WO2024094771A1

WO2024094771A1 - Oleic acid sunflower mutants and methods for detection

Info

Publication number: WO2024094771A1
Application number: PCT/EP2023/080503
Authority: WO
Inventors: Silke WIECKHORST; Brigitte POPPENBERGER-SIEBERER; Veronica RAMIREZ; Wilfried Rozhon; Volker Hahn
Original assignee: KWS SAAT SE & Co. KGaA; Universität Hohenheim; Technische Universität München
Priority date: 2022-11-02
Filing date: 2023-11-02
Publication date: 2024-05-10

Abstract

The present invention relates to a plant of the species Helianthus annuus comprising a mutant allele of a gene which encodes an endogenous FAD2-1 enzyme as well as to a cell, tissue, organ, seed, or part of a plant of the species Helianthus annuus comprising a mutant allele of a gene which encodes an endogenous FAD2-1 enzyme, and further relates to methods for producing the same. Additionally, the present invention relates to a method of detecting and/or selecting a cell, tissue, organ, seed, part or a plant of the species Helianthus annuus comprising a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme. Furthermore, provided is a use of the plant of the species Helianthus annuus, or the cell, tissue, organ, seed, or part of the plant for producing an oil product having an optimized oil fatty acid profile.

Description

Oleic acid sunflower mutants and methods for detection

Technical Field

The present invention relates to a plant of the species Helianthus annuus comprising a mutant allele of a gene which encodes an endogenous FAD2-1 enzyme as well as to a cell, tissue, organ, seed, or part of a plant of the species Helianthus annuus comprising a mutant allele of a gene which encodes an endogenous FAD2-1 enzyme, and further relates to methods for producing the same. Additionally, the present invention relates to a method of detecting and/or selecting a cell, tissue, organ, seed, or a plant of the species Helianthus annuus or a part thereof comprising a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme. Furthermore, provided is a use of the plant of the species Helianthus annuus, orthe cell, tissue, organ, seed, or part of the plant for producing an oil product having an optimized oil fatty acid profile.

Background of the invention

Sunflower (Helianthus annuus L.) is a large annual forb grown as a crop for its edible oil and edible fruits. It is one of the most important oil crops in Europe (Zhou et al., 2020). Sunflower oil is extracted from the seeds and is used for cooking, as a carrier oil and for production of margarine and biodiesel, since it is generally cheaper than olive oil. Sunflower oil is obtained by cold-pressing the seeds that are formed in the centre of the large flower.

Sunflower oil is rich in the polyunsaturated fatty acid linoleic acid (C18:2) and very popular as an edible oil. In addition, it has potential for use in the chemical-technical industry as a replacement for petroleum-based products in case the content of the monounsaturated fatty acid oleic acid (C18:1) is high. High oleic acid (HO) oils, with an oleic acid content of > 85%, have high oxidation and heat stability. This prevents the formation of trans-fats during high heating (such as deep frying), which is why HO oils are considered premium cooking and frying oils.

In the non-food sector, HO oils are used as biodiesel and biolubricants, among other things. Since the fatty acid profile of sunflower seeds with an oleic acid content of < 30% does not meet the requirements for these applications in terms of oleic acid content, a central aim of sunflower breeding is to create new varieties that are richer in oleic acid.

Oleic acid is biosynthesized in plants from stearic acid (C18:0) and converted to linoleic acid by the activity of the enzyme oleate desaturase FAD2 (Figure 1A). The isoform FAD2-1 is active in seeds. If oleate desaturase activity is suppressed, oleic acid accumulates because its conversion into linoleic acid does not take place. Thus, oleate desaturase represents a promising target for sunflower breeding.

The most important, currently available HO varieties of sunflower derive from the line Pervenets, which has been obtained by chemically induced duplication of the promoter region of FAD2-1 (Soldatov, 1976). Via silencing, this leads to a reduction in the FAD2- 1 transcript and thus to reduced FAD2-1 enzyme activity (Schuppert et al. 2006). However, since the FAD2-1 promoter is still active and strongly regulated by environmental influences, the HO trait of these Pervenets-based varieties is unstable and the oleic acid content is subject to strong fluctuations depending on the environmental conditions (Grunvald et al., 2013). In addition, the genetic background (probably containing several modifier loci) affects the oleic acid content in Pervenets- based lines. An oleic acid content of >85% can only be achieved if particular requirements are met (Miller et al. 1987; Fernandez-Martinez et al. 1989), which makes it rather difficult to breed HO varieties with Pervenets material. However, in the Pervenets line no single-nucleotide polymorphisms (SNPs) between elite lines could be identified in the FAD2-1 region, which would allow the development of cost-efficient, diagnostic, codominant markers to distinguish between wild-type and mutant (Schuppert et al. 2006).

WO 2013/004281 A1 and Alberio et al., 2015 describe two FAD2-1 mutants in sunflower containing large insertions of 785 nucleotides or 4,872 nucleotides, respectively, changing the reading frame and leading to truncated proteins. The mutants have been obtained by mutagenesis methods based on X-ray treatment of seeds or ethyl methanesulfonate (EMS) injection into the flower head followed by X-ray irradiation on harvested seeds. WO 2013/004281 A1 highlighted explicitly that the truncated FAD2-1 protein should exhibit not more than 110 amino acids of the N-terminal end. It is assumed that thereby the complete loss of function shall be ensured.

However, large insertions might tend to be instable and to get lost. Thereby, introduced early stop codons as part of the insertion gets lost too and functionality of FAD2-1 protein might be re-established at least partially. This would reduce then the content of oleic acid in the sunflower plants. Hence, insertions, especially large insertions, are not a safe solution for establishing a durable HO trait.

WO 2013/004280 A1 discloses fad2-1 mutants generated by EMS-induced mutagenesis. The mutants contain point mutations causing amino acid exchanges between amino acid positions 103 and 265 of fad2-1 protein or the substitution of amino acid for a stop codon. The latter results in truncation of the protein. However, there is only one mutation exemplified exhibiting the first 80 amino acids of the N-terminal end.

It was thus the primary object of the present invention to provide new mutant sunflower lines that have a stable oleic acid content of > 85 weight-%, preferably > 87 weight-%, particularly preferably > 90 weight-% and at most about 99 weight-% and up to but below (indicated as “<” in the following) < 100 weight-%, based on the combined weight of all fatty acid residues present in at least one seed, preferably a pool of seeds, in sunflower plants which are part of these mutant sunflower lines.

Moreover, it was an object of the present invention to provide new mutant sunflower lines that have a stable oleic acid content of > 85 weight-%, preferably > 87 weight-%, particularly preferably > 90 weight-%, based on the combined weight of all fatty acid residues present in at least one seed, preferably a pool of seeds, in sunflower plants which are part of these mutant sunflower lines, wherein this stable oleic acid content is only minimally, preferably not at all, affected and/or regulated by environmental conditions, such as weather, climate, humidity, soil quality, and parasites.

It was a further object of the present invention to provide new mutant sunflower lines which do not comprise any transgenes and which have a stable oleic acid content of > 85 weight-%, preferably > 87 weight-%, particularly preferably > 88 weight-%, based on the combined weight of all fatty acid residues present in at least one seed, preferably a pool of seeds, in sunflower plants which are part of these mutant sunflower lines.

Moreover, it was an object of the present invention to provide new mutant sunflower lines which do not comprise any transgenes and which have a stable linoleic acid content of < 4 weight.-%, preferably, < 3 weight.-%, especially preferably < 2 weight.-%.

It was also an objective of the present invention to develop cost-efficient, diagnostic, and codominant markers allowing for distinguishing mutant sunflower lines according to the present invention from respective wildtype sunflower plants.

Furthermore, it was an objective of the present invention to develop cost-efficient assays for detection of mutations present in the mutant sunflower lines according to the present invention.

It was also an objective of the present invention to develop cost-efficient assays allowing for marker-assisted selection of heterozygous as well as homozygous carriers and the distinction of these carriers from non-carriers.

Moreover, it was an object of the present invention to provide reliable and stable markers for identification of a mutant sunflower line according to the present invention.

Summary of the invention

The above objectives have been achieved by providing, in a first aspect, a plant of the species Helianthus annuus comprising a mutant allele of a gene which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence the mutant allele has a single nucleotide deletion between nucleotide position 916 and 944 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6 leading to a modified C- terminal end of the enzyme from position 315 onwards having an amino acid sequence of SEQ ID NO: 14. Further provided is a plant of the species Helianthus annuus as described herein, wherein the modified C-terminal end of the enzyme from position 307 onwards has an amino acid sequence of SEQ ID NO: 15.

In one embodiment, there is provided a plant of the species Helianthus annuus as described herein, wherein the enzyme encoded by the mutant allele has an amino acid sequence of SEQ ID NOs: 1 or 2, or a sequence having an identity of at least 95%, 96%, 97%, 98%, 99%, 99,5% or 100% to the sequence of SEQ ID NOs: 1 or 2; or wherein the enzyme is encoded by a nucleic acid molecule that has a sequence of SEQ ID NOs: 4 or 5, or a sequence having an identity of at least 95%, 96%, 97%, 98%, 99%, 99,5% or 100% to the sequence of SEQ ID NOs: 4 or 5.

In another embodiment, there is provided a plant of the species Helianthus annuus as described herein, wherein the mutant allele is present homozygously or heterozygously in the plant, preferably homozygously.

In yet another embodiment, there is provided a plant of the species Helianthus annuus as described herein, wherein at least one seed, preferably a pool of seeds, originating from or grown on the plant has an oil fatty acid profile characterized by

(i) an oleic acid (C18:1) content of from 85 weight-% to < 100 weight-%, preferably of from 87 weight-% to < 100 weight-%, particularly preferably of from 89 weight-% to < 100 weight-%, especially preferably of from 90 weight-% to < 100 weight -%, and

(ii) a linoleic acid (C18:2) content of 5 weight-% or less, more preferably of 3 weight-% or less, and

(iii) a stearic acid (C18:0) content of 6 weight-% or less, more preferably of 5 weight-% or less, and

(iv) a palmitic acid (C16:0) content of 6 weight-% or less, more preferably of 4.8 weight-% or less; wherein all weight-% being (w/w) values based on the combined weight of all fatty acid residues present in and/or obtainable from the at least one seed, preferably the pool of seeds, of said sunflower plant. In still another embodiment, there is provided a plant of the species Helianthus annuus as described herein, wherein the nucleic acid molecule encoding said enzyme having the function of converting stearic acid (C18:0) to oleic acid (C18:1) is inserted as a non- transgenic modification, including a modification inserted by irradiation, chemical treatment, including ethyl methanesulfonate (EMS) treatment, as a genome editing modification using at least one site-directed nuclease-, nickase-, base editor-, or prime editor-based system, or a combination thereof.

In another embodiment, there is provided a plant of the species Helianthus annuus as described herein, wherein the plant comprises one of the following marker haplotypes (I) or (II) of single nucleotide polymorphisms associated with the oil fatty acid profile as defined herein:

(I) a T at position 151343763 referenced to map XRQv2 on linkage group 14, preferably detectable by molecular marker having nucleotide sequence of SEQ ID NO: 7, a single nucleotide deletion at position 151343789 referenced to map XRQv2 on linkage group 14, preferably detectable by molecular marker having nucleotide sequence of SEQ ID NO: 8, a T at position 151348075 referenced to map XRQv2 on linkage group 14, preferably detectable by molecular marker having nucleotide sequence of SEQ ID NO: 11 , a G at position 151348095 referenced to map XRQv2 on linkage group 14, preferably detectable by molecular marker having nucleotide sequence of SEQ ID NO: 10, and an A at position 151348167 referenced to map XRQv2 on linkage group 14, preferably detectable by molecular marker having nucleotide sequence of SEQ ID NO: 9; or

(II) a single nucleotide deletion at position 151343763 referenced to map XRQv2 on linkage group 14, preferably detectable by molecular marker having nucleotide sequence of SEQ ID NO: 7, a T at position 151343789 referenced to map XRQv2 on linkage group 14, preferably detectable by molecular marker having nucleotide sequence of SEQ ID NO: 8, a T at position 151348075 referenced to map XRQv2 on linkage group 14, preferably detectable by molecular marker having nucleotide sequence of SEQ ID NO: 11 , a G at position 151348095 referenced to map XRQv2 on linkage group 14, preferably detectable by molecular marker having nucleotide sequence of SEQ ID NO: 10, and an A at position 151348167 referenced to map XRQv2 on linkage group 14, preferably detectable by molecular marker having nucleotide sequence of SEQ ID NO: 9.

In a further embodiment, there is provided a plant of the species Helianthus annuus as described herein, wherein the plant comprises one of the following single nucleotide polymorphisms: a single nucleotide deletion at position 149223165 referenced to map XRQv2 on linkage group 14, preferably detectable by molecular marker having nucleotide sequence of SEQ ID NO: 12, or a A at position 149310377 referenced to map XRQv2 on linkage group 14, preferably detectable by molecular marker having nucleotide sequence of SEQ ID NO: 13.

In one aspect of the present invention, there is provided a cell, tissue, organ, seed, or part of a plant of the species Helianthus annuus as described herein.

In one embodiment, there is provided a seed of a plant of the species Helianthus annuus as described herein, wherein the mutant allele is present homozygously or heterozygously in the seed, preferably homozygously.

In a further aspect of the present invention, there is provided a method for producing a cell, tissue, organ, seed or a whole plant of the species Helianthus annuus as described herein, the method comprising: (i) providing at least one cell of a plant of the species Helianthus annuus',

(ii) mutagenizing the at least one cell by irradiation or chemical treatment, including ethyl methanesulfonate (EMS) treatment or by introducing into the at least one cell at least one site-directed nuclease-, nickase-, base editor-, or prime editorbased system, or the sequence(s) encoding the same;

(iii) regenerating and thus obtaining at least one whole plant of the species Helianthus annuus as described herein or a cell, tissue, organ or seed thereof.

Generally, a variety of transformation techniques for sunflowers applicable for different cultivars is available to the skilled person. These technologies can be used for introducing at least one site-directed nuclease-, nickase-, base editor-, or prime editorbased system, or a construct encoding the same, into a cell or tissue of interest according to the present disclosure. For example, transformation of sunflower as it is well-known in the art is disclosed in Dagustu, N; Fraser, P; Enfissi, E; Bramley, P (2008) BIOTECHNOLOGY & BIOTECHNOLOGICAL EQUIPMENT 22: 933-937), or in Rao, KS; Rohini, VK (1999) Agrobacterium-mediated transformation of sunflower (Helianthus annus L.): A simple protocol. ANNALS OF BOTANY 83: 347-354.

In yet another aspect of the present invention, there is provided a method of detecting and/or selecting a cell, tissue, organ, seed, or a plant of the species Helianthus annuus or a part thereof having an oil fatty acid profile as defined herein, comprising the mutant allele as defined herein, and/or a haplotype as defined herein, and/or a single nucleotide polymorphism as defined herein, wherein the method comprises:

(i) providing at least one plant or seed or a pool of plants or seeds of the species Helianthus annuus comprising genomic DNA;

(ii) detecting the presence of the mutant allele as defined herein, or a haplotype as defined herein, or a single nucleotide polymorphism as defined herein; and

(iii) optionally selecting at least one cell, tissue, organ, seed, part of a plant or a whole plant of the species Helianthus annuus based on the detection in step (ii).

In one embodiment, there is provided a method, wherein the mutant allele as defined herein, or a haplotype as defined herein, or a single nucleotide polymorphism as defined herein has been detected by means of a molecular marker or a set of molecular markers selected from the group consisting of SEQ ID NOs: 7 to 13, or a sequence having at least 95%, 96%, 97%, 98%, or at least 99% sequence identity to the respective marker sequence.

In a further embodiment, there is provided a method, wherein molecular markers) used or suitable to be used for detection are at least one of SEQ ID NO: 7 and SEQ ID NO: 8, optionally combined with at least one of SEQ ID NOs: 9 to 11 , or at least one of SEQ ID NO: 7 and SEQ ID NO: 8, optionally combined with at least one of SEQ ID NO: 12 or 13.

In another aspect of the present invention, there is provided a use of the plant of the species Helianthus annuus as defined herein, or the cell, tissue, organ, seed, or part of the plant of the species Helianthus annuus as defined herein for producing an oil product having an oil fatty acid profile characterized by

(i) an oleic acid (C18:1) content of from about 85 weight-% to < 100 weight-%, preferably of from about 87 weight-% to < 100 weight-%, particularly preferably of from about 89 weight-% to < 100 weight-%, especially preferably of from about 90 weight-% to < 100 weight-%, and

(ii) a linoleic acid (C18:2) content of less than 5 weight-% or less, more preferably less than of 3 weight-% or less, and

(iii) a stearic acid (C18:0) content of less than 6 weight-% or less, more preferably less than of 5 weight-% or less, and

(iv) a palmitic acid (C16:0) content of less than 6 weight-% or less, more preferably less than of 4.8 weight-% or less; wherein all weight-% being (w/w) values based on the combined weight of all fatty acid residues present in the oil product. Brief Description of Sequences

Table 1 Brief description of sequences disclosed in the sequence listing.

Whenever the present disclosure relates to the percentage of identity of nucleic acid or amino acid sequences to each other these values define those values as obtained by using the EMBOSS Water Pairwise Sequence Alignments (nucleotide) programme (www.ebi.ac.uk/Tools/psa/ emboss_water/nucleotide.html) nucleic acids or the EMBOSS Water Pairwise Sequence Alignments (protein) programme (www.ebi.ac.uk/Tools/psa/emboss_water/) for amino acid sequences. 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.

Definitions

Nucleic acid sequences disclosed herein may be "codon-optimized". "Codon optimization" implies that a DNA or RNA synthetically produced or isolated from a donor organism is adapted to the codon usage of different acceptor organism to improve transcription rates, mRNA processing and/or stability, and/or translation rates, and/or subsequent protein folding of said recombinant nucleic acid in the cell or organism of interest. The skilled person is well aware of the fact that a target nucleic acid can be modified at one position due to the codon degeneracy, whereas this modification will still lead to the same amino acid sequence at that position after translation, which is achieved by codon optimization to take into consideration the species-specific codon usage of a target cell or organism. In turn, nucleic acid sequences as defined herein may have a certain degree of identity to a different sequence, encoding the same protein, but having been codon optimized.

"Complementary" or "complementarity" as used herein describes the relationship between two (c)DNA, two RNA, or between an RNA and a (c)DNA nucleic acid region. Defined by the nucleobases of the DNA or RNA, two nucleic acid regions can hybridize to each other in accordance with the lock-and-key model. To this end the principles of Watson-Crick base pairing have the basis adenine and thymine/uracil as well as guanine and cytosine, respectively, as complementary bases apply. Furthermore, also non-Watson-Crick pairing, like reverse-Watson-Crick, Hoogsteen, reverse-Hoogsteen and Wobble pairing are comprised by the term "complementary" as used herein as long as the respective base pairs can build hydrogen bonding to each other, i.e. two different nucleic acid strands can hybridize to each other based on said complementarity.

A "gene" as used herein refers to a DNA region encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions.

The term "gene expression" or "expression" as used herein refers to the conversion of the information, contained in a gene, into a "gene product". A "gene product" can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of an mRNA. Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristilation, and glycosylation.

A "genome" as used herein includes both the genes (the coding regions), the noncoding DNA and, if present, the genetic material of the mitochondria and/or chloroplasts, or the genomic material encoding a virus, or part of a virus. The "genome" or "genetic material" of an organism usually consists of DNA, wherein the genome of a virus may consist of RNA (single-stranded or double-stranded).

The terms "genome editing", “gene editing” and "genome engineering" are used interchangeably herein and refer to strategies and techniques for the targeted, specific modification of any genetic information or genome of a living organism at least one position. As such, the terms comprise gene editing, but also the editing of regions other than gene encoding regions of a genome. It further comprises the editing or engineering of the nuclear (if present) as well as other genetic information of a cell. Furthermore, the terms "genome editing", “gene editing” and "genome engineering" also comprise an epigenetic editing or engineering, i.e. the targeted modification of, e.g. methylation, histone modification or of non-coding RNAs possibly causing heritable changes in gene expression. The term “3’-region” as used herein, e.g. in the context of nucleic acid sequences and nucleic acid molecules, describes a nucleotide or a group of nucleotides of a given nucleotide sequence, which are separated by fewer nucleotides from the 3’-end than from the 5’-end of the respective nucleotide sequence. For example, when starting from the 3’-end of a given nucleotide sequence, the first 50% of nucleotides, with regard to the overall number of nucleotides present in said nucleotide sequence, constitute the 3’-region of said nucleotide sequence.

The terms “nucleotide sequence” and “nucleic acid sequence” as used herein can be used interchangeably.

The terms “C-terminal end” or “C-terminal region” or “C-proximal portion”, as used herein, e.g. in the context of amino acid sequences or amino acid molecules, such as proteins and/or enzymes, describe an amino acid or a group of amino acids of a given amino acid sequence, which are separated by fewer amino acids from the C-terminus than from the N-terminus of the respective amino acid sequence. For example, when starting from the C-terminus of a given amino acid sequence, the first 50% of amino acids, with regard to the overall number of amino acids present in said amino acid sequence, constitute the C-terminal end or C-terminal region of said amino acid sequence.

A nucleic acid molecule or gene that is “endogenous” to a cell or organism refers to a nucleic acid molecule or gene that naturally occurs in the genome of this cell or organism. On the other hand, a nucleic acid molecule or gene that is “exogenous” to a cell or organism refers to a nucleic acid molecule or gene that does not naturally occur in this cell or organism but has been introduced by a transgenic event.

An amino acid molecule, or protein, or enzyme that is “endogenous” to a cell or organism refers to an amino acid molecule or protein or enzyme that naturally occurs in the genome of this cell or organism. On the other hand, an amino acid molecule, or protein, or enzyme that is “exogenous” to a cell or organism refers to an amino acid molecule or protein or enzyme that does not naturally occur in this cell or organism but has been introduced by a transgenic event.

A “genome modification” in the context of the present invention refers to any change of a (nucleic acid) sequence that results in at least one difference in the (nucleic acid) sequence distinguishing it from the original sequence. In particular, a modification can be achieved by insertion or addition of one or more nucleotide(s), or substitution or deletion of one or more nucleotide(s) of the original sequence or any combination of these.

“Regenerating” a plant, tissue, organ or seed is done by culturing a modified or edited cell in a way that may include steps of de-differentiation and differentiation to obtain specialized tissue or a whole plant, which carries the modification or edit, preferably in every cell. Techniques for regeneration of a plant are well known to the skilled person.

A “reliable" marker as used herein defines a marker that functions under various circumstances in different backgrounds and has distinctiveness. A “stable” marker as used herein as a further characteristic of a marker defines a marker that shows a low risk to get lost, e.g., via recombination, without simultaneously losing a trait of interest as identified by the marker.

A “nucleic acid construct”, “construct” or “expression construct” refers to a nucleic acid molecule encoding or comprising one or more genetic elements, which upon introduction into a target cell can be transcribed and/or translated into a functional form, e.g. RNA(s) or polypeptide(s) or protein(s). A nucleic acid construct may also comprise regulatory sequences such as promoter and terminator sequences facilitating expression of the genetic element(s) as well as spacers and introns. The genetic elements of the present invention can also be encoded on a set of constructs, which constructs can be introduced into a cell simultaneously or consecutively.

The term “vector”, as used herein, 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. 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 or female gametophytes, sporophytes, pollen, pollen tubes and microspores and protoplasts.

The term “mutagenesis” or “mutagenizing”, as used herein, refer to a technique, by which modifications or mutations are introduced into a nucleic acid sequence in a random or non- site-specific 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 or X-rays. “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.

A ’’genome editing system” refers to a combination of a site-specific nuclease or sitespecific 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 non-homologous 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. The genome editing system may be a CRISPR/Cas system including CRISPR/Cas9 systems, CRISPR/Cas13 systems, CRISPRZCpfl (CRISPR/Cas12a) systems, CRISPR/C2C2 systems, CRISPR/CasX systems, CRISPR/CasY systems, CRISPR/Cmr systems, CRISPR/Csm systems, CRISPR/MAD2 systems, CRISPR/MAD7 systems, CRISPR/CasZ systems, or catalytically active fragment thereof. The “guide molecule”, in particular the “guide RNA” (gRNA) may be a transactivating CRISPR RNA (tracrRNA) plus a synthetic CRISPR RNA (crRNA) or a single guide RNA (sgRNA), which comprises the sequence information targeting the genomic sequence for cleavage by the nuclease.

A "CRISPR nuclease", as used herein, is any nuclease which has been identified in a naturally occurring CRISPR system, which has subsequently been isolated from its natural context, and which preferably has been modified or combined into a recombinant construct of interest to be suitable as tool for targeted genome engineering. Any CRISPR nuclease can be used and optionally reprogrammed or additionally mutated to be suitable for the various embodiments according to the present invention as long as the original wild-type CRISPR nuclease provides for DNA recognition, i.e., binding properties. Said DNA recognition can be PAM (protospacer adjacent motif) dependent. CRISPR nucleases having optimized and engineered PAM recognition patterns can be used and created for a specific application. The expansion of the PAM recognition code can be suitable to target site-specific effector complexes to a target 5 site of interest, independent of the original PAM specificity of the wild-type CRISPR-based nuclease. Cpf1 variants can comprise at least one of a S542R, K548V, N552R, or K607R mutation, preferably mutation S542R/K607R or S542R/K548V/N552R in AsCpfl from Acidaminococcus. Furthermore, modified Cas or Cpf1 variants or any other modified CRISPR effector variants, e.g., Cas9 variants, can be used according to the methods of the present invention as part of a base editing complex, e.g. BE3, VQR-BE3, EQR- BE3, VRER-BE3, SaBE3, SaKKH-BE3 (see Kim et al., Nat. Biotech., 2017, doi:10.1038/nbt.3803). Therefore, according to the present invention, artificially modified CRISPR nucleases are envisaged, which might indeed not be any "nucleases" in the sense of double-strand cleaving enzymes, but which are nickases or nuclease- dead variants, which still have inherent DNA recognition and thus binding ability. Suitable Cpf1 -based effectors for use in the methods of the present invention are derived from Lachnospiraceae bacterium (LbCpfl , e.g., NCBI Reference Sequence: WP_051666128.1), or from Francisella tularensis (FnCpfl , e.g., UniProtKB/Swiss-Prot: A0Q7Q2.1). Variants of Cpf1 are known (cf. Gao et al., BioRxiv, dx.doi.org/10.1101/091611). Variants of AsCpfl with the mutations S542R/K607R and S542R/K548V/N552R that can cleave target sites with TYCV/CCCC and TATV PAMs, respectively, with enhanced activities in vitro and in vivo are thus envisaged as sitespecific effectors according to the present invention. Genome-wide assessment of off- target activity indicated that these variants retain a high level of DNA targeting specificity, which can be further improved by introducing mutations in non-PAM- interacting domains. Together, these variants increase the targeting range of AsCpfl to one cleavage site for every ~8.7 base pairs (bp) in non-repetitive regions of the human genome, providing a useful addition to the CRISPR/Cas genome engineering toolbox (see Gao et al., supra).

A "base editor" as used herein refers to a protein or a complex comprising at least one protein or a fragment thereof having the capacity to mediate a targeted base modification, i.e., the conversion of a base of interest resulting in a point mutation of interest. Preferably, the at least one base editor in the context of the present invention comprises at least one nucleic acid recognition domain for targeting the base editor to a specific site of a nucleic acid sequence and at least one nucleic acid editing domain, which performs the conversion of at least one nucleobase at the specific target site. The nucleic acid recognition domain can additionally comprise at least one nucleic acid molecule, e.g., a guide RNA, or any other single- or double-stranded nucleic acid molecule. A “base edit” therefore refers to at least one specific nucleotide carrying a different nucleobase than previously. Based on the above, a "predetermined location" according to the present invention means the location or site in a genomic material in a cellular system, or within a genome of a cell of interest to be modified, where a targeted edit is to be introduced. The base editor may comprise further components besides the nucleic acid recognition domain and the nucleic acid editing domain, such as spacers, localization signals and components inhibiting naturally occurring DNA or RNA repair mechanisms to ensure the desired editing outcome. The term “nucleic acid recognition domain” refers to the component of the base editor, which ensures the site-specificity of the base editor by directing it to a target site within the predetermined location. A nucleic acid recognition domain may be based on a CRISPR system, which specifically recognizes a target sequence within the nucleic acid molecule of the cellular system using a guide RNA (gRNA) or single guide RNA (sgRNA), may be a synthetic fusion of a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA).

The term “nucleic acid editing domain” refers to the component of the base editor, which initiates the nucleotide conversion to result in the desired edit. The catalytic function of the nucleic acid editing domain may be a cytidine deaminase or an adenine deaminase function.

In general, base editors are composed of at least one nucleic acid recognition domain and at least one nucleic acid editing domain that deaminates cytidine or adenine. Nucleic acid editing domains which deaminate cytidine are able to convert C to T (G to A), and they are called BEs; nucleic acid editing domain which deaminate adenine can convert A to G (T to C), and they are called ABEs.

Base editors usually are composed of cytidine deaminase domain (such as APOBEC1 , APOBEC3A, APOBEC3G, PmCDAI , AID), linker (usually XTEN), CRISPR domain (d/nCas9, dCpfl , CasX, CasY, or other suitable domains) and uracil DNA glycosylase inhibitor (UGI). In a modified system, the number of UGI domain or NLS can vary, so does the length of the linker. It can also include other domains such as Gam (e.g. in BE4). There can be variants with amino acid point mutations in the cytidine deaminase domain for different editing window, such as YE-BE3, YEE-BE3 and also mutations in the CRISPR domain for different PAM recognition, such as VQR-BE3, EQR-BE3, VRER-BE3, and SaKKH-BE3. In the BE-PLUS system, the CRISPR domain and cytidine deaminase domain is not expressed as fusion protein but instead linked together using a Suntag system for broadening the editing window. More details on preferred base editors, including cytidine deaminase-based DNA base editors, adenine deaminase-based DNA base editors, can be derived from Eid A et al. (Ayman Eid, Sahar Alshareef and Magdy M. Mahfouz (2018), CRISPR base editors: genome editing without double-strand breaks, Biochemical Journal (2018) 475 1955-1964).

“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 or using irradiation with UV or gamma rays or X-rays. Then, a sensitive DNA screening technique is used to identify single base mutations. Methods for performing TILLING are known to the skilled person.

Brief Description of Figures

Fig. 1 High oleic acid (HO) sunflower lines. A, Representation of the fatty acid biosynthetic pathway with key enzymatic steps. Oleic acid (C18:1) is synthesized from stearic acid (C18:0) in plastids and converted into linoleic acid (C18:2) after transport into the endoplasmic reticulum by FAD2. The resulting triacylglycerols (TAGs) are stored in oil bodies. Abbreviations: Ac-CoA, acetyl coenzyme A; ACP, acyl carrier protein; DAG, diacylglycerols; G3P, 3-phosphoglycerate; LPA, lysophosphatidic acids; Mal-CoA, malonyl coenzyme A; PA, phosphatidic acids; PC, phosphatidylchoins; TAG, triacylglycerols. B, Fatty acid measurements in individual sunflower seeds using HPLC. HPLC chromatograms show parental line (in grey; wild type) in comparison with mutant B line (in black; mutant). An increase in the C18:1 content with a simultaneous decrease in the C18:2 content is evident. X-axis, Retention time [min]; Y-axis, Absorption [mAU] at 260 nm.

Fig. 2 Alignment of the coding sequences (DNA) of wild type FAD2-1 protein from Helianthus annuus (top row; wildtype; SEQ-ID NO: 6), a first mutant FAD2-1 protein (middle row; mutant_A; SEQ-ID NO: 4), and a second mutant FAD2-1 protein (bottom row; mutant_B; SEQ-ID NO: 5). In comparison to the wildtype FAD2-1 protein, mutants A and B each show a single SNP (deletion) at positions 943 and 917, respectively.

Fig. 3 Alignment of the amino acid sequences of wild type FAD2-1 protein from Helianthus annuus (top row; wildtype; SEQ-ID NO: 3), a first mutant FAD2-1 protein (middle row; mutant_A; SEQ-ID NO: 1), and a second mutant FAD2-1 protein (bottom row; mutant_B; SEQ-ID NO: 2). In comparison with the wildtype protein, mutant A shows the first amino acid substitution at position 315 (L315C) and mutant B shows the first amino acid substitution at position 307 (T307P).

Fig. 4 Chromosomal map of Helianthus annuus indicating chromosomal loci of markers (cf. SEQ-ID NOs: 7 to 13) for identification of FAD2-1 mutants as well as the start and end positions of the FAD2-1 gene according to the reference map XRQv2 on linkage group 14. Marker ha93318d01 specifically detects mutant T307P and marker ha93318d02 specifically detects mutant L315C.

Detailed Description

The present invention provides a plant of the species Helianthus annuus, whose seeds comprise an oil with a stable oleic acid content, which is markedly higher as compared to wildtype sunflower plants, namely a stable oleic acid content of from 86 weight-% to 90 weight-%.

In a first aspect, the present invention relates to a plant of the species Helianthus annuus comprising a mutant allele of a gene which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence the mutant allele has a single nucleotide deletion between nucleotide position 916 and 944 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6 leading to a modified C-terminal end of the enzyme from position 315 onwards having an amino acid sequence of SEQ ID NO: 14.

It was established in the context of the present invention that a plant of the species Helianthus annuus according to the present invention comprising a mutant allele of a gene which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence the mutant allele has a single nucleotide deletion between nucleotide position 916 and 944 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6 has an oil fatty acid profile with a markedly increased oleic acid content (C18:1) as compared to wildtype plants of the species Helianthus annuus. It was also established in the context of the present invention that a plant of the species Helianthus annuus according to the present invention comprising a mutant allele of a gene which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence the mutant allele has a single nucleotide deletion between nucleotide position 916 and 944 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6 has an oil fatty acid profile with a markedly increased oleic acid content (C18:1) as compared to a population of sunflower plants of the species Helianthus annuus, which were subjected to mutagenesis.

Furthermore, it was established in the context of the present invention that a single nucleotide deletion between nucleotide position 916 and 944 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6 in an endogenous FAD2-1 enzyme of a plant of the species Helianthus annuus results in a markedly reduced FAD2-1 activity.

This markedly reduced FAD2-1 activity, in turn, leads to significantly lower conversion rate of oleic acid (C18:1) to linoleic acid (C18:2) in the seeds of the mutant sunflower plants of the species Helianthus annuus according to the present invention as compared to seeds of the wildtype sunflower plants of the species Helianthus annuus.

Furthermore, it was established in the context of the present invention that a single nucleotide deletion between nucleotide position 916 and 944 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6 in an endogenous FAD2-1 enzyme of a plant of the species Helianthus annuus harbours a minimized risk of these genetic modifications and the associated molecular and/or phenotypical trait or traits to get lost due to recombination events. The risk of losing these genetic modifications (single nucleotide deletions) is particularly reduced in comparison to insertions, especially in comparison to large insertions, i.e. insertions that are larger than 100 base pairs, particularly insertions that are larger than 500 base pairs, especially insertions that are larger than 1000 base pairs.

Accordingly, plants of the species Helianthus annuus according to the present invention comprising a mutant allele of a gene which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence the mutant allele has a single nucleotide deletion between nucleotide position 916 and 944 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6 can reliably be used for breeding purposes as the single nucleotide deletion between nucleotide position 916 and 944 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6 of a gene which encodes an endogenous FAD2-1 enzyme is stably passed on to the progeny.

Furthermore, it was established in the context of the present invention that single nucleotide polymorphisms, preferably single nucleotide deletions, in the 3’-region of the coding sequence of a gene, which encodes an endogenous FAD2-1 enzyme, leading to a modified C-terminal end of the enzyme, preferably from position 315 onwards, minimize the risk of a re-gain of function of the mutated endogenous FAD2-1 gene as compared to genetic modifications, particularly insertions, especially large insertions, i.e. insertions that are larger than 100 base pairs, particularly insertions that are larger than 500 base pairs, especially insertions that are larger than 1000 base pairs.

In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion between nucleotide position 917 and 943 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6.

In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 916 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6.

In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 917 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6.

In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 918 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6. In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 919 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6.

In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 920 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6.

In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 921 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6.

In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 922 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6.

In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 923 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6.

In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 924 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6. In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 925 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6.

In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 926 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6.

In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 927 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6.

In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 928 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6.

In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 929 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6.

In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 930 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6. In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 931 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6.

In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 932 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6.

In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 933 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6.

In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 934 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6.

In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 935 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6.

In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 936 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6. In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 937 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6.

In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 938 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6.

In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 939 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6.

In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 940 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6.

In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 941 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6.

In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 942 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6. In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 943 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6.

In one embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence of the mutant allele has a single nucleotide deletion at nucleotide position 944 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6.

Preferably, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the modified C-terminal end of the enzyme from position 307 onwards has an amino acid sequence of SEQ ID NO: 15.

The coding sequence of the wildtype FAD2-1 enzyme from Helianthus annuus is represented by the sequence of SEQ ID NO: 6. The protein (i.e. amino acid) sequence of FAD2-1 in sunflower (Helianthus annuus) translated from this coding sequence is represented by the sequence with SEQ ID NO: 3.

In one embodiment, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the modified C-terminal end of the enzyme from position 308 onwards has an amino acid sequence corresponding to the last 23 amino acids of SEQ ID NO: 15.

In one embodiment, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the modified C-terminal end of the enzyme from position 309 onwards has an amino acid sequence corresponding to the last 22 amino acids of SEQ ID NO: 15.

In one embodiment, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the modified C-terminal end of the enzyme from position 310 onwards has an amino acid sequence corresponding to the last 21 amino acids of SEQ ID NO: 15.

In one embodiment, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the modified C-terminal end of the enzyme from position 311 onwards has an amino acid sequence corresponding to the last 20 amino acids of SEQ ID NO: 15.

In one embodiment, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the modified C-terminal end of the enzyme from position 312 onwards has an amino acid sequence corresponding to the last 19 amino acids of SEQ ID NO: 15.

In one embodiment, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the modified C-terminal end of the enzyme from position 313 onwards has an amino acid sequence corresponding to the last 18 amino acids of SEQ ID NO: 15.

In one embodiment, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the modified C-terminal end of the enzyme from position 314 onwards has an amino acid sequence corresponding to the last 17 amino acids of SEQ ID NO: 15.

In one embodiment, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the modified C-terminal end of the enzyme from position 315 onwards has an amino acid sequence corresponding to the last 16 amino acids of SEQ ID NO: 15.

In one embodiment, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the modified C-terminal end of the enzyme from position 316 onwards has an amino acid sequence corresponding to the last 15 amino acids of SEQ ID NO: 15.

In one embodiment, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the modified C-terminal end of the enzyme from position 317 onwards has an amino acid sequence corresponding to the last 14 amino acids of SEQ ID NO: 15.

In one embodiment, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the modified C-terminal end of the enzyme from position 318 onwards has an amino acid sequence corresponding to the last 13 amino acids of SEQ ID NO: 15.

In one embodiment, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the modified C-terminal end of the enzyme from position 319 onwards has an amino acid sequence corresponding to the last 12 amino acids of SEQ ID NO: 15.

In one embodiment, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the modified C-terminal end of the enzyme from position 320 onwards has an amino acid sequence corresponding to the last 11 amino acids of SEQ ID NO: 15.

In one embodiment, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the modified C-terminal end of the enzyme from position 321 onwards has an amino acid sequence corresponding to the last 10 amino acids of SEQ ID NO: 15.

In one embodiment, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the modified C-terminal end of the enzyme from position 322 onwards has an amino acid sequence corresponding to the last 9 amino acids of SEQ ID NO: 15.

In one embodiment, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the modified C-terminal end of the enzyme from position 323 onwards has an amino acid sequence corresponding to the last 8 amino acids of SEQ ID NO: 15.

In one embodiment, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the modified C-terminal end of the enzyme from position 324 onwards has an amino acid sequence corresponding to the last 7 amino acids of SEQ ID NO: 15.

In one embodiment, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the modified C-terminal end of the enzyme from position 325 onwards has an amino acid sequence corresponding to the last 6 amino acids of SEQ ID NO: 15.

In one embodiment, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the modified C-terminal end of the enzyme from position 326 onwards has an amino acid sequence corresponding to the last 5 amino acids of SEQ ID NO: 15.

In one embodiment, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the modified C-terminal end of the enzyme from position 327 onwards has an amino acid sequence corresponding to the last 4 amino acids of SEQ ID NO: 15.

In one embodiment, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the modified C-terminal end of the enzyme from position 328 onwards has an amino acid sequence corresponding to the last 3 amino acids of SEQ ID NO: 15.

In one embodiment, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the modified C-terminal end of the enzyme from position 329 onwards has an amino acid sequence corresponding to the last 2 amino acids of SEQ ID NO: 15.

Preferably, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene which encodes an endogenous FAD2-1 enzyme, wherein the enzyme encoded by the mutant allele has an amino acid sequence of SEQ ID NOs: 1 or 2, or a sequence having an identity of at least 95%, 96%, 97%, 98%, 99%, 99,5% or 100% to the sequence of SEQ ID NOs: 1 or 2; or wherein the enzyme is encoded by a nucleic acid molecule that has a sequence of SEQ ID NOs: 4 or 5, or a sequence having an identity of at least 95%, 96%, 97%, 98%, 99%, 99,5% or 100% to the sequence of SEQ ID NOs: 4 or 5.

It was established in the context of the present invention that a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene which encodes an endogenous FAD2-1 enzyme, wherein the enzyme encoded by the mutant allele has an amino acid sequence of SEQ ID NOs: 1 or 2 has an oil fatty acid profile with a markedly increased oleic acid content (C18:1) as compared to the average value of a population of 3269 sunflower plants of the species Helianthus annuus, which were subjected to mutagenesis.

Preferably, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the mutant allele is present homozygously or heterozygously in the plant, preferably homozygously.

In one embodiment, the mutant allele of a gene, which encodes an endogenous FAD2- 1 enzyme may be present homozygously in a plant or seed. This may have advantages to fully profit from FAD2-1 mutant enzyme phenotype as disclosed herein.

In other embodiments, the mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme may be present heterozygously in a plant or seed. This can have advantages in case a dosage effect of the mutant allele of the present invention in combination with other alleles, for example, as combined with the mutant allele in the course of breeding to obtain an optimum fatty acid profile for a certain purpose, may be of interest.

Preferably, a plant of the species Helianthus annuus according to the present invention comprises at least one seed, preferably a pool of seeds, originating from or grown on the plant has an oil fatty acid profile characterized by

(i) an oleic acid (C18:1) content of from 85 weight-% to < 100 weight-%, preferably of from 87 weight-% to < 100 weight-%, particularly preferably of from 89 weight-% to < 100 weight-%, especially preferably of from 90 weight-% to < 100 weight-%, and

(iv) a palmitic acid (C16:0) content of 6 weight-% or less, more preferably of 4.8 weight-% or less; wherein all weight-% being (w/w) values based on the combined weight of all fatty acid residues present in the at least one seed, preferably the pool of seeds, of said plant.

The term “pool of seeds” as used herein describes a group consisting of at least two or more seeds from a plant of the species Helianthus annuus according to the present invention. The pool of seeds can be a random sample of at least two or more seeds from one and the same plant of the species Helianthus annuus, or from different Helianthus annuus plants.

Additionally or alternatively, the pool of seeds can be a random sample of at least two or more seeds from two or more different plants of the species Helianthus annuus according to the present invention, preferably from one or more plants of the species Helianthus annuus according to the present invention. Additionally or alternatively, the pool of seeds can be a pre-selected group of at least two or more seeds from a plant of the species Helianthus annuus according to the present invention.

Additionally or alternatively, the pool of seeds can be a pre-selected group of at least two or more seeds from one or more plants of the species Helianthus annuus according to the present invention.

The term “oil fatty acid profile” as used herein describes the relative amounts in weight- % of the different fatty acid residues, which are present in and/or obtainable from at least one seed, preferably a pool of seeds, originating from or grown on a sunflower plant, preferably on a sunflower plant of the species Helianthus annuus according to the present invention. If not explicitly indicated otherwise, the amounts given in weight-% are based on the combined weight of all fatty acid residues present in and/or obtainable from the at least one seed, preferably the pool of seeds, of said sunflower plant.

The term “fatty acid residues” as used herein describes fatty acid molecules, which are not and/or no longer attached to a glycerol molecule or glycerol backbone of a monoglyceride, diglyceride, or triglyceride via an ester bond (cf. formula II). In case the term “fatty acid residues” is used in the context of monoglycerides, diglycerides, or triglycerides, which are present in or were obtained from the seeds of plants of the species Helianthus annuus according to the present invention, the term is to be understood as referring to a fatty acid tail (cf. formula I), whose ester bond to a glycerol backbone (cf. formula I) was hydrolysed resulting in a resulting in the respective fatty acid residue according to formula II.

(formula I) Monoglyceride ot diglvf tilde Fatty acid residue i t . . . .

(formula II)

In both formulae I and II, R can either be a hydroxy group (-OH) or a fatty acid tail. In both formulae I and II, n can be any number in the range of from 4 to 40, preferably of from 8 to 35, particularly preferably of from 10 to 32, especially preferably of from 10 to 25. In both formulae I and II, fatty acid tails and fatty acid residues may comprise a number of carbon-carbon double bonds, which is in the range of from 0 to 10, preferably of from 0 to 7, particularly preferably of from 0 to 5, especially preferably of from 0 to 3.

It was established in the context of the present invention that the oil fatty acid profile, as described herein, of a plant of the species Helianthus annuus according to the present invention is very stable, meaning that is reliably produced irrespectively of environmental conditions, such as biotic and/or abiotic stresses, for example weather, climate, humidity, heat, cold, drought, soil quality, and parasites.

It was also established in the context of the present invention that the oil fatty acid profile, as described herein, of a plant of the species Helianthus annuus according to the present invention is apparently not affected by any modifier loci.

Preferably, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene which encodes an endogenous FAD2-1 enzyme, wherein the nucleic acid molecule encoding said enzyme having the function of converting stearic acid (C18:0) to oleic acid (C18:1) is inserted as a non-transgenic modification, including a modification inserted by irradiation, chemical treatment, including ethyl methanesulfonate (EMS) treatment, as a genome editing modification using at least one site-directed nuclease-, nickase-, base editor-, or prime editor-based genome editing system, or a combination thereof.

In one preferred embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the mutation in the mutant allele is introduced by means of gamma radiation.

In one preferred embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the mutation in the mutant allele is introduced by means of UV radiation.

In one preferred embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the mutation in the mutant allele is introduced by means of X-rays.

In one preferred embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the mutation in the mutant allele is introduced by means of a CRISPR/Cas9 system.

In one preferred embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the mutation in the mutant allele is introduced by means of a CRISPR/Cas12a (CRISPR/Cpf1) system.

In one preferred embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the mutation in the mutant allele is introduced by means of an adenine base editor (ABE).

In one preferred embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the mutation in the mutant allele is introduced by means of a cytosine base editor (CBE).

In one embodiment, a plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene which encodes an endogenous FAD2-1 enzyme, wherein the nucleic acid molecule encoding said enzyme having the function of converting stearic acid (C18:0) to oleic acid (C18:1) is inserted as a transgenic modification.

In one preferred embodiment, the plant of the species Helianthus annuus according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the mutation in the mutant allele is introduced by means of co-cultivation and transformation using Agrobacterium.

Preferably, a plant of the species Helianthus annuus according to the present invention comprises one of the following marker haplotypes (I) or (II) of single nucleotide polymorphisms associated with the oil fatty acid profile as defined herein:

The term “associated with” as used herein, e.g. in the context of single nucleotide polymorphisms, describes the causal connection between the presence and/or absence of a given single nucleotide polymorphism and a given phenotypical and/or molecular trait, which can be observed and/or detected in vivo and/or in vitro.

It was established in the context of the present invention that the respective haplotypes as described herein can be reliably and cost-efficiently identified by the above-described combinations of markers, despite the fact that the neighbouring genomic regions of the fad2-1 gene are generally hard to target with markers due to a large number of repetitive sequences and monomorphic regions.

It was also established in the context of the present invention that the above-described markers are stably linked to the high-oleic-acid trait (HO trait), as they are located in close proximity to the fad2-1 gene. Thus, the above described markers allow for a reliable and cost-efficient selection and detection of the HO trait in the species Helianthus annuus.

Preferably, a plant of the species Helianthus annuus according to the present invention comprises one of the following single nucleotide polymorphisms: a single nucleotide deletion at position 149223165 referenced to map XRQv2 on linkage group 14, preferably detectable by molecular marker having nucleotide sequence of SEQ ID NO: 12, or an A at position 149310377 referenced to map XRQv2 on linkage group 14, preferably detectable by molecular marker having nucleotide sequence of SEQ ID NO: 13.

In a further aspect, the present invention relates to a cell, tissue, organ, seed, or part of a plant of the species Helianthus annuus according to the present invention as defined herein.

Preferably, a seed of a plant according to the present invention comprises a mutant allele of a gene, which encodes an endogenous FAD2-1 enzyme, wherein the mutant allele is present homozygously or heterozygously in the seed, preferably homozygously.

In another aspect, the present invention relates to a method for producing a cell, tissue, organ, seed or a whole plant of the species Helianthus annuus as defined herein, wherein the method comprises:

(i) providing at least one cell of a plant of the species Helianthus annuus;

(iii) regenerating and thus obtaining at least one whole plant of the species Helianthus annuus as defined herein or a cell, tissue, organ or seed thereof.

In a further aspect, the present invention relates to method of detecting and/or selecting a cell, tissue, organ, seed, part or a plant of the species Helianthus annuus having an oil fatty acid profile as defined herein, comprising the mutant allele of a gene which encodes an endogenous FAD2-1 enzyme as defined herein, and/or a haplotype as defined herein, and/or a single nucleotide polymorphism as defined herein, wherein the method comprises: (i) providing at least one plant or seed or a pool of plants or seeds of the species Helianthus annuus comprising genomic DNA;

(ii) detecting the presence of the mutant allele as defined herein, and/or a haplotype as defined herein, and/or a single nucleotide polymorphism as defined herein; and

Surprisingly, it was found that a method according to the present invention is a costefficient assay for detection of mutations present in the mutant sunflower lines according to the present invention.

Surprisingly, it was also found that a method according to the present invention allows for distinguishing mutant sunflower lines according to the present invention from respective wildtype sunflower plants utilizing the cost-efficient, diagnostic, and codominant markers, which were developed within the scope of the present invention.

Preferably, the method of detecting and/or selecting a cell, tissue, organ, seed, part or a plant of the species Helianthus annuus having an oil fatty acid profile as defined herein, comprises detection of the mutant allele as defined herein, and/or a haplotype as defined herein, and/or a single nucleotide polymorphism as defined herein by means of a molecular marker or a set of molecular markers selected from the group consisting of SEQ ID NOs: 7 to 1 3, or a sequence having at least 95%, 96%, 97%, 98%, or at least 99% sequence identity to the respective marker sequence.

Preferably, the molecular marker(s) used for detection are at least one of SEQ ID NO: 7 and SEQ ID NO: 8, optionally combined with at least one of SEQ ID NOs: 9 to 11 , or at least one of SEQ ID NO: 7 and SEQ ID NO: 8, optionally combined with at least one of SEQ ID NO: 12 or 13.

Preferably, the molecular marker(s) used for detection are at least one of SEQ ID NO: 7 and SEQ ID NO: 8, optionally combined with at least one of SEQ ID NOs: 9 to 11 and at least one of SEQ ID NO: 12 or 13. In a further aspect, the present invention also relates to a use of the plant of the species Helianthus annuus as defined herein, orthe cell, tissue, organ, seed, or part of the plant as defined herein for producing an oil product having an oil fatty acid profile characterized by

(iv) a palmitic acid (C16:0) content of 6 weight-% or less, more preferably of 4.8 weight-% or less; wherein all weight-% being (w/w) values based on the combined weight of all fatty acid residues present in the oil product.

The skilled person is well aware of techniques to determine the fatty acid content and the overall fatty acid profile of an oil product obtained from sunflowers. For example, as it was performed for the purpose of the present application, the oil was hydrolysed with KOH and subsequently derivatized. Analysis was performed using free acids as standards. Thus, the weight% refers to the total amount of fatty acids (calculated as free fatty acids, i.e. R-COOH). For analysis as disclosed herein, free fatty acids were used as standards and the hydrolysed fatty acids were calculated as free acids i.e. protonated (R-COOH), which is known as common practise to the skilled person. The glycerol backbone was thus ignored for calculation of weight-% values relating to fatty acid residues presented above.

In a further aspect, the present invention also relates to a non-naturally occurring FAD2- 1 enzyme or a nucleic acid molecule encoding the same, wherein the FAD2-1 enzyme has the function of converting stearic acid (C18:0) to oleic acid (C18:1), and wherein (a) said enzyme contains at least one or more amino acid substitution(s) in comparison to SEQ ID NO: 3 as reference sequence, wherein the at least one or more amino acid substitution(s) is/are located within the C-proximal portion of said enzyme from position 307 onwards in comparison to SEQ ID NO: 3 as reference sequence; and wherein

(b) said enzyme contains at least 1 , at least 5, at least 15, at least 20 at least 25, at least 30, at least 35, at least 40, at least 45 and preferably up to 48 amino acid deletions located within the C-proximal portion of said enzyme from position 330 onwards in comparison to SEQ ID NO: 3 as reference sequence; and

(c) wherein at least one of the amino acid substitution(s), is/are located at position 307 and/or 315 in comparison to SEQ ID NO: 3 as reference sequence, preferably wherein the at least one or more amino acid mutation(s), is/are T307P or L315C in comparison to SEQ ID NO: 3 as reference sequence.

In one embodiment, a non-naturally occurring FAD2-1 enzyme according to the present invention contains at least one, two, or all three substitution(s) Q328R, K329R and/or A330R and an amino acid deletion of up to 48 amino acids located in the C-proximal portion of said enzyme in comparison to SEQ ID NO: 3 as reference sequence.

In a further embodiment, a non-naturally occurring FAD2-1 enzyme according to the present invention contains at least one, two, or all three substitution(s) Q328R, K329R and/or A330R and an amino acid deletion of up to 48 amino acids located in the C- proximal portion of said enzyme in comparison to SEQ ID NO: 3 as reference sequence, wherein the last 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, or 49 amino acids as viewed from the C-terminus of sequence according to SEQ ID NO: 3 or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 3.

In another embodiment, a non-naturally occurring FAD2-1 enzyme according to the present invention has an amino acid sequence of SEQ ID NOs: 1 or 2, or a sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% a sequence identity to SEQ ID NO: 1 or 2 as reference sequence, respectively. In a further aspect, the present invention relates to a nucleic acid molecule encoding a non-naturally occurring FAD2-1 enzyme, as described herein, or a functional fragment thereof, wherein this enzyme, or functional fragment thereof, have the function of converting stearic acid (C18:0) to oleic acid (C18:1), and wherein the nucleic acid molecule has a sequence of SEQ ID NOs: 4 and 5, or a sequence having at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% a sequence identity to any of the SEQ ID NOs: 4 or 5, respectively.

In a further aspect, the present invention relates to an expression construct or vector comprising a nucleic acid molecule, or functional fragment thereof, as described herein.

In a further aspect, the present invention relates to an oil and/or oil product obtained and/or obtainable from at least one seed, preferably a pool of seeds, from a plant of the of the species Helianthus annuus according to the present invention.

In one embodiment, the oil and/or oil product obtained and/or obtainable from at least one seed, preferably a pool of seeds, from a plant of the of the species Helianthus annuus according to the present invention has an oleic acid (C18:1) content of from 85 weight-% to < 100 weight-%, preferably of from 87 weight-% to < 100 weight-%, particularly preferably of from 89 weight-% to < 100 weight-%, especially preferably of from 90 weight-% to < 100 weight-%.

In one embodiment, the oil obtained and/or obtainable from at least one seed, preferably a pool of seeds, from a plant of the of the species Helianthus annuus according to the present invention has an oleic acid (C18:1) content of from 85 weight-% to < 100 weight- %, preferably of from 87 weight-% to < 100 weight-%, particularly preferably of from 89 weight-% to < 100 weight-%, especially preferably of from 90 weight-% to < 100 weight- % and a linoleic acid (C18:2) content of 4 weight-% or less, preferably 3 weight-% or less, more preferably of 2 weight-% or less.

In one embodiment, the oil obtained and/or obtainable from at least one seed, preferably a pool of seeds, from a plant of the species Helianthus annuus according to the present invention has an oleic acid (C18:1) content of from 85 weight-% to < 100 weight-%, preferably of from 87 weight-% to < 100 weight-%, particularly preferably of from 89 weight-% to < 100 weight-%, especially preferably of from 90 weight-% to < 100 weight- % and a stearic acid (C18:0) content of 6 weight- % or less, more preferably of 5 weight- % or less.

In one embodiment, the oil obtained and/or obtainable from at least one seed, preferably a pool of seeds, from a plant of the of the species Helianthus annuus according to the present invention has an oleic acid (C18:1) content of from 85 weight-% to < 100 weight- %, preferably of from 87 weight-% to < 100 weight-%, particularly preferably of from 89 weight-% to < 100 weight-%, especially preferably of from 90 weight-% to < 100 weight- % and a palmitic acid (C16:0) content of 6 weight-% or less, more preferably of 5 weight- % or less.

In one embodiment, the oil obtained and/or obtainable from at least one seed, preferably a pool of seeds, from a plant of the of the species Helianthus annuus according to the present invention has an oleic acid (C18:1) content of from 85 weight-% to < 100 weight- %, preferably of from 87 weight-% to < 100 weight-%, particularly preferably of from 89 weight-% to < 100 weight-%, especially preferably of from 90 weight-% to < 100 weight- % and a stearic acid (C18:0) content of 6 weight-% or less, more preferably of 5 weight- % or less and a linoleic acid (C18:2) content of 4 weight-% or less, preferably of 3 weight-% or less, more preferably of 2 weight-% or less.

In one embodiment, the oil obtained and/or obtainable from at least one seed, preferably a pool of seeds, from a plant of the of the species Helianthus annuus according to the present invention has an oleic acid (C18:1) content of from 85 weight-% to < 100 weight- %, preferably of from 87 weight-% to < 100 weight-%, particularly preferably of from 89 weight-% to < 100 weight-%, especially preferably of from 90 weight-% to < 100 weight- % and a stearic acid (C18:0) content of 6 weight-% or less, more preferably of 5 weight- % or less and a palmitic acid (C16:0) content of 6 weight-% or less, more preferably of 4 weight-% or less.

In one embodiment, the oil obtained and/or obtainable from at least one seed, preferably a pool of seeds, from a plant of the species Helianthus annuus according to the present invention has an oleic acid (C18:1) content of from 85 weight-% to < 100 weight-%, preferably of from 87 weight-% to < 100 weight-%, particularly preferably of from 89 weight-% to < 100 weight-%, especially preferably of from 90 weight-% to < 100 weight- % and a palmitic acid (C16:0) content of 6 weight-% or less, more preferably of 4 weight- % or less and a linoleic acid (C18:2) content of 4 weight-% or less, preferably 3 weight- % or less, more preferably of 2 weight-% or less. In one embodiment, the oil obtained and/or obtainable from at least one seed, preferably a pool of seeds, from a plant of the of the species Helianthus annuus according to the present invention has an oleic acid (C18:1) content of from 85 weight-% to < 100 weight- %, preferably of from 87 weight-% to < 100 weight-%, particularly preferably of from 89 weight-% to < 100 weight-%, especially preferably of from 90 weight-% to < 100 weight- % and a stearic acid (C18:0) content of 6 weight-% or less, more preferably of 5 weight- % or less and a palmitic acid (C16:0) content of 6 weight-% or less, more preferably of 4 weight-% or less and a linoleic acid (C18:2) content of 4 weight-% or less, preferably 3 weight-% or less, more preferably of 2 weight-% or less.

Examples

1. Mutant A:

Sunflower seeds of a parental line (RHA857) were mutagenized using gamma rays. Subsequently, M2 progeny were screened forfad2-1 mutants vie two screening method: (i) TILLING (Targeting Induced Local Lesions in Genomes) and (ii) HPLC (high performance liquid chromatography)-based high-throughput fatty acid analysis.

For the TILLING, the FAD2-1 region was amplified by PCR, and mutations were then detected using the endonuclease CELI, which cleaves DNA at mismatched bases (Kurowska et al. 2011).

For high-throughput fatty acid analysis, oil was pressed from seeds before analysis. Before analysis to obtain oil, seeds derived from 5,235 mutant plants were collected. Seeds came from one single plant. Not all seeds were used, only a subset of 10 to 20 kernels per line in view of the fact that analysis is destructive and remaining seeds are needed for seed increase.

Oil was then obtained in a hydraulic press which was operated at room temperature and approximately 3 t were applied. A preselection of lines was performed based on the available number of kernels. A minimum of 10 kernels was needed to start the analysis. The analysis was repeated for the homozygous mutant after seed increase (seeds came again from single plants).

The fatty acids contained in the oil were converted into potassium salts using a hydrolysis reagent and derivatized with 4-bromophenacyl bromide. The fatty acids were quantified by means of HPLC using a Nucleodur C8 Gravity 1 .8 pM 50x3.0 mm column and acetonitrile/methanol/water = 50/36/14 as the eluent. Detection was by UV absorbance at 260 nm. A total of 5,235 independent lines were screened using these two methods, allowing 5 candidate lines to be identified (mutants A-E). These were verified by fatty acid measurements of the offspring and molecularly characterized by FAD2-1 sequencing.

The identified mutant A shows a frame shift mutation, i.e. a single nucleotide deletion of a T at position 943 in comparison to SEQ ID NO: 3 (cf. Fig. 2). This single nucleotide deletion leads to the specific amino acid exchange L315C, i.e. leucine is replaced by cystein at position 315 in comparison to SEQ ID NO: 3 (cf. Figure 3). Thus, truncation of the C-terminus is induced, which leads to a loss of FAD2-1 activity. Therefore, mutant A shows a stable oleic acid (C18:1) content of from 88.16 to 89.09 weight-%, a stable linoleic acid (C18:2) content of from 1 .83 to 2.53 weight-%, a stable stearic acid (C18:0) content of from 4.17 to 4.78 weight-%, and a stable palmitic acid (C16:0) content of from 3.74 to 4.53 weight-%.

Table 2 below shows the oil fatty acid profile of mutant A as compared to the total TILLING population, wherein the TILLING population represents the wild-type data as starting point for the subsequent mutagenesis. The average and the standard deviation (SD) of the measured oil fatty acid content for all progeny not showing a change in the overall fatty acid profile are represented below.

Table 2 Oil fatty acid profile of mutant A as compared to the total TILLING population.

2. Mutant B:

Sunflower seeds of a parental line (RHA857) were mutagenized using gamma rays. Different conditions for mutagenizing with gamma rays have been tested empirically in sensitivity tests and the parameters which work best in the respective germplasm were then used for generation of the respective TILLING population.

Subsequently, M2 progeny were screened forfad2-1 mutants vie two screening method: (i) TILLING (Targeting Induced Local Lesions in Genomes) and (ii) HPLC (high performance liquid chromatography)-based high-throughput fatty acid analysis.

For high-throughput fatty acid analysis, oil was pressed from seeds (see description for Mutant A above for the method of obtaining the oil).

Next, the fatty acids contained in the oil were converted into potassium salts using a hydrolysis reagent and derivatized with 4-bromophenacyl bromide. The fatty acids were quantified by means of HPLC using a Nucleodur C8 Gravity 1 .8 pM 50x3.0 mm column and acetonitrile/methanol/water = 50/36/14 as the eluent. Detection was by UV absorbance at 260 nm. A total of 5,235 independent lines were screened using these two methods, allowing 5 candidate lines to be identified (mutants A-E). These were verified by fatty acid measurements of the offspring and molecularly characterized by FAD2-1 sequencing.

The identified mutant B shows a frame shift mutation, i.e. a single nucleotide deletion of a T at position 917 in comparison to SEQ ID NO: 3 (cf. Fig. 2). This single nucleotide deletion leads to the specific amino acid exchange T307P, i.e. threonine is replaced by proline at position 307 in comparison to SEQ ID NO: 3 (cf. Figure 3). Thus, truncation of the C-terminus is induced, which leads to a loss of FAD2-1 activity. Therefore, mutant B shows a stable oleic acid (C18:1) content of 86.03 to 89.57 weight-%, a stable linoleic acid (C18:2) content of from 1 .49 to 3.77 weight-%, a stable stearic acid (C18:0) content of from 4.53 to 5.52 weight-%, and a stable palmitic acid (C16:0) content of from 3.89 to 5.68 weight-%. Table 3 below shows the oil fatty acid profile of mutant A as compared to the total TILLING population, wherein the TILLING population represents the wild-type data as starting point for the subsequent mutagenesis. The average and the standard deviation (SD) of the measured oil fatty acid content for all progeny not showing a change in the overall fatty acid profile are represented below.

Table 3 Oil fatty acid profile of mutant B as compared to the total TILLING population.

Claims

1 . A plant of the species Helianthus annuus comprising a mutant allele of a gene which encodes an endogenous FAD2-1 enzyme, wherein in the coding sequence the mutant allele has a single nucleotide deletion between nucleotide position 916 and 944 with reference to the wildtype coding sequence as set forth in SEQ ID NO: 6 leading to a modified C-terminal end of the enzyme from position 315 onwards having an amino acid sequence of SEQ ID NO: 14.

2. The plant of claim 1 , wherein the modified C-terminal end of the enzyme from position 307 onwards has an amino acid sequence of SEQ ID NO: 15.

3. The plant of claim 1 or 2, wherein the enzyme encoded by the mutant allele has an amino acid sequence of SEQ ID NOs: 1 or 2, or a sequence having an identity of at least 95%, 96%, 97%, 98%, 99%, 99,5% or 100% to the sequence of SEQ ID NO: 1 or 2; or wherein the enzyme is encoded by a nucleic acid molecule that has a sequence of SEQ ID NOs: 4 or 5, or a sequence having an identity of at least 95%, 96%, 97%, 98%, 99%, 99,5% or 100% to the sequence of SEQ ID NOs: 4 or 5.

4. The plant of any of the claims 1 to 3, wherein the mutant allele is present homozygously or heterozygously in the plant, preferably homozygously.

5. The plant of claim 4, wherein at least one seed, preferably a pool of seeds, originating from or grown on the plant has an oil fatty acid profile characterized by

(iii) a stearic acid (C18:0) content of 6 weight-% or less, more preferably of 5 weight-% or less, and (iv) a palmitic acid (C16:0) content of 6 weight-% or less, more preferably of 4.8 weight-% or less; wherein all weight-% being (w/w) values based on the combined weight of all fatty acid residues present in and/or obtainable from the at least one seed, preferably the pool of seeds, of said sunflower plant. The plant of any one of claims 1 to 5, wherein the nucleic acid molecule encoding said enzyme having the function of converting stearic acid (C18:0) to oleic acid (C18:1) is inserted as a non-transgenic modification, including a modification inserted by irradiation, chemical treatment, including ethyl methanesulfonate (EMS) treatment, as a genome editing modification using at least one site- directed nuclease-, nickase-, base editor-, or prime editor-based genome editing system, or a combination thereof. The plant of any one of claims 1 to 5, wherein the plant comprises one of the following marker haplotypes (I) or (II) of single nucleotide polymorphisms associated with the oil fatty acid profile as defined in claim 5:

(I) a T at position 151343763 referenced to map XRQv2 on linkage group 14, preferably detectable by molecular marker having nucleotide sequence of SEQ ID NO: 7, a single nucleotide deletion at position 151343789 referenced to map XRQv2 on linkage group 14, preferably detectable by molecular marker having nucleotide sequence of SEQ ID NO: 8, a T at position 151348075 referenced to map XRQv2 on linkage group 14, preferably detectable by molecular marker having nucleotide sequence of SEQ ID NO: 1 1 , a G at position 151348095 referenced to map XRQv2 on linkage group 14, preferably detectable by molecular marker having nucleotide sequence of SEQ ID NO: 10, and an A at position 151348167 referenced to map XRQv2 on linkage group 14, preferably detectable by molecular marker having nucleotide sequence of SEQ ID NO: 9; or

(II) a single nucleotide deletion at position 151343763 referenced to map XRQv2 on linkage group 14, preferably detectable by molecular marker having nucleotide sequence of SEQ ID NO: 7, a T at position 151343789 referenced to map XRQv2 on linkage group 14, preferably detectable by molecular marker having nucleotide sequence of SEQ ID NO: 8, a T at position 151348075 referenced to map XRQv2 on linkage group 14, preferably detectable by molecular marker having nucleotide sequence of SEQ ID NO: 1 1 , a G at position 151348095 referenced to map XRQv2 on linkage group 14, preferably detectable by molecular marker having nucleotide sequence of SEQ ID NO: 10, and an A at position 151348167 referenced to map XRQv2 on linkage group 14, preferably detectable by molecular marker having nucleotide sequence of SEQ ID NO: 9. The plant of any one of claims 1 to 5, wherein the plant comprises one of the following single nucleotide polymorphisms: a single nucleotide deletion at position 149223165 referenced to map XRQv2 on linkage group 14, preferably detectable by molecular marker having nucleotide sequence of SEQ ID NO: 12, or an A at position 149310377 referenced to map XRQv2 on linkage group 14, preferably detectable by molecular marker having nucleotide sequence of SEQ ID NO: 13. A cell, tissue, organ, seed, or part of a plant of any one of claims 1 to 8. A seed of a plant of any one of claims 1 to 8, wherein the mutant allele is present homozygously or heterozygously in the seed, preferably homozygously. A method for producing a cell, tissue, organ, seed or a whole sunflower plant of the species Helianthus annuus of any one of claims 1 to 10, the method comprising:

(i) providing at least one cell of a plant of the species Helianthus annuus;

(ii) mutagenizing the at least one cell by irradiation or chemical treatment, including ethyl methanesulfonate (EMS) treatment or by introducing into the at least one cell at least one site-directed nuclease-, nickase-, base editor-, or prime editor-based system, or the sequence(s) encoding the same;

(iii) regenerating and thus obtaining at least one whole sunflower plant of the species Helianthus annuus of any one of claims 1 to 8. A method of detecting and/or selecting a cell, tissue, organ, seed, part or a plant of the species Helianthus annuus having an oil fatty acid profile as defined in claim 5, comprising the mutant allele as defined in any one of claims 1 to 3, and/or a haplotype as defined in claim 7, and/or a single nucleotide polymorphism as defined in claim 8, wherein the method comprises:

(ii) detecting the presence of the mutant allele as defined in any one of claims 1 to 3, or a haplotype as defined in claim 7, or a single nucleotide polymorphism as defined in claim 8; and

(iii) optionally selecting at least one cell, tissue, organ, seed, part of a plant or a whole plant of the species Helianthus annuus based on the detection in step (ii). The method of claim 12, wherein the the mutant allele as defined in any one of claims 1 to 3, or a haplotype as defined in claim 7, or a single nucleotide polymorphism as defined in claim 8 in step (ii) has been detected by means of a molecular marker or a set of molecular markers selected from the group consisting of SEQ ID NOs: 7 to 13, or a sequence having at least 95%, 96%, 97%, 98%, or at least 99% sequence identity to the respective marker sequence. The method of claim 13, wherein molecular marker(s) used for detection are at least one of SEQ ID NO: 7 and SEQ ID NO: 8, optionally combined with at least one of SEQ ID NOs: 9 to 11 , or at least one of SEQ ID NO: 7 and SEQ ID NO: 8, optionally combined with at least one of SEQ ID NO: 12 or 13. A use of the plant of any one of claims 1 to 8, or the cell, tissue, organ, seed, or part of the plant of any one of claims 1 to 8 for producing an oil product having an oil fatty acid profile characterized by

(i) an oleic acid (C18:1) content of from about 85 weight-% to < 100 weight- %, preferably of from about 87 weight-% to < 100 weight-%, particularly preferably of from about 89 weight-% to < 100 weight-%, especially preferably of from about 90 weight-% to < 100 weight-%, and

(iv) a palmitic acid (C16:0) content of less than 6 weight-% or less, more preferably less than of 4.8 weight-% or less; wherein all weight-% being (w/w) values based on the combined weight of all fatty acid residues present in the oil product.