WO2021014010A1 - Sunflower with high oleic acid content and method for obtaining same - Google Patents

Sunflower with high oleic acid content and method for obtaining same Download PDF

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Publication number
WO2021014010A1
WO2021014010A1 PCT/EP2020/071026 EP2020071026W WO2021014010A1 WO 2021014010 A1 WO2021014010 A1 WO 2021014010A1 EP 2020071026 W EP2020071026 W EP 2020071026W WO 2021014010 A1 WO2021014010 A1 WO 2021014010A1
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plant
sunflower
nucleotide sequence
seeds
oleic acid
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PCT/EP2020/071026
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French (fr)
Inventor
Jordi COMADRAN TRABAN
Jean-Baptiste Laffaire
Elena Albrecht
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Soltis
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/14—Asteraceae or Compositae, e.g. safflower, sunflower, artichoke or lettuce
    • A01H6/1464—Helianthus annuus [sunflower]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/19—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with oxidation of a pair of donors resulting in the reduction of molecular oxygen to two molecules of water (1.14.19)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/19—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with oxidation of a pair of donors resulting in the reduction of molecular oxygen to two molecules of water (1.14.19)
    • C12Y114/19006—DELTA12-fatty-acid desaturase (1.14.19.6), i.e. oleoyl-CoA DELTA12 desaturase

Definitions

  • the present invention provides isolated nucleotide sequences which encode a truncated sunflower A12-oleate desaturase protein.
  • these nucleotide sequences contain a premature stop codon which leads to the production of this truncated protein.
  • the presence of these nucleotide sequences thus makes it possible to lead to sunflower plants having, in a stable manner, a high content of oleic acid in the seed oil.
  • the present invention therefore also relates to the plants, parts of plants or seeds which contain these sequences, their obtaining and their uses in introgression processes and / or for obtaining oil with a high oleic acid content.
  • Sunflower is most often cultivated to obtain oils that contain saturated and unsaturated fatty acids.
  • Saturated fatty acids have 16 or 18 carbon atoms (C16 or C18).
  • C16 fatty acids are generally saturated like palmitic acid (16: 0).
  • C18 fatty acids are either saturated, such as stearic acid (18: 0), or unsaturated with 1, 2 or 3 double bonds, such as oleic acid (18: 1), linoleic acid (18: 2 ), linolenic acid (18: 3).
  • LO low oleic
  • HO high oleic
  • the Pervenets mutant was obtained by chemical mutagenesis by Soldatov in 1976 on a sunflower population.
  • the average 18: 1 content in seeds from this population is over 65%, with individual contents ranging between 60-90%, compared to an average content of 20% in normal LO varieties.
  • the Pervenets population has been distributed worldwide and used in numerous breeding programs to convert selected genotypes with low 18: 1 content to genotypes with 18: 1 content greater than 80% in their seeds.
  • the HO phenotype has been shown to be associated with a marked decrease in the activity of the enzyme A12-oleate desaturase, which catalyzes the desaturation of 18: 1 to 18: 2 during critical stages of stock building. lipids, which explains the accumulation of 18: 1 (Garces R. and Mancha M., 1991).
  • the enzyme A12-oleate desaturase (EC 1.3.1.35) is also known under the names of: oleic acid desaturase, linoleate synthase, oleoyl CoA desaturase, oleoylphosphatidylcholine desaturase, oleoyl-PC desaturase, FAD2.
  • three fad2 genes have been identified (fad2-1, fad2-2, and fad2-3) encoding microsomal D12-desaturase.
  • the A12-oleate desaturase enzyme (EC 1.3.1.35) is encoded by the A12-desaturase gene corresponding to the fad2-1 gene.
  • a knock-down mutation in the A12-desaturase gene was isolated by the inventors.
  • the documents WO2013004280 and WO2013004281 describe a mutation obtained by chemical treatment in the A12-desaturase gene selected for a phenotype of increased oleic content compared to that of the Pervenets mutation. Different mutations have been obtained in sunflower, creating a STOP codon in the reading frame, and leading to a truncated protein. All of these mutations, however, result in a truncated protein less than or equal to 110 amino acids in length.
  • the present invention relates to an isolated nucleotide sequence which comprises a stop codon at positions 484 to 486 with reference to the wild-type nucleotide sequence SEQ ID NO: 1, and which encodes a D12-oleate desaturase protein which exhibits in less 95% identity with SEQ ID NO 4:
  • the nucleotide sequence of the invention is SEQ ID NO: 2: ATGGGTGCAGGAG AAT ACACGTCTGTGACCAACG AAAACAACCCACTCGATCGAGTCCC T CATGCAAAACCACCCTT CACCAT CGGCGAT CT GAAAAAAGCCAT CCCACCACACTGCTT CCAGCGGTCGCTAACCCGTTCGTTCTCCTACGTGCTGTCTGACCTCACCATAACCGCTG T CTC CT ACCACATTGCCACCACCT ACTT CCACCACCT CCCCACCCCTTTGTCATCCAT CG
  • the present invention relates to a truncated sunflower A12-oleate desaturase protein of sequence SEQ ID NO: 4.
  • the present invention relates to a sunflower plant comprising a sequence of nucleotides according to the invention, which can be present either in the heterozygous state or in the homozygous state in the genome of said plant.
  • the sequence of nucleotides according to the invention is present in the homozygous state.
  • the seeds produced by the sunflower plants according to the invention also constitute another aspect of the present invention, as well as their progeny provided that they comprise at least one sequence of nucleotides according to the present invention.
  • Such seeds have an oleic acid content of at least 85%, relative to the total percentage of fatty acids of said seeds, and preferably an oleic acid content in a range from 85% to 95%, in particular when they comprise the sequence according to the invention in the homozygous state.
  • the present invention relates to a process for obtaining a sunflower plant comprising a sequence of nucleotides according to the invention, said process comprising the following steps:
  • step b) Generate a plant from said plant part which has undergone said mutagenesis, c) Obtain at least one progeny from the plant generated in step b), and d) Identify and select at least one plant obtained in step b) step b) or c) comprising said nucleotide sequence according to the invention.
  • the present invention relates to a method for identifying the presence, or the absence, of a nucleotide or amino acid sequence according to the invention in a sunflower plant, comprising the extraction of genomic DNA, RNA or all the proteins of the proteome of the sunflower plant and the use of means for identifying said sequence.
  • the means of identifying a plant, part of a plant according to the invention also constitute one of the aspects of the present invention.
  • the use of these means thus makes it possible to identify and / or select sunflower plants or seeds according to the invention.
  • the production process according to the invention necessarily includes a step of voluntary and human-induced mutagenesis, which explicitly excludes natural production and essentially biological processes.
  • the present invention relates to a method of crossing by so-called "forward" selection of a sunflower plant comprising a sequence of nucleotides according to the invention; said method comprising the following steps:
  • step b) Repeating step b) to obtain progeny comprising said nucleotide sequence according to the invention and exhibiting at least one of said agronomic characteristics.
  • the invention relates to a process for introgression by backcrossing of a genome fragment comprising a nucleotide sequence according to the invention as described above, from a donor sunflower plant to a recipient sunflower plant which does not contain said nucleotide sequence in its genome, said method comprising the following steps: a) Crossing of the donor sunflower plant with the recipient plant,
  • step d) Selection of a plant comprising said nucleotide sequence according to the invention, e) Backcrossing of the plant selected in step d) with the recipient plant
  • the present invention relates to the use of sunflower seeds according to the invention as described above, to obtain oil, and in particular oil having a high oleic acid content. , that is to say at least 85% of oleic acid relative to the total quantity of fatty acids present in this oil.
  • Figure 1 shows the structure of the fad2-1 gene and the position of the mutation according to the invention leading to the STOP codon.
  • the present invention relates to an isolated nucleotide sequence which comprises a stop codon at positions 484 to 486 with reference to the wild-type nucleotide sequence SEQ ID NO: 1, and which encodes a D12-oleate desaturase protein which exhibits at least 95% identity with SEQ ID NO: 4.
  • the sunflower A12-oleate desaturase (Helianthus annuus) according to the present invention is expressed from the mutated fad2-1 gene whose structure is shown in Figure 1.
  • the mutation corresponds to the change of the codon corresponding to the amino acid tryptophan " W ”into a STOP codon.
  • This STOP codon is premature with respect to the wild-type sequence SEQ ID NO: 1.
  • the nucleotide sequence according to the invention encodes a truncated protein D12-sunflower oleate desaturase which comprises more than 110 amino acids, in particular more than 130 acids. amino acids, and preferably more than 160 amino acids.
  • the nucleotide sequence which codes for a truncated sunflower A12-oleate desaturase protein of sequence SEQ ID NO: 4 according to the invention can be any sequence of nucleotides of sequence SEQ ID: 1, in which any STOP codon is present at positions 484 to 486, and in particular a codon chosen from TAA, TAG and TGA.
  • the nucleotide sequence according to the invention is the sequence SEQ ID NO: 2.
  • the present invention relates to a nucleotide sequence comprising a stop codon at positions 484 to 486 with reference to the wild-type nucleotide sequence SEQ ID NO: 1 and which encodes an A12-oleate desaturase protein which exhibits at least 95% identity with SEQ ID NO: 4, and preferably, a D12-oleate desaturase protein which has at least 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 4.
  • the sunflower protein according to the invention is the sequence of 161 amino acids of sequence SEQ ID NO: 4.
  • the wild-type protein is the sequence SEQ ID NO: 3 ( Figure 1) of 378 amino acids encoded by the wild-type nucleotide sequence SEQ ID NO: 1.
  • the percentages of identity to which reference is made in the context of the disclosure of the present invention are determined after optimal alignment of the sequences to be compared, which may therefore include one or more additions, deletions, truncations and / or substitutions.
  • This percentage identity can be calculated by any method of sequence analysis well known to those skilled in the art.
  • the percentage of identity defined in the context of the present invention is determined by means of an overall alignment of the sequences to be compared over their entire length. It can in particular be evaluated using the BLASTP program, more specifically BLASTP 2.2.29, Altschul et al, 1997 and Altschul et al, 2005, with the truncated mutant sequence SEQ ID NO: 4. Thus, it is in particular possible to use the following parameters of the algorithm:
  • Compositional adjustments adjustment of the score matrix on the conditional composition (Conditional compositional score matrix adjustment)
  • the present invention relates to a sunflower plant comprising a nucleotide sequence comprising a STOP codon at positions 484 to 486 with reference to the wild-type nucleotide sequence SEQ ID NO: 1 and which encodes a D12-oleate desaturase protein. which has at least 95% identity with SEQ ID NO: 4, and preferably, a D12-oleate desaturase protein which has at least 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 4.
  • the present invention relates to any sunflower plant which has at least one mutated allele of the gene encoding the D12-oleate desaturase protein corresponding to a sequence of nucleotides according to the invention.
  • the sunflower plant according to the invention has this nucleotide sequence in its genome, in the heterozygous or homozygous state.
  • the sunflower plant according to the invention can be a line or a hybrid.
  • said plant may optionally comprise another allele which leads to an inhibition of the gene encoding the D12-oleate desaturase protein and which, in combination with the mutation according to the invention, to produce seeds having an oleic acid content of at least 85% relative to the total percentage of fatty acids of said seeds.
  • another allele which leads to inhibition of the gene encoding the D12-oleate desaturase protein
  • mention may in particular be made of the Pervenets mutation mention may in particular be made of the Pervenets mutation.
  • the nucleotide sequence according to the invention is present in the homozygous state in the sunflower plant of the invention, said plant producing seeds comprising this nucleotide sequence according to the invention and having an oleic acid content. at least 85% with respect to the total percentage of fatty acids of said seeds.
  • the oleic acid content of these seeds is within a range from 85% to 95%, relative to the total percentage of fatty acids of said seeds.
  • Another object of the present invention therefore also relates to sunflower seeds capable of being obtained from a sunflower plant as described above.
  • These seeds contain at least one mutated allele of the gene encoding the D12-oleate desaturase protein corresponding to a sequence of nucleotides according to the invention.
  • said seed can optionally comprise another allele which leads to inhibition of the gene encoding the A12-desaturase protein and which, in combination with the nucleotide sequence according to invention, to obtain seeds having an oleic acid content of at least 85% relative to the total percentage of fatty acids of said seeds.
  • the Pervenets mutation mention may in particular be made of the Pervenets mutation.
  • the seeds have the nucleotide sequence according to the invention in the homozygous state and have an oleic acid content of at least 85% relative to the percentage. total fatty acids of said seeds.
  • these seeds have an oleic acid content within a range of from 85% to 95%.
  • Any progeny of a seed according to the invention as described above constitutes another object of the present invention since it comprises a sequence of nucleotides according to the invention as described above.
  • the plants and parts of plants according to the invention can be obtained by a process that is not exclusively biological.
  • the present invention relates to a process for obtaining a sunflower plant comprising a sequence of nucleotides according to the invention, comprising the following steps:
  • step b) Generate a plant from said plant part which has undergone said mutagenesis, c) Obtain at least one progeny from the plant generated in step b), and d) Identify and select at least one plant obtained in step b) step b) or c) comprising said nucleotide sequence according to the invention.
  • the process will result in a sunflower plant with a high oleic acid content.
  • This process according to the invention which necessarily includes a step of voluntary and human-induced mutagenesis, therefore does not constitute an essentially biological process.
  • the production process may include an additional step, such as for example self-pollination, to make it possible to obtain a plant with a high oleic acid content comprising the nucleotide sequence according to the invention in the homozygous state.
  • the term “sunflower plant with a high oleic acid content” is understood to mean a plant in which the oleic acid content of the seeds is at least 85%, relative to the total fatty acid content of these seeds and preferably in a range ranging from at least 85 to 95% or more preferably from at least 86 to 95%, or from at least 87 to 95% or from at least 88 to 95% or from at least 89 to 95%. More specifically, the content may be within a range going from at least 89 to 94% or from at least 89 to 93% or from at least 89 to 92% or from at least 89 to 91% or at least 89 to 90%.
  • any technique well known to those skilled in the art can be used. It is in particular possible to use random mutagenesis techniques such as mutagenesis induced by a chemical or physical agent, such as treatment with ethyl-methanesulfonate (EMS) or irradiation, or else methods such as TILLING (Targeting Induced Local Lesions in Genomes) (Till et al, 2003), or even such as the random recombination technique or “DNA shuffling” (Stemmer, 1994).
  • EMS ethyl-methanesulfonate
  • TILLING Targeting Induced Local Lesions in Genomes
  • DNA shuffling Stepmmer, 1994.
  • the mutation according to the invention can be generated directly on elite sunflower lines which will be used as progenitors in the production of hybrids.
  • a cell, a cellular tissue, a callus or a seed obtained from the plant is used as part of the sunflower plant to carry out step a) of mutagenesis in the context of the process for obtaining a plant from sunflower according to the invention.
  • the identification and selection of a plant obtained in step b) can be carried out by any technique well known to those skilled in the art. In particular, by PCR, Northern Blot or Southern Blot amplification techniques, microarrays, "ligase chain reaction” (LCR), and “genotyping-by-sequence” (GBS), or a combination of these techniques.
  • PCR methods are known to those skilled in the art, such as RT-PCR or the method using primers of the Kaspar type (KBioscience (LGC Group, Teddington, Middlesex, UK).
  • the Kompetitive Allele Specifies PCR "KASP TM" genotyping method uses three specific target primers: two primers, each specific for each allelic form of a given SNP (Single Nucleotide Poiymorphism) and a third primer to allow reverse amplification for each. allelic form.
  • the means of identification according to the invention which are described below are advantageously used in the process for obtaining or identifying a sunflower plant with a high oleic acid content according to the invention.
  • the present invention relates to a method for identifying the presence, or the absence, of a nucleotide or amino acid sequence according to the invention in a sunflower plant, which comprises the extraction of the genetic material of the plant such as genomic DNA, RNA or proteome, and the use of means for identifying the sequence of nucleotides or amino acids according to the invention.
  • molecular marker is meant, for example, a specific fragment of a sequence that can be identified within the genome of a plant using various means identification such as probes or primers.
  • the marker can be a fragment of a protein sequence which can be identified within all the proteins of a cell or proteome of a plant, in particular using antibodies.
  • a specific molecular marker of the nucleotide sequence according to the invention corresponds to a fragment of at least 20 nucleotides of the nucleotide sequence according to the invention, said fragment comprising the mutation corresponding to the STOP codon at positions 484 to 486 with reference to the wild-type sequence SEQ ID NO: 1.
  • the marker can be dominant, co-dominant.
  • the identification of the sequence of interest, or its non-identification makes it possible to select the plants exhibiting the gene or allele of interest or the particular characteristic, or on the contrary, not to select the plants not exhibiting the sequence of interest.
  • the means of identification can be probes or primers of at least 12 nucleotides up to 30 nucleotides complementary to the DNA strand that it is desired to amplify, hybridize or even sequence.
  • These means of identification are in this case specific for the marker to be identified and make it possible to rapidly detect the presence of the mutation according to the invention in the genome of plants resulting from given crosses, or parts of plants.
  • identification means can constitute means of identification and / or selection of sunflower plants which contain at least one allele of the sequence according to the invention described above. Preferably, they make it possible to identify and / or to select sunflower plants which produce seeds having an oleic acid content of at least 85%, and preferably within a range of from 85% to 95%.
  • said plant when said plant comprises a nucleotide sequence according to the invention in the heterozygous state, said plant may comprise another allele which leads to inhibition of the gene encoding the D12-desaturase protein and which, in combination with the nucleotide sequence, allows according to the invention, to obtain seeds having an oleic acid content of at least 85% relative to the total percentage of fatty acids of said seeds.
  • the Pervenets mutation mention may in particular be made of the Pervenets mutation.
  • these identification means make it possible to identify and / or select sunflower plants which produce seeds which have the nucleotide sequence according to the invention in the homozygous state and have an oleic acid content of at least less 85% relative to the total percentage of fatty acids of said seeds, and preferably within a range from 85% to 95%.
  • the means for identifying a plant, part of a plant according to the invention also constitute one of the aspects of the present invention.
  • these means identification are capable of specifically detecting the mutation in the nucleotide sequence according to the invention, namely the presence of a STOP codon at positions 484 to 486 with reference to the sequence SEQ ID NO: 1.
  • these means may consist of primers or probes specific for the mutation described above according to the invention.
  • the set of primers which can be used in the methods according to the invention is the set of KASP primers of sequences SEQ ID NO: 7, 8 and 9.
  • the present invention also covers a method of crossing by so-called "forward" selection of a sunflower plant comprising a sequence of nucleotides according to the invention; said method comprising the following steps:
  • step b) Repeating step b) to obtain progeny comprising said nucleotide sequence according to the invention and exhibiting at least one of said agronomic characteristics.
  • the progeny have the nucleotide sequence according to the invention in the homozygous state.
  • Step c) can be repeated as many times as necessary to obtain progeny comprising the nucleotide sequence according to the invention.
  • agronomic characteristic is meant any trait of interest making it possible to improve a plant or a variety, for example resistance to herbicides or pathogens or any other trait making it possible to improve quantitative or qualitative characteristics.
  • this progeny comprises a gene encoding a protein of sequence SEQ ID NO: 4.
  • Forward selection is generally used to improve the agronomic performance of a variety or line carrying a specific mutation or trait while backcross selection is used to introduce a specific mutation or trait into the particular genotypic context. of a variety or line.
  • backcross selection is used to introduce a specific mutation or trait into the particular genotypic context. of a variety or line. A comparison of the two methods is described in Mumm et al, 2007.
  • the invention relates to a process for introgression by backcrossing of a genome fragment comprising a nucleotide sequence according to the invention as described above, from a donor sunflower plant to a recipient sunflower plant which does not contain said nucleotide sequence in its genome, said method comprising the following steps:
  • step d) Selection of a plant comprising said nucleotide sequence according to the invention, e) Backcrossing of the plant selected in step d) with the recipient plant, f) Optionally, self-pollination of the plants obtained in e) to obtain a plant comprising said nucleotide sequence according to the invention in the homozygous state.
  • the recipient plant does not contain in its genome the sequence of nucleotides according to the invention, that is to say a sequence of nucleotides having a STOP codon at positions 484 to 486 with reference to the wild-type nucleotide sequence SEQ ID NO: 1 and encoding an A12-desaturase protein which has at least 95% identity with SEQ ID NO: 4, and preferably, which has at least 96%, 97%, 98% or at least 99% identity with SEQ ID NO : 4.
  • the recipient plant does not contain in its genome the mutation described above, namely a STOP codon at positions at positions 484 to 486 with reference to the sequence SEQ ID NO: 1.
  • the backcrosses carried out aim to increase the percentage of the genome of the recurrent or recipient plant.
  • the number of inbreeding or backcrosses performed is repeated as many times to obtain a percentage of at least 80% of the genome of the recipient plant used in the cross, preferably at least 98%.
  • the present invention relates to the use of sunflower seeds according to the invention as described above or their descendants, to obtain oil, and in particular oil having a high content.
  • oleic acid that is to say at least 85% of oleic acid relative to the total quantity of fatty acids present in this oil.
  • EXAMPLE 1 Creation and screening of a sunflower TILLING population.
  • the TILLING population was obtained in 2015 from 2 varieties of sunflower according to the KumarA protocol. et al (2013).
  • the population initially comprises 18,407 M2 families from which 10,000 M2 families with more than 50 seeds per family were selected for isolate genomic DNA and perform screening.
  • DNA extraction was carried out from leaf material from 5 individual plants per M2 families.
  • the DNA is normalized to 5 ng / mI for the following experiments.
  • EXAMPLE 2 Identification in silico of potential STOP codons in the wild-type sequence of the fad2-1 gene.
  • the sequence of the wild-type HanXRQChr14g0452931 fad2-1 gene or otherwise described in SEQ ID NO: 1) was analyzed to identify all the STOP codons that can be generated by the EMS treatment. 34 potential EMS point mutations each generating a STOP codon capable of producing a truncated FAD2 protein have been identified. Specific primers specifically targeting each of the 34 mutations were generated for screening M2 families.
  • the identification of the mutation was carried out from a pool of DNAs from 96 M2 families by PCR amplification. Then, thanks to the analysis of the pools, the presence of potential mutations was checked in 10 individual M2 seeds. A single mutant family (HABT-3816 M2) was confirmed and the mutation was identified by Sanger sequencing. The mutation identified corresponds to a modification G to A located at nucleotide position 485 in the sequence described in SEQ ID NO: 1. The point mutation creates a STOP codon which produces a truncated protein of 161 amino acids described in the sequence SEQ ID NO: 4, instead of the 378 amino acids of the wild-type protein described in the sequence SEQ ID NO: 3 ( Figure 1).
  • mutant and wild-type context sequences used for the identification of the STOP mutation in the mutant family are described in the sequences SEQ ID NO: 5 (ACCACCGCCACCATTCCAACACTGGATCACTCGAGCGGGACGAGGTTTTCGTCCCCAA AT CCCGATCGAAAGTCCCGTAGT ACT CG AAATCC ACTTGATTGGGTAGT ACT CG AAATCC ACTTGATTGGGTAGT ACT CG AAATCC ACTT
  • the saturated oil composition was measured from 73 seeds segregated from a heterozygous plant at the HABT-3816 locus. For each seed, one half is used to measure the saturated acid composition by
  • GC Gas Chromatography
  • the GC is carried out using the GC Thermo 1310 GC_1 and GC_2 device according to the following protocol:
  • the fatty acids are dissolved in isooctane (2,2,4 -Trimethyl pentane) and then converted into methyl esters by adding methanolic potassium hydroxide (2N KOH / MeOH).
  • the potassium hydroxide is neutralized with sodium hydrogen sulphate to prevent the saponification of the methyl esters.
  • leaves of young plants were sampled for genotyping using the Ha_FAD2-1_2439_E1_485 KASP markers described in Example 3.29 seeds are homozygous mutant plants (A: A), 44 seeds are heterozygous (A: G) and 10 seeds are homozygous wild plants (G: G) were respectively analyzed for their fatty acid content
  • A A
  • G heterozygous wild plants
  • G G
  • standard deviation The mutation is recessive with a major effect on the oleic and linoleic acid content.
  • Homozygous seeds which carry the mutation have a stable oleic acid content greater than 90% (average 92.8 +/- 0.17%) and a very low level of linoleic acid.

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Abstract

The present invention relates to isolated nucleotide sequences that code a truncated sunflower oleate desaturase protein. The presence of such nucleotide sequences thus leads to sunflower plants that have, in a stable manner, a high oleic acid content. The present invention also relates to plants, parts of plants or seeds that contain these sequences, production and use thereof in introgression methods and/or to obtain oil with a high oleic acid content.

Description

TOURNESOL A TENEUR ELEVEE EN ACIDE OLEIQUE ET PROCEDE D’OBTENTION SUNFLOWER WITH A HIGH OLEIC ACID CONTENT AND PROCESS FOR OBTAINING
DOMAINE TECHNIQUE DE L'INVENTION TECHNICAL FIELD OF THE INVENTION
L'invention concerne des séquences de nucléotides isolées qui codent une protéine A12-oléate désaturase de tournesol tronquée. En particulier ces séquences de nucléotides contiennent un codon stop prématuré qui conduit à la production de cette protéine tronquée. La présence de ces séquences de nucléotides permet ainsi de conduire à des plantes de tournesol ayant, de manière stable, une teneur élevée en acide oléique dans l’huile de graines. La présente invention concerne donc également les plantes, parties de plantes ou graines qui contiennent ces séquences, leur obtention et leurs utilisations dans des procédés d’introgression et/ou pour obtenir de l’huile à teneur élevée en acide oléique. The present invention provides isolated nucleotide sequences which encode a truncated sunflower A12-oleate desaturase protein. In particular, these nucleotide sequences contain a premature stop codon which leads to the production of this truncated protein. The presence of these nucleotide sequences thus makes it possible to lead to sunflower plants having, in a stable manner, a high content of oleic acid in the seed oil. The present invention therefore also relates to the plants, parts of plants or seeds which contain these sequences, their obtaining and their uses in introgression processes and / or for obtaining oil with a high oleic acid content.
ARRIERE-PLAN TECHNIQUE TECHNICAL BACKGROUND
Le tournesol est le plus souvent cultivé pour obtenir des huiles qui contiennent des acides gras saturés et insaturés. Les acides gras saturés possèdent 16 ou 18 atomes de carbones (C16 ou C18). Les acides gras en C16 sont généralement saturés comme l’acide palmitique (16:0). Les acides gras en C18 sont soit saturés, comme l’acide stéarique (18:0), soit insaturés avec 1 , 2 ou 3 doubles liaisons, comme l’acide oléique (18: 1), l’acide linoléique (18:2), l’acide linolénique (18:3). Sunflower is most often cultivated to obtain oils that contain saturated and unsaturated fatty acids. Saturated fatty acids have 16 or 18 carbon atoms (C16 or C18). C16 fatty acids are generally saturated like palmitic acid (16: 0). C18 fatty acids are either saturated, such as stearic acid (18: 0), or unsaturated with 1, 2 or 3 double bonds, such as oleic acid (18: 1), linoleic acid (18: 2 ), linolenic acid (18: 3).
Les huiles provenant de variétés de tournesol dits oléique faible (low oleic ou LO) contiennent principalement de l’acide linoléique. Des lignées et des hybrides, dits oléique élevé ( high oleic ou HO) qui possèdent une teneur 18:1 élevée dans leurs graines ont été obtenues par sélection à partir du mutant Pervenets. Oils from so-called low oleic (LO) sunflower varieties mainly contain linoleic acid. Lines and hybrids, known as high oleic (HO) which have a high 18: 1 content in their seeds were obtained by selection from the mutant Pervenets.
Le mutant Pervenets a été obtenu par mutagenèse chimique par Soldatov en 1976 sur une population tournesol. Le contenu moyen en 18:1 dans les graines de cette population est supérieur à 65 %, les contenus individuels allant entre 60 et 90 %, comparé à un contenu moyen de 20 % dans les variétés normales LO. The Pervenets mutant was obtained by chemical mutagenesis by Soldatov in 1976 on a sunflower population. The average 18: 1 content in seeds from this population is over 65%, with individual contents ranging between 60-90%, compared to an average content of 20% in normal LO varieties.
La population Pervenets a été distribuée dans le monde entier et utilisée dans de nombreux programmes de sélection afin de convertir des génotypes sélectionnés avec un contenu 18: 1 faible en des génotypes ayant un contenu 18:1 supérieur à 80 % dans leurs graines. The Pervenets population has been distributed worldwide and used in numerous breeding programs to convert selected genotypes with low 18: 1 content to genotypes with 18: 1 content greater than 80% in their seeds.
Il a été montré que le phénotype HO est associé à une diminution marquée de l’activité de l’enzyme A12-oléate désaturase, qui catalyse la désaturation du 18: 1 vers 18:2 au cours des stades critiques de la construction du stock de lipides, ce qui explique l’accumulation de 18:1 (Garces R. et Mancha M., 1991). L’enzyme A12-oléate désaturase (EC 1.3.1.35) est également connue sous les noms de : acide oléique désaturase, linoléate synthase, oléoyle CoA désaturase, oleoylphosphatidylcholine désaturase, oleoyl-PC désaturase, FAD2. Chez le tournesol, trois gènes fad2 ont été identifiés ( fad2-1 , fad2-2, et fad2-3) codant pour des D12- désaturase microsomales. The HO phenotype has been shown to be associated with a marked decrease in the activity of the enzyme A12-oleate desaturase, which catalyzes the desaturation of 18: 1 to 18: 2 during critical stages of stock building. lipids, which explains the accumulation of 18: 1 (Garces R. and Mancha M., 1991). The enzyme A12-oleate desaturase (EC 1.3.1.35) is also known under the names of: oleic acid desaturase, linoleate synthase, oleoyl CoA desaturase, oleoylphosphatidylcholine desaturase, oleoyl-PC desaturase, FAD2. In sunflower, three fad2 genes have been identified (fad2-1, fad2-2, and fad2-3) encoding microsomal D12-desaturase.
L’enzyme A12-oléate désaturase (EC 1.3.1.35) est codée par le gène A12-désaturase correspondant au gène fad2-1. The A12-oleate desaturase enzyme (EC 1.3.1.35) is encoded by the A12-desaturase gene corresponding to the fad2-1 gene.
Par ailleurs, l’analyse moléculaire a montré que la mutation Pervenets est associée à une duplication du gène A12-désaturase, conduisant à une mise sous silence génétique ou silencing. Cette diminution de transcription explique la diminution du niveau d’enzyme et ainsi la diminution de l’activité D12- oléate désaturase qui produit une plus grande accumulation d’acide oléique dans les graines de tournesol (Hongtrakul et al., 1998). Des marqueurs moléculaires associés à cette mutation ont été développés pour être utilisés dans les programmes de croisement basé sur la sélection des génotypes HO (WO 2005/106022 ; Lacombe et al. 2001). Furthermore, molecular analysis has shown that the Pervenets mutation is associated with duplication of the A12-desaturase gene, leading to genetic silencing or silencing. This decrease in transcription explains the decrease in the enzyme level and thus the decrease in D12-oleate desaturase activity which produces a greater accumulation of oleic acid in sunflower seeds (Hongtrakul et al., 1998). Molecular markers associated with this mutation have been developed for use in breeding programs based on the selection of HO genotypes (WO 2005/106022; Lacombe et al. 2001).
Pour obtenir une teneur à la fois plus élevée en acide oléique et aussi plus stable, une mutation knock-down dans le gène A12-désaturase a été isolée par les inventeurs. Les documents WO2013004280 et WO2013004281 décrivent une mutation obtenue par traitement chimique dans le gène A12-désaturase sélectionnée pour un phénotype d’augmentation du contenu oléique comparé à celui de la mutation Pervenets. Différentes mutations ont été obtenues chez le tournesol, créant un codon STOP dans la phase de lecture, et conduisant à une protéine tronquée. Toutes ces mutations conduisent cependant à une protéine tronquée d’une longueur inférieure ou égale à 1 10 acides aminés. To obtain a content both higher in oleic acid and also more stable, a knock-down mutation in the A12-desaturase gene was isolated by the inventors. The documents WO2013004280 and WO2013004281 describe a mutation obtained by chemical treatment in the A12-desaturase gene selected for a phenotype of increased oleic content compared to that of the Pervenets mutation. Different mutations have been obtained in sunflower, creating a STOP codon in the reading frame, and leading to a truncated protein. All of these mutations, however, result in a truncated protein less than or equal to 110 amino acids in length.
Il est donc intéressant d’explorer si d’autres mutations peuvent aboutir à une teneur élevée en acide oléique et/ou à une meilleure stabilité chez le tournesol. It is therefore interesting to explore whether other mutations may lead to a high oleic acid content and / or better stability in sunflower.
RESUME DE L’INVENTION SUMMARY OF THE INVENTION
Selon un premier aspect, la présente invention concerne une séquence de nucléotides isolée qui comprend un codon stop aux positions 484 à 486 en référence à la séquence de nucléotides sauvage SEQ ID NO : 1 , et qui code une protéine D12- oléate désaturase qui présente au moins 95% d’identité avec SEQ ID NO 4 : According to a first aspect, the present invention relates to an isolated nucleotide sequence which comprises a stop codon at positions 484 to 486 with reference to the wild-type nucleotide sequence SEQ ID NO: 1, and which encodes a D12-oleate desaturase protein which exhibits in less 95% identity with SEQ ID NO 4:
MGAGEYTSVTNENNPLDRVPHAKPPFTIGDLKKAIPPHCFQRSLTRSFSYVLSDLTITAVLYHI ATTYFHHLPTPLSSIAWASYWVVQGCVLTGVWVIAHECGHHAFSDYQWVDDTVGFVLHSSL LVPYFSWKYSHHRHHSNTGSLERDEVFVPKSRSKVP. De préférence, la séquence de nucléotides selon l’invention est de séquence SEQ ID NO : 2 : ATGGGTGCAGGAG AAT ACACGTCTGTGACCAACG AAAACAACCCACTCGATCGAGTCCC T CATGCAAAACCACCCTT CACCAT CGGCGAT CT GAAAAAAGCCAT CCCACCACACTGCTT CCAGCGGTCGCTAACCCGTTCGTTCTCCTACGTGCTGTCTGACCTCACCATAACCGCTG T CCT CT ACCACATTGCCACCACCT ACTT CCACCACCT CCCCACCCCTTTGTCATCCAT CGMGAGEYTSVTNENNPLDRVPHAKPPFTIGDLKKAIPPHCFQRSLTRSFSYVLSDLTITAVLYHI ATTYFHHLPTPLSSIAWASYWVVQGCVLTGVWVIAHECGHHAFSDYQWVDDDTVGFVLHSSLKSHHRDEHFSVHSWK. Preferably, the nucleotide sequence of the invention is SEQ ID NO: 2: ATGGGTGCAGGAG AAT ACACGTCTGTGACCAACG AAAACAACCCACTCGATCGAGTCCC T CATGCAAAACCACCCTT CACCAT CGGCGAT CT GAAAAAAGCCAT CCCACCACACTGCTT CCAGCGGTCGCTAACCCGTTCGTTCTCCTACGTGCTGTCTGACCTCACCATAACCGCTG T CTC CT ACCACATTGCCACCACCT ACTT CCACCACCT CCCCACCCCTTTGTCATCCAT CG
CATGGGCCTCTTACTGGGTAGTCCAAGGCTGCGTCCTCACCGGAGTCTGGGTCATCGC CCACGAATGTGGTCACCATGCGTTTAGTGATTATCAATGGGTCGACGACACTGTGGGCT TTGTT CT CCACT CGTCTTT ACT CGTCCCTT ACTTTT CGTGG AAAT AT AGT CACCACCGCCA CCATT CCAACACTGG AT CACT CGAGCGGG ACG AGGTTTT CGTCCCCAAAT CCCGATCGA AAGTCCCGTAGTACT CG AAAT ACTTT AACAACACAGT G GG CCG CATTGTCAGT ATGTTCGCATGGGCCTCTTACTGGGTAGTCCAAGGCTGCGTCCTCACCGGAGTCTGGGTCATCGC CCACGAATGTGGTCACCATGCGTTTAGTGATTATCAATGGGTCGACGACACTGTGGGCT TTGTT CT CCACT CGTCTTT ACT CGTCCCTT ACTTTT CGTGG STW AT AGT CACCACCGCCA CCATT CCAACACTGG AT CACT CGAGCGGG ACG AGGTTTT CGTCCCCAAAT CCCGATCGA AAGTCCCGTAGTACT CG STW CADTC AACAACACAGT G GG CGC CATTGTCAGT ATGTTCG
TCACTCTCACTCTCGGCTGGCCCTTGTACTTAGCTTTCAATGTGTCGGGCCGACCCTAT G ACCGTTT CGCCTGCCACT ACGTCCCAACCAGCCCT AT GT ACAAT G AACGT AAACGTT A CCAG AT AGT CAT GT CCG ACAT CGGG ATTGTT AT CACAT CGTT CAT CCTTT ATCGTGTTGC TATGGCAAAAGGGTTGGTTTGGGTGATTTGCGTCTATGGGGTTCCGTTGATGGTTGTGA ACG CGTTT CTG GTGTT G AT C ACTT AT CTT CAACAT ACTCACCCT G GCTT G CCG CATT AT GTCACTCTCACTCTCGGCTGGCCCTTGTACTTAGCTTTCAATGTGTCGGGCCGACCCTAT G ACCGTTT CGCCTGCCACT ACGTCCCAACCAGCCCT AT GT ACAAT G AACGT AAACGTT A GCC AT AGT CAT CGC GT ACAT CGGG ATTGTT AT cacat CGTT CAT CCTTT ATCGTGTTGC TATGGCAAAAGGGTTGGTTTGGGTGATTTGCGTCTATGGGGTTCCGTTGATGGTTGTGA ACG CTG CGTTT GTGTT G AT C ACTT AT CTT CAACAT ACTCACCCT GCTT G G G CGC AT CATT
ATAGCTCGGAATGGGAATGGTTAAAGGGAGCATTGGCGACAGTGGACCGTGACTATGG TGTGTTGAACAAGGTGTTCCATCATATTACCGACACACATGTGGTGCACCATTTGTTTTC GACAATGCCTCATTATAATGCGATGGAAGCACAGAAGGCGCTGAGACCGGTGCTTGGG G AGT ATT AT CGGTTT G ACAAG ACCCCGTTTT ATGTAGCCAT GTGG AG AG AG AT G AAGGA ATGTTTGTTTGTGGAGCAAGATGATGAAGGGAAAGGAGGTGTGTTTTGGTACAAGAATAA GATGAATTAA. ATAGCTCGGAATGGGAATGGTTAAAGGGAGCATTGGCGACAGTGGACCGTGACTATGG TGTGTTGAACAAGGTGTTCCATCATATTACCGACACACATGTGGTGCACCATTTGTTTTC GACAATGCCTCATTATAATGCGATGGAAGCACAGAAGGCGCTGAGACCGGTGCTTGGG G AGT ATT AT CGGTTT G ACAAG ACCCCGTTTT ATGTAGCCAT GTGG AG AG AG AT G AAGGA ATGTTTGTTTGTGGAGCAAGATGATGAAGGGAAAGGAGGTGTGTTTTGGTACAAGAATAA GATGAATTAA.
Selon un autre aspect, la présente invention concerne une protéine A12-oléate désaturase de tournesol tronquée de séquence SEQ ID NO : 4. According to another aspect, the present invention relates to a truncated sunflower A12-oleate desaturase protein of sequence SEQ ID NO: 4.
Selon encore un autre aspect, la présente invention se rapporte à une plante de tournesol comprenant une séquence de nucléotides selon l’invention, qui peut être présente soit à l’état hétérozygote soit à l’état homozygote dans le génome de ladite plante. De préférence, la séquence des nucléotides selon l’invention est présente à l’état homozygote. According to yet another aspect, the present invention relates to a sunflower plant comprising a sequence of nucleotides according to the invention, which can be present either in the heterozygous state or in the homozygous state in the genome of said plant. Preferably, the sequence of nucleotides according to the invention is present in the homozygous state.
Les graines produites par les plantes de tournesol selon l’invention constituent également un autre aspect de la présente invention, ainsi que leur descendance dès lors qu’elles comprennent au moins une séquence de nucléotides selon la présente invention. De telles graines possèdent une teneur en acide oléique d’au moins 85 %, par rapport au pourcentage total d’acides gras desdites graines, et de préférence une teneur en acide oléique comprise dans une gamme allant de 85 % à 95 %, en particulier lorsqu’elles comprennent la séquence selon l’invention à l’état homozygote. Selon encore un autre aspect, la présente invention concerne un procédé d’obtention d’une plante de tournesol comprenant une séquence de nucléotides selon l’invention, ledit procédé comprenant les étapes suivantes : The seeds produced by the sunflower plants according to the invention also constitute another aspect of the present invention, as well as their progeny provided that they comprise at least one sequence of nucleotides according to the present invention. Such seeds have an oleic acid content of at least 85%, relative to the total percentage of fatty acids of said seeds, and preferably an oleic acid content in a range from 85% to 95%, in particular when they comprise the sequence according to the invention in the homozygous state. According to yet another aspect, the present invention relates to a process for obtaining a sunflower plant comprising a sequence of nucleotides according to the invention, said process comprising the following steps:
a) Procéder à la mutagenèse d’une partie de plante de tournesol pour qu’elle comprenne une séquence de nucléotides selon l’invention, a) Mutagenize a part of a sunflower plant so that it comprises a sequence of nucleotides according to the invention,
b) Générer une plante à partir de ladite partie de plante ayant subi ladite mutagenèse, c) Obtenir au moins une descendance à partir de la plante générée à l’étape b), et d) Identifier et sélectionner au moins une plante obtenue à l’étape b) ou c) comprenant ladite séquence de nucléotides selon l’invention. b) Generate a plant from said plant part which has undergone said mutagenesis, c) Obtain at least one progeny from the plant generated in step b), and d) Identify and select at least one plant obtained in step b) step b) or c) comprising said nucleotide sequence according to the invention.
Selon un autre aspect, la présente invention concerne un procédé d’identification de la présence, ou de l’absence, d’une séquence de nucléotides ou d’acides aminés selon l’invention dans une plante de tournesol, comprenant l’extraction de l’ADN génomique, de l’ARN ou de l’ensemble des protéines du protéome de la plante de tournesol et l’utilisation de moyens d’identification de ladite séquence. According to another aspect, the present invention relates to a method for identifying the presence, or the absence, of a nucleotide or amino acid sequence according to the invention in a sunflower plant, comprising the extraction of genomic DNA, RNA or all the proteins of the proteome of the sunflower plant and the use of means for identifying said sequence.
Les moyens d’identification d’une plante, partie de plante selon l’invention constituent également un des aspects de la présente invention. L’utilisation de ces moyens permet ainsi d’identifier et/ou de sélectionner des plantes de tournesol ou des graines selon l’invention.The means of identifying a plant, part of a plant according to the invention also constitute one of the aspects of the present invention. The use of these means thus makes it possible to identify and / or select sunflower plants or seeds according to the invention.
Le procédé d’obtention selon l’invention inclut nécessairement une étape de mutagenèse volontaire et induite par l’homme, ce qui exclut explicitement la réalisation naturelle et les procédés essentiellement biologiques. The production process according to the invention necessarily includes a step of voluntary and human-induced mutagenesis, which explicitly excludes natural production and essentially biological processes.
Dans un autre aspect, la présente invention concerne un procédé de croisement par sélection dite « forward » d’une plante de tournesol comprenant une séquence de nucléotides selon l’invention ; ledit procédé comprenant les étapes suivantes : In another aspect, the present invention relates to a method of crossing by so-called "forward" selection of a sunflower plant comprising a sequence of nucleotides according to the invention; said method comprising the following steps:
a) Croisement de ladite plante avec toute autre plante de tournesol présentant des caractéristiques agronomiques pour obtenir une plante F 1 , a) Crossing of said plant with any other sunflower plant having agronomic characteristics to obtain an F 1 plant,
b) Autofécondation d’une plante F1 comprenant ladite séquence de nucléotides selon l’invention, b) Self-fertilization of an F1 plant comprising said nucleotide sequence according to the invention,
c) Répétition de l’étape b) pour obtenir une descendance comprenant ladite séquence de nucléotides selon l’invention et présentant au moins une desdites caractéristiques agronomiques. c) Repeating step b) to obtain progeny comprising said nucleotide sequence according to the invention and exhibiting at least one of said agronomic characteristics.
Selon encore un autre aspect, l’invention concerne un procédé d’introgression par rétrocroisement d’un fragment de génome comprenant une séquence de nucléotides selon l’invention telle que décrite ci-dessus, à partir d’une plante de tournesol donneuse vers une plante de tournesol receveuse qui ne contient pas ladite séquence de nucléotides dans son génome, ledit procédé comprenant les étapes suivantes : a) Croisement de la plante de tournesol donneuse avec la plante receveuse, According to yet another aspect, the invention relates to a process for introgression by backcrossing of a genome fragment comprising a nucleotide sequence according to the invention as described above, from a donor sunflower plant to a recipient sunflower plant which does not contain said nucleotide sequence in its genome, said method comprising the following steps: a) Crossing of the donor sunflower plant with the recipient plant,
b) Obtention d’un hybride F1 , b) Obtaining an F1 hybrid,
c) Rétrocroisement dudit hybride F1 avec la plante receveuse, c) Backcrossing of said F1 hybrid with the recipient plant,
d) Sélection d’une plante comprenant ladite séquence de nucléotides selon l’invention, e) Rétrocroisement de la plante sélectionnée à l’étape d) avec la plante receveuse d) Selection of a plant comprising said nucleotide sequence according to the invention, e) Backcrossing of the plant selected in step d) with the recipient plant
f) Optionnellement, autofécondation des plantes obtenues en e) pour obtenir une plante comprenant ladite séquence de nucléotides selon l’invention à l’état homozygote. f) Optionally, self-fertilization of the plants obtained in e) to obtain a plant comprising said nucleotide sequence according to the invention in the homozygous state.
Enfin, selon un dernier aspect, la présente invention concerne l’utilisation de graines de tournesol selon l’invention telles que décrites ci-dessus, pour obtenir de l’huile, et en particulier de l’huile ayant une teneur élevée en acide oléique, c’est-à-dire d’au moins 85 % d’acide oléique par rapport à la quantité totale d’acides gras présents dans cette huile. Finally, according to a last aspect, the present invention relates to the use of sunflower seeds according to the invention as described above, to obtain oil, and in particular oil having a high oleic acid content. , that is to say at least 85% of oleic acid relative to the total quantity of fatty acids present in this oil.
BREVE DESCRIPTION DES DESSINS La figure 1 représente la structure du gène fad2-1 et la position de la mutation selon l’invention conduisant au codon STOP. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the structure of the fad2-1 gene and the position of the mutation according to the invention leading to the STOP codon.
DESCRIPTION DETAILLEE DE L'INVENTION La présente invention concerne une séquence de nucléotides isolée qui comprend un codon stop aux positions 484 à 486 en référence à la séquence de nucléotides sauvage SEQ ID NO : 1 , et qui code une protéine D12- oléate désaturase qui présente au moins 95% d’identité avec SEQ ID NO : 4. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an isolated nucleotide sequence which comprises a stop codon at positions 484 to 486 with reference to the wild-type nucleotide sequence SEQ ID NO: 1, and which encodes a D12-oleate desaturase protein which exhibits at least 95% identity with SEQ ID NO: 4.
La A12-oleate désaturase de tournesol ( Helianthus annuus) selon la présente invention est exprimée à partir du gène fad2-1 muté dont la structure est représentée sur la Figure 1. La mutation correspond au changement du codon correspondant à l’acide aminé tryptophane « W » en un codon STOP. Ce codon STOP est prématuré par rapport à la séquence sauvage SEQ ID NO : 1. La séquence de nucléotides selon l’invention code une protéine tronquée D12- oleate désaturase de tournesol qui comprend plus de 1 10 acides aminés, en particulier plus de 130 acides aminés, et de manière préférée plus de 160 acides aminés. The sunflower A12-oleate desaturase (Helianthus annuus) according to the present invention is expressed from the mutated fad2-1 gene whose structure is shown in Figure 1. The mutation corresponds to the change of the codon corresponding to the amino acid tryptophan " W ”into a STOP codon. This STOP codon is premature with respect to the wild-type sequence SEQ ID NO: 1. The nucleotide sequence according to the invention encodes a truncated protein D12-sunflower oleate desaturase which comprises more than 110 amino acids, in particular more than 130 acids. amino acids, and preferably more than 160 amino acids.
La séquence de nucléotides qui code une protéine A12-oleate désaturase de tournesol tronquée de séquence SEQ ID NO : 4 selon l’invention peut être toute séquence de nucléotides de séquence SEQ ID : 1 , dans laquelle un quelconque codon STOP est présent aux positions 484 à 486, et notamment un codon choisi parmi TAA, TAG et TGA. De préférence, la séquence de nucléotides selon l’invention est la séquence SEQ ID NO : 2. Dans ce contexte, selon un premier aspect, la présente invention concerne une séquence de nucléotides comprenant un codon stop aux positions 484 à 486 en référence à la séquence nucléotidique sauvage SEQ ID NO :1 et, qui code une protéine A12-oleate désaturase qui présente au moins 95% d’identité avec SEQ ID NO : 4, et préférentiellement, une protéine D12- oleate désaturase qui présente au moins 96%, 97%, 98% ou au moins 99% d’identité avec SEQ ID NO : 4. The nucleotide sequence which codes for a truncated sunflower A12-oleate desaturase protein of sequence SEQ ID NO: 4 according to the invention can be any sequence of nucleotides of sequence SEQ ID: 1, in which any STOP codon is present at positions 484 to 486, and in particular a codon chosen from TAA, TAG and TGA. Preferably, the nucleotide sequence according to the invention is the sequence SEQ ID NO: 2. In this context, according to a first aspect, the present invention relates to a nucleotide sequence comprising a stop codon at positions 484 to 486 with reference to the wild-type nucleotide sequence SEQ ID NO: 1 and which encodes an A12-oleate desaturase protein which exhibits at least 95% identity with SEQ ID NO: 4, and preferably, a D12-oleate desaturase protein which has at least 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 4.
De manière particulièrement préférée, la protéine de tournesol selon l’invention est la séquence de 161 acides aminés de séquence SEQ ID NO : 4. La protéine sauvage est la séquence SEQ ID NO : 3 (Figure 1 ) de 378 acides-aminés codée par la séquence nucléotidique sauvage SEQ ID NO : 1. In a particularly preferred way, the sunflower protein according to the invention is the sequence of 161 amino acids of sequence SEQ ID NO: 4. The wild-type protein is the sequence SEQ ID NO: 3 (Figure 1) of 378 amino acids encoded by the wild-type nucleotide sequence SEQ ID NO: 1.
Les pourcentages d’identité auxquels il est fait référence dans le cadre de l’exposé de la présente invention sont déterminés après alignement optimal des séquences à comparer, qui peuvent donc comprendre une ou plusieurs additions, délétions, troncatures et/ou substitutions. Ce pourcentage d’identité peut être calculé par toute méthode d’analyse de séquences bien connue de l’homme du métier. The percentages of identity to which reference is made in the context of the disclosure of the present invention are determined after optimal alignment of the sequences to be compared, which may therefore include one or more additions, deletions, truncations and / or substitutions. This percentage identity can be calculated by any method of sequence analysis well known to those skilled in the art.
De préférence, le pourcentage d’identité défini dans le cadre de la présente invention est déterminé au moyen d’un alignement global des séquences à comparer sur toute leur longueur. Il peut notamment être évalué à l’aide du programme BLASTP, plus spécifiquement BLASTP 2.2.29, Altschul et al, 1997 et Altschul et al, 2005, avec la séquence mutante tronquée SEQ ID NO : 4. Ainsi, on peut notamment utiliser les paramètres suivants de l’algorithme : Preferably, the percentage of identity defined in the context of the present invention is determined by means of an overall alignment of the sequences to be compared over their entire length. It can in particular be evaluated using the BLASTP program, more specifically BLASTP 2.2.29, Altschul et al, 1997 and Altschul et al, 2005, with the truncated mutant sequence SEQ ID NO: 4. Thus, it is in particular possible to use the following parameters of the algorithm:
“Seuil attendu (Expected threshold) : 10 “Expected threshold: 10
Longueur de mot (Word size) : 3 Word size: 3
Nombre maximum de concordances dans une zone de la requête ( Max matches in a query range) : 0 Maximum number of matches in a query range: 0
Matrice ( Matrix ) : BLOSUM62 Matrix: BLOSUM62
Coûts des trous (Gap Costs) : Existence 1 1 , Extension 1. Gap Costs: Existence 1 1, Extension 1.
Ajustements des compositions ( Compositional adjustments) : ajustement de la matrice de scores sur la composition conditionnelle ( Conditional compositional score matrix adjustment) Compositional adjustments: adjustment of the score matrix on the conditional composition (Conditional compositional score matrix adjustment)
Pas de filtre des régions de faible complexité (No filterforlow complexity régions)”. No filterforlow complexity regions ”.
Selon un autre aspect, la présente invention se rapporte à une plante de tournesol comprenant une séquence de nucléotides comprenant un codon STOP aux positions 484 à 486 en référence à la séquence nucléotidique sauvage SEQ ID NO : 1 et qui code une protéine D12- oleate désaturase qui présente au moins 95% d’identité avec SEQ ID NO : 4, et préférentiellement, une protéine D12-oleate désaturase qui présente au moins 96%, 97%, 98% ou au moins 99% d’identité avec SEQ ID NO : 4. According to another aspect, the present invention relates to a sunflower plant comprising a nucleotide sequence comprising a STOP codon at positions 484 to 486 with reference to the wild-type nucleotide sequence SEQ ID NO: 1 and which encodes a D12-oleate desaturase protein. which has at least 95% identity with SEQ ID NO: 4, and preferably, a D12-oleate desaturase protein which has at least 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 4.
La présente invention est relative à toute plante de tournesol qui possède au moins un allèle muté du gène codant la protéine D12-oleate désaturase correspondant à une séquence de nucléotides selon l’invention. En particulier, la plante de tournesol selon l’invention possède cette séquence de nucléotides dans son génome, à l’état hétérozygote ou homozygote. The present invention relates to any sunflower plant which has at least one mutated allele of the gene encoding the D12-oleate desaturase protein corresponding to a sequence of nucleotides according to the invention. In particular, the sunflower plant according to the invention has this nucleotide sequence in its genome, in the heterozygous or homozygous state.
La plante de tournesol selon l’invention peut être une lignée ou un hybride. The sunflower plant according to the invention can be a line or a hybrid.
Lorsque la séquence nucléotides selon l’invention est présente à l’état hétérozygote, ladite plante peut éventuellement comprendre un autre allèle qui conduit à une inhibition du gène codant la protéine D12-oleate désaturase et qui permet, en combinaison avec la mutation selon l’invention, de produire des graines ayant une teneur en acide oléique d’au moins 85 % par rapport au pourcentage total d’acides gras desdites graines. Parmi les exemples d’autre allèle connu de l’art antérieur qui conduit à une inhibition du gène codant la protéine D12-oleate désaturase, on peut notamment citer la mutation Pervenets. Par « inhibition du gène codant la protéine D12-oleate désaturase », on entend une inhibition de l’expression du transcrit, de sa traduction, ou l’absence de protéine fonctionnelle. De manière avantageuse, la séquence de nucléotides selon l’invention est présente à l’état homozygote dans la plante de tournesol de l’invention, ladite plante produisant des graines comprenant cette séquence de nucléotides selon l’invention et ayant une teneur en acide oléique d’au moins 85 % par rapport au pourcentage total d’acides gras desdites graines. When the nucleotide sequence according to the invention is present in the heterozygous state, said plant may optionally comprise another allele which leads to an inhibition of the gene encoding the D12-oleate desaturase protein and which, in combination with the mutation according to the invention, to produce seeds having an oleic acid content of at least 85% relative to the total percentage of fatty acids of said seeds. Among the examples of other allele known from the prior art which leads to inhibition of the gene encoding the D12-oleate desaturase protein, mention may in particular be made of the Pervenets mutation. By "inhibition of the gene encoding the D12-oleate desaturase protein" is meant an inhibition of the expression of the transcript, its translation, or the absence of a functional protein. Advantageously, the nucleotide sequence according to the invention is present in the homozygous state in the sunflower plant of the invention, said plant producing seeds comprising this nucleotide sequence according to the invention and having an oleic acid content. at least 85% with respect to the total percentage of fatty acids of said seeds.
De préférence, la teneur en acide oléique de ces graines est comprise dans une gamme allant de 85 % à 95 %, par rapport au pourcentage total d’acides gras desdites graines. Preferably, the oleic acid content of these seeds is within a range from 85% to 95%, relative to the total percentage of fatty acids of said seeds.
Un autre objet de la présente invention concerne donc également des graines de tournesol susceptible d’être obtenues à partir d’une plante de tournesol telle que décrite ci-dessus. Ces graines contiennent au moins un allèle muté du gène codant la protéine D12-oleate désaturase correspondant à une séquence de nucléotides selon l’invention. Lorsque la séquence nucléotides selon l’invention est présente à l’état hétérozygote, ladite graine peut éventuellement comprendre un autre allèle qui conduit à une inhibition du gène codant la protéine A12-désaturase et qui permet, en combinaison avec la séquence nucléotides selon l’invention, d’obtenir des graines ayant une teneur en acide oléique d’au moins 85 % par rapport au pourcentage total d’acides gras desdites graines. Parmi les exemples d’autre allèle connu de l’art antérieur qui conduit à une inhibition du gène codant la protéine A12-désaturase, on peut notamment citer la mutation Pervenets. Another object of the present invention therefore also relates to sunflower seeds capable of being obtained from a sunflower plant as described above. These seeds contain at least one mutated allele of the gene encoding the D12-oleate desaturase protein corresponding to a sequence of nucleotides according to the invention. When the nucleotide sequence according to the invention is present in the heterozygous state, said seed can optionally comprise another allele which leads to inhibition of the gene encoding the A12-desaturase protein and which, in combination with the nucleotide sequence according to invention, to obtain seeds having an oleic acid content of at least 85% relative to the total percentage of fatty acids of said seeds. Among the examples of other allele known from the prior art which leads to an inhibition of the gene encoding the A12-desaturase protein, mention may in particular be made of the Pervenets mutation.
De façon préférée, les graines possèdent la séquence de nucléotides selon l’invention à l’état homozygote et ont une teneur en acide oléique d’au moins 85 % par rapport au pourcentage total d’acides gras desdites graines. De préférence, ces graines possèdent une teneur en acide oléique comprise dans une gamme allant de 85 % à 95 %. Preferably, the seeds have the nucleotide sequence according to the invention in the homozygous state and have an oleic acid content of at least 85% relative to the percentage. total fatty acids of said seeds. Preferably, these seeds have an oleic acid content within a range of from 85% to 95%.
Toute descendance d’une graine selon l’invention telle décrite ci-dessus constitue un autre objet de la présente invention dès lors qu’elle comprend une séquence de nucléotides selon l’invention comme décrit ci-dessus. Any progeny of a seed according to the invention as described above constitutes another object of the present invention since it comprises a sequence of nucleotides according to the invention as described above.
Les plantes et parties de plantes selon l’invention peuvent être obtenues par un procédé non exclusivement biologique. The plants and parts of plants according to the invention can be obtained by a process that is not exclusively biological.
Selon encore un autre aspect, la présente invention concerne un procédé d’obtention d’une plante de tournesol comprenant une séquence de nucléotides selon l’invention, comprenant les étapes suivantes : According to yet another aspect, the present invention relates to a process for obtaining a sunflower plant comprising a sequence of nucleotides according to the invention, comprising the following steps:
a) Procéder à la mutagenèse d’une partie de plante de tournesol pour qu’elle comprenne une séquence de nucléotides selon l’invention, a) Mutagenize a part of a sunflower plant so that it comprises a sequence of nucleotides according to the invention,
b) Générer une plante à partir de ladite partie de plante ayant subi ladite mutagenèse, c) Obtenir au moins une descendance à partir de la plante générée à l’étape b), et d) Identifier et sélectionner au moins une plante obtenue à l’étape b) ou c) comprenant ladite séquence de nucléotides selon l’invention. b) Generate a plant from said plant part which has undergone said mutagenesis, c) Obtain at least one progeny from the plant generated in step b), and d) Identify and select at least one plant obtained in step b) step b) or c) comprising said nucleotide sequence according to the invention.
En particulier, le procédé permettra d’obtenir une plante de tournesol à teneur élevée en acide oléique. Ce procédé selon l’invention qui inclut nécessairement une étape de mutagenèse volontaire et induite par l’homme ne constitue donc pas un procédé essentiellement biologique. Plus particulièrement, le procédé d’obtention pourra comprendre une étape supplémentaire, comme par exemple une autofécondation, pour permettre d’obtenir une plante à teneur élevée en acide oléique comprenant la séquence de nucléotides selon l’invention à l’état homozygote. Dans ce cadre, on entend par « plante de tournesol à teneur élevée en acide oléique », une plante dont la teneur en acide oléique des graines est d’au moins 85 %, par rapport à la teneur totale en acide gras de ces graines et préférentiellement dans une gamme allant d’au moins 85 à 95% ou plus préférentiellement d’au moins 86 à 95%, ou d’au moins 87 à 95% ou d’au moins 88 à 95% ou d’au moins 89 à 95%. Plus spécifiquement, le teneur pourra être comprise dans une gamme allant d’au moins 89 à 94% ou d’au moins 89 à 93% ou d’au moins 89 à 92% ou d’au moins 89 à 91 % ou d’au moins 89 à 90%. In particular, the process will result in a sunflower plant with a high oleic acid content. This process according to the invention, which necessarily includes a step of voluntary and human-induced mutagenesis, therefore does not constitute an essentially biological process. More particularly, the production process may include an additional step, such as for example self-pollination, to make it possible to obtain a plant with a high oleic acid content comprising the nucleotide sequence according to the invention in the homozygous state. In this context, the term “sunflower plant with a high oleic acid content” is understood to mean a plant in which the oleic acid content of the seeds is at least 85%, relative to the total fatty acid content of these seeds and preferably in a range ranging from at least 85 to 95% or more preferably from at least 86 to 95%, or from at least 87 to 95% or from at least 88 to 95% or from at least 89 to 95%. More specifically, the content may be within a range going from at least 89 to 94% or from at least 89 to 93% or from at least 89 to 92% or from at least 89 to 91% or at least 89 to 90%.
Pour procéder à la mutagenèse d’une partie de plante de tournesol, toute technique bien connue de l’homme du métier peut être utilisée. On peut notamment utiliser des techniques de mutagenèse aléatoire telles que la mutagenèse induite par un agent chimique ou physique comme le traitement au éthyl-méthanesulfonate (EMS) ou l’irradiation ou encore des méthodes telles que le TILLING ( Targeting Induced Local Lésions in Genomes) (Till et al, 2003), ou encore telle que la technique de recombinaison aléatoire ou « DNA shuffling » (Stemmer, 1994). Il est également possible d’utiliser toute technique d’édition de gènes, permettant une modification ciblée des gènes, telle que TALENs, telle que décrite dans WO201 1072246 ou la technologie CRISPR comme décrite par exemple dans W02013181440, ou grâce aux technologies comme celles décrites dans WO2017004375 ou WO2018015956 couplant la technologie d’édition et l’induction d’haploïdes. To carry out the mutagenesis of a part of a sunflower plant, any technique well known to those skilled in the art can be used. It is in particular possible to use random mutagenesis techniques such as mutagenesis induced by a chemical or physical agent, such as treatment with ethyl-methanesulfonate (EMS) or irradiation, or else methods such as TILLING (Targeting Induced Local Lesions in Genomes) (Till et al, 2003), or even such as the random recombination technique or “DNA shuffling” (Stemmer, 1994). It is also possible to use any gene editing technique, allowing targeted modification of genes, such as TALENs, as described in WO201 1072246 or CRISPR technology as described for example in WO2013181440, or using technologies such as those described. in WO2017004375 or WO2018015956 coupling the editing technology and the induction of haploids.
Dans le cas d’utilisation de technologie d’édition ciblée, la mutation selon l’invention pourra être générée directement sur des lignées élites de tournesol qui seront utilisées comme géniteurs dans la production d’hybrides. In the case of using targeted editing technology, the mutation according to the invention can be generated directly on elite sunflower lines which will be used as progenitors in the production of hybrids.
De préférence, on utilise une cellule, un tissu cellulaire, un cal ou une graine issue de la plante comme partie de plante du tournesol pour procéder à l’étape a) de mutagenèse dans le cadre du procédé d’obtention d’une plante de tournesol selon l’invention. Preferably, a cell, a cellular tissue, a callus or a seed obtained from the plant is used as part of the sunflower plant to carry out step a) of mutagenesis in the context of the process for obtaining a plant from sunflower according to the invention.
Les méthodes permettant de générer une plante à partir d’une partie de plante ayant subi une mutagenèse, ainsi que pour produire une descendance à partir de celle-ci, sont bien connues de l’homme du métier. Methods for generating a plant from a mutagenized plant part, as well as for producing progeny therefrom, are well known to those skilled in the art.
L’identification et la sélection d’une plante obtenue à l’étape b) peuvent être réalisées par toute technique bien connue de l’homme du métier. Notamment, par des techniques d’amplification PCR, Northern Blot ou Southern Blot, puces, « ligase chain reaction » (LCR), et « genotyping- by-sequence » (GBS), ou une combinaison de ces techniques. The identification and selection of a plant obtained in step b) can be carried out by any technique well known to those skilled in the art. In particular, by PCR, Northern Blot or Southern Blot amplification techniques, microarrays, "ligase chain reaction" (LCR), and "genotyping-by-sequence" (GBS), or a combination of these techniques.
Différentes méthodes de PCR sont connues de l’homme du métier, comme la RT-PCR ou la méthode mettant en oeuvre des amorces de type Kaspar (KBioscience (LGC Group, Teddington, Middlesex, UK). Various PCR methods are known to those skilled in the art, such as RT-PCR or the method using primers of the Kaspar type (KBioscience (LGC Group, Teddington, Middlesex, UK).
La méthode de génotypage Kompetitive Allele Spécifie PCR « KASP™ » utilise trois amorces cibles spécifiques : deux amorces, chacune étant spécifique de chaque forme allélique d’un SNP donné ( Single Nucléotide Poiymorphism) et une troisième amorce pour permettre l’amplification reverse pour chaque forme allélique. The Kompetitive Allele Specifies PCR "KASP ™" genotyping method uses three specific target primers: two primers, each specific for each allelic form of a given SNP (Single Nucleotide Poiymorphism) and a third primer to allow reverse amplification for each. allelic form.
Les moyens d’identification selon l’invention qui sont décrits ci-dessous sont avantageusement utilisés dans le procédé d’obtention ou d’identification d’une plante de tournesol à teneur élevée en acide oléique selon l’invention. The means of identification according to the invention which are described below are advantageously used in the process for obtaining or identifying a sunflower plant with a high oleic acid content according to the invention.
Selon un autre aspect, la présente invention concerne un procédé d’identification de la présence, ou de l’absence, d’une séquence de nucléotides ou d’acides d’aminés selon l’invention dans une plante de tournesol, qui comprend l’extraction du matériel génétique de la plante tel que l’ADN génomique, l’ARN ou le protéome, et l’utilisation de moyens d’identification de la séquence de nucléotides ou d’acides aminés selon l’invention. According to another aspect, the present invention relates to a method for identifying the presence, or the absence, of a nucleotide or amino acid sequence according to the invention in a sunflower plant, which comprises the extraction of the genetic material of the plant such as genomic DNA, RNA or proteome, and the use of means for identifying the sequence of nucleotides or amino acids according to the invention.
Par marqueur moléculaire, on entend par exemple un fragment spécifique d'une séquence pouvant être identifiée au sein du génome d’une plante à l’aide de différents moyens d’identification tels que des sondes ou amorces. Le marqueur peut être un fragment de séquence de protéine, pouvant être identifiée au sein de l’ensemble des protéines d’une cellule ou protéome d’une plante, notamment à l’aide d’anticorps. En particulier, un marqueur moléculaire spécifique de la séquence nucléotides selon l’invention correspond à un fragment d’au moins 20 nucléotides de la séquence nucléotides selon l’invention, ledit fragment comprenant la mutation correspondant au codon STOP aux positions 484 à 486 en référence à la séquence sauvage SEQ ID NO : 1. Le marqueur peut être dominant, co-dominant. By molecular marker is meant, for example, a specific fragment of a sequence that can be identified within the genome of a plant using various means identification such as probes or primers. The marker can be a fragment of a protein sequence which can be identified within all the proteins of a cell or proteome of a plant, in particular using antibodies. In particular, a specific molecular marker of the nucleotide sequence according to the invention corresponds to a fragment of at least 20 nucleotides of the nucleotide sequence according to the invention, said fragment comprising the mutation corresponding to the STOP codon at positions 484 to 486 with reference to the wild-type sequence SEQ ID NO: 1. The marker can be dominant, co-dominant.
L’identification de la séquence d’intérêt, ou sa non-identification, permet de sélectionner les plantes présentant le gène ou l’allèle d'intérêt ou la caractéristique particulière, ou au contraire, de ne pas sélectionner les plantes ne présentant pas la séquence d'intérêt. The identification of the sequence of interest, or its non-identification, makes it possible to select the plants exhibiting the gene or allele of interest or the particular characteristic, or on the contrary, not to select the plants not exhibiting the sequence of interest.
Dans la présente invention, les moyens d’identification peuvent être des sondes ou des amorces d’au moins 12 nucléotides jusqu’à 30 nucléotides complémentaires du brin d’ADN que l’on souhaite amplifier, hybrider ou encore séquencer. Ces moyens d’identification sont en l’occurrence spécifique du marqueur que l’on souhaite identifier et permettent de détecter rapidement la présence de la mutation selon l’invention dans le génome de plantes issues de croisements donnés, ou les parties de plantes. In the present invention, the means of identification can be probes or primers of at least 12 nucleotides up to 30 nucleotides complementary to the DNA strand that it is desired to amplify, hybridize or even sequence. These means of identification are in this case specific for the marker to be identified and make it possible to rapidly detect the presence of the mutation according to the invention in the genome of plants resulting from given crosses, or parts of plants.
Ces moyens d’identification peuvent constituer des moyens d’identification et/ou de sélection de plantes de tournesol qui contiennent au moins un allèle de la séquence selon l’invention décrite ci-dessus, De préférence, ils permettent d’identifier et/ou de sélectionner des plantes de tournesol qui produisent des graines ayant une teneur en acide oléique d’au moins 85%, et de préférence comprise dans une gamme allant de 85 % à 95%. En particulier, lorsque ladite plante comprend une séquence nucléotides selon l’invention à l’état hétérozygote, ladite plante peut comprendre un autre allèle qui conduit à une inhibition du gène codant la protéine D12- désaturase et qui permet, en combinaison avec la séquence nucléotides selon l’invention, d’obtenir des graines ayant une teneur en acide oléique d’au moins 85 % par rapport au pourcentage total d’acides gras desdites graines. Parmi les exemples d’autre allèle connu de l’art antérieur qui conduit à une inhibition du gène codant la protéine A12-désaturase, on peut notamment citer la mutation Pervenets. These identification means can constitute means of identification and / or selection of sunflower plants which contain at least one allele of the sequence according to the invention described above. Preferably, they make it possible to identify and / or to select sunflower plants which produce seeds having an oleic acid content of at least 85%, and preferably within a range of from 85% to 95%. In particular, when said plant comprises a nucleotide sequence according to the invention in the heterozygous state, said plant may comprise another allele which leads to inhibition of the gene encoding the D12-desaturase protein and which, in combination with the nucleotide sequence, allows according to the invention, to obtain seeds having an oleic acid content of at least 85% relative to the total percentage of fatty acids of said seeds. Among the examples of other allele known from the prior art which leads to an inhibition of the gene encoding the A12-desaturase protein, mention may in particular be made of the Pervenets mutation.
De façon préférée, ces moyens d’identification permettent d’identifier et/ou de sélectionner des plantes de tournesol qui produisent des graines qui possèdent la séquence nucléotides selon l’invention à l’état homozygote et ont une teneur en acide oléique d’au moins 85 % par rapport au pourcentage total d’acides gras desdites graines, et de préférence comprise dans une gamme allant de 85 % à 95%. Preferably, these identification means make it possible to identify and / or select sunflower plants which produce seeds which have the nucleotide sequence according to the invention in the homozygous state and have an oleic acid content of at least less 85% relative to the total percentage of fatty acids of said seeds, and preferably within a range from 85% to 95%.
Les moyens d’identification d’une plante, partie de plante selon l’invention, constituent également un des aspects de la présente invention. Préférentiellement, ces moyens d’identification sont capables de détecter spécifiquement la mutation dans la séquence nucléotidique selon l’invention, à savoir la présence d’un codon STOP aux positions 484 à 486 en référence à la séquence SEQ ID NO : 1. Comme indiqué ci-dessus, ces moyens peuvent consister en des amorces ou des sondes spécifiques de la mutation décrite ci-dessus selon l’invention. Plus particulièrement, le jeu d’amorces qui peut être utilisé dans les procédés selon l’invention est le jeu d’amorces KASP de séquences SEQ ID NO : 7, 8 et 9. The means for identifying a plant, part of a plant according to the invention, also constitute one of the aspects of the present invention. Preferably, these means identification are capable of specifically detecting the mutation in the nucleotide sequence according to the invention, namely the presence of a STOP codon at positions 484 to 486 with reference to the sequence SEQ ID NO: 1. As indicated above, these means may consist of primers or probes specific for the mutation described above according to the invention. More particularly, the set of primers which can be used in the methods according to the invention is the set of KASP primers of sequences SEQ ID NO: 7, 8 and 9.
Selon un autre aspect, la présente invention couvre également un procédé de croisement par sélection dite « forward » d’une plante de tournesol comprenant une séquence de nucléotides selon l’invention ; ledit procédé comprenant les étapes suivantes : According to another aspect, the present invention also covers a method of crossing by so-called "forward" selection of a sunflower plant comprising a sequence of nucleotides according to the invention; said method comprising the following steps:
a) Croisement de ladite plante avec toute autre plante de tournesol présentant des caractéristiques agronomiques pour obtenir une plante F1 , a) Crossing of said plant with any other sunflower plant having agronomic characteristics to obtain an F1 plant,
b) Autofécondation d’une plante F1 comprenant ladite séquence de nucléotides selon l’invention, b) Self-fertilization of an F1 plant comprising said nucleotide sequence according to the invention,
c) Répétition de l’étape b) pour obtenir une descendance comprenant ladite séquence de nucléotides selon l’invention et présentant au moins une desdites caractéristiques agronomiques. c) Repeating step b) to obtain progeny comprising said nucleotide sequence according to the invention and exhibiting at least one of said agronomic characteristics.
De préférence, la descendance présente la séquence nucléotides selon l’invention à l’état homozygote. Preferably, the progeny have the nucleotide sequence according to the invention in the homozygous state.
Le croisement peut être réalisé par toute méthode classique bien connue dans le domaine. L’étape c) peut être répétée autant de fois que nécessaire pour obtenir une descendance comprenant la séquence de nucléotides selon l’invention. Par caractéristique agronomique, on entend tout trait d’intérêt permettant d’améliorer une plante ou une variété, comme par exemple des résistances aux herbicides ou à des pathogènes ou tout autre trait permettant d’améliorer des caractères quantitatifs ou qualitatifs. The crossing can be carried out by any conventional method well known in the field. Step c) can be repeated as many times as necessary to obtain progeny comprising the nucleotide sequence according to the invention. By agronomic characteristic is meant any trait of interest making it possible to improve a plant or a variety, for example resistance to herbicides or pathogens or any other trait making it possible to improve quantitative or qualitative characteristics.
De préférence, cette descendance comprend un gène codant une protéine de séquence SEQ ID NO : 4. Preferably, this progeny comprises a gene encoding a protein of sequence SEQ ID NO: 4.
La sélection « forward » est généralement utilisée pour améliorer les performances agronomiques d’une variété ou d’une lignée portant une mutation ou un trait spécifique alors que la sélection par rétrocroisement est utilisée pour introduire une mutation ou un trait spécifique dans le contexte génotypique particulier d’une variété ou lignée. Une comparaison des deux procédés est décrite dans Mumm et al, 2007. Forward selection is generally used to improve the agronomic performance of a variety or line carrying a specific mutation or trait while backcross selection is used to introduce a specific mutation or trait into the particular genotypic context. of a variety or line. A comparison of the two methods is described in Mumm et al, 2007.
Selon encore un autre aspect, l’invention concerne un procédé d’introgression par rétrocroisement d’un fragment de génome comprenant une séquence de nucléotides selon l’invention telle que décrite ci-dessus, à partir d’une plante de tournesol donneuse vers une plante de tournesol receveuse qui ne contient pas ladite séquence de nucléotides dans son génome, ledit procédé comprenant les étapes suivantes : According to yet another aspect, the invention relates to a process for introgression by backcrossing of a genome fragment comprising a nucleotide sequence according to the invention as described above, from a donor sunflower plant to a recipient sunflower plant which does not contain said nucleotide sequence in its genome, said method comprising the following steps:
a) Croisement de la plante de tournesol donneuse avec la plante receveuse, a) Crossing of the donor sunflower plant with the recipient plant,
b) Obtention d’un hybride F1 , b) Obtaining an F1 hybrid,
c) Rétrocroisement dudit hybride F1 avec la plante receveuse, c) Backcrossing of said F1 hybrid with the recipient plant,
d) Sélection d’une plante comprenant ladite séquence de nucléotides selon l’invention, e) Rétrocroisement de la plante sélectionnée à l’étape d) avec la plante receveuse, f) Optionnellement, autofécondation des plantes obtenues en e) pour obtenir une plante comprenant ladite séquence de nucléotides selon l’invention à l’état homozygote. d) Selection of a plant comprising said nucleotide sequence according to the invention, e) Backcrossing of the plant selected in step d) with the recipient plant, f) Optionally, self-pollination of the plants obtained in e) to obtain a plant comprising said nucleotide sequence according to the invention in the homozygous state.
La plante receveuse ne contient pas dans son génome la séquence de nucléotides selon l’invention, c’est-à-dire une séquence de nucléotides présentant un codon STOP aux positions 484 à 486 en référence à la séquence nucléotidique sauvage SEQ ID NO : 1 et codant une protéine A12-désaturase qui présente au moins 95% d’identité avec SEQ ID NO : 4, et préférentiellement, qui présente au moins 96%, 97%, 98% ou au moins 99% d’identité avec SEQ ID NO : 4. Ainsi, la plante receveuse ne contient pas dans son génome la mutation décrite ci-dessus, à savoir un codon STOP aux positions aux positions 484 à 486 en référence à la séquence SEQ ID NO : 1. The recipient plant does not contain in its genome the sequence of nucleotides according to the invention, that is to say a sequence of nucleotides having a STOP codon at positions 484 to 486 with reference to the wild-type nucleotide sequence SEQ ID NO: 1 and encoding an A12-desaturase protein which has at least 95% identity with SEQ ID NO: 4, and preferably, which has at least 96%, 97%, 98% or at least 99% identity with SEQ ID NO : 4. Thus, the recipient plant does not contain in its genome the mutation described above, namely a STOP codon at positions at positions 484 to 486 with reference to the sequence SEQ ID NO: 1.
Les rétrocroisements effectués visent à accroître le pourcentage de génome de la plante récurrente ou receveuse. Le nombre d’autofécondation ou rétrocroisements effectués est répété autant de fois pour obtenir un pourcentage d'au moins 80% du génome de la plante receveuse utilisée dans le croisement, de préférence d’au moins 98%. The backcrosses carried out aim to increase the percentage of the genome of the recurrent or recipient plant. The number of inbreeding or backcrosses performed is repeated as many times to obtain a percentage of at least 80% of the genome of the recipient plant used in the cross, preferably at least 98%.
Enfin, selon un dernier aspect, la présente invention concerne l’utilisation de graines de tournesol selon l’invention telles que décrites ci-dessus ou leur descendance, pour obtenir de l’huile, et en particulier de l’huile ayant une teneur élevée en acide oléique, c’est-à-dire d’au moins 85 % d’acide oléique par rapport à la quantité totale d’acides gras présents dans cette huile. Finally, according to a final aspect, the present invention relates to the use of sunflower seeds according to the invention as described above or their descendants, to obtain oil, and in particular oil having a high content. oleic acid, that is to say at least 85% of oleic acid relative to the total quantity of fatty acids present in this oil.
Les techniques d’obtention d’huile à partir de graines de tournesol sont bien connue dans le domaine de l’invention. EXEMPLES Techniques for obtaining oil from sunflower seeds are well known in the field of the invention. EXAMPLES
EXEMPLE 1 : Création et criblage d’une population TILLING de tournesol. EXAMPLE 1: Creation and screening of a sunflower TILLING population.
La population TILLING a été obtenue en 2015 à partir de 2 variétés de tournesol selon le protocole KumarA. ét al (2013). La population comprend initialement 18407 familles M2 à partir desquelles 10000 familles M2 ayant plus de 50 graines par famille ont été sélectionnées pour isoler l’ADN génomique et réaliser le criblage. L’extraction d’ADN a été réalisée à partir de matériel foliaire de 5 plantes individuelles par familles M2. L’ADN est normalisé à 5 ng/mI pour les expérimentations suivantes. EXEMPLE 2 : Identification in silico de codons STOP potentiels dans la séquence sauvage du gène fad2-1. The TILLING population was obtained in 2015 from 2 varieties of sunflower according to the KumarA protocol. et al (2013). The population initially comprises 18,407 M2 families from which 10,000 M2 families with more than 50 seeds per family were selected for isolate genomic DNA and perform screening. DNA extraction was carried out from leaf material from 5 individual plants per M2 families. The DNA is normalized to 5 ng / mI for the following experiments. EXAMPLE 2 Identification in silico of potential STOP codons in the wild-type sequence of the fad2-1 gene.
La séquence du gène fad2-1 sauvage HanXRQChr14g0452931 ou autrement décrite dans SEQ ID NO :1) a été analysées pour identifier l’ensemble des codons STOP pouvant être générés par le traitement EMS. 34 potentielles mutations ponctuelles EMS générant chacune un codon STOP pouvant produire une protéine FAD2 tronquée ont été identifiées. Des amorces spécifiques ciblant spécifiquement chacune des 34 mutations ont été générés pour le criblage des familles M2. The sequence of the wild-type HanXRQChr14g0452931 fad2-1 gene or otherwise described in SEQ ID NO: 1) was analyzed to identify all the STOP codons that can be generated by the EMS treatment. 34 potential EMS point mutations each generating a STOP codon capable of producing a truncated FAD2 protein have been identified. Specific primers specifically targeting each of the 34 mutations were generated for screening M2 families.
EXEMPLE 3 : Identification de la mutation EXAMPLE 3: Identification of the mutation
L’identification de la mutation a été réalisée à partir de pool d’ADNs issus de 96 familles M2 par amplification PCR. Puis, grâce à l’analyse des pools, la présence de mutations potentielles a été vérifiée chez 10 graines individuelles M2. Une seule famille mutante (HABT-3816 M2) a été confirmée et la mutation a été identifiée par séquençage Sanger. La mutation identifiée correspond à une modification G en A localisée à la position nucléotidique 485 dans la séquence décrite dans SEQ ID NO : 1. La mutation ponctuelle créé un codon STOP qui produit une protéine tronquée de 161 acides-aminés décrites dans la séquence SEQ ID NO : 4, au lieu des 378 acides-aminés de la protéine sauvage décrite dans la séquence SEQ ID NO : 3 (Figure 1). Les séquences contextes mutante et sauvage utilisées pour l’identification de la mutation STOP dans la famille mutante sont décrites dans les séquences SEQ ID NO : 5 (ACCACCGCCACCATTCCAACACTGGATCACTCGAGCGGGACGAGGTTTTCGTCCCCAA AT CCCGATCGAAAGTCCCGTAGT ACT CG AAAT ACTTT AACAACACAGTGGGCCGCATT GT The identification of the mutation was carried out from a pool of DNAs from 96 M2 families by PCR amplification. Then, thanks to the analysis of the pools, the presence of potential mutations was checked in 10 individual M2 seeds. A single mutant family (HABT-3816 M2) was confirmed and the mutation was identified by Sanger sequencing. The mutation identified corresponds to a modification G to A located at nucleotide position 485 in the sequence described in SEQ ID NO: 1. The point mutation creates a STOP codon which produces a truncated protein of 161 amino acids described in the sequence SEQ ID NO: 4, instead of the 378 amino acids of the wild-type protein described in the sequence SEQ ID NO: 3 (Figure 1). The mutant and wild-type context sequences used for the identification of the STOP mutation in the mutant family are described in the sequences SEQ ID NO: 5 (ACCACCGCCACCATTCCAACACTGGATCACTCGAGCGGGACGAGGTTTTCGTCCCCAA AT CCCGATCGAAAGTCCCGTAGT ACT CG AAATCC ACTTGATTGGGTAGT ACT CG AAATCC ACTTGATTGGGTAGT ACT CG AAATCC ACTT
CAGTATGTTCGTCACTCTCACTCTCGGCTGGCCCTTGTACTTAGCTTTCAATGTGTCGGG CAGTATGTTCGTCACTCTCACTCTCGGCTGGCCCTTGTACTTAGCTTTCAATGTGTCGGG
) et SEQ ID NO : 6) and SEQ ID NO: 6
(ACCACCGCCACCATTCCAACACTGGATCACTCGAGCGGGACGAGGTTTTCGTCCCCAA AT CCCGAT CG AAAGTCCCGTGGT ACT CG AAAT ACTTT AACAACACAGTGGGCCGCATT G TCAGTATGTTCGTCACTCTCACTCTCGGCTGGCCCTTGTACTTAGCTTTCAATGTGTCGG (ACCACCGCCACCATTCCAACACTGGATCACTCGAGCGGGACGAGGTTTTCGTCCCCAA AT CCCGAT CG AAAGTCCCGTGGT ACT CG AAAT ACTTT AACAACACAGTGGGCCGCATT G TCAGTATGTTCGTCGTCACTCTCACTCTCGTCGTCGTGTAGTAGCT
G). G).
Ces séquences génomiques flanquants la mutation d’intérêt G485A peuvent être utilisées pour développer un test de génotypage pour suivre la mutation dans une procédure de sélection assistée par marqueur. Dans cet exemple, des marqueurs codominants“KASP” (Ha_FAD2- 1_2439_E1_485) ont été développés et les amorces pour l’identification de la mutation G485A dans le gène fad2-1 sont décrites dans le tableau 1 . Ils permettent de différentier entre elles, les plantes homozygotes pour l’allèle mutant, les plantes homozygotes pour l’allèle normal et les plantes hétérozygotes. TABLEAU 1 These genomic sequences flanking the G485A mutation of interest can be used to develop a genotyping test to track the mutation in a marker-assisted selection procedure. In this example, codominant “KASP” markers (Ha_FAD2-1_2439_E1_485) were developed and the primers for the identification of the G485A mutation. in the fad2-1 gene are described in Table 1. They make it possible to differentiate among themselves, plants homozygous for the mutant allele, plants homozygous for the normal allele and heterozygous plants. TABLE 1
Figure imgf000015_0001
Figure imgf000015_0001
EXEMPLE 4 : Validation phénotypique et utilisation en sélection. EXAMPLE 4 Phenotypic validation and use in selection.
Afin d’évaluer l’effet de la mutation, la composition en huile saturée a été mesurée à partir de 73 graines ségrégées à partir d’une plante hétérozygote au locus HABT-3816. Pour chaque graine, lune moitié est utilisée pour mesurer la composition en acides saturés par In order to assess the effect of the mutation, the saturated oil composition was measured from 73 seeds segregated from a heterozygous plant at the HABT-3816 locus. For each seed, one half is used to measure the saturated acid composition by
Chromatographie en Phase Gazeuse (CPG) et l’autre moitié est semée pour réaliser le génotypage sur feuilles. Gas Chromatography (GC) and the other half is sown for leaf genotyping.
La CPG est réalisée à l’aide du dispositif GC Thermo 1310 GC_1 et GC_2 selon le protocole suivant : The GC is carried out using the GC Thermo 1310 GC_1 and GC_2 device according to the following protocol:
Des broyais de graines de tournesol sont aliquotés dans des boites 96 de type Collection Microtubes racked 10x96. Crushed sunflower seeds are aliquoted in 96 boxes of the Microtubes Racked 10x96 Collection type.
Dans une première étape les acides gras sont dissolus dans l’isooctane (2,2,4 -Trimethyl pentane) puis conversion en esters méthyliques par ajout de l’hydroxyde de potassium méthanolique (2N KOH/MeOH). In a first step, the fatty acids are dissolved in isooctane (2,2,4 -Trimethyl pentane) and then converted into methyl esters by adding methanolic potassium hydroxide (2N KOH / MeOH).
Une fois la réaction terminée, l’hydroxyde de potassium est neutralisé avec de l’hydrogénosulfate de sodium, afin d’éviter la saponification des esters méthyliques. After the reaction is complete, the potassium hydroxide is neutralized with sodium hydrogen sulphate to prevent the saponification of the methyl esters.
Enfin, les esters méthyliques sont séparés et quantifiés par CPG. Finally, the methyl esters are separated and quantified by GPC.
Pour le génotypage, des feuilles de jeunes plantes ont été échantillonnées en vue du génotypage à l’aide des marqueurs Ha_FAD2-1_2439_E1_485 KASP décrits dans l’exemple 3. 29 graines sont des plantes mutantes homozygotes (A : A), 44 graines sont hétérozygotes (A : G) et 10 graines sont des plantes sauvages homozygotes (G : G) ont été respectivement analysées pour leur teneur en acides gras For genotyping, leaves of young plants were sampled for genotyping using the Ha_FAD2-1_2439_E1_485 KASP markers described in Example 3.29 seeds are homozygous mutant plants (A: A), 44 seeds are heterozygous (A: G) and 10 seeds are homozygous wild plants (G: G) were respectively analyzed for their fatty acid content
Les résultats de correspondance entre génotype et phénotype sont présentés dans le tableau 2 qui regroupe les moyennes des teneurs en acides gras des plantes mutantes homozygotes The results of correspondence between genotype and phenotype are presented in Table 2 which groups together the means of the fatty acid contents of the homozygous mutant plants.
(A:A), hétérozygotes (A:G) et des plantes sauvages homozygotes (G:G) (s.e.: écart-type). La mutation est récessive avec un effet majeur sur la teneur en acide Oléique et linoléique. Les graines homozygotes qui portent la mutation ont une teneur stable en acide oléique supérieure à 90% (moyenne de 92.8 +/- 0.17 %) et un très faible niveau en acide linoléique. (A: A), heterozygous (A: G) and homozygous wild plants (G: G) (s.e .: standard deviation). The mutation is recessive with a major effect on the oleic and linoleic acid content. Homozygous seeds which carry the mutation have a stable oleic acid content greater than 90% (average 92.8 +/- 0.17%) and a very low level of linoleic acid.
TABLEAU 2 TABLE 2
Figure imgf000016_0001
Figure imgf000016_0001
REFERENCES REFERENCES
Altschul et al, (1997), Nucleic Acids Res. 25:3389-3402 Altschul et al, (1997), Nucleic Acids Res. 25: 3389-3402
Altschul et al, (2005) FEBS J. 272:5101 -5109) Altschul et al, (2005) FEBS J. 272: 5101-5109)
Garces R, Mancha M (1991) "In vitro oleate desaturase in sunflower seeds developing" Phytochemistry 30:2127-2130 Garces R, Mancha M (1991) "In vitro oleate desaturase in sunflower seeds developing" Phytochemistry 30: 2127-2130
Hongtrakul et al. (1998) « A seed spécifie D 12 oleate-desaturase gene is duplicated, rearranged and weakly expressed in high oleic acid sunflower lines”. Crop Sci 38:1245-1249 Kumar A et al. (2013) SMART - Sunflower Mutant population And Reverse genetic Tool for crop improvement. BMC Plant Biol. 13: 38. Lacombe et al. (2001) « An oleate-desaturase and a suppressor locus direct high oleic acid content of sunflower (Helianthus annuus L.) oil in the Pervenets mutant”. C.R. Acad. Sci, Life Sci 324:839-845. Hongtrakul et al. (1998) “A seed specifies D 12 oleate-desaturase gene is duplicated, rearranged and weakly expressed in high oleic acid sunflower lines”. Crop Sci 38: 1245-1249 Kumar A et al. (2013) SMART - Sunflower Mutant population And Reverse genetic Tool for crop improvement. BMC Plant Biol. 13:38. Lacombe et al. (2001) “An oleate-desaturase and a suppressor locus direct high oleic acid content of sunflower (Helianthus annuus L.) oil in the Pervenets mutant”. C.R. Acad. Sci, Life Sci 324: 839-845.
Mumm et al (2007)“Backcross versus Forward Breeding in the Development of Transgenic Maize Hybrids: Theory and Practice”. Crop Sci (Suppl 3) 47: S164-S171. Mumm et al (2007) “Backcross versus Forward Breeding in the Development of Transgenic Maize Hybrids: Theory and Practice”. Crop Sci (Suppl 3) 47: S164-S171.
Soldatov (1976)“Chemical mutagenesis in sunflower breeding”. InProc. Vllthlnt. Sunflower Conférence, June 27th - July 3, Krasnodar. Soldatov (1976) “Chemical mutagenesis in sunflower breeding”. InProc. Vllthlnt. Sunflower Conference, June 27th - July 3, Krasnodar.

Claims

REVENDICATIONS
1. Séquence de nucléotides isolée qui comprend un codon stop aux positions 484 à 486 en référence à la séquence de nucléotides sauvage SEQ ID NO : 1 et qui code une protéine D12- oleate désaturase qui présente au moins 95% d’identité avec SEQ ID NO : 4. 1. Isolated nucleotide sequence which comprises a stop codon at positions 484 to 486 with reference to the wild-type nucleotide sequence SEQ ID NO: 1 and which encodes a D12-oleate desaturase protein which has at least 95% identity with SEQ ID NO: 4.
2. Séquence de nucléotides selon la revendication 1 de séquence SEQ ID NO : 2. 2. Nucleotide sequence according to claim 1 of sequence SEQ ID NO: 2.
3. Protéine A12-oleate désaturase de tournesol tronquée de séquence SEQ ID NO : 4. 3. Truncated sunflower A12-oleate desaturase protein of sequence SEQ ID NO: 4.
4. Plante de tournesol comprenant la séquence de nucléotides selon la revendication 1 ou 2. 4. Sunflower plant comprising the nucleotide sequence according to claim 1 or 2.
5. La plante de tournesol selon la revendication 4, dans laquelle la séquence de nucléotides selon la revendication 1 ou 2 est présente dans le génome de ladite plante à l’état hétérozygote ou homozygote. 5. The sunflower plant according to claim 4, wherein the nucleotide sequence according to claim 1 or 2 is present in the genome of said plant in the heterozygous or homozygous state.
6. La plante de tournesol selon la revendication 4 ou 5, dans laquelle la plante est une lignée ou un hybride. 6. The sunflower plant according to claim 4 or 5, wherein the plant is a line or a hybrid.
7. La plante de tournesol selon l’une quelconque des revendications 4 à 6, caractérisée en ce qu’elle produit des graines comprenant la séquence de nucléotides selon la revendication 1 ou 2 et que lesdites graines ont une teneur en acide oléique d’au moins 85 %, de préférence une teneur comprise dans une gamme allant de 85 % à 95% par rapport au pourcentage total d’acides gras desdites graines. 7. The sunflower plant according to any one of claims 4 to 6, characterized in that it produces seeds comprising the nucleotide sequence according to claim 1 or 2 and that said seeds have an oleic acid content of at less 85%, preferably a content within a range from 85% to 95% relative to the total percentage of fatty acids of said seeds.
8. Graine de tournesol susceptible d’être obtenues à partir d’une plante selon l’une quelconque des revendications 4 à 7, caractérisée en ce qu’elle comprend la séquence de nucléotides selon la revendication 1 ou 2 et qu’elle possède une teneur en acides oléiques d’au moins 85 %, de préférence une teneur comprise dans une gamme allant de 85 % à 95% par rapport au pourcentage total d’acides gras desdites graines. 8. Sunflower seed obtainable from a plant according to any one of claims 4 to 7, characterized in that it comprises the nucleotide sequence according to claim 1 or 2 and that it has a oleic acid content of at least 85%, preferably a content within a range from 85% to 95% relative to the total percentage of fatty acids of said seeds.
9. Descendance d’une graine selon la revendication 8, caractérisée en ce qu’elle comprend la séquence de nucléotides selon la revendication 1 ou 2. 9. Progeny of a seed according to claim 8, characterized in that it comprises the sequence of nucleotides according to claim 1 or 2.
10. Procédé d’obtention d’une plante de tournesol comprenant une séquence nucléotides selon l’une des revendications 1 à 2, ledit procédé comprenant les étapes suivantes : 10. A method of obtaining a sunflower plant comprising a nucleotide sequence according to one of claims 1 to 2, said method comprising the following steps:
a) Procéder à la mutagenèse d’une partie de plante de tournesol pour qu’elle comprenne une séquence de nucléotides selon l’une des revendications 1 ou 2 a) Mutagenize a part of a sunflower plant so that it comprises a nucleotide sequence according to one of claims 1 or 2
b) Générer une plante à partir de ladite partie de plante ayant subi ladite mutagenèse c) Obtenir au moins une descendance à partir de la plante générée à l’étape b), et d) Identifier et sélectionner au moins une plante obtenue à l’étape b) ou c) comprenant ladite séquence de nucléotides selon la revendication 1 ou 2. b) Generate a plant from said plant part which has undergone said mutagenesis c) Obtain at least one progeny from the plant generated in step b), and d) Identify and select at least one plant obtained by step b) or c) comprising said nucleotide sequence according to claim 1 or 2.
1 1 . Le procédé d’obtention selon la revendication 10, dans lequel l’étape a) de mutagenèse est réalisée par un agent chimique ou physique ou par l’utilisation de toute technique d’édition de gènes. 1 1. The production process according to claim 10, wherein step a) of mutagenesis is carried out by a chemical or physical agent or by the use of any gene editing technique.
12. Procédé d’identification de la présence d’une séquence selon l’une quelconque des revendications 1 à 3 dans une plante de tournesol, comprenant l’extraction de l’ADN génomique, de l’ARN ou du protéome de la plante et l’utilisation de moyens d’identification de ladite séquence. 12. A method of identifying the presence of a sequence according to any one of claims 1 to 3 in a sunflower plant, comprising extracting the genomic DNA, RNA or proteome of the plant and the use of means for identifying said sequence.
13. Moyens d’identification d’une plante, partie de plante selon l’une quelconque des revendications 4 à 9. 13. Means of identifying a plant, plant part according to any one of claims 4 to 9.
14. Procédé de croisement par sélection dite « forward » d’une plante de tournesol comprenant une séquence de nucléotides selon l’une des revendications 1 ou 2 ; ledit procédé comprenant les étapes suivantes : 14. A method of crossing by so-called "forward" selection of a sunflower plant comprising a nucleotide sequence according to one of claims 1 or 2; said method comprising the following steps:
a) Croisement de ladite plante avec toute autre plante de tournesol présentant des caractéristiques agronomiques pour obtenir une plante F1 , a) Crossing of said plant with any other sunflower plant having agronomic characteristics to obtain an F1 plant,
b) Autofécondation d’une plante F1 comprenant ladite séquence de nucléotides, b) Self-fertilization of an F1 plant comprising said nucleotide sequence,
c) Répétition de l’étape b) pour obtenir une descendance comprenant ladite séquence de nucléotides et présentant au moins une desdites caractéristiques agronomiques. c) Repeating step b) to obtain progeny comprising said nucleotide sequence and exhibiting at least one of said agronomic characteristics.
15. Procédé d’introgression par rétrocroisement d’un fragment de génome comprenant une séquence de nucléotides selon la revendication 1 ou 2, à partir d’une plante de tournesol donneuse vers une plante de tournesol receveuse qui ne contient pas ladite séquence de nucléotides dans son génome, ledit procédé comprenant les étapes suivantes : 15. A method of introgression by backcrossing a genome fragment comprising a nucleotide sequence according to claim 1 or 2, from a donor sunflower plant to a recipient sunflower plant which does not contain said nucleotide sequence in its genome, said method comprising the following steps:
a) Croisement de la plante de tournesol donneuse avec la plante receveuse, b) Obtention d’un hybride F1 , a) Crossing of the donor sunflower plant with the recipient plant, b) Obtaining an F1 hybrid,
c) Rétrocroisement dudit hybride F1 avec la plante receveuse, c) Backcrossing of said F1 hybrid with the recipient plant,
d) Sélection d’une plante comprenant ladite séquence de nucléotides selon la revendication 1 ou 2, d) Selection of a plant comprising said nucleotide sequence according to claim 1 or 2,
e) Rétrocroisement de la plante sélectionnée à l’étape d) avec la plante receveuse f) Optionnellement, autofécondation des plantes obtenues en e) pour obtenir une plante comprenant ladite séquence de nucléotides à l’état homozygote. e) Backcrossing of the plant selected in step d) with the recipient plant f) Optionally, self-pollination of the plants obtained in e) to obtain a plant comprising said sequence of nucleotides in the homozygous state.
16. Utilisation de graines de tournesol selon la revendication 8 ou 9, pour obtenir de l’huile, en particulier de l’huile ayant une teneur élevée en acide oléique. 16. Use of sunflower seeds according to claim 8 or 9, to obtain oil, in particular oil having a high content of oleic acid.
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Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6342658B1 (en) * 1995-12-14 2002-01-29 Cargill, Incorporated Fatty acid desaturases and mutant sequences thereof
WO2005106022A2 (en) 2004-04-29 2005-11-10 Institut National De La Recherche Agronomique Method of selecting sunflower genotypes with high oleic acid content in seed oil
WO2011072246A2 (en) 2009-12-10 2011-06-16 Regents Of The University Of Minnesota Tal effector-mediated dna modification
WO2013004281A1 (en) 2011-07-01 2013-01-10 Advanta International Bv Nucleotide sequences mutated by insertion that encode a truncated oleate desaturase protein, proteins, methods and uses
WO2013004280A1 (en) 2011-07-01 2013-01-10 Advanta International Bv Isolated mutated nucleotide sequences that encode a modified oleate destaurase sunflower protein, modified protein, methods and uses
WO2013181440A1 (en) 2012-05-30 2013-12-05 Baylor College Of Medicine Supercoiled minivectors as a tool for dna repair, alteration and replacement
WO2017004375A1 (en) 2015-06-30 2017-01-05 Regents Of The University Of Minnesota Haploid inducer line for accelerated genome editing
WO2018015956A1 (en) 2016-07-21 2018-01-25 Kaiima Bio Agritech Ltd. Compositions and methods for generating a haploid of a target plant

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6342658B1 (en) * 1995-12-14 2002-01-29 Cargill, Incorporated Fatty acid desaturases and mutant sequences thereof
WO2005106022A2 (en) 2004-04-29 2005-11-10 Institut National De La Recherche Agronomique Method of selecting sunflower genotypes with high oleic acid content in seed oil
WO2011072246A2 (en) 2009-12-10 2011-06-16 Regents Of The University Of Minnesota Tal effector-mediated dna modification
WO2013004281A1 (en) 2011-07-01 2013-01-10 Advanta International Bv Nucleotide sequences mutated by insertion that encode a truncated oleate desaturase protein, proteins, methods and uses
WO2013004280A1 (en) 2011-07-01 2013-01-10 Advanta International Bv Isolated mutated nucleotide sequences that encode a modified oleate destaurase sunflower protein, modified protein, methods and uses
WO2013181440A1 (en) 2012-05-30 2013-12-05 Baylor College Of Medicine Supercoiled minivectors as a tool for dna repair, alteration and replacement
WO2017004375A1 (en) 2015-06-30 2017-01-05 Regents Of The University Of Minnesota Haploid inducer line for accelerated genome editing
WO2018015956A1 (en) 2016-07-21 2018-01-25 Kaiima Bio Agritech Ltd. Compositions and methods for generating a haploid of a target plant

Non-Patent Citations (11)

* Cited by examiner, † Cited by third party
Title
ALBERIO CONSTANZA ET AL: "A new sunflower high oleic mutation confers stable oil grain fatty acid composition across environments", EUROPEAN JOURNAL OF AGRONOMY, ELSEVIER, AMSTERDAM, NL, vol. 73, 11 November 2015 (2015-11-11), pages 25 - 33, XP029377029, ISSN: 1161-0301, DOI: 10.1016/J.EJA.2015.10.003 *
ALTSCHUL ET AL., FEBS J., vol. 272, 2005, pages 5101 - 5109
ALTSCHUL ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402
GARCES RMANCHA M: "In vitro oleate desaturase in sunflower seeds developing", PHYTOCHEMISTRY, vol. 30, 1991, pages 2127 - 2130, XP026631299, DOI: 10.1016/0031-9422(91)83599-G
HONGTRAKUL ET AL.: "A seed specific Δ 12 oleate-desaturase gene is duplicated, rearranged and weakly expressed in high oleic acid sunflower lines", CROP SCI, vol. 38, 1998, pages 1245 - 1249, XP008035350
KUMAR A ET AL.: "SMART - Sunflower Mutant population And Reverse genetic Tool for crop improvement", BMC PLANT BIOL., vol. 13, 2013, pages 38, XP021140940, DOI: 10.1186/1471-2229-13-38
LACOMBE ET AL.: "An oleate-desaturase and a suppressor locus direct high oleic acid content of sunflower (Helianthus annuus L.) oil in the Pervenets mutant", C.R. ACAD. SCI, LIFE SCI, vol. 324, 2001, pages 839 - 845
MUMM ET AL.: "Backcross versus Forward Breeding in the Development of Transgenic Maize Hybrids: Theory and Practice", CROP SCI, vol. 47, no. 3, 2007, pages S164 - S171, XP002503385, DOI: 10.2135/CROPSCI2007.04.0014IPBS
SÃ VERINE LACOMBE ET AL: "An insertion of oleate desaturase homologous sequence silences via siRNA the functional gene leading to high oleic acid content in sunflower seed oil", MOLECULAR GENETICS AND GENOMICS, SPRINGER, BERLIN, DE, vol. 281, no. 1, 28 October 2008 (2008-10-28), pages 43 - 54, XP019658427, ISSN: 1617-4623 *
SOLDATOV: "Chemical mutagenesis in sunflower breeding", PROC. VLLTHLNT. SUNFLOWER CONFÉRENCE, 1976
UNKNOWN: "Tour", 10 May 2015 (2015-05-10), XP055693603, Retrieved from the Internet <URL:http://www.open-source-biology.com/Synthese_high-oleic_final_27-06-2015.pdf> [retrieved on 20200511] *

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