymorphic KASP markers with the corresponding mutation markers. Homozygous plants were analyzed using Illumina microarrays. All individual mutants showed zero induction rate on both the male and female sides.

[0064] In the following steps, multiple mutations of interest were combined. For CENH3, knockout mutants of both copies were obtained. The HA88509 mutant HA304m004k lost the start codon, while the HA880508 mutant HA304m003j (where glutamine at position 94 of the sequence SEQ ID NO: 5 was replaced, resulting in a stop codon) prematurely terminated at position 94. Combining these mutations failed to identify dihomozygous plants. However, plants used as pollen donors (containing one mutation fixed in a homozygous state and another maintaining a heterozygous state) were able to induce female haploid seeds (halploids were identified in two out of three hybridization experiments: one haploid was detected in 110 seeds, one in 23 seeds, and none in 192 seeds).

[0065] In one embodiment of the above-described plant, at least one mutation in the first CENH3 protein, preferably at least one knockdown or knockout mutation, is selected from: (a) The glutamine at position 94 of the sequence of SEQ ID NO: 5 is substituted, preferably resulting in the formation of a stop codon; (b) The tryptophan at position 93 in the sequence of SEQ ID NO: 5 is substituted, preferably resulting in the formation of a stop codon; (c) The glutamic acid at position 103 in the sequence of SEQ ID NO: 5 is substituted, preferably with lysine; (d) The histidine at position 47 of the sequence of SEQ ID NO: 5 is substituted, preferably with tyrosine; (e) The asparagine at position 43 of the sequence of SEQ ID NO: 5 is substituted, preferably with lysine; (f) The glycine at position 41 of the sequence of SEQ ID NO: 5 is substituted, preferably with glutamic acid; (g) The leucine at position 56 in the sequence of SEQ ID NO: 5 is substituted, preferably with phenylalanine; (h) The serine at position 87 in the sequence of SEQ ID NO: 5 is substituted, preferably with threonine; (i) The threonine at position 53 in the sequence of SEQ ID NO: 5 is substituted, preferably with methionine; (j) The isoleucine at position 70 of SEQ ID NO: 5 is substituted, preferably with threonine; (k) The lysine at position 9 of the sequence SEQ ID NO: 5 is substituted, preferably with arginine; and wherein At least one mutation in the second CENH3 protein, preferably at least one knockdown or knockout mutation selected from: (l) One or more nucleotides in the ATG nucleic acid sequence encoding methionine at position 1 of the sequence in SEQ ID NO: 6 are deleted, inserted, or substituted, resulting in a knockout mutation, preferably achieved by frameshift insertion or deletion or nonsense mutation, or resulting in an exchange of the encoded amino acid at position 1, for example, replacing methionine with isoleucine. (m) The glutamine at position 96 of the sequence SEQ ID NO: 6 is substituted, preferably resulting in the formation of a stop codon. (n) In the sequence of SEQ ID NO: 6, isoleucine at position 118 is substituted, preferably with phenylalanine, and (o) According to public reference XRQ2, a mutation occurs at position 93963324 on chromosome 15.

[0066] In a preferred embodiment, the plant carries at least the above-defined mutation (a) and one mutation selected from (l), (m), (n), and (o); or the plant carries at least the above-defined mutation (b) and one mutation selected from (l), (m), (n), and (o); or the plant carries at least the above-defined mutation (c) and one mutation selected from (l), (m), (n), and (o); or the plant carries at least the above-defined mutation (d) and one mutation selected from (l), (m), (n), and (o); or the plant carries at least the above-defined mutation (e) and one mutation selected from (l), (m), (n), and (o); or the plant carries at least the above-defined mutation (f) and one mutation selected from (l), (m), (n), and (o); or the plant carries at least the above-defined mutation (g) and one mutation selected from (l), (m), (n), and (o); or the plant carries at least the above-defined mutation (h) and one mutation selected from (l), (m), and (o). The plant carries at least one of the mutations (n) and (o), or the plant carries at least the (i) mutation as defined above and one of the mutations selected from (l), (m), (n), and (o), or the plant carries at least the (j) mutation as defined above and one of the mutations selected from (l), (m), (n), and (o), or the plant carries at least the (k) mutation as defined above and one of the mutations selected from (l), (m), (n), and (o), preferably the plant carries at least the (a) and (l) mutations as defined above, or the plant carries at least the (a) and (l) mutations as defined above. The plant carries at least the (a) and (m) mutations defined above, or the plant carries at least the (a) and (n) mutations defined above, or the plant carries at least the (a) and (o) mutations defined above, or the plant carries at least the (b) and (l) mutations defined above, or the plant carries at least the (b) and (m) mutations defined above, or the plant carries at least the (b) and (o) mutations defined above, or the plant carries at least one of the (f), (g), (h), (i) and (k) mutations defined above, as well as the (m) mutation.

[0067] In another aspect, the present invention relates to a polynucleotide or polynucleotide group that encodes a protein represented by the amino acid sequence of SEQ ID NO: 5 and / or 6, or a protein having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5 or 6 as their respective reference sequences, and carrying at least one knockout mutation, preferably selected from mutations defined above or preferably combinations of at least two knockout combinations as defined above.

[0068] In a further aspect, the present invention also relates to polypeptides encoded by polynucleotides or polynucleotide groups as defined above.

[0069] In another aspect, the present invention relates to a vector comprising a polynucleotide or a group of polynucleotides as defined above.

[0070] In a further aspect, the present invention relates to cells comprising polynucleotides or polynucleotide groups as defined above, polypeptides as defined above, or carriers as defined above.

[0071] The cells may be plant cells, preferably sunflower plant cells, or, for example, bacterial host cells.

[0072] The present invention also relates to a method for cultivating plants or plant parts, wherein the plants are particularly plants of the genus *Helianthus*, preferably as defined in any of the embodiments described above, the method comprising: (a) Using gene editing technology, or through random or directed mutagenesis, at least one mutation is introduced into the genome of a plant or part of a plant (preferably sunflower) in each of the first nucleotide sequence encoding the first CENH3 protein and the second nucleotide sequence encoding the second CENH3 protein, preferably at least one knockdown or knockout mutation, such that three of the four alleles encoding the first and second CENH3 proteins are knocked down or knocked out; or (b) To achieve stable or transient integration through transformation, or to insert through gene editing technology, an RNAi molecule or RNAi molecule group is introduced into the plant or plant part, the RNAi molecule being targeted, able to hybridize with a first nucleotide sequence encoding a first CENH3 protein and a second nucleotide sequence encoding a second CENH3 protein; Furthermore, optionally, a plant is regenerated from the plant portion obtained in (a) or (b) above.

[0073] Preferably, the first CENH3 protein is the amino acid sequence shown in SEQ ID NO: 5, or a sequence representative having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO: 5; and / or the second CENH3 protein is the amino acid sequence shown in SEQ ID NO: 6, or a sequence representative having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence of SEQ ID NO: 6.

[0074] The plant described in this invention can be obtained by introducing mutations into both copies of the endogenous CENH3 gene, or by stably / transiently introducing a gene silencing construct targeting both copies of the CENH3 gene.

[0075] Genome editing technologies allow the introduction of double-strand breaks (DSBs) at one or more predetermined target sites, such as at or within the CENH3 locus, thereby disrupting the locus and optionally inserting exogenous sequences or replacing endogenous sequences. These DSBs are introduced by site-specific nucleases, such as large-scale nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or clustered regularly spaced short palindromic repeats (CRISPR) nucleases. These nucleases can generate DSBs at specific cleavage sites, which are then repaired by cells via non-homologous end joining (NHEJ) or homologous recombination (HR). The use of a repair template guides the cellular repair process, ensuring error-free and predictable repair outcomes. The repair template preferably contains symmetrical or asymmetrical homologous arms complementary to the sequences flanking the DSB, allowing for controlled sequence insertion or closure of the break.

[0076] In embodiments where the site-directed nuclease or its variants are nucleic acid-guided site-directed nucleases, the at least one genome editing system further comprises at least one guide molecule or a sequence encoding the guide molecule. The “guide molecule” or “guide nucleic acid sequence” (typically referred to and abbreviated as guide RNA, crRNA, crRNA+tracrRNA, gRNA, sgRNA, depending on the representative prototype nucleic acid-guided site-directed nuclease system—the CRISPR system) recognizes the target sequence to be cleaved by the nuclease. The at least one “guide nucleic acid sequence” or “guide molecule” comprises a “scaffold region” and a “target region.” The “scaffold region” is a sequence that the nucleic acid-guided nuclease binds to to form a targetable nuclease complex. The scaffold region may contain a direct repeat sequence that can be recognized and processed by the nucleic acid-guided nuclease to provide mature crRNA. In the guide molecule, the pegRNA may also contain another region, the so-called “primer binding site.” The “target region” determines its complementarity with the target site to be cleaved. Therefore, the term crRNA, used in this paper, can be used interchangeably with the term guide RNA when it also functions as a guide RNA for a mature CRISPR nuclease. Some CRISPR nucleases (such as Cas9) can be used by providing two independent guide RNA sequences, namely tracrRNA and crRNA, which can be provided individually or linked via covalent / non-covalent bonds / interactions. The guide RNA can also be pegRNA in the lead editing system further disclosed below. The at least one guide molecule can be provided as a single, coherent molecular form or as a sequence encoding it, or as two independent molecular forms (such as crRNA and tracrRNA) or as a sequence encoding it.

[0077] Mutagenesis can be carried out using a variety of techniques known to those skilled in the art. For example, mutations can be induced by chemical reagents such as EMS (ethyl methanesulfonate), ENU (N-ethyl-N-nitrosourea), or by physical methods such as ultraviolet radiation or gamma ray irradiation.

[0078] In one implementation of the above method, after (non-specific) mutagenesis is completed, TILLING (targeted induction of local lesions in the genome) is used to identify mutations in the two copies of the CENH3 gene.

[0079] RNAi technology for targeted gene silencing is well known in the art. For this purpose, an RNAi construct containing the sequence information of the genomic target to be silenced can be introduced into plant cells. The construct is preferably introduced in the form of a DNA sequence, which is then transcribed into functional RNA within the cell. Specifically, the RNAi construct encodes a sense sequence and an antisense sequence, both representing fragments of the genomic target. The complementary sense and antisense sequences inversely present in the construct are transcribed to form a double-stranded RNA, resulting in an RNA hairpin structure with spacer intron loop sequences. The presence of the RNAi construct ultimately leads to a decrease in the expression of the target gene, i.e., knockdown.

[0080] In one embodiment of the above method, at least one mutation in the first CENH3 protein (preferably at least one knockdown or knockout mutation) is selected from: (a) The glutamine at position 94 of the sequence of SEQ ID NO: 5 is substituted, preferably resulting in a stop codon; (b) The tryptophan at position 93 in the sequence of SEQ ID NO: 5 is substituted, preferably resulting in a stop codon; (c) The glutamic acid at position 103 in the sequence of SEQ ID NO: 5 is substituted, preferably with lysine; (d) The histidine at position 47 of the sequence of SEQ ID NO: 5 is substituted, preferably with tyrosine; (e) The asparagine at position 43 of the sequence of SEQ ID NO: 5 is substituted, preferably with lysine; (f) The glycine at position 41 of the sequence of SEQ ID NO: 5 is substituted, preferably with glutamic acid; (g) The leucine at position 56 in the sequence of SEQ ID NO: 5 is substituted, preferably with phenylalanine; (h) The serine at position 87 in the sequence of SEQ ID NO: 5 is substituted, preferably with threonine; (i) The threonine at position 53 in the sequence of SEQ ID NO: 5 is substituted, preferably with methionine; (j) The isoleucine at position 70 of SEQ ID NO: 5 is substituted, preferably with threonine; (k) The lysine residue at position 9 of the sequence SEQ ID NO: 5 is substituted, preferably with arginine; and At least one mutation in the second CENH3 protein, preferably at least one knockdown or knockout mutation, selected from: (l) At least one nucleotide in the ATG nucleic acid sequence encoding methionine at position 1 of the sequence SEQ ID NO: 6 is deleted, inserted, or substituted, resulting in a knockout mutation, preferably achieved by frameshift insertion or deletion or nonsense mutation, or resulting in an exchange of the encoded amino acid at position 1, for example, replacing methionine with isoleucine. (m) The glutamine at position 96 of the sequence SEQ ID NO: 6 is substituted, preferably resulting in a stop codon, and (n) In the sequence of SEQ ID NO: 6, isoleucine at position 118 is substituted, preferably with phenylalanine, and (o) According to public reference XRQ2, a mutation occurs at position 93963324 on chromosome 15.

[0081] In a preferred embodiment, step (a) introduces at least one mutation (a) as defined above and one mutation selected from mutations (l), (m), (n), and (o), or at least one mutation (b) as defined above and one mutation selected from mutations (l), (m), (n), and (o), or at least one mutation (c) as defined above and one mutation selected from mutations (l), (m), (n), and (o), or at least one mutation (d) as defined above and one mutation selected from mutations (l), (m), (n), and (o), or at least one mutation (e) as defined above and one mutation selected from mutations (l), (m), (n), and (o), or at least one mutation (f) as defined above and one mutation selected from mutations (l), (m), (n), and (o), or at least one mutation (g) as defined above and one mutation selected from mutations (l), (m), (n), and (o), or at least one mutation (h) as defined above and one A mutation selected from mutations (l), (m), (n) and (o), or at least mutation (i) as defined above and a mutation selected from mutations (l), (m), (n) and (o), or at least mutation (j) as defined above and a mutation selected from mutations (l), (m), (n) and (o), or at least mutation (k) as defined above and a mutation selected from mutations (l), (m), (n) and (o), preferably at least mutations (a) and (l) as defined above, or at least mutations (a) and (m) as defined above, or at least mutations (a) and (n) as defined above, or at least mutations (a) and (o) as defined above, or at least mutations (b) and (l) as defined above, or at least mutations (b) and (m) as defined above, or at least mutations (b) and (o) as defined above, or at least one of mutations (f), (g), (h), (i) and (k) as defined above and mutation (m).

[0082] In another aspect, the present invention relates to the use of the above-defined mutations or combinations of the above-defined mutations or the above-defined polynucleotides or the above-defined polypeptides or the above-defined vectors for generating plants having haploid inducer activity, preferably sunflower plants.

[0083] Finally, the present invention relates to a method for identifying sunflower plants with haploid inducer activity, preferably sunflower plants as defined in any of the above embodiments, the method comprising: (a) Screening for mutations in the first nucleotide sequence encoding the first CENH3 protein and the second nucleotide sequence encoding the second CENH3 protein, preferably the presence of at least one functional knockdown or knockout mutation, or (b) Screening for reduced expression of the first and second nucleotide sequences encoding the first and second CENH3 proteins.

[0084] Preferably, the first nucleotide sequence is represented by the sequence of SEQ ID NO:1 or 2 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO:1 or 2, and / or the second nucleotide sequence is represented by the sequence of SEQ ID NO:3 or 4 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO:3 or 4. and / or The first CENH3 protein is represented by the amino acid sequence of SEQ ID NO:5 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO:5, and / or the second CENH3 protein is represented by the amino acid sequence of SEQ ID NO:6 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO:6.

[0085] In a preferred embodiment of the above method for identifying sunflower plants with haploid inducer activity, at least one mutation in the first CENH3 protein, preferably at least one knockdown or knockout mutation, is selected from: (a) The glutamine at position 94 of SEQ ID NO:5 is substituted, preferably resulting in a stop codon; (b) The tryptophan at position 93 of SEQ ID NO:5 is substituted, preferably resulting in a stop codon; (c) The glutamic acid at position 103 of SEQ ID NO:5 is substituted, preferably with lysine; (d) Histidine at position 47 of SEQ ID NO:5 is substituted, preferably with tyrosine; (e) Asparagine at position 43 of SEQ ID NO:5 is substituted, preferably with lysine; (f) Glycine at position 41 of SEQ ID NO:5 is substituted, preferably with glutamic acid; (g) Leucine at position 56 of SEQ ID NO:5 is substituted, preferably with phenylalanine; (h) The serine at position 87 of SEQ ID NO:5 is substituted, preferably with threonine; (i) Threonine at position 53 of SEQ ID NO:5 is substituted, preferably with methionine; (j) The isoleucine at position 70 of SEQ ID NO:5 is substituted, preferably with threonine; (k) The 9th lysine residue in SEQ ID NO:5 is substituted, preferably with arginine; and At least one mutation in the second CENH3 protein, preferably at least one knockdown or knockout mutation, selected from: (l) At least one nucleotide of the ATG nucleic acid sequence encoding methionine at position 1 of the sequence SEQ ID NO:6 is deleted, inserted, or substituted, resulting in a knockout mutation (preferably by frameshift insertion or deletion or nonsense mutation) or resulting in the exchange of the amino acid encoded at position 1 (e.g., methionine is exchanged for isoleucine). (m) The glutamine at position 96 of SEQ ID NO:6 is substituted, preferably resulting in a stop codon. (n) The isoleucine at position 118 of SEQ ID NO:6 is substituted, preferably with phenylalanine. (o) A mutation occurs at position 93963324 on chromosome 15 according to the public reference XRQ2.

[0086] More preferably, in the above method, the plant contains at least one mutation as defined above (a) and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation as defined above (b) and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation as defined above (c) and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation as defined above (d) and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation as defined above (e) and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation as defined above (f) and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation as defined above (g) and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation as defined above (h) and one mutation selected from mutation (l). The mutation is one of the above-defined mutations (i) and one of the above-defined mutations (l), (m), (n), (o), or at least the above-defined mutation (j) and one of the above-defined mutations (l), (m), (n), (o), or at least the above-defined mutation (k) and one of the above-defined mutations (l), (m), (n), (o), preferably at least the above-defined mutations (a) and (l), or at least the above-defined mutations (a) and (m), or at least the above-defined mutations (a) and (n), or at least the above-defined mutations (a) and (o), or at least the above-defined mutations (b) and (l), or wherein at least the above-defined mutations (b) and (m), or at least the above-defined mutations (b) and (o), or at least the above-defined mutations (f), (g), (h), (i), (k) and mutation (m).

[0087] Screening can be accomplished using any sequencing method or by applying markers at the desired locations. The plant material to be screened can be a mutagenic population (e.g., obtained by EMS mutagenesis or other methods) or a T0 plant from genome-edited plant material.

[0088] The markers used to detect specific mutations are disclosed in the sequence listing, and the mutations are detected as follows: The marker ha88508s17 (SEQ ID NO: 17) was used to detect the gln94STOP mutation in the sequence of SEQ ID NO: 5.

[0089] The marker ha88508s10 (SEQ ID NO: 13) was used to detect the trp93STOP mutation in the sequence of SEQ ID NO: 5.

[0090] The marker ha88508s18 (SEQ ID NO: 18) was used to detect the glu103lys mutation in the sequence of SEQ ID NO: 5.

[0091] The marker ha88508s02 (SEQ ID NO: 9) was used to detect the his47tyr mutation in the sequence of SEQ ID NO: 5.

[0092] The marker ha88508s12 (SEQ ID NO: 14) was used to detect the ans43lys mutation in the sequence of SEQ ID NO: 5.

[0093] The marker ha88508s09 (SEQ ID NO: 12) was used to detect the gly41glu mutation in the sequence of SEQ ID NO: 5.

[0094] The marker ha88508s08 (SEQ ID NO: 11) was used to detect the leu56phe mutation in the sequence of SEQ ID NO: 5.

[0095] The marker ha88508s25 (SEQ ID NO:20) was used to detect the ser87thr mutation in the sequence of SEQ ID NO:5.

[0096] The marker ha88508s13 (SEQ ID NO:15) was used to detect the thr53met mutation in the sequence of SEQ ID NO:5.

[0097] The marker ha88508s14 (SEQ ID NO:16) was used to detect the ile70thr mutation in the sequence of SEQ ID NO:5.

[0098] The marker ha88508s20 (SEQ ID NO:19) was used to detect the lys9arg mutation in the sequence of SEQ ID NO:5.

[0099] The marker ha0000ac07 (SEQ ID NO:8) was used to detect metaile mutations in the sequence of SEQ ID NO:6.

[0100] The marker ha0000ab98 (SEQ ID NO:7) was used to detect the gln96STOP mutation in the sequence of SEQ ID NO:6.

[0101] The marker ha88509s15 (SEQ ID NO:22) was used to detect the ile118phe mutation in the sequence of SEQ ID NO:6.

[0102] The marker ha88509s06 (SEQ ID NO:21) was used to detect the mutation at position 93963324 on chromosome 15 according to public reference XRQ2.

[0103] Example 1: Mutation Generation and Screening The sunflower strain HA304 was subjected to EMS mutagenesis using the KeyPointMB method provided by Keygene NV. Two copies of the CENH3 gene were amplified from the DNA of the mutagenized population by PCR, followed by sequencing using Illumina short-read technology. All plants carrying mutations in either copy were screened and self-pollinated. In progeny and testcross experiments, plants with amino acid exchange mutations were selected based on KASP marker analysis.

[0104] Example 2: Mutant Combinations The mutations in each copy of the CENH3 gene were ranked according to the predicted mutation intensity, and the strongest mutation from one copy was combined with a mutation from another copy. Potential knockout mutations (carrying early stop codons) were given priority. For each hybridization, plants with a homozygous or heterozygous mutation in one CENH3 copy were selected, artificially emasculated, and pollinated with pollen from plants carrying a homozygous or heterozygous mutation in the second CENH3 copy. F1 plants with heterozygous mutations in both CENH3 copies were screened and self-pollinated. Plants from the F2 generation with homozygous mutations in both copies, or with a homozygous mutation in one copy and a heterozygous mutation in the other, were selected for testcross experiments or self-pollination propagation.

[0105] Example 3: Testcross experiment to evaluate the haploid induction ability of plants with double-copy CENH3 gene mutants Testcross experiments were conducted using selected F2 or F3 plants with homozygous CENH3 double-copy mutations, or with one copy mutation being homozygous and the other heterozygous. The pollen from the selected plants was used to pollinate the hybrid test material carrying sterile cytoplasm (CMS) and lacking the restorer gene, thus sterilizing the female plants without the need for artificial emasculation.

[0106] F1 seeds obtained from testcrosses were germinated, and DNA was extracted from each plant and analyzed using 4-8 KASP marker sets. Some markers could distinguish the alleles of male genotypes from those of female test materials, while others could distinguish the alleles of the test parent lines. Mutation markers were also included. For all plant DNA showing homozygosity to all markers and where the mutation marker was a wild-type allele, additional DNA microarray analysis was performed to allow assessment of conjugation at a large number of genome-wide loci.

[0107] Presumed haploids were detected in testcrosses with the HA304m004k_m003j mutant. Table 1 below summarizes the results of the analyzed testcrosses. Each testcross used a single male plant. All testcrosses used the same hybridization test material.

[0108] Table 1:

[0109] The above results were validated using an additional induced hybridization with the mutant HA304m004k_m003j. Unlike the previous results, this experiment analyzed the ploidy status of the plant rather than the grain and calculated the haploid induction rate.

[0110] The results are summarized in the table below:

Claims

1. Plants of the genus *Helianthus* that possess haploid inducer activity and contain: (i) The first nucleotide sequence encoding the first CENH3 protein, and (ii) The second nucleotide sequence encoding the second type of CENH3 protein. in, Compared with wild-type sunflower plants, the expression and / or activity of the first and second CENH3 proteins are reduced by about 60% to about 95%, preferably by about 65% to about 90%, and particularly preferably by about 75% to about 80%; preferably, the sunflower plant is not a plant obtained through basic biological processes or is not obtainable through basic biological processes.

2. The plant according to claim 1, wherein three of the four alleles encoding the first and second CENH3 proteins carry at least one mutation, preferably at least one knockdown or knockout mutation.

3. The plant of claim 1, wherein the expression of the first and second CENH3 proteins is reduced by the presence of a double-stranded RNA molecule or group of molecules that perform gene silencing by targeting the first and second nucleotide sequences.

4. The plant according to any one of claims 1 to 3, wherein the first nucleotide sequence is represented by the sequence of SEQ ID NO: 1 or 2 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 3 or 4, which is the respective reference sequence; and / or wherein the second nucleotide sequence is represented by the sequence of SEQ ID NO: 3 or 4 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 3 or 4, which is the respective reference sequence.

5. The plant according to any one of claims 1 to 4, wherein the first CENH3 protein is represented by the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 5 as its respective reference sequence, and / or wherein the second CENH3 protein is represented by the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 6 as its respective reference sequence.

6. The plant according to any one of claims 1, 2, 4 and 5, wherein at least one mutation in the first CENH3 protein, preferably at least one knockdown or knockout mutation, is selected from: (a) The glutamine at position 94 of the sequence of SEQ ID NO: 5 is substituted, preferably resulting in a stop codon; (b) The tryptophan at position 93 in the sequence of SEQ ID NO: 5 is substituted, preferably resulting in a stop codon; (c) The glutamic acid at position 103 in the sequence of SEQ ID NO: 5 is substituted, preferably with lysine; (d) The histidine at position 47 of the sequence of SEQ ID NO: 5 is substituted, preferably with tyrosine; (e) The asparagine at position 43 of the sequence of SEQ ID NO: 5 is substituted, preferably with lysine; (f) The glycine at position 41 of the sequence of SEQ ID NO: 5 is substituted, preferably with glutamic acid; (g) The leucine at position 56 in the sequence of SEQ ID NO: 5 is substituted, preferably with phenylalanine; (h) The serine at position 87 in the sequence of SEQ ID NO: 5 is substituted, preferably with threonine; (i) The threonine at position 53 in the sequence of SEQ ID NO: 5 is substituted, preferably with methionine; (j) The isoleucine at position 70 of SEQ ID NO: 5 is substituted, preferably with threonine; (k) The lysine at position 9 of the sequence SEQ ID NO: 5 is substituted, preferably with arginine; and wherein At least one mutation in the second CENH3 protein, preferably at least one knockdown or knockout mutation, selected from: (l) At least one nucleotide in the ATG nucleic acid sequence encoding methionine at position 1 of the sequence encoding SEQ ID NO: 6 is deleted, inserted, or substituted, resulting in a knockout mutation, preferably achieved by frameshift insertion or deletion or by nonsense mutation, or resulting in an exchange of the amino acid encoded at position 1, for example, methionine is exchanged for isoleucine. (m) In the sequence of SEQ ID NO: 6, the glutamine at position 96 is substituted, preferably resulting in a stop codon. (n) In the sequence of SEQ ID NO: 6, isoleucine at position 118 is substituted, preferably with phenylalanine, and (o) According to public reference XRQ2, a mutation occurs at position 93963324 on chromosome 15.

7. The plant according to claim 6, wherein the plant carries at least one mutation (a) as defined in claim 6 and one mutation selected from mutations (l), (m), (n), and (o); or wherein the plant carries at least one mutation (b) as defined in claim 6 and one mutation selected from mutations (l), (m), (n), and (o); or wherein the plant carries at least one mutation (c) as defined in claim 6 and one mutation selected from mutations (l), (m), (n), and (o); or wherein the plant carries at least one mutation (d) as defined in claim 6 and one mutation selected from mutations (l), (m), (n), and (o). The plant carries at least one mutation of (e) as defined in claim 6 and one mutation selected from mutations (l), (m), (n), and (o); or the plant carries at least one mutation of (f) as defined in claim 6 and one mutation selected from mutations (l), (m), (n), and (o); or the plant carries at least one mutation of (g) as defined in claim 6 and one mutation selected from mutations (l), (m), (n), and (o); or the plant carries at least one mutation of (h) as defined in claim 6 and one mutation selected from mutations (l), (m), (n), and (o). The plant carries at least one mutation as defined in claim 6, or at least one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation as defined in claim 6, or at least one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation as defined in claim 6, or at least one mutation selected from mutations (l), (m), (n), and (o); preferably, the plant carries at least one mutation as defined in claim 6, or at least one mutation as defined in claim 6, or at least one mutation selected from mutations (l), (m), (n), and (o); a) and (m), or the plant carries at least the mutations (a) and (n) as defined in claim 6, or the plant carries at least the mutations (a) and (o) as defined in claim 6, or the plant carries at least the mutations (b) and (l) as defined in claim 6, or the plant carries at least the mutations (b) and (m) as defined in claim 6, or the plant carries at least the mutations (b) and (o) as defined in claim 6, or the plant carries at least one of the mutations (f), (g), (h), (i) and (k) as defined in claim 6, and mutation (m).

8. A polynucleotide or polynucleotide sequence encoding a protein represented by the following sequence: the amino acid sequence of SEQ ID NO: 5 and / or 6 and / or having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5 or 6 as their respective reference sequences and carrying at least one mutation, including point mutations, insertion mutations, and / or deletion mutations, preferably selected from the mutations defined in claim 6, or a combination of at least two point mutations, preferably a sequence of a combination of at least two point mutations as defined in claim 7.

9. The polypeptide encoded by the polynucleotide or polynucleotide group according to claim 8.

10. A vector comprising the polynucleotide or polynucleotide group according to claim 8.

11. A cell comprising a polynucleotide or polynucleotide group according to claim 8, a polypeptide according to claim 9, or a carrier according to claim 10, preferably wherein the cell is not obtained by or cannot be obtained by a basic biological process.

12. A method for producing a plant or plant part, particularly a sunflower, preferably a plant or plant part as defined in any one of claims 1 to 7, comprising: (a) By gene editing techniques or by using random or directed mutagenesis, at least one mutation, preferably at least one knockdown or knockout mutation, is introduced into each of the first nucleotide sequence encoding a first CENH3 protein and the second nucleotide sequence encoding a second CENH3 protein in the genome of a plant or plant part, preferably a sunflower, such that three of the four alleles encoding the first and second CENH3 proteins are knocked down or knocked out; or (b) Introducing an RNAi molecule or a group of RNAi molecules, which targets, hybridizes with a first nucleotide sequence encoding a first CENH3 protein and a second CENH3 protein, into a plant or plant part by means of stable or transient integration through transformation, or by insertion using gene editing techniques. Furthermore, optionally, a plant can be regenerated from either (a) or (b). Preferably, the first CENH3 protein is represented by the amino acid sequence of SEQ ID NO: 5, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO: 5, and / or the second CENH3 protein is represented by the amino acid sequence of SEQ ID NO: 6, or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO:

6.

13. The method of claim 12, wherein at least one mutation in the first CENH3 protein, preferably at least one knockdown or knockout mutation, is selected from: (a) The glutamine at position 94 of the sequence of SEQ ID NO: 5 is substituted, preferably resulting in a stop codon; (b) The tryptophan at position 93 in the sequence of SEQ ID NO: 5 is substituted, preferably resulting in a stop codon; (c) The glutamic acid at position 103 in the sequence of SEQ ID NO: 5 is substituted, preferably with lysine; (d) The histidine at position 47 of the sequence of SEQ ID NO: 5 is substituted, preferably with tyrosine; (e) The aspartic acid at position 43 of the sequence of SEQ ID NO: 5 is substituted, preferably with lysine; (f) The glycine at position 41 of the sequence of SEQ ID NO: 5 is substituted, preferably with glutamic acid; (g) The leucine at position 56 in the sequence of SEQ ID NO: 5 is substituted, preferably with phenylalanine; (h) The serine at position 87 in the sequence of SEQ ID NO: 5 is substituted, preferably with threonine; (i) The threonine at position 53 in the sequence of SEQ ID NO: 5 is substituted, preferably with methionine; (j) The isoleucine at position 70 of SEQ ID NO: 5 is substituted, preferably with threonine; (k) The lysine residue at position 9 of SEQ ID NO: 5 is substituted, preferably with arginine; and At least one mutation in the second CENH3 protein, preferably a knockout or knockdown mutation, is selected from: (l) At least one nucleotide in the ATG nucleic acid sequence encoding methionine at position 1 of the sequence in SEQ ID NO: 6 is deleted, inserted, or substituted, resulting in a knockout mutation, preferably achieved by frameshift insertion or deletion or nonsense mutation, or resulting in the exchange of the encoded amino acid at position 1, for example, methionine is exchanged for isoleucine. (m) In the sequence of SEQ ID NO: 6, the glutamine at position 96 is substituted, preferably resulting in a stop codon. (n) In the sequence of SEQ ID NO: 6, isoleucine at position 118 is substituted, preferably with phenylalanine. (o) According to public reference XRQ2, a mutation occurs at position 93963324 on chromosome 15; Preferably, the mutation includes at least one mutation (a) as defined in claim 6 and one mutation selected from mutations (l), (m), (n), and (o); or the mutation includes at least one mutation (b) as defined in claim 6 and one mutation selected from mutations (l), (m), (n), and (o); or the mutation includes at least one mutation (c) as defined in claim 6 and one mutation selected from mutations (l), (m), (n), and (o); or the mutation includes at least one mutation (d) as defined in claim 6 and one mutation selected from mutations (l), (m), (n), and (o); or the mutation includes at least one mutation (e) as defined in claim 6 and one mutation selected from mutations (l), (m), (n), and (o); or the mutation includes at least one mutation (f) as defined in claim 6 and one mutation selected from mutations (l), (m), (n), and (o); or the mutation includes at least one mutation (g) as defined in claim 6 and one mutation selected from mutations (l), (m), (n), and (o); or the mutation includes at least one mutation (h) as defined in claim 6 and one mutation selected from mutations (l), (m), (n), and (o); or Wherein at least mutation (i) as defined in claim 6 and a mutation selected from mutations (l), (m), (n), (o), or wherein at least mutation (j) as defined in claim 6 and a mutation selected from mutations (l), (m), (n), (o), or wherein at least mutation (k) as defined in claim 6 and a mutation selected from mutations (l), (m), (n), (o), preferably wherein at least mutations (a) and (l) as defined in claim 6, or wherein at least mutations (a) and (m) as defined in claim 6, or wherein at least mutations (a) and (n) as defined in claim 6, or wherein at least mutations (a) and (o) as defined in claim 6, or wherein at least mutations (b) and (l) as defined in claim 6, or wherein at least mutations (b) and (m) as defined in claim 6, or wherein at least mutations (b) and (o) as defined in claim 6, or wherein at least one of mutations (f), (g), (h), (i), and (k) as defined in claim 6 and mutation (m) are introduced in step (a).

14. Use of the mutation as defined in claim 6 or a combination of mutations as defined in claim 7 or a polynucleotide as defined in claim 8 or a polypeptide as defined in claim 9 or a vector as defined in claim 10 for the production of plants having haploid inducer activity, preferably sunflower plants.

15. A method for identifying sunflower plants with haploid inducer activity, preferably sunflower plants as defined in any one of claims 1 to 7, comprising: (a) Screening for at least one mutation in the first nucleotide sequence encoding the first CENH3 protein and the second nucleotide sequence encoding the second CENH3 protein, preferably the presence of at least one knockdown or knockout mutation, or (b) Screening for reduced expression of the first and second nucleotide sequences encoding the first and second CENH3 proteins. Preferably, the first nucleotide sequence is represented by the sequence of SEQ ID NO:1 or 2 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO:1 or 2, and / or the second nucleotide sequence is represented by the sequence of SEQ ID NO:3 or 4 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO:3 or 4. and / or The first CENH3 protein is represented by the amino acid sequence of SEQ ID NO:5 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO:5, and / or the second CENH3 protein is represented by the amino acid sequence of SEQ ID NO:6 or a sequence having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequence of SEQ ID NO:

6. More preferably, at least one mutation in the first CENH3 protein, preferably at least one knockdown or knockout mutation, is selected from: (a) The glutamine at position 94 of SEQ ID NO:5 is substituted, preferably resulting in a stop codon; (b) The tryptophan at position 93 of SEQ ID NO:5 is substituted, preferably resulting in a stop codon; (c) The glutamic acid at position 103 of SEQ ID NO:5 is substituted, preferably with lysine; (d) Histidine at position 47 of SEQ ID NO:5 is substituted, preferably with tyrosine; (e) Asparagine at position 43 of SEQ ID NO:5 is substituted, preferably with lysine; (f) Glycine at position 41 of SEQ ID NO:5 is substituted, preferably with glutamic acid; (g) Leucine at position 56 of SEQ ID NO:5 is substituted, preferably with phenylalanine; (h) The serine at position 87 of SEQ ID NO:5 is substituted, preferably with threonine; (i) Threonine at position 53 of SEQ ID NO:5 is substituted, preferably with methionine; (j) The isoleucine at position 70 of SEQ ID NO:5 is substituted, preferably with threonine; (k) The 9th lysine residue in SEQ ID NO:5 is substituted, preferably with arginine; and At least one mutation in the second CENH3 protein, preferably at least one knockdown or knockout mutation, selected from: (l) At least one nucleotide of the ATG nucleic acid sequence encoding methionine at position 1 of SEQ ID NO:6 is deleted, inserted, or substituted, resulting in a knockout mutation, preferably achieved by frameshift insertion or deletion or nonsense mutation, or resulting in the exchange of the amino acid encoded at position 1, for example, methionine is exchanged for isoleucine. (m) The glutamine at position 96 of SEQ ID NO:6 is substituted, preferably resulting in a stop codon. (n) The isoleucine at position 118 of SEQ ID NO:6 is substituted, preferably with phenylalanine, and (o) A mutation occurs at position 93963324 on chromosome 15 according to the public reference XRQ2; More preferably, the plant contains at least one mutation (a) as defined in claim 6 and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation (b) as defined in claim 6 and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation (c) as defined in claim 6 and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation (d) as defined in claim 6 and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation (e) as defined in claim 6 and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation (f) as defined in claim 6 and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation (g) as defined in claim 6 and one mutation selected from mutations (l), (m), (n), and (o); or at least one mutation (h) as defined in claim 6 and one mutation selected from mutations (l), (m), (n), and (o). A mutation, or wherein at least mutation (i) as defined in claim 6 and a mutation selected from mutations (l), (m), (n), (o), or wherein at least mutation (j) as defined in claim 6 and a mutation selected from mutations (l), (m), (n), (o), or wherein at least mutation (k) as defined in claim 6 and a mutation selected from mutations (l), (m), (n), (o), preferably wherein at least mutations (a) and (l) as defined in claim 6, or wherein at least mutations (a) and (m) as defined in claim 6, or wherein at least mutations (a) and (n) as defined in claim 6, or wherein at least mutations (a) and (o) as defined in claim 6, or wherein at least mutations (b) and (l) as defined in claim 6, or wherein at least mutations (b) and (m) as defined in claim 6, or wherein at least mutations (b) and (o) as defined in claim 6, or wherein at least one of mutations (f), (g), (h), (i), and (k) and mutation (m) as defined in claim 6.

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