WO2025078496A1

WO2025078496A1 - In vivo haploid inducer for sunflower

Info

Publication number: WO2025078496A1
Application number: PCT/EP2024/078504
Authority: WO
Inventors: Alevtina RUBAN; Silke WIECKHORST; Markus Niessen; Milena OUZUNOVA; Monika KLOIBER-MAITZ
Original assignee: KWS SAAT SE & Co. KGaA
Priority date: 2023-10-12
Filing date: 2024-10-10
Publication date: 2025-04-17

Abstract

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

Description

In vivo Haploid Inducer for Sunflower

Technical Field

The present invention provides a plant of the genus Helianthus having the activity of a haploid inducer. In particular, haploid inducer activity is obtained by targeting the two copies of CENH3 present in Helianthus in order to reduce their expression and/or activity. The present invention also provides polynucleotides and polypeptides carrying certain knockdown or knock-out mutations as well as methods of generating and identifying a plant according to the invention.

Background of the invention

The generation and use of haploids is one of the most powerful biotechnological means to improve cultivated plants. The advantage of haploids for breeders is that homozygosity can be achieved already in the first generation after dihaploidization, creating doubled haploid plants, without the need of multiple selfing/backcrossing steps to obtain a high degree of homozygosity. Furthermore, the value of haploids in plant research and breeding lies in the fact that the founder cells of doubled haploids are products of meiosis, so that resultant populations constitute pools of diverse recombinant and at the same time genetically fixed individuals. The generation of doubled haploids thus provides not only perfectly useful genetic variability to select from with regard to crop improvement but is also a valuable means to produce mapping populations, recombinant inbreds as well as instantly homozygous mutants and transgenic lines.

Haploid plants can be produced in vitro or in vivo. Haploid production in vitro is performed in cell culture by androgenesis through anther or pollen culture or by gynogenesis though ovule or ovary culture. Suitable techniques have to be established for every plant species individually.

Haploid plants can be obtained in vivo by inter- and intraspecific crosses, in which one parental genome is eliminated after fertilization. It was shown that genome elimination after fertilization could be induced by modifying a centromere protein, the centromere-specific histone CENH3 in Arabidopsis thaliana (Ravi and Chan, Haploid plants produced by centromere-mediated genome elimination, Nature, Vol. 464, 2010, 615-619). With the modified haploid inducer lines, haploidization occurred in the progeny when a haploid inducer plant was crossed with a wild type plant. Interestingly, the haploid inducer line was stable upon selfing, suggesting that a competition between modified and wild type centromere in the developing hybrid embryo results in centromere inactivation of the inducer parent and consequently in uniparental chromosome elimination. However, Ravi and Chan only used a transgenic system and did not show any data on transferability of this system to crops. Although many scientists worked on this transfer, it showed to be more difficult than expected.

Wang et al. (Haploid induction by a maize cenh3 null mutant, Sci. Adv., 2021 , 7(4), eabe2299) showed the use of heterozygous CenH3 knock out mutants to produce paternal haploids in maize.

For sunflower (Helianthus) no efficient doubled haploid (DH) technology is currently available, neither in vitro nor in vivo. All in vivo systems based on mutations in known candidate genes such as CenH3, patatin-like-phospholipase or KNL2 failed until recently.

Therefore, it was an objective of the present invention to provide an efficient way to produce haploid sunflower plants for improved crop production.

More specifically, it was an objective of the present invention to identify mutations in the sunflower genome, which confer the activity of a haploid inducer.

It was also an objective of the present invention to provide sunflower plants, exhibiting haploid induction rates to allow the development of improved plants.

Summary of the invention

In one aspect, the present invention relates to a plant of the genus Helianthus having activity of a haploid inducer and comprising

(i) a first nucleotide sequence encoding a first CENH3 protein, and (ii) a second nucleotide sequence encoding a second CENH3 protein, wherein the expression and/or activity of the first and the second CENH3 protein in sum is reduced by about 60 to about 95%, preferably by about 65 to about 90%, particularly preferably by about 75 to about 80% compared to a wild type plant of the genus Helianthus.

In one embodiment of the plant described above, three of the four alleles encoding the first and second CENH3 protein carry at least one mutation, including at least one knock-down or knock-out mutation.

In another embodiment of the plant described above, the expression of the first and the second CENH3 protein is reduced by the presence of a double stranded RNA molecule or set of molecules targeting the first and the second nucleotide sequence for gene silencing.

In one embodiment of the plant according to any of the embodiments described above, 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 to the sequence of SEQ ID NO: 1 or 2 as respective reference sequence, 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 to the sequence of SEQ ID NO: 3 or 4 as respective reference sequence.

In one embodiment of the plant according to any of the embodiments described above, 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 to the sequence of SEQ ID NO: 5 as 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 to the sequence of SEQ ID NO: 6 as respective reference sequence.

In one embodiment of the plant described above, the at least one mutation, preferably resulting in at least one knock-down or knock-out mutation, in the first CENH3 protein is selected from: (a) the glutamine at position 94 of the sequence of SEQ ID NO: 5 is substituted, preferably to result in a stop codon;

(b) the tryptophan at position 93 of the sequence of SEQ ID NO: 5 is substituted, preferably to result in a stop codon;

(c) the glutamic acid at position 103 of the sequence of SEQ ID NO: 5 is substituted, preferably by lysine;

(d) the histidine at position 47 of the sequence of SEQ ID NO: 5 is substituted, preferably by tyrosine;

(e) the asparagine at position 43 of the sequence of SEQ ID NO: 5 is substituted, preferably by lysine;

(f) the glycine at position 41 of the sequence of SEQ ID NO: 5 is substituted, preferably by glutamic acid;

(g) the leucine at position 56 of the sequence of SEQ ID NO: 5 is substituted, preferably by phenylalanine;

(h) the serine at position 87 of the sequence of SEQ ID NO: 5 is substituted, preferably by threonine;

(i) the threonine at position 53 of the sequence of SEQ ID NO: 5 is substituted, preferably by methionine;

(j) the isoleucine at position 70 of the sequence of SEQ ID NO: 5 is substituted, preferably by threonine;

(k) the lysine at position 9 of the sequence of SEQ ID NO: 5 is substituted, preferably by arginine; and wherein the at least one mutation, preferably resulting in a functional knock-down or knock-out, in the second CENH3 protein is selected from:

(l) at least one nucleotide of the ATG nucleic acid sequence encoding the methionine at position 1 of the sequence of SEQ ID NO: 6 is deleted, inserted or substituted, resulting either in a knock-out mutation, preferably by a frame-shifting insertion or deletion or by a nonsense mutation, or resulting in an exchange of the encoded amino acid at position 1 , for example, an exchange of methionine by isoleucine, (m) the glutamine at position 96 of the sequence of SEQ ID NO: 6 is substituted, preferably to result in a stop codon, and

(n) the isoleucine at position 1 18 ofthe sequence of SEQ ID NO: 6 is substituted, preferably by phenylalanine, and

(o) position 93963324 on chromosome 15 according to public reference XRQ2 is mutated.

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

In another aspect, the present invention relates to a polynucleotide or set of polynucleotides encoding a protein represented by the amino acid sequence of SEQ ID NO: 5 and/or 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 to SEQ ID NO: 5 or 6 as respective reference sequence and carrying at least one knockout mutation, preferably selected from the mutations defined above or a combination of at least two knock-out combinations, preferably as defined above. In yet another aspect, the present invention relates to a polypeptide encoded by a polynucleotide or set of polynucleotides as defined above.

In a further aspect, the present invention relates to a vector comprising a polynucleotide or set of polynucleotides as defined above.

In one aspect, the present invention also relates to a cell comprising a polynucleotide or set of polynucleotides as defined above, a polypeptide according as defined above or a vector as defined above.

In another aspect, the present invention relates to a method of generating a plant or plant part, in particular of the genus Helianthus, preferably as defined in any of the embodiments described above, comprising:

(a) introducing through gene editing technology or modification using random or targeted mutagenesis into the genome of a plant or plant part, preferably of the genus Helianthus, at least one mutation, preferably a knock-down or knock-out mutation, in each of a first nucleotide sequence encoding a first CENH3 protein and a second nucleotide sequence encoding a second CENH3 protein, so that three of the four alleles encoding the first and the second CENH3 protein are knocked-down or knocked-out; or

(b) introducing through stable or transient integration by means of transformation or insertion using gene editing technology into the plant or the plant part an RNAi molecule or a set of RNAi molecules directed against, targeting, or hybridizing with a first nucleotide sequence encoding a first CENH3 protein and a second nucleotide sequence encoding a second CENH3 protein, and, optionally, regenerating a plant from the plant part of any of (a) or (b), preferably, wherein 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 to the sequence of SEQ ID NO: 5, and/or wherein 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 to the sequence of SEQ ID NO: 6.

In one embodiment of the method described above, the at least one mutation, preferably a mutation functionally resulting in a knock-down or knock-out, in the first CENH3 protein is selected from: (a) the glutamine at position 94 of the sequence of SEQ ID NO: 5 is substituted, preferably to result in a stop codon;

(k) the lysine at position 9 of the sequence of SEQ ID NO: 5 is substituted, preferably by arginine; and the at least one mutation, preferably a knock-down or knock-out mutation, in the second CENH3 protein is selected from:

(o) position 93963324 on chromosome 15 according to public reference XRQ2 is mutated; preferably, at least mutations (a) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (b) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (c) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (d) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (e) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (f) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (g) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (h) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (i) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (j) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (k) and one mutation selected from mutations (I), (m), (n) and (o) as defined above, preferably at least mutations (a) and (I) 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 (I) 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) and mutation (m) as defined above are introduced in step (a).

In another aspect, the present invention relates to the use of a mutation as defined above or a combination of mutations as defined above or a polynucleotide as defined above or a polypeptide as defined above or a vector as defined above for generating a plant, preferably of genus Helianthus, having activity of a haploid inducer.

In a further aspect, the present invention relates to a method of identifying a Helianthus plant having activity of a haploid inducer, preferably as defined in any of the embodiments described above, comprising (a) screening for the presence of a mutation, preferably a knock-down or knock-out mutation, in a first nucleotide sequence encoding a first CENH3 protein and in a second nucleotide sequence encoding a second CENH3 protein, or

(b) screening for reduced expression of a first and a second nucleotide sequence encoding a first CENH3 protein and a second CENH3 protein, preferably wherein the first nucleotide sequence is represented by a 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 to the sequence of SEQ ID NO: 1 or 2, and/or wherein the second nucleotide sequence is represented by a 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 to the sequence of SEQ ID NO: 3 or 4, and/or wherein 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 to the sequence of SEQ ID NO: 5, and/or wherein 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 to the sequence of SEQ ID NO: 6, further preferably, the at least one knock-down or knock-out mutation in the first CENH3 protein is selected from:

(a) the glutamine at position 94 of the sequence of SEQ ID NO: 5 is substituted, preferably to result in a stop codon;

(d) the histidine at position 47 of the sequence of SEQ ID NO: 5 is substituted, preferably by tyrosine; (e) the asparagine at position 43 of the sequence of SEQ ID NO: 5 is substituted, preferably by lysine;

(l) at least one nucleotide of the ATG nucleic acid sequence encoding the methionine at position 1 of the sequence of SEQ ID NO: 6 is deleted, inserted or substituted, resulting either in a knock-out mutation, preferably by a frame-shifting insertion or deletion or by a nonsense mutation, or resulting in an exchange of the encoded amino acid at position 1 , for example, an exchange of methionine by isoleucine,

(m) the glutamine at position 96 of the sequence of SEQ ID NO: 6 is substituted, preferably to result in a stop codon, and

(n) the isoleucine at position 118 ofthe sequence of SEQ ID NO: 6 is substituted, preferably by phenylalanine, and

(o) position 93963324 on chromosome 15 according to public reference XRQ2 is mutated; more preferably, at least mutations (a) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (b) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (c) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (d) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (e) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (f) and one mutation selected from mutations (I), (m), (n) and (o) as above or at least mutations (g) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (h) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (i) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (j) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (k) and one mutation selected from mutations (I), (m), (n) and (o) as defined above, preferably at least mutations (a) and (I) 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 (I) as defined above or wherein 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) and mutation (m) as defined above are present in the plant.

List of sequences

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 marker ha0000ac07

SEQ ID NO: 9 marker ha88508s02

SEQ ID NO: 10 intentionally skipped sequence

SEQ ID NO: 11 marker ha88508s08 SEQ ID NO: 12 marker ha88508s09

SEQ ID NO: 13 marker ha88508s10

SEQ ID NO: 14 marker ha88508s12

SEQ ID NO: 15 marker 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 marker ha88509s15

Definitions

A "haploid plant" or "haploid plant cell" herein refers to a plant or a plant cell having only one set of chromosomes each one not being part of a pair. The number of chromosomes in a single set is called the haploid number, given the symbol n. "Gametes" are haploid cells, of which two combine in fertilization to form a "zygote" with n pairs of chromosomes, i.e. 2n chromosomes in total. Each chromosome pair comprises one chromosome from each gamete, called homologous chromosomes. Cells and organisms with pairs of homologous chromosomes are "diploid".

"Doubled haploid" in the context of the present disclosure refers to a genotype formed when haploid cells undergo chromosome doubling. Doubled haploids may be produced spontaneously or by chromosome doubling from haploid cells, organisms or material. Therefore, doubled haploids are homozygous. For polyploid cell (2n= 3x, 4x, 5, 6x, or more), the corresponding polyhaploids may contain more than one copy of the same haploid genome.

A "plant having activity of a haploid inducer" or a "haploid inducer" or a "haploid inducer line" in the context of the present invention is a plant or plant line, which was genetically modified to have the capability to produce 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%, 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 1 1 .0%, at least 12.0%, at least 13.0%, at least 14.0%, at least 15.0% of cases when combined in fertilization with a wild-type plant. Since the chromosomes of the haploid inducer are eliminated, the resulting haploid progeny only comprises genetic material of the wild-type parent.

As used herein, “reduced (protein) expression” refers to a reduction in the expression rate of a nucleotide sequence encoding the protein by a certain percentage in comparison to the specified reference, such as a plant not comprising the genetic or otherwise modifications according to the invention as described herein elsewhere, or a reference plant, such as a wild type plant.

As used herein, “reduced (protein) activity” refers to decreased activity by a certain percentage compared to a reference such as a wild type plant. (Protein) activity of CENH3 may be assessed by quantifying localization of CENH3 on the centromeres using antibodies against CENH3 and detection by confocal microscopy.

A "knock-out" or "knock-down" of a target gene in the context of the present disclosure refers to a full or partial inactivation of the target gene, respectively. In case of a knockdown, the expression of the target gene is usually reduced, while in case of a "knock-out", no (functional) gene is expressed at all.

A “double stranded RNA molecule or set of molecules targeting a certain nucleic acid sequence” refers to an RNAi, microRNA (miRNA) or small interfering RNA (siRNA), which are involved in gene silencing (RNA interference) and post-transcriptional regulation of gene expression.

The terms "RNA interference" or "RNAi" or "RNA silencing" or "gene silencing" as used herein interchangeably refer to a gene down-regulation (or knock-down) mechanism meanwhile demonstrated to exist in all eukaryotes. The mechanism was first recognized in plants where it was called "post-transcriptional gene silencing" or "PTGS". In RNAi, small RNAs function to guide specific effector proteins to a target nucleotide sequence by complementary base pairing resulting in degradation of the target. A "gene silencing construct" or "RNAi construct" usually comprises so called "sense" and "antisense" sequences. A sense and an antisense sequence both are complementary sequences, which are present in reverse orientation in a nucleic acid sequence. If a nucleic acid construct comprises a sense and a corresponding antisense sequence, the two complementary sequences form an RNA double strand upon transcription, which results in an "RNA hairpin". In an RNA hairpin, sense sequences and corresponding antisense sequences, together form a double strand and are separated by an "intervening intron loop sequence" forming the loop of the hairpin structure. An "RNAi construct" may also comprise more than one sense and antisense pair and form several loops. An "RNAi construct" may be provided in the form of an expression construct or as a vector/plasmid.

A nucleic acid (construct) “targets” a genomic sequence or a gene when it contains sequence information, which allows recognition of the genomic sequence or gene and can thus interfere with the sequence, e.g., by site-specific cleavage or silencing. The targeting can be affected either by direct interaction with the genomic sequence itself or by interaction with the transcript of the genomic sequence. For example, if the nucleic acid construct of the present invention comprises or encodes a sense and a corresponding antisense sequence targeting a genomic sequence, an RNA silencing or RNA interference (RNAi) mechanism is activated upon transcription of the construct, which leads to the destruction of the transcript of the genomic target sequence and thus suppresses expression of the target. In another case, the nucleic acid construct may encode a sitespecific nuclease and a guide RNA, which results in cleavage of the target sequence.

A “stop codon” is a nucleotide triplet, which signals the termination of the translation process of a protein. A “start codon” is the first nucleotide triplet of a messenger RNA transcript, which is translated by a ribosome.

“Gene editing technology” refers to the use of a site-specific nuclease or site-specific nickase or a functional active fragment or variant thereof together with the cognate guide RNA (or pegRNA or crRNA) guiding the relevant CRISPR nuclease to its target site to be cleaved. A “site-specific nuclease” refers to a nuclease or an active fragment thereof, which is capable to specifically recognize and cleave DNA at a certain target site. Such nucleases typically produce a double strand break (DSB), which is then repaired by nonhomologous end-joining (NHEJ) or homologous recombination (HR). The nucleases include zinc-finger nucleases, transcription activator-like effector nucleases, engineered homing endonucleases, recombinases, transposases and meganucleases and CRISPR nucleases and/or any combination, variant or active fragment thereof.

As used herein “introducing” certain constructs or effectors, such as genome editing systems or gene silencing constructs, can be performed by any method known to the skilled person such as transformation or transfection. “Transformation” or “Transfection” of a plant cell with a construct or set of constructs or an effector molecule refers to any established technique to introduce nucleic acid molecules into a cell, such as biolistic approaches (e.g., particle bombardment), microinjection, permeabilising the cell membrane with various treatments such as electroporation or PEG treatment or Agrobacterium tumefaciens mediated transformation. Generally, incorporating (a) nucleic acid construct(s), for example by way of transformation, may be accomplished with techniques that are basically known to the person skilled in the art. For example, the nucleic acid construct can be incorporated into the plant cells by infecting a plant tissue or a plant cell with Agrobacterium tumefaciens containing the nucleic acid sequence to be transferred in its plasmid that can be integrated into the plant genome. Incorporating by means of a biolistic transfer is another option, wherein the nucleic acid construct to be incorporated into the plant cell is applied to gold particles or tungsten particles, which are then shot into the cells at a high speed. Another option known to the person skilled in the art for incorporating a nucleic acid construct into a plant cell, is the protoplast transformation, wherein either polyethylene glycol is added to the protoplasts in the presence of the nucleic acid molecules to be incorporated, or the protoplasts are exposed to a short current impulse, so that the protoplast membrane transiently becomes permeable for the nucleic acid construct(s).

“Introducing” a modification in the genome of a plant can also be performed by mutagenesis. “Random Mutagenesis” refers to a technique, by which modifications or mutations are introduced into a nucleic acid sequence in a random or non- site-specific (non-targeted) way. For example, mutations can be induced by certain chemicals such as EMS (ethyl methanesulfonate) or ENU (N-ethyl-N-nitrosourea) or physically, e.g., by irradiation with UV or gamma rays. “Targeted” or “site-specific modifications”, on the other hand, rely on the action of site-specific effectors such as nucleases, nickases, recombinases, transposases, base editors. These tools recognize a certain target sequence and allow to introduce a modification at a specific location within the target sequence.

“TILLING” (Targeting Induced Local Lesions in Genomes) is a process, which allows to identify mutations in a specific gene after an (unspecific) mutagenesis has been performed. Mutagenesis may e.g., be performed using a chemical mutagen such as EMS. Then, a sensitive DNA screening technique is used to identify single base mutations. Methods for performing TILLING are known to the skilled person.

The term "transient integration" as used herein refers to the transient introduction of at least one nucleic acid and/or amino acid sequence according to the present disclosure, preferably incorporated into a delivery vector and/or into a recombinant construct, with or without the help of a delivery vector, into a target structure, for example, a plant cell or cellular system, wherein the at least one nucleic acid or nucleotide sequence is introduced under suitable reaction conditions so that no integration of the at least one nucleic acid sequence into the endogenous nucleic acid material of a target structure, the genome as a whole, occurs, so that the at least one nucleic acid sequence will not be integrated into the endogenous DNA of the target cell. As a consequence, in the case of transient integration, the introduced genetic construct will not be inherited to a progeny of the target structure, for example a plant cell. The at least one nucleic acid and/or amino acid sequence or the products resulting from transcription, translation, processing, post-translational modifications or complex building thereof are only present temporarily, i.e., in a transient way, in constitutive or inducible form, and thus can only be active in the target cell for exerting their effect for a limited time. Therefore, the at least one sequence introduced via transient integration will not be heritable to the progeny of a cell. The effect mediated by at least one sequence or effector introduced in a transient way can, however, potentially be inherited to the progeny of the target cell. A "stable integration” therefore implies the integration of a nucleic acid or nucleotide sequence into the genome of a target cell or cellular system of interest, wherein the genome comprises the nuclear genome as well as the genome comprised by further organelles.

The term “vector” refers to an element used for introducing a nucleic acid construct or set of nucleic acid constructs into a cellular system. The vector may be a plasmid or plasmid vector, cosmid, artificial yeast artificial chromosomes (YAC), bacterial artificial chromosome (BAC) or P1 artificial chromosomes (PACs), phagemid, bacterial phage based vector, an isolated single-stranded or double-stranded nucleic acid sequence, comprising DNA and RNA sequences in linear or circular form, or a mixture thereof, for introduction or transformation into a plant, plant cell, tissue, organ or material according to the present disclosure.

The terms "plant" or "plant cell" or “part of a plant” as used herein refer to a plant organism, a plant organ, differentiated and undifferentiated plant tissues, plant cells, seeds, and derivatives and progeny thereof, including in particular plant structures, plant protoplast, plant cell or tissue culture from which plants can be regenerated, plant calli, plant clumps and plant cells that are intact in plants or parts of plants, such as seeds, kernels, cobs, fruits, flowers, cotyledons, leaves, stems, buds, roots, root tips, stover, and the like. Plant cells include without limitation, for example, cells from seeds, from mature and immature cells or organs, including embryos, meristematic tissues, seedlings, callus tissues in different differentiation states, leaves, flowers, roots, shoots, male orfemale gametophytes, sporophytes, pollen, pollen tubes and microspores and protoplasts.

Whenever the present disclosure relates to the percentage of identity of nucleic acid or amino acid sequences to each otherthese values define those values as obtained by using the EMBOSS Water Pairwise Sequence Alignments (https://www.ebi.ac.uk/Tools/psa/emboss_water/). Alignments or sequence comparisons as used herein refer to an alignment over the whole length of two sequences compared to each other. Those tools provided by the European Molecular Biology Laboratory (EMBL) European Bioinformatics Institute (EBI) for local sequence alignments use a modified Smith-Waterman algorithm (see www.ebi.ac.uk/Tools/psa/ and Smith, T.F. & Waterman, M.S. "Identification of common molecular subsequences" Journal of Molecular Biology, 1981 147 (1 ): 195-197). When conducting an alignment, the default parameters defined by the EMBL-EBI are used. Those parameters are (i) for amino acid sequences: Matrix = BLOSUM62, gap open penalty = 10 and gap extend penalty = 0.5 or (ii) for nucleic acid sequences: Matrix = DNAfull, gap open penalty = 10 and gap extend penalty = 0.5. The skilled person is well aware of the fact that, for example, a sequence encoding a protein can be "codon-optimized" if the respective sequence is to be used in another organism in comparison to the original organism a molecule originates from.

Detailed description

A gene encoding CENH3 (HanXRQr2_Chr15g0703621) in sunflower was published in 2015 (Nagaki et al, 2015, Front Plant Sci. 2015; 6: 912.). In the context of the present invention, it was found that, surprisingly, a second gene encoding another copy of CENH3 is present in sunflower (HanXRQr2_Chr14g0649211), which significantly differs at the N- terminus from the previously published CENH3. Both copies were expressed transiently in different biological systems. Localization of the protein on the centromeres was assessed using antibodies against sunflower CENH3 and detected by confocal microscopy.

Similarly to the rest of the plant kingdom, Helianthus exhibits considerable variation in genome size, and much of this variation is attributable to differences between ploidy levels. Indeed, Helianthus used as agricultural plant usually contains diploid (2n = 2x = 34) species. Still, tetrapioid (2n = 4x = 68) and hexapioid species (2n = 6x = 102) that formed during neopolyploidization events occurring since the radiation of the genus million years ago were observed and characterized (Kantar et al., Briefings in Functional Genomics, Volume 13, Issue 4, July 2014, Pages 328-340, https://doi.org/10.1093/bfgp/elu004).

The present inventors now found that the expression of both different CENH3 proteins observed in diploid Helianthus germplasm needs to be reduced in order to achieve haploid inducer activity in a sunflower plant. However, there still has to be a residual activity, otherwise the plant is not viable. Therefore, in a first aspect, the present invention relates to a plant of the genus Helianthus having activity of a haploid inducer and comprising

(i) a first nucleotide sequence encoding a first CENH3 protein, and

(ii) a second nucleotide sequence encoding a second CENH3 protein, said second CENH3 protein being different from the first CENH3 protein as naturally occurring, wherein the expression and/or activity of the first and the second CENH3 protein in sum is reduced by about 60 to about 95%, preferably by about 65 to about 90%, particularly preferably by about 75 to about 80% compared to a wild type plant of the genus Helianthus.

In a diploid Helianthus plant, several possibilities exist to reduce expression and/or activity of the first and the second CENH3 protein. Even more possibilities exist in polyploidy Helianthus species. In a diploid species, at least one allele encoding one of the two different CENH3 proteins, should thus carry at least one mutation, so that the total dosage of the resulting CENH3 protein is reduced.

Therefore, in certain embodiments, at least one mutation in only one allele encoding one of the two different CENH3 proteins might already have a strong effect.

In preferred embodiments, at least one mutation in at least one allele encoding the first and the second CENH3 protein will be present to achieve a reduction of the expression and/or activity of the first and the second CENH3 protein so that the remaining activity in sum is reduced by about 60 to about 95%, preferably by about 65 to about 90%, particularly preferably by about 75 to about 80% compared to a wild type plant of the genus Helianthus.

To achieve the reduction, mutations, preferably knock-down or knock-out mutations on both CENH3 genes, or alleles thereof, can be introduced. However, they need to be in a heterozygous state for one copy in the resulting plant in order to retain enough activity for the plant to be viable. As used herein, a “mutation” is used to define at least one change of a nucleotide position in a DNA molecule in comparison to a reference molecule not carrying the mutation(s), which may include a point mutation, a deletion mutation or an insertion mutation, or, in certain embodiments, a combination thereof. A “knock-out” mutation and a “knock-down” mutation as used herein represent the functional outcome of a mutation, including at least one point, insertion, or deletion mutation, which can be a complete or almost complete loss-of function of the original gene product (RNA or protein) in the case of a knock-out, or which may be a reduction of the original function of a gene product in the case of a “knock-down” mutation. In one embodiment of the plant described above, three of the four alleles encoding the first and second CENH3 protein carry at least one mutation, preferably at least one knock-down or knock-out mutation.

Preferably, the Helianthus plant of the above first aspect is a plant not obtained or obtainable by an essentially biological process.

The present invention therefore also relates to a plant of the genus Helianthus having activity of a haploid inducer and comprising

(i) a first nucleotide sequence encoding a first CENH3 protein, and

(ii) a second nucleotide sequence encoding a second CENH3 protein, wherein three of the four alleles encoding the first and second CENH3 protein carry at least one mutation, preferably at least one knock-down or knock-out mutation.

Alternatively, the expression of the two CENH3 genes can be reduced using gene silencing while maintaining enough residual activity.

The molecule or set of molecules targeting the first and the second nucleotide sequence for gene silencing may be an RNAi construct or a microRNA (miRNA) or small interfering RNA (siRNA) which recognizes the CENH3 sequence by base pairing. Two molecules may be used, one recognizing each CENH3 sequence. The transcribed CENH3 sequences, which are base paired with the molecule(s) are then degraded resulting in reduced expression of the CENH3 genes. Advantageously, this method strongly knocks-down CENH3 to provide haploid induction activity but maintains a level of activity sufficient to circumvent lethality.

As mentioned above, in the context of the present invention, a second copy of CENH3 was identified in sunflower. The genomic sequence of this copy is represented by the sequence of SEQ ID NO: 1 and the cDNA sequence is represented by the sequence of SEQ ID NO: 2. The previously published copy of CENH3 is represented by the sequences of SEQ ID NO: 3 (genomic sequence) and SEQ ID NO: 4 (cDNA). In one embodiment of the plant according to any of the embodiments described above, 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 to the sequence of SEQ ID NO: 1 or 2 as 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 to the sequence of SEQ ID NO: 3 or 4 as respective reference sequence.

The amino acid sequence of the newly identified copy of CENH3 is represented the sequence of SEQ ID NO: 5 and the amino acid sequence of the previously published copy is represented by the sequence of SEQ ID NO: 6.

In the context of the present invention, knock-down and knock-out mutations on both copies of CENH3 were assessed for haploid inducer activity.

A TILLING reverse screen was performed for both copies of CENH3. 23 individual mutations were identified and 19 were tested for performance to induce haploid plants (HIR, haploid induction rate). HIR was tested by crossing the mutant with a heterozygous tester. The offspring was screened with 4-8 polymorphic KASP markers plus corresponding mutation markers. Homozygous plants were analyzed by Illumina chip. The induction rate, both on male and female side was zero for all individual mutants.

In the following step, several mutations of interest were combined. For CENH3, knock out mutants for both copies were available. The mutant HA304m004k of HA88509 has lost the start codon and the mutant HA304m003j (the glutamine at position 94 of the sequence of SEQ ID NO: 5 is substituted to result in a stop codon) of HA880508 has a premature stop at position 94. Combining these mutations, no double homozygous plants could be identified. But plants used as pollen donor, containing one mutation fixed in homozygous state and the other mutation kept in heterozygous state, are able to induce female haploid kernels (among 3 crosses performed, haploids were identified in two of them: 1 haploid among 110 kernels, 1 haploid among 23 kernels and 0 among 192 kernels).

In one embodiment of the plant described above, the at least one mutation, preferably the at least one knock-down or knock-out mutation in the first CENH3 protein is selected from:

(k) the lysine at position 9 of the sequence of SEQ ID NO: 5 is substituted, preferably by arginine; and wherein the at least one mutation, preferably the at least one knock-down or knock-out mutation, in the second CENH3 protein is selected from:

In a preferred embodiment, the plant carries at least mutations (a) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or the plant carries at least mutations (b) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or the plant carries at least mutations (c) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or the plant carries at least mutations (d) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or the plant carries at least mutations (e) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or the plant carries at least mutations (f) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or the plant carries at least mutations (g) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or the plant carries at least mutations (h) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or the plant carries at least mutations (i) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or the plant carries at least mutations (j) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or the plant carries at least mutations (k) and one mutation selected from mutations (I), (m), (n) and (o) as defined above, preferably the plant carries at least mutations (a) and (I) as defined above or the plant carries at least mutations (a) and (m) as defined above or the plant carries at least mutations (a) and (n) as defined above or the plant carries mutations (a) and (o) as defined above or wherein the plant carries at least mutations (b) and (I) as defined above or the plant carries at least mutations (b) and (m) as defined above or the plant carries mutations (b) and (o) as defined above or the plant carries at least one of mutations (f), (g), (h), (i) and (k) and mutation (m) as defined above. In another aspect, the present invention relates to a polynucleotide or set of polynucleotides encoding a protein represented by the amino acid sequence of SEQ ID NO: 5 and/or 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 to SEQ ID NO: 5 or 6 as respective reference sequence and carrying at least one knockout mutation, preferably selected from the mutations defined above or a combination of at least two knock-out combinations, preferably as defined above.

In a further aspect, the present invention also relates to a polypeptide encoded by a polynucleotide or set of polynucleotides as defined above.

In yet another aspect, the present invention relates to a vector comprising a polynucleotide or set of polynucleotides as defined above.

In a further aspect, the present invention relates to a cell comprising a polynucleotide or set of polynucleotides as defined above, a polypeptide as defined above or a vector as defined above.

The cell may be a plant cell, preferably of genus Helianthus, or e.g. a bacterial host cell.

The present invention also relates to a method of generating a plant or plant part, in particular of the genus Helianthus, preferably as defined in any of the embodiments described above, comprising:

(a) introducing through gene editing technology or modification using random or targeted mutagenesis into the genome of a plant or plant part, preferably of the genus Helianthus, at least one mutation, preferably at least one knock-down or knock-out mutation, in each of a first nucleotide sequence encoding a first CENH3 protein and a second nucleotide sequence encoding a second CENH3 protein, so that three of the four alleles encoding the first and the second CENH3 protein are knocked-down or knocked-out; or

A plant of the present invention can be produced either by introducing mutations in both copies of the endogenous CENH3 gene or by stably or transiently introducing a gene silencing construct targeting both copies of CENH3.

Genome editing technology allows to introduce a double strand break at one or more predetermined target site(s), e.g., by or within the CENH3 locus thereby disrupting the locus and, optionally, inserting an exogenous sequence or replacing an endogenous sequence. The double strand break is introduced by a site-specific nuclease such as meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or the clustered regularly interspaced short palindromic repeat (CRISPR) nucleases. The nucleases cause double strand breaks (DSBs) at specific cleaving sites, which are repaired by non-homologous end-joining (NHEJ) or homologous recombination (HR). The use of a repair template guides the cellular repair process so that the results of the repair are error- free and predictable. A repair template preferably comprises symmetric or asymmetric homology arms, which are complementary to the sequences flanking a double strand break and therefore allow to insert a sequence or close the break in a controlled manner.

In embodiments, wherein the site-directed nuclease or variant thereof is a nucleic acid- guided site-directed nuclease, the at least one genome editing system additionally includes at least one guide molecule, or a sequence encoding the same. The "guide molecule" or "guide nucleic acid sequence" (usually called and abbreviated as guide RNA, crRNA, crRNA+tracrRNA, gRNA, sgRNA, depending on the corresponding CRISPR system representing a prototypic nucleic acid-guided site-directed nuclease system), which recognizes a target sequence to be cut 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, to which the nucleic acid guided nuclease binds to form a targetable nuclease complex. The scaffold region may comprise direct repeats, which are recognized and processed by the nucleic acid guided nuclease to provide mature crRNA. A pegRNAs may comprise a further region within the guide molecule, the so-called "primer-binding site". The "target region" defines the complementarity to the target site, which is intended to be cleaved. A crRNA as used herein may thus be used interchangeably herein with the term guide RNA in case it unifies the effects of meanwhile well-established CRISPR nuclease guide RNA functionalities. Certain CRISPR nucleases, e.g., Cas9, may be used by providing two individual guide nucleic acid sequences in the form of a tracrRNA and a crRNA, which may be provided separately, or linked via covalent or non-covalent bonds/interactions. The guide RNA may also be a pegRNA of a Prime Editing system as further disclosed below. The at least one guide molecule may be provided in the form of one coherent molecule, orthe sequence encoding the same, or in the form of two individual molecules, e.g., crRNA and tracr RNA, or the sequences encoding the same.

Mutagenesis can be performed by a number of techniques known to the skilled person. For example, mutations can be induced by certain chemicals such as EMS (ethyl methanesulfonate) or ENU (N-ethyl-N-nitrosourea) or physically, e.g., by irradiation with UV or gamma rays.

In one embodiment of the method described above, TILLING (Targeting Induced Local Lesions in Genomes) is used to identify mutations in both copies of the CENH3 gene after an (unspecific) mutagenesis has been performed.

RNAi techniques fortargeted gene silencing are well known in the art. To this end, an RNAi construct is introduced into a plant cell, which contains sequence information of the genomic target to be silenced in the cell. The construct is preferably introduced in form of a DNA sequence, which is then transcribed into functional RNA in the cell. In particular, the RNAi construct encodes sense and antisense sequences, which represent (a fragment of) the genomic target. The complementary sense and antisense sequences, which are present in reverse orientation in the construct form an RNA double strand upon transcription, which results in an RNA hairpin with an intervening intron loop sequence. The presence of the RNAi construct ultimately results in a reduced expression of the target, i.e., a knock-down.

In one embodiment of the method described above, the at least one mutation, preferably the at least one knock-down or knock-out mutation, in the first CENH3 protein is selected from:

(b) the tryptophan at position 93 of the sequence of SEQ ID NO: 5 is substituted, preferably to result in a stop codon; (c) the glutamic acid at position 103 of the sequence of SEQ ID NO: 5 is substituted, preferably by lysine;

(k) the lysine at position 9 of the sequence of SEQ ID NO: 5 is substituted, preferably by arginine; and the at least one mutation, preferably the at least one knock-down or knock-out mutation, in the second CENH3 protein is selected from:

(n) the isoleucine at position 118 of the sequence of SEQ ID NO: 6 is substituted, preferably by phenylalanine, and (o) position 93963324 on chromosome 15 according to public reference XRQ2 is mutated.

In a preferred embodiment, at least mutations (a) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (b) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (c) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (d) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (e) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (f) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (g) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (h) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (i) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (j) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (k) and one mutation selected from mutations (I), (m), (n) and (o) as defined above, preferably at least mutations (a) and (I) 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 (I) 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) and mutation (m) as defined above are introduced in step (a).

In a further aspect, the present invention relates to the use of a mutation as defined above or a combination of mutations as defined above or a polynucleotide as defined above or a polypeptide as defined above or a vector as defined above for generating a plant, preferably of genus Helianthus, having activity of a haploid inducer.

Finally, the present invention also provides a method for identifying a Helianthus plant having activity of a haploid inducer, preferably as defined in any of the embodiments described above, comprising

(a) screening for the presence of a mutation, preferably at least one functional knock-down or knock-out mutation, in a first nucleotide sequence encoding a first CENH3 protein and in a second nucleotide sequence encoding a second CENH3 protein, or

(b) screening for reduced expression of a first and a second nucleotide sequence encoding a first CENH3 protein and a second CENH3 protein. Preferably, the first nucleotide sequence is represented by a 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 to the sequence of SEQ ID NO: 1 or 2, and/or wherein the second nucleotide sequence is represented by a 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 to the sequence of SEQ ID NO: 3 or 4, and/or wherein 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 to the sequence of SEQ ID NO: 5, and/or wherein 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 to the sequence of SEQ ID NO: 6.

In a preferred embodiment of the method for identifying a Helianthus plant having activity of a haploid inducer described above, the at least one mutation, preferably at least one knock-down or knock-out mutation, in the first CENH3 protein is selected from:

(f) the glycine at position 41 of the sequence of SEQ ID NO: 5 is substituted, preferably by glutamic acid; (g) the leucine at position 56 of the sequence of SEQ ID NO: 5 is substituted, preferably by phenylalanine;

(n) the isoleucine at position 118 of the sequence of SEQ ID NO: 6 is substituted, preferably by phenylalanine, and

Further preferably, in the method described above, at least mutations (a) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (b) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (c) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (d) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (e) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (f) and one mutation selected from mutations (I), (m), (n) and (o) as above or at least mutations (g) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (h) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (i) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (j) and one mutation selected from mutations (I), (m), (n) and (o) as defined above or at least mutations (k) and one mutation selected from mutations (I), (m), (n) and (o) as defined above, preferably at least mutations (a) and (I) 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 (I) as defined above or wherein 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) and mutation (m) as defined above are present in the plant.

Screening can be done by any sequencing method or by marker application using markers on desired positions. Plant material to screen can be mutagenized populations (e.g. obtained by EMS mutagenesis or any other methods) or TO plants in genome editing plant material.

Markers for the detection of the specified mutations are disclosed in the sequence listing and detect the mutations as follows:

Marker ha88508s17 (SEQ ID NO: 17) detects a gln94STOP mutation in the sequence of SEQ ID NO: 5.

Marker ha88508s10 (SEQ ID NO: 13) detects a trp93STOP mutation in the sequence of SEQ ID NO: 5.

Marker ha88508s18 (SEQ ID NO: 18) detects a glu 103lys mutation in the sequence of SEQ ID NO: 5.

Marker ha88508s02 (SEQ ID NO: 9) detects a his47tyr mutation in the sequence of SEQ ID NO: 5.

Marker ha88508s12 (SEQ ID NO: 14) detects a ans43lys mutation in the sequence of SEQ ID NO: 5.

Marker ha88508s09 (SEQ ID NO: 12) detects a gly41glu mutation in the sequence of SEQ ID NO: 5.

Marker ha88508s08 (SEQ ID NO: 11) detects a Ieu56phe mutation in the sequence of SEQ ID NO: 5. Marker ha88508s25 (SEQ ID NO: 20) detects a ser87thr mutation in the sequence of SEQ ID NO: 5.

Marker ha88508s13 (SEQ ID NO: 15) detects a thr53met mutation in the sequence of SEQ ID NO: 5.

Marker ha88508s14 (SEQ ID NO: 16) detects a ile70thr mutation in the sequence of SEQ ID NO: 5.

Marker ha88508s20 (SEQ ID NO: 19) detects a Iys9arg mutation in the sequence of SEQ ID NO: 5.

Marker ha0000ac07 (SEQ ID NO: 8) detects a metl ile mutation in the sequence of SEQ ID NO: 6.

Marker ha0000ab98 (SEQ ID NO: 7) detect a gln96STOP mutation in the sequence of SEQ ID NO: 6.

Marker ha88509s15 (SEQ ID NO: 22) detects a ile118phe mutation in the sequence of SEQ ID NO: 6.

Marker ha88509s06 (SEQ ID NO: 21) detects a mutation at position 93963324 on chromosome 15 according to public reference XRQ2.

Example 1 : Mutation generation and screening

Sunflower accession HA304 was mutagenized with EMS using the KeyPointMB method provided by Keygene N.V. Two copies of CENH3 gene were amplified by PCR from DNA of plants from the mutagenized populations, then sequenced by Illumina short read technology. All plants carrying mutation in either copy were selected and self-pollinated. In the next generations and test crosses plants with amino acid exchange mutations were selected based on the results of KASP marker analysis.

Example 2: Mutation combination

Mutations in each copy of CENH3 gene were ranked according to predicted strength of the mutation and the strongest mutations in one copy were selected to be combined with mutations in the other copy. Potential knockouts (carrying premature stop codon) were selected with the first priority. For each crossing combination plants homozygous or heterozygous for the mutation in one CENH3 copy were selected, hand emasculated, and pollinated with the pollen from plants carrying homozygous or heterozygous mutation in second CENH3 copy. F1 plants heterozygous for mutations in both CENH3 copies were selected and self-pollinated. Plants of F2 generation homozygous for mutations in both copies, or otherwise homozygous for mutation in one copy and heterozygous for the mutation in the other copy, were selected for test crosses or self-pollinated for seed increase.

Example 3: Test crosses to evaluate haploid induction ability of plants with mutations in both copies of CENH3 gene

Test crosses were performed with selected F2 or F3 plants homozygous for mutations in both CENH3 copies, or otherwise homozygous for mutation in one copy and heterozygous for the mutation in the other copy. Pollen from selected plants was used to pollinate hybrid tester which carried sterile cytoplasm (CMS) and no restorer genes, so that female plants were sterile and did not require hand emasculation.

F1 kernels from test crosses were germinated, DNA was extracted from each plant and analyzed with the set of 4-8 KASP markers, some allowing to discriminate alleles of male genotype from alleles of female tester, and some differentiating alleles of tester parental lines. Mutation markers were included as well. DNA of all plants showing homozygosity for all markers and wild type alleles for mutation markers was additionally analyzed with DNA microarray allowing to assess zygosity of large number of genome-wide loci.

Putative haploids were detected in test crosses with HA304m004k_m003j mutant. Summary of analyzed test crosses is presented in table 1 below. Each cross was performed with individual male plant. For all crosses the same hybrid tester was used.

Table 1 :

The above results were verified in an additional induction cross with mutant HA304m004k_m003j. Differing from the results shown above, for this experiment, plants, not kernels, were analyzed for ploidy status and calculation of the haploid induction rate. The results are summarized in the Table below:

Claims

1 . Plant of the genus Helianthus having activity of a haploid inducer and comprising

(i) a first nucleotide sequence encoding a first CENH3 protein, and

(ii) a second nucleotide sequence encoding a second CENH3 protein, wherein the expression and/or activity of the first and the second CENH3 protein in sum is reduced by about 60 to about 95%, preferably by about 65 to about 90%, particularly preferably by about 75 to about 80% compared to a wild type plant of the genus Helianthus, preferably wherein the Helianthus plant is a plant not obtained or obtainable by an essentially biological process.

2. Plant according to claim 1 , wherein three of the four alleles encoding the first and second CENH3 protein carry at least one mutation, preferably at least one knockdown or knock-out mutation.

3. Plant according to claim 1 , wherein the expression of the first and the second CENH3 protein is reduced by the presence of a double stranded RNA molecule or set of molecules targeting the first and the second nucleotide sequence for gene silencing.

4. Plant according to any 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 to the sequence of SEQ ID NO: 1 or 2 as 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 to the sequence of SEQ ID NO: 3 or 4 as respective reference sequence.

5. Plant according to any 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 to the sequence of SEQ ID NO: 5 as 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 to the sequence of SEQ ID NO: 6 as respective reference sequence.

6. Plant according to any of claims 1 , 2, 4 and 5, wherein the at least one mutation, preferably the at least one knock-down or knock-out mutation, in the first CENH3 protein is selected from:

(k) the lysine at position 9 of the sequence of SEQ ID NO: 5 is substituted, preferably by arginine; and wherein the at least one mutation, preferably at least one knock-down or knock-out mutation, in the second CENH3 protein is selected from:

(m) the glutamine at position 96 of the sequence of SEQ ID NO: 6 is substituted, preferably to result in a stop codon,

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

8. Polynucleotide or set of polynucleotides encoding a protein represented by the amino acid sequence of SEQ ID NO: 5 and/or 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 to SEQ ID NO: 5 or 6 as respective reference sequence and carrying at least one mutation, including a point, insertion, and/or a deletion mutation, preferably selected from the mutations defined in claim 6 or a combination of at least two point mutations, preferably as defined in claim 7.

9. Polypeptide encoded by a polynucleotide or set of polynucleotides according to claim 8.

10. Vector comprising a polynucleotide or set of polynucleotides according to claim 8.

11 . Cell, comprising a polynucleotide or set of polynucleotides according to claim 8, a polypeptide according to claim 9 or a vector according to claim 10, preferably wherein the cell not obtained or obtainable by an essentially biological process.

12. Method of generating a plant or plant part, in particular a plant of the genus Helianthus, preferably as defined in any of claims 1 to 7, comprising:

(b) introducing through stable or transient integration by means of transformation or insertion using gene editing technology into the plant or the plant part an RNAi molecule or a set of RNAi molecules directed against, targeting, or hybridizing with a first nucleotide sequence encoding a first CENH3 protein and a second nucleotide sequence encoding a second CENH3 protein, and, optionally, regenerating a plant from the plant part of any of (a) or (b), preferably, wherein 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 to the sequence of SEQ ID NO: 5, and/orwherein 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 to the sequence of SEQ ID NO: 6.

13. Method according to claim 12, wherein the at least one mutation, preferably the at least one knock-down or knock-out mutation, in the first CENH3 protein is selected from:

(i) the threonine at position 53 of the sequence of SEQ ID NO: 5 is substituted, preferably by methionine; (j) the isoleucine at position 70 of the sequence of SEQ ID NO: 5 is substituted, preferably by threonine;

(o) position 93963324 on chromosome 15 according to public reference XRQ2 is mutated; preferably wherein at least mutations (a) and one mutation selected from mutations (I), (m), (n) and (o) as defined in claim 6 or at least mutations (b) and one mutation selected from mutations (I), (m), (n) and (o) as defined in claim 6 or wherein at least mutations (c) and one mutation selected from mutations (I), (m), (n) and (o) as defined in claim 6 or wherein at least mutations (d) and one mutation selected from mutations (I), (m), (n) and (o) as defined in claim 6 or wherein at least mutations (e) and one mutation selected from mutations (I), (m), (n) and (o) as defined in claim 6 or wherein at least mutations (f) and one mutation selected from mutations (I), (m), (n) and (o) as defined in claim 6 or wherein at least mutations (g) and one mutation selected from mutations (I), (m), (n) and (o) as defined in claim 6 or wherein at least mutations (h) and one mutation selected from mutations (I), (m), (n) and (o) as defined in claim 6 or wherein at least mutations (i) and one mutation selected from mutations (I), (m), (n) and (o) as defined in claim 6 or wherein at least mutations (j) and one mutation selected from mutations (I), (m), (n) and (o) as defined in claim 6 or wherein at least mutations (k) and one mutation selected from mutations (I), (m), (n) and (o) as defined in claim 6, preferably wherein at least mutations (a) and (I) 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 (I) 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 are introduced in step (a).

14. Use of a mutation as defined in claim 6 or a combination of mutations as defined in claim 7 or a polynucleotide according to claim 8 or a polypeptide according to claim 9 or a vector according to claim 10 for generating a plant, preferably of a plant of the genus Helianthus, having activity of a haploid inducer.

15. A method for identifying a Helianthus plant having activity of a haploid inducer, preferably as defined in any of claims 1 to 7, comprising

(a) screening forthe presence of at least one mutation, preferably at least one knockdown or knock-out mutation, in a first nucleotide sequence encoding a first CENH3 protein and in a second nucleotide sequence encoding a second CENH3 protein, or

(b) screening for reduced expression of a first and a second nucleotide sequence encoding a first CENH3 protein and a second CENH3 protein, preferably wherein the first nucleotide sequence is represented by a 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 to the sequence of SEQ ID NO: 1 or 2, and/or wherein the second nucleotide sequence is represented by a 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 to the sequence of SEQ ID NO: 3 or 4, and/or wherein 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 to the sequence of SEQ ID NO: 5, and/or wherein 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 to the sequence of SEQ ID NO: 6, further preferably, wherein the at least one mutation, preferably the at least one knock-down or knock-out mutation, in the first CENH3 protein is selected from:

(k) the lysine at position 9 of the sequence of SEQ ID NO: 5 is substituted, preferably by arginine; and the at least one mutation, preferably at least one knock-down or knock-out mutation, in the second CENH3 protein is selected from: (l) at least one nucleotide of the ATG nucleic acid sequence encoding the methionine at position 1 of the sequence of SEQ ID NO: 6 is deleted, inserted or substituted, resulting either in a knock-out mutation, preferably by a frame-shifting insertion or deletion or by a nonsense mutation, or resulting in an exchange of the encoded amino acid at position 1 , for example, an exchange of methionine by isoleucine,

(o) position 93963324 on chromosome 15 according to public reference XRQ2 is mutated; more preferably wherein at least mutations (a) and one mutation selected from mutations (I), (m), (n) and (o) as defined in claim 6 or at least mutations (b) and one mutation selected from mutations (I), (m), (n) and (o) as defined in claim 6 or wherein at least mutations (c) and one mutation selected from mutations (I), (m), (n) and (o) as defined in claim 6 or wherein at least mutations (d) and one mutation selected from mutations (I), (m), (n) and (o) as defined in claim 6 orwherein at least mutations (e) and one mutation selected from mutations (I), (m), (n) and (o) as defined in claim 6 or wherein at least mutations (f) and one mutation selected from mutations (I), (m), (n) and (o) as defined in claim 6 or wherein at least mutations (g) and one mutation selected from mutations (I), (m), (n) and (o) as defined in claim 6 or wherein at least mutations (h) and one mutation selected from mutations (I), (m), (n) and (o) as defined in claim 6 or wherein at least mutations (i) and one mutation selected from mutations (I), (m), (n) and (o) as defined in claim 6 or wherein at least mutations (j) and one mutation selected from mutations (I), (m), (n) and (o) as defined in claim 6 or wherein at least mutations (k) and one mutation selected from mutations (I), (m), (n) and (o) as defined in claim 6, preferably wherein at least mutations (a) and (I) 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 (I) as defined in claim 6 or wherein at least mutations (b) and (m) as defined in claim 6 orwherein at least mutations (b) and (o) as defined in claim 6 orwherein at least one of mutations (f), (g), (h), (i) and (k) and mutation (m) as defined in claim 6 are present in the plant.