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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology

Definitions

• the present invention relates to a method for increasing meiotic recombination in plants.
• Meiotic recombination is a DNA exchange between homologous chromosomes during meiosis; it intervenes during the prophase of the first meiotic division.
• One of the products of recombination is the crossing-over, which causes a reciprocal exchange of continuity between two homologous chromatids.
• This prophase comprises 5 successive stages: leptotene, zygotene, pachytene, diplotene, and diacinesis.
• leptotene chromosomes become individualized, each chromosome being formed by two sister chromatids resulting from the duplication that occurred prior to prophase.
• homologous chromosomes pair together, forming a structure called "divalent” that contains four chromatids, two maternal sister chromatids, and two paternal sister chromatids, homologous to maternal chromatids.
• the chromosomes are completely paired, and recombinant nodules are formed between the homologous chromatids closely related to each other by the synaptonemal complex (SC); at diplotene, the SC dissociates progressively, and the homologous chromosomes start to separate, but remain joined at the level of the chiasmas, which correspond to the crossing-overs (COs) sites.
• SC synaptonemal complex
• COs crossing-overs
• Meiotic recombination is triggered by the formation of double-strand breaks (DSBs) in either homologous chromatid, and results from the repair of these breaks using a homologous chromosome chromatid as a template.
• DSBs double-strand breaks
• Meiotic recombination results in a reassortment of paternal and maternal alleles in the genome, which contributes to generating genetic diversity. It is therefore of particular interest in plant breeding programs (WIJNKER & de Jong, Trends in Plant Science, 13, 640-646, 2008).
• an improvement in the recombination rate may make it possible to increase the genetic mixing, and thus the probabilities of obtaining new combinations of characteristics; it can also facilitate the introgression of genes of interest, as well as genetic mapping and positional cloning of genes of interest.
• a resolvase a single-stranded DNA binding protein, a protein involved in chromatin remodeling, or a protein of the synaptonemal complex, to increase the frequency of recombination between homologous chromatids
• PCT Application WO 2004016795 proposes to increase the recombination between homologous chromosomes by expressing a SPOll protein fused to a DNA binding domain
• PCT Application WO 03104451 proposes to increase the recombination potential between homologous chromosomes by overexpression of a protein (MutS) involved in the repair of mismatches.
• COs type I COs type I
• ZMM genes ZIP1, ZIP2, ZIP3, ZIP4, MER3, MSH4, MSH5
• the inventors have now discovered that the inactivation of the Arabidopsis thaliana Fanconi Anemia Complementation Group M (FANCM) gene compensated for the effects of inactivation of AtMSH5, SHOC1, or AtZIP4, and allowed increase the number of meiotic COs and restore fertility in zmm mutants Atzip4 - / -, Atmsh5 - / - and Atshocl - / -. They further found that the effects of inactivating the FANCM gene on increasing meiotic COs occurred not only in zmm mutants, but also in wild type plants with functional ZMM genes.
• FANCM Fanconi Anemia Complementation Group M
• the FANCM protein was initially identified in humans as part of the search for mutations associated with Fanconi anemia, which is a genetic disorder characterized by genomic instability and susceptibility to cancer. This protein participates in the repair of DNA lesions; the human FANC protein contains two helicase domains in its N-terminal half: a DEXDc domain (cd00046, and a HELICc domain (cd00079) as well as an ERCC1 / XPF endonuclease domain in its C-terminal half, the latter domain being however, probably not functional because it is degenerate for several residues essential for endonuclease activity (MEETEI et al, Nat. Genet., 37, 958-963, 2005).
• the FANCM protein appears conserved, and various homologues of this protein have been identified in eukaryotes, based on sequence homologies.
• the plant FANCMs, represented by the Arabidopsis thaliana protein, are also lacking the endonuclease domain.
• AtFANCM is encoded by the AT1G35530 gene; two predicted isoforms of this protein are described in the sequence databases: one (GenBank: NP_001185141; UniProtKB: F4HYE4) represented in the attached sequence listing under the number SEQ ID NO: 1, has a size of 1390 amino acids ; the other (GenBank: NP_174785; UniProtKB: F4HYE5), has a size of 1324 amino acids. These two isoforms differ from each other by the presence of two insertions in the NP_001185141 / F4HYE4 sequence: one of 45 amino acids between positions 575 and 576 of the NP_174785 / F4HYE5 sequence, and the other of 21 amino acids between the positions. 1045 and 1046 of the sequence NP_174785 / F4HYE5.
• AtFANCM has in its N-terminal half the two helicase domains DEXDc (amino acids 129-272 of SEQ ID NO: 1) and HELICc (amino acids 445-570 of SEQ ID NO: 1); these two domains are respectively described under the references cd00046 and cd00079 in the CDD database (MARCHLER-BAUER et al., Nucleic Acids Res .39 (D) 225-9, 2011).
• AtFANCM orthologs in a large panel of eukaryotes, and it can be assumed that this protein is conserved in all higher plants.
• AtFANCM orthologues include:
• Vitis vinifera protein whose polypeptide sequence is available in the GenBank database under access number CBI18266;
• the subject of the present invention is a method for increasing the frequency of meiotic COs in a plant, characterized in that it comprises the inhibition in said plant of a protein hereinafter called FANCM, said protein having at least 30% and in order of increasing preference, at least 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence identity, or at least 45%, and in order of increasing preference, at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence similarity with the AtFANCM protein of sequence SEQ ID NO: 1, and containing a DEXDc helicase domain (cd00046) and HELICc helicase domain (cd00079).
• FANCM protein hereinafter called FANCM
• the DEXDc helicase domain of said FANCM protein has at least 65%, and in order of preference at least 70,75, 80, 85, 90, 95 or 98% sequence identity, or at least 75%, and in order of increasing preference, at least 80, 85, 90, 95 or 98% of sequence similarity with DEXDc domain of AtFANCM protein (amino acids 129-272 of SEQ ID NO: 1).
• the HELICc helicase domain of said FANCM protein has at least 60%, and in order of increasing preference, at least 65, 70, 75, 80, 85, 90, 95 or 98% sequence identity, or at least 70%, and in order of increasing preference, at least 75, 80, 85, 90, 95, or 98% sequence similarity to the HELICc domain of the AtFANCM protein ( Amines 445-570 of SEQ ID NO: 1).
• Inhibition of the FANCM protein can be achieved by suppressing or decreasing its activity or by suppressing or decreasing the expression of the corresponding gene.
• the inhibition can in particular be obtained by mutagenesis of the FANCM gene.
• a mutation in the coding sequence may induce, depending on the nature of the mutation, the expression of an inactive protein, or of a reduced activity protein; a mutation at a splice site may also alter or abolish the function of the protein; a mutation in the promoter sequence may induce the absence of expression of said protein, or the decrease of its expression.
• the mutagenesis may be carried out for example by removing all or part of the coding sequence or the FANCM promoter, or by insertion of an exogenous sequence, for example a transposon or a T-DNA, within said coding sequence or said promoter. It can also be carried out by inducing point mutations, for example by EMS mutagenesis, or by radiation.
• the mutated alleles can be detected for example by PCR, using primers specific for the FANCM gene.
• Mutant plants containing a mutation in the FANCM gene which induces the inhibition of the FANCM protein are also part of the subject of the present invention, with the exception of the plants of the species Arabidopsis thaliana wherein said mutation is an insertion. T-DNA in said gene.
• This mutation may be, for example, a deletion of all or part of the coding sequence or the FANCM promoter, or a point mutation of this coding sequence or this promoter.
• the inhibition of the FANCM protein is obtained by silencing the FANCM gene.
• Different techniques of gene silencing in plants are known per se (for review see for example: WATSON & GRIERSON, Transgenic Plants: Fundamentals and Applications (Hiatt, A, ed) New York: Marcel Dekker, 255-281, 1992, CHICAS & MACINO, EMBO reports, 21, 992-996, 2001).
• antisense inhibition or co-suppression described for example in US Pat. Nos. 5,190,065 and 5,283,323. It is also possible to use ribozymes targeting the mRNA of the FANCM protein.
• RNAi interfering RNA
• siRNAs small interfering RNAs
• miRNAs microRNAs
• DNA constructs for expressing interfering RNAs in plants contained a 300 bp or larger fragment (typically 300-800 bp) of the targeted gene cDNA under transcriptional control of a suitable promoter.
• the most widely used constructs are those that can produce a hairpin RNA transcript (hpRNA).
• the target gene fragment is inversely repeated, generally with a spacer region between repeats (for review, see WATSON et al., FEBS Letters, 579, 5982-5987, 2005).
• amiRNAs Artificial microRNAs directed against the FANCM gene can also be used (OSSOWSKI et al., The Plant Journal, 53, 674-690, 2008; SCHWAB et al., Methods Mol Biol., 592, 71-88 , WEI et al., Funct Integr Genomics., 9, 499-511, 2009).
• the present invention relates to recombinant DNA constructs, and in particular expression cassettes, producing RNAi for extinguishing the FANCM gene.
• An expression cassette in accordance with the invention comprises a recombinant DNA sequence whose transcript is an RNAi, in particular a hpRNA or a amiRNA, targeting the FANCM gene, placed under transcriptional control of a functional promoter in a plant cell.
• RNAi in particular a hpRNA or a amiRNA
• promoters can be obtained for example from plants, plant viruses, or bacteria such as Agrobacterium. They include constitutive promoters, namely promoters that are active in most tissues and cells and under most environmental conditions, as well as specific tissue-specific or cell promoters, which are active only or mainly in certain tissues or types of cells, and inducible promoters that are activated by physical or chemical stimuli.
• constitutive promoters examples include the 35S promoter of cauliflower mosaic virus (CaMV) described by KAY et al. (Science, 236, 4805, 1987), or its derivatives, the cassava mosaic virus virus promoter (CsVMV) described in International Application WO 97/48819, the corn ubiquitin promoter or the promoter Actin-Intron-actin of rice (McELROY et al., Mol Gen. Genet., 231, 150-160, 1991, GenBank accession number S 44221).
• a promoter specific for meiosis that is to say active exclusively or preferentially in the cells in meiosis.
• DMC1 promoter KLIMYUK & JONES, Plant J., 11, 1-14, 1997.
• the expression cassettes of the invention generally comprise a transcriptional terminator, for example the 3 'NOS terminator of nopaline synthase (DEPICKER et al., J. Mol Appl. Genet., 1, 561-573, 1982), or the 3'CaMV terminator (FRANCK et al., Cell, 21, 285-294, 1980). They may also include other transcriptional regulatory elements such as amplifiers.
• a transcriptional terminator for example the 3 'NOS terminator of nopaline synthase (DEPICKER et al., J. Mol Appl. Genet., 1, 561-573, 1982), or the 3'CaMV terminator (FRANCK et al., Cell, 21, 285-294, 1980).
• They may also include other transcriptional regulatory elements such as amplifiers.
• the recombinant DNA constructs in accordance with the invention also include recombinant vectors containing an expression cassette according to the invention. These recombinant vectors may also include one or more marker genes, which allow the selection of transformed cells or plants.
• the choice of the most appropriate vector depends in particular on the intended host and the method envisaged for the transformation of the host in question.
• Many methods for the genetic transformation of plant or plant cells are available in the art for many plant species, dicotyledonous or monocotyledonous.
• viruses-mediated transformation include virus-mediated transformation, microinjection transformation, electroporation, micropro ectile transformation, Agrobacterium transformation, and the like.
• the subject of the invention is also a host cell comprising a recombinant DNA construct according to the invention.
• Said host cell may be a prokaryotic cell, for example an Agrobacterium cell, or a eukaryotic cell, for example a plant cell genetically transformed with a DNA construct of the invention.
• the construct may be transiently expressed, it may also be incorporated into a stable extrachromosomal replicon, or integrated into the chromosome.
• the invention also provides a process for producing a transgenic plant having a higher meiotic CO 2 level than that of the wild plant from which it originates, characterized in that it comprises the following steps:
• the invention also encompasses plants genetically transformed by a DNA construct of the invention.
• said plants are transgenic plants, wherein said construct is contained in a transgene integrated into the genome of the plant, so that it is transmitted to successive generations of plants.
• the expression of the DNA construct of the invention results in a downregulation of the expression of the FANCM gene, which confers on said transgenic plants a meiotic CO 2 level higher than that of wild plants (not containing the construction of DNA of the invention) from which they are derived.
• this rate of meiotic COs is at less than 2 times higher, preferably at least 3 times higher than that of the wild plants from which they are derived.
• the present invention can be applied in particular in the field of plant breeding, in order to accelerate the obtaining of new varieties. It also facilitates the crossing between related species, and thus the introgression of characters of interest. It also speeds up genetic mapping and positional cloning.
• the present invention is applicable to a wide range of monocotyledonous or dicotyledonous crops of agronomic interest.
• rapeseed sunflower, potato, maize, wheat, barley, rye, sorghum, rice, beans, carrots and tomatoes.
• EXAMPLE 1 OBTAINING MUTANTS SUPPRESSING MUTATIONS OF GENES ZMM
• Arabidopsis thaliana seeds homozygous for insertion of T-DNA into ZIP4, SHOC1, or MSH5 were mutated by EMS (ethylmethanesulfonate).
• Plants derived from mutated seeds (Ml population, heterozygous for EMS-induced mutations, and homozygous for ZMM gene mutation) have an identical phenotype resulting from inactivation of ZMM gene, which results in a sharp decrease.
• Ml plants were self-pollinated to produce a population of offspring (M2 population) potentially containing homozygous plants for EMS-induced mutations. Plants of the M2 population with a silique longer than that of the homozygous Ml generation plants were selected and genotyped to verify their homozygous status for the insertion of T-DNA into the relevant ZMM gene. The segregation of their chromosomes during meiosis was compared with that of wild plants and homozygous zmm plants not mutated to EMS.
• FIG. 1 The top of the figure shows the length of the siliques of the different plants compared.
• the boxes at the bottom of the figure illustrate the segregation of chromosomes during metaphase I.
• D ZMM / suppressor: plant not carrying the zmm mutation and homozygous for the mutation induced by EMS.
• the fertility of ZMM / suppressor plants is identical to that of wild plants. They do not present any obvious phenotypic differences with wild plants.
• the mutations induced by the EMS are recessive. Indeed, they do not result in a heterozygous detectable phenotype in the Ml mutant population, and the plants resulting from backcrossing of the zmm / suppressor mutants with the initial zmm mutants which they are respectively derived, have a phenotype that does not differ from that of the original mutants.
• Meiotic recombination frequency in FANCM / ZIP4 plants wild plants
• FANCM / zip4 homozygous for the zip4 mutation and non-mutated in FANCM
• fancm-1 / ZIP4 homozygous for the fancm-1 mutation and non-mutated in ZIP4
• fancm-1 / zip4 homozygous for the 2 mutations zip4 and fancm-1
• the meiotic recombination frequency was measured by tetrad analysis using fluorescent markers, as described by BERCHOWITZ & COPENHAVER (Nat., Protocols, 3, 41-50, 2008). )
• X axis genotype of the plants tested; The number of tetrads tested for each genotype is indicated in parentheses.
• Y axis genetic distance (in cM).
• I5c and I5d measure respectively 6.19 cM and 5.65 cM in FANCM / ZIP4 plants and only 2.98 cM and 1.86 cM in FANCM / zip4 plants.
• the genetic distance is very strongly increased in plants fancm / zip4 (18.45 cM and 14.57 cM for I5c and I5d respectively) and even more in the case of fancm / ZIP4 (respectively 21.06 cM and 17, 20cM for I5c and I5d).
• X axis interval tested
• Y axis genetic distance (in cM).
• Recombination is reduced by a factor of 2 to 3 in the FANCM / zip4 mutant compared to the wild type.
• the genetic distances are increased by a factor of 1.9 to 3.1 relative to the wild type (P ⁇ 10 ⁇ 5 ).
• This confirms that the FANCM mutation not only restores the formation of COs in the absence of ZIP4, but also increases the frequency of COs well beyond that seen in wild plants In fancm-1 / ZIP4), recombination is increased even more than in fancm-1 / zip4, averaging 12% (P ⁇ 0.05 in 3 of 6 individual intervals).
• genetic distance is increased by a factor of 2 to 3.6 (P ⁇ 10 "8) on the 8 tested intervals, which stresses the importance of FANCM in limiting the frequency of COs.

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