Increased meiotic recombination in plants by inhibition of a RecQ4 or Top3A protein of the RTR complex

Description

[0001] The present invention relates to a method for increasing meiotic recombination in plants.

[0002] Meiotic recombination is an exchange of DNA between homologous chromosomes during meiosis; it occurs during prophase I of the first meiotic division. One of the products of recombination is crossing-over, which results in a reciprocal exchange of continuity between two homologous chromatids. This prophase (prophase I) comprises five successive stages: leptotene, zygotene, pachytene, diplotene, and diakinesis. During leptotene, the chromosomes differentiate, each chromosome being formed from two sister chromatids resulting from the duplication that occurred prior to prophase I. During zygotene, the homologous chromosomes pair up, forming a structure called a "bivalent" which contains four chromatids: two maternal sister chromatids and two paternal sister chromatids, homologous to the maternal chromatids.During pachytene, the chromosomes are fully paired, and recombination nodules form between homologous chromatids closely linked by the synaptonemal complex (SC). During diplotene, the SC gradually dissociates, and homologous chromosomes begin to separate but remain joined at chiasmata, which correspond to crossing-over sites (COs). The chromosomes condense during diakinesis; the chiasmata persist until metaphase I, during which they maintain the pairing of bivalents on either side of the equatorial plate.

[0003] Meiotic recombination is triggered by the formation of double-strand breaks (DSBs) in either of the homologous chromatids, and results from the repair of these breaks, using as a template a chromatid from the homologous chromosome.

[0004] Meiotic recombination results in a reassortment of paternal and maternal alleles in the genome, thereby contributing to genetic diversity. It is therefore of particular interest in plant breeding programs (Wijnker & de Jong, Trends in Plant Science, 2008, 13, 640-646; Crismani et al., Journal of Experimental Botany, 2013, 64, 55-65). Specifically, increasing the recombination rate can lead to new combinations of traits; it can also facilitate the introgression of genes of interest from one line to another, as well as genetic mapping and positional cloning of genes of interest.

[0005] Various methods for controlling meiotic recombination have been proposed, based on the overexpression or silencing of one or more of the many genes identified as being involved or potentially involved in this mechanism. For example, PCT Application WO / 0208432 proposes the overexpression of the RAD51 protein, which is involved in homologous recombination, to stimulate meiotic recombination; US Application 2004 / 023388 proposes to overexpress a meiotic recombination activator selected from: SPO11, MRE11, RAD50, XRS2 / NBS1, DMC1, RAD51, RPA, MSH4, MSHS, MLH1, RAD52, RAD54, TID1, RAD5S, RADS7, RADS9, a resolvase, a single-stranded DNA-binding protein, a protein involved in chromatin remodeling, or a synaptonemal complex protein, to increase the recombination frequency between homologous chromatids;PCT Application WO 200 / 4016795 proposes to increase recombination between homologous chromosomes by expressing an SPO11 protein fused to a DNA-binding domain; PCT Application WO 03 / 104451 proposes to increase the recombination potential between homologous chromosomes by overexpressing a protein (MutS) involved in mismatch repair; PCT Application WO 2010 / 071418 proposes to reduce the expression or activity of the RecQ15 protein (RECQ5).

[0006] However, in most cases, the effect of these candidate genes on the formation of meiotic COs in planta has not been confirmed, and it is therefore necessary to identify other genes involved in this phenomenon.

[0007] One of the known factors influencing the meiotic recombination rate is interference: the formation of a chromosome complement (CO) at one location on the chromosome inhibits the formation of other COs nearby. However, it has been shown that there are actually two distinct pathways for the formation of meiotic COs (Hollingsworth & Brill, Genes Dev., 2004, 18, 117-125; Berchowitz & Copenhaver, Current Genomics, 2010, 11, 91-102). The first generates interfering COs called type I COs (COIs) and involves a set of genes collectively referred to as the genomics genes. ZMM ( ZIP1, ZIP2 / SHOC1, ZIP3, HEI10, ZIP4, MER3, MSH4, MSH5, PTD) as well as proteins MLH1 And MLH3 ; The second pathway generates non-interfering COs, called type II COs (COII), and depends on the gene MUS81.

[0008] The use of one or both of these two pathways varies from one organism to another. In higher plants, represented by the model plant Arabidopsis thaliana, The two pathways coexist, with the type I CO pathway being predominant; it has been observed that gene inactivation AtMSH4, AtMSH5, AtMER3, SHOC1, PTD, AtHEI10 Or AtZIP4 induced up to 85% reduction in CO frequency (Higgins et al., Genes Dev., 2004, 18, 2557-2570; Mercier et al., Curr. Biol., 2005, 15, 692-701; Chelysheva et al., PLoS Genet. 2007, 3, e83; Higgins et al., Plant J., 2008, 55, 28-39; Macaisne et al., Current Biology, 2008, 18, 1432-1437; Wijeratne et al., Molecular Biology of the Cell, 2006, 17, 1331-43; Chelysheva et al., PLoS Genetics, 2012, 8, e1002799). Consequently, some homologous chromosomes are no longer paired as bivalents, but appear as univalents. This decrease in the number of bivalents is accompanied by a significant reduction in fertility.

[0009] Previously, the inventors identified a gene, named FANCM (For « Fanconi Anemia Complementation Group M ") whose inhibition compensated for the effects of that of AtMSH5, of SHOC1, or 'AtZIP4 and allowed to increase the number of meiotic COs (PCT Request WO 2013 / 038376; Crismani et al., Science 2012, 336, 6088, 1588-90).

[0010] By continuing their research, the inventors have now identified two other genes whose inhibition produces effects similar to that of FANCM.

[0011] These are the genes TOP3A (for DNA TOPOISOMERASE III alpha) and RECQ4, encoding two of the proteins of the RTR complex.

[0012] The RTR complex is a conserved complex, consisting of a RecQ family helicase, a DNA Topoisomerase III (Type I Topoisomerase, subtype IA) and a structural protein of the RMI1 family. This complex, which is involved in the resolution of DNA recombination intermediates in all eukaryotes, is essential for maintaining genome integrity (Mankouri and Hickson, Trends Biochem Sci., 2007, 32, 538-46).

[0013] RecQ family helicases are proteins involved in genome maintenance and stability in all organisms, such as bacteria, yeast, animals, and plants. In eukaryotes, the number of RecQ genes (or RECQ The structure of RecQ (or RECQ) proteins varies enormously between organisms. Arabidopsis thaliana, There are 7 genes RECQ of which AtRECQ4A which codes for a protein homologous to the RecQ helicase of Escherichia coli, Yeast Sgs1 and human BLM (Hartung F. and Puchta, H., J. Plant. Physiol., 2006, 163, 287-296; Hartung et al., Nucleic Acids Res., 2000, 28, 4275-4282; Bagherieh-Najjar et al., The Plant Journal, 2005, 43, 789-798; Crismani et al., Cell Cycle, 2012, 11, 3527-3528). The gene AtRECQ4B, a paralogue of 'AtRECQ4A probably resulting from a gene duplication that occurred only in Brassicacea, codes for a protein that has 70% identity with AtRECQ4A ( Figure 1 ). The AtRECQ4A and AtRECQ4B proteins comprise 3 conserved domains; a helicase domain, which is the most important, and comprises eight motifs (0, I, Ia, II, III, IV, V and VI), with a total length of approximately 300 to 450 amino acids, containing the sequences necessary for ATP binding, hydrolysis and DNA unwinding (NCBI cd00079 and cd00046); an RQC domain (smart00956), conserved in almost all RECQ proteins; an HRDC domain (pfam00570), conserved in more than 50% of RECQ proteins and also at the C-terminus of RNase D.

[0014] Whereas in yeast S. cerevisiae, Meiotic recombination is increased in the absence of the Sgs1 helicase by a factor of 1.17–1.6 (Jessop et al., PLoS Genetics, 2006, 2, e155; Oh et al., Cell, 2007, 130(2), 259–272), the mutation of 'AtRECQ4A ( recq4a-5 / SALK_069672 ) Or AtRECQ4B is described as producing no significant effect on the formation of meiotic COs (Higgins et al., The Plant Journal, 2011, 65, 492-502). In particular, it has been observed that the mutation of d 'AtRECQ4A ( recq4a- 5 / SALK_069672) or d 'AtRECQ4B was unable to restore fertility and bivalent formation in a mutant msh4 of the Columbia strain. On the other hand, it has been suggested that RECQ4A plays a role in telomere maintenance, a role in genome integrity, previously unknown for a protein of the RecQ family.

[0015] The AtTOP3A protein (TOP3a, TOP3, Top3a, Top3 alpha, TOP3 alpha, or TOP3α, for DNA TOPOISOMERASE III alpha or DNA TOPOISOMERASE 3-alpha) contains 4 conserved domains: in its N-terminal region, a TOPRIM domain (NCBI cd03362) and a DNA Topoisomerase subtype IA domain (NCBI cd00186), both conserved in all TOP3 homologs; in its C-terminal region, two zinc finger domains (pfam01396 and pfam06839), which are conserved in plants and animals but not in yeasts ( Figure 2 ).

[0016] Two mutant alleles of top3a ( top3a-1 and top3a-2 ) have previously been described in Arabidopsis (Hartung et al., PLoS Genet., 2008, 4, e1000285, Hartung et al., PNAS 2007, 104, 47, 18836-41). The mutation top3a-1 is likely negligible given that it leads to early lethality during development. The second allele, top3a-2, is a hypomorphic mutant that is viable but exhibits somatic growth defects and is completely sterile (Hartung et al., PLoS Genet., 2008, 4, e1000285). Severe phenotypes associated with the inactivation of TOP3A at the house of Arabidopsis and various species (Goodwin et al., Nucleic Acids Res., 1999, 27, 4050-8; Kim et al., Nucleic Acids Res., 2000, 28, 2012-7; Li and Wang, , Proc. Natl. Acad. Sci. USA, 1998, 95, 1010-3 Plank et al., J. Biol. Chem., 2005, 280, 3564-73) highlight an essential role of TOP3A in the resolution of mitotic and meiotic DNA repair intermediates but do not suggest an anti-MEiotic CO activity of this protein.

[0017] On the contrary, as demonstrated in the present invention, the inhibition of genes AtRECQ4A et AtRECQ4B Or TOP3A increases the number of meiotic COs, and not only in mutants zmm, but also in wild-type plants for ZMM genes.

[0018] Indeed, the inventors have shown that the inhibition of 'AtRECQ4A and 'AtRECQ4B compensates for the effects of that of 'AtMSH4 or 'AtZIP2 / SHOC1 and allows for an increase in the number of meiotic COs and the restoration of fertility in mutants zmm Atmsh4- / - And shoc1- / -.

[0019] Inhibition of TOP3A compensates for the effects of that of 'AtHEI10 or AtMSH5 and allows for an increase in the number of meiotic COs and the restoration of fertility in mutants zmm Athei10- / -et Atmsh5- / - .

[0020] The inventors also discovered the effects of gene inhibition TOP3A Or RECQ4 the increase in the number of meiotic COs occurred not only in mutants zmm, but also in plants possessing genes ZMM functional.

[0021] FIDG proteins belong to the AAA-ATPase (ATPases Associated with various Activities) family, and more specifically to subgroup 7 (Beyer, Protein Sci., 1997, 6, 2043-58; Frickey & Lupas, J. Struct. Biol., 2004, 146, 2-10). Only one representative of this family has been identified in the genome of most plants. FIDG proteins have two conserved protein domains: an AAA-ATPase domain, with a putative role in ATP hydrolysis; and a VSP4 domain, known to play an important role in microtubule binding. These two domains are listed under the references PF00004 and PF09336, respectively, in the PFAM database (Punta et al., Nucleic Acids Res., 2012, Database Issue 40:D290-D301).

[0022] FIDG proteins are described as microtubule-cutting enzymes involved in regulating the number and size of microtubules in many animal and plant species ( C. elegans (Yakushiji et al., FEBS Lett., 2004, 578, 191-7); D. melanogaster (Zhang et al., J. Cell. Biol., 2007, 177, 231-42); H. sapiens (Mukherjee et al., Cell Cycle, 2012, 11, 2359-66); HAS. thaliana (Stoppin-Mellet et al., Biochem J., 2002, 365, 337-42)).

[0023] The inventors further discovered that when the gene is inhibited TOP3A Or RECQ4 was combined with that of the gene FANCM ou FIDG, the number of meiotic COs was further increased compared to those observed when these genes were inactivated separately.

[0024] The invention is as defined in claims 1 to 13.

[0025] The invention relates to a method for increasing the frequency of meiotic crossovers in a plant, characterized in that it comprises the inhibition in said plant of the expression or function of at least one protein of the RTR complex called RECQ4, by mutagenesis of the gene encoding said protein or of its promoter, by silencing of the gene encoding said protein or by an inhibitor binding specifically to a functional domain of said protein, said RECQ4 protein having at least 40% sequence identity with the RECQ4 protein of sequence SEQ ID NO: 1 and further comprising a region having at least 60% sequence identity with the region extending from positions 407 to 959 of sequence SEQ ID NO: 1.

[0026] In one embodiment, the invention relates to a method as described above, characterized in that it further comprises the inhibition in said plant of the expression or function of a protein of the RTR complex called TOP3A, by mutagenesis of the gene encoding said protein or of its promoter, by silencing of the gene encoding said protein or by an inhibitor binding specifically to a functional domain of said protein, said TOP3A protein having at least 50% sequence identity with the TOP3A protein of sequence SEQ ID NO: 2.

[0027] In one embodiment, the invention relates to a method as described above, characterized in that it further comprises the inhibition in said plant of the expression or function of at least one protein selected from: a protein hereinafter referred to as FIDG, said protein having at least 45% sequence identity, or at least 60% sequence similarity with the AtFIDG protein of sequence SEQ ID NO: 46 and containing an AAA-ATPase domain and a VSP4 domain, and a protein hereinafter referred to as FANCM, said protein having at least 30% sequence identity, or at least 45% sequence similarity with the AtFANCM protein of sequence SEQ ID NO: 45, and containing a DEXDc helicase domain and a HELICc helicase domain, by mutagenesis of the gene encoding said protein or of its promoter, by silencing of the gene encoding said protein or by an inhibitor binding specifically to a functional domain of said protein.

[0028] The RECQ4 protein may further comprise a region having at least 60%, and in increasing order of preference at least 65, 70, 75, 80, 85, 90, 95 or 98% sequence identity with the region extending from positions 407 to 959 of the SEQ ID NO: 1 sequence.

[0029] Alternatively, the RECQ4 protein can be defined as a protein comprising a region having at least 60%, and in increasing order of preference at least 65, 70, 75, 80, 85, 90, 95 or 98% sequence identity with the region extending from positions 407 to 959 of the SEQ ID NO: 1 sequence.

[0030] The sequence identities of the RECQ4 and TOP3 proteins are calculated over the entire length of the longer of the two proteins, which are compared after alignment using T-Coffee (v6.85) with default parameters ( http: / / toolkit.tuebingen.mpg.de / t_coffee The percentage of identity for the RECQ4 and TOP3 proteins is obtained from this alignment using BioEdit 7.2.5 (Hall, TA 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95 / 98 / NT. Nucl. Acids. Symp. Ser. 41:95-98). The percentage of identity for the RECQ4 protein region is calculated on the sequence from positions 407-959 of the SEQ ID NO: 1 sequence after aligning the RECQ4 protein with the SEQ ID NO: 1 sequence.

[0031] The sequences of the RECQ4 and TOP3A genes and proteins are available in public databases, such as, but not limited to, the PLAZA database ( http: / / bioinformatics.psb.ugent.be / plaza / ) and phytozome http: / / www.phytozome.net / (phytosome v9.1).

[0032] For the purposes of the present invention, the expression RECQ4 protein corresponds to the RECQ4A and RECQ4B proteins in plants of the Brassicaceae family ( Brassica sp. ) which possess two RECQ4 genes ( RECQ4A And RECQ4B ), and to the RECQ4 protein in plants other than Brassicaceae, which possess only one gene RECQ4. However, in some Brassicacae lines in which one of the RECQ4 proteins is not functional, only the RECQ4 protein that is functional is inhibited.

[0033] The SEQ ID NO: 1 sequence represents the RECQ4A protein sequence d'Arabidopsis thaliana (AT1G10930 in the PLAZA database). The RECQA ortholog and RECQB paralog sequences in different flowering plant species are shown in the table below: Tableau I RECQ4 Sequences Plante Numéro d'accès* SEQ ID NO : Arabidopsis thaliana AT1G10930 1 Arabidopsis lyrata AL1G11090 3 Arabidopsis lyrata AL2G05030 4 Arabidopsis thaliana AT1G60930** 5 Brachypodium distachyon BD5G10432 6 Brassica rapa Bra031741 7 Brassica rapa Bra027115 8 Brassica rapa Bra018418 9 Carica papaya CP00031G00490 10 Thellungiella halophila Thhalv10006602m 11 Thellungiella halophila Thhalv10023226m 12 Eucalyptus grandis Eucgr.G00562.1 13 Glycine max Glyma12g29151 14 Glycine max Glyma08g20070 15 Gossypium raimondii Gorai.013G038600 16 Manihot esculenta ME02817G00160 17 Oryza sativa OS04G35420 18 Phaseolus vulgaris Phvul.006G216200 19 Prunus persica ppa000416m.g 20 Ricinus communis RC28725G00120 21 Sorghum bicolor Sb04g003070.1 22 Italian silkworm Si016138m.g 23 Solanum lycopersicum 01g103960.2 24 Theobroma cacao Thecc1EG006933t1 25 Vitis vinifera VV12G07620 26 *on the basis of women PLAZA or phytozome **RECQ4B

[0034] According to the invention, said RECQ4 protein comprises 3 conserved domains: a helicase domain, which is the most important, and comprises eight motifs (O, I, Ia, II, III, IV, V, and VI), with a total length of approximately 300 to 450 amino acids, containing the sequences necessary for ATP binding, hydrolysis, and DNA unwinding (NCBI). cd00079 And cd00046 ); an RQC domain (smart00956); an HRDC domain (pfam00570).

[0035] The SEQ ID NO: 2 sequence represents the TOP3A protein sequence. Arabidopsis thaliana (AT5G63920 in the PLAZA database). Ortholog sequences in different species of flowering plants are shown in the table below: Tableau II: TOP3A Sequences Plants Access number* SEQ ID NO : Arabidopsis thaliana AT5G63920 2 Arabidopsis lyrata AL8G30570 27 Brachypodium distachyon BD1G74120 28 Fragaria vesca FV0G21450 29 Oryza sativa OS03G06900 30 Populus trichocarpa PT05G06620 31 Ricinus communis RC30149G00070 32 Sorghum bicolor SB01G046210 33 Zea mays ZM01G04670 34 Thellungiella halophila Thhalv10003618m.g 35 Capsella rubella Carubv10025845m 36 Brassica rapa Bra031944 37 Gossypium raimondii Gorai.007G066700. 38 Theobroma cacao Thecc1EG001553t1 39 Eucalyptus grandis Eucgr.H05141.1 40 Glycine max Glyma06g16175.1 41 Mimulus guttatus mgv1a000984m.g 42 Aquilegia caerula Aquca_069_00036.1 43 Setaria italica Si034218m.g 44 *in the PLAZA or phytozome database

[0036] According to the invention, the TOP3A protein comprises 4 conserved domains: in its N-terminal region, a TOPRIM domain (NCBI cd03362) and a DNA Topoisomerase subtype IA domain (NCBI cd00186); in its C-terminal region, two zinc finger domains (pfam01396 and pfam06839); Figure 2 The first zinc finger domain is located in the region of said TOP3A protein that extends from positions 639 to 677 of the SEQ ID NO: 2 sequence. The second zinc finger domain is located in the region of said TOP3A protein that extends from positions 804 to 844 of the SEQ ID NO: 2 sequence.

[0037] The invention encompasses the simultaneous inhibition of RECQ4 and / or TOP3A proteins.

[0038] Inhibition of the RECQ4 and / or TOP3A protein is achieved by abolishing, blocking, or inhibiting gene expression. RECQ4Or TOP3A or the function of said RECQ4 or TOP3A protein. Furthermore, the inhibition of the TOP3A protein in the plant is carried out in such a way that the plant necessarily expresses, from the gene TOP3A mutated or from a transgene, a mutated TOP3A protein comprising a mutation in its C-terminal region that inhibits at least one of the two zinc finger domains while its N-terminal region comprising the TOPRIM and DNA topoisomerase IA domains is intact.

[0039] Inhibition can be achieved, in particular, by gene mutagenesis RECQ4 Or TOP3A.For example, a mutation in the coding sequence can induce, depending on the nature of the mutation, the expression of an inactive protein, or a protein with reduced activity; a mutation at a splice site can also alter or abolish the function of the protein; a mutation in the promoter sequence can induce the absence of expression of said protein, or the reduction of its expression.

[0040] Mutagenesis can be carried out, for example, by deleting all or part of the coding sequence or promoter of RECQ4 Or TOP3A,or by the 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, by radiation, or by site-directed mutagenesis, for example using TALENs (Transcription Activator-Like Effector Nucleases), CRISPR-Cas systems or zinc finger nucleases (CHRISTIAN et al., Genetics, 186, 757-61, 2010; CURTIN et al., Plant Gen., 5, 42-50, 2012).

[0041] Mutated alleles can be detected, for example, by PCR, using gene-specific primers. RECQ4 or TOP3A.

[0042] Various high-throughput mutagenesis and screening methods are described in the prior art. Examples include TILLING (Targeting Induced Local Lesions In Genome) methods, described by McCallum et al. (Plant Physiol., 2000, 123, 439-442). The lack of RECQ4 or TOP3A functionality in mutants can be verified based on the phenotypic characteristics of their offspring; plants homozygous for a gene-inactivating mutation RECQ4 Or TOP3A have a higher level of meiotic COs than wild plants (not carrying the mutation in the gene) RECQ4 Or TOP3A ) from which they originate. Generally, this meiotic COs level is at least 50% higher, preferably at least twice higher than that of the wild plants from which they originate.

[0043] According to an advantageous implementation of said method, it comprises the introduction, into an allele of the gene TOP3A,of a mutation that results in the inhibition of at least one zinc finger domain of the TOP3A protein, said domain being located in the region of said protein extending from positions 639 to 677 or 804 to 844 of the SEQ ID NO: 2 sequence. The mutation preserves the TOPRIM and DNA topoisomerase IA domains but inhibits, and preferably inactivates, at least one of the two zinc finger domains. This includes, in particular, an insertion or deletion in the C-terminal region of TOP3A comprising said domains. Preferably, said mutation is a deletion of the C-terminal sequence of said TOP3A protein, from one of the residues of said protein located from positions 610 to 803 of the SEQ ID NO: 2 sequence, preferably from the residue of said protein located at position 640 of the SEQ ID NO: 2 sequence (production of a C-terminally truncated TOP3A protein).

[0044] Alternatively, inhibition of the RECQ4 or TOP3A protein is achieved by silencing the gene RECQ4 Or TOP3A. Several gene silencing techniques in plants are known (for a 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, 2001, 21, 992-996; Puchta, H and Fauser, F, Int. J. Dev. Biol., 2013, 57, 629-37; Ali et al., GM crops, 2010, 1, 207-213). Antisense inhibition or co-suppression, described, for example, in US Patents 5190065 and 5283323, can be cited. It is also possible to use ribozymes targeting the mRNA of the RECQ4 or TOP3A protein.

[0045] Preferably, gene extinction RECQ4 Or TOP3A is induced by RNA interference targeting said gene.

[0046] In one embodiment, the invention relates to a method as described above, characterized in that the inhibition of the RECQ4 and / or TOP3A protein and where applicable of the FIDG and / or FANCM protein, is obtained by silencing the gene encoding said protein by expressing in said plant an interfering RNA targeting said gene.

[0047] An interfering RNA (RNAi) is a small RNA that can silence a target gene in a sequence-specific manner. Interfering RNAs include, in particular, small interfering RNAs (siRNAs) and microRNAs (miRNAs).

[0048] Initially, DNA constructs for expressing interfering RNAs in plants contained a 100 bp or longer fragment (typically 100–800 bp) of the target gene's cDNA, under transcriptional control by a suitable promoter. Widely used constructs are those that can produce a hairpin RNA transcript (hpRNA). In these constructs, the target gene fragment is inverted, usually with a gap region between the repeats (for review, see Watson et al., FEBS Letters, 2005, 579, 5982–5987). Artificial microRNAs (amiRNAs) targeting the gene can also be used. RECQ4 Or TOP3A (Ossowski et al., The Plant Journal, 2008, 53, 674-690, ; Schwab et al., Methods Mol Biol., 2010, 592, 71-88; Wei et al., Funct Integr Genomics., 2009, 9, 499-511).

[0049] To inhibit the TOP3A protein, the method of the invention is advantageously implemented with one of the following genetically modified plants: (i) a mutant plant homozygous for the mutation of TOP3A, as defined above, resulting in the expression of a TOP3A protein mutated in the C-terminal domain, for example a truncated TOP3A protein, in which the TOPRIM and Topoisomerase IA domains are intact but at least one or both of the zinc finger domains are inhibited, and preferably inactivated, (ii) a mutant and transgenic plant, homozygous for a mutation that inhibits the TOP3A gene and comprising a transgene TOP3A encoding a C-terminally mutated TOP3A protein, for example a truncated TOP3A protein, as defined above, and (iii) a double transgenic plant, comprising a first transgene encoding an RNAi that targets the gene TOP3A and a second transgene comprising a gene TOP3Arecombinant encoding a mutated TOP3A protein, for example a TOP3A protein truncated at the C-terminus, as defined above; the second transgene not being sensitive to the RNAi produced by the first transgene. To achieve this, one can, without limitation, use an RNAi targeting the C-terminal region of TOP3AInhibition of the RECQ4 or TOP3A protein can also be achieved by abolishing, blocking, or reducing the function of this protein. To inhibit the activity of this protein, inhibitors can be used that bind specifically to a functional domain of the protein and block its activity. These include protein inhibitors, such as peptides, antibodies or functional antibody fragments, or small molecules. As a non-limiting example, small molecule inhibitors of RecQ are described in Nguyen et al., Chemistry & Biology, 2013, 20, 1, 24, 55-62. Advantageously, these inhibitors are applied to plants.

[0050] The present invention also relates to an expression cassette, characterized in that it comprises a recombinant DNA sequence whose transcript is an interfering RNA targeting the gene RECQ4placed under the transcriptional control of a functional promoter in a plant cell, said gene RECQ4 encoding a RECQ4 protein having at least 40% sequence identity with the RECQ4 protein of sequence SEQ ID NO: 1 and further comprising a region having at least 60% sequence identity with the region extending from positions 407 to 959 of sequence SEQ ID NO: 1.

[0051] In one embodiment, the invention relates to an expression cassette as described above, characterized in that it further comprises a recombinant DNA sequence encoding a TOP3A protein comprising an unaltered TOPRIM domain and Topoisomerase IA domain and at least one zinc finger domain which is inactivated, placed under transcriptional control of a functional promoter in a plant cell, said TOP3A protein having at least 50% sequence identity with the TOP3A protein of sequence SEQ ID NO: 2.

[0052] In one embodiment, the invention relates to an expression cassette as described above, characterized in that it further comprises a recombinant DNA sequence whose transcript is an interfering RNA targeting the gene FANCM Or FIDG, placed under the transcriptional control of a functional promoter in a plant cell, said gene FANCM encoding a FANCM protein having at least 30% sequence identity, or at least 45% sequence similarity with the AtFANCM protein of sequence SEQ ID NO: 45, and containing a DEXDc helicase domain and a HELICc helicase domain and said gene FIDG encoding a FIDG protein having at least 45% sequence identity, or at least 60% sequence similarity with the AtFIDG protein of sequence SEQ ID NO: 46 and containing an AAA-ATPase domain and a VSP4 domain.

[0053] The mutated TOP3A protein can, in particular, be expressed from a gene promoter TOP3A,particularly in plants, such as the gene promoter TOP3A of the transgenic plant in which said C-terminally mutated TOP3A protein is expressed.

[0054] A wide selection of suitable promoters for the expression of heterologous genes in plant cells or plants is available in art.

[0055] These 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 tissue-specific or cell-specific promoters, which are active only or primarily in certain tissues or cell types, and inducible promoters that are activated by physical or chemical stimuli.

[0056] Examples of constitutive promoters commonly used in plant cells include the 35S promoter of cauliflower mosaic virus (CaMV) described by Kay et al. (Science, 1987, 236, 4805) or its derivatives, the promoter of cassava vein mosaic virus (CsVMV) described in International Application WO 97 / 48819, the maize ubiquitin promoter, and the rice actin-intron-actin promoter (McElroy et al., Mol. Gen. Genet., 1991, 231, 150-160; GenBank accession number S 44221). For the implementation of the present invention, a functional promoter in meiocytes will be selected.

[0057] Within the framework of the present invention, a meiosis-specific promoter (i.e., one that is active exclusively or preferentially in cells undergoing meiosis) may also be used. As a non-limiting example, the DMC1 promoter may be cited (Klimyuk & Jones, Plant J., 1997, 11, 1-1).

[0058] The expression cassettes of the invention generally include a transcriptional terminator, for example the 3'NOS terminator of nopaline synthase (Depicker et al., J. Mol. Appl. Genet., 1982, 1, 561-573), the 3'CaMV terminator (Franck et al., Cell, 1980, 21, 285-294) or the terminator of a gene TOP3A, particularly in plants, such as the gene terminator TOP3A of the transgenic plant in which the mutated TOP3A protein is expressed. They may also include other transcriptional regulators such as activators.

[0059] The recombinant DNA constructs according to the invention may comprise several expression cassettes encoding respectively one of the RNAi targeting the gene RECQ4 Or TOP3A and a C-terminally mutated TOP3A protein, including two expression cassettes, one encoding an RNAi targeting the gene TOP3Aand the other for a TOP3A protein mutated at the C-terminus.

[0060] The present invention also relates to a recombinant vector containing an expression cassette as described above.

[0061] The recombinant DNA constructs according to the invention also include recombinant vectors containing one or more expression cassettes according to the invention as defined above. These recombinant vectors may also include one or more marker genes, which allow for the selection of transformed cells or plants.

[0062] The choice of the most appropriate vector depends in particular on the intended host and the method planned for transforming that host. Numerous methods for the genetic transformation of plant cells or plants are available in the art, for many plant species, both dicotyledonous and monocotyledonous. Non-limiting examples include virus-mediated transformation, microinjection transformation, electroporation transformation, microprojectile transformation, and transformation by Agrobacterium, etc.

[0063] The present invention also relates to a host cell transformed by a recombinant vector as described above.

[0064] The invention also relates to a host cell comprising at least one recombinant DNA construct according to the invention. This cell may comprise two recombinant DNA constructs, one encoding an RNAi targeting the gene TOP3Aand the other for a TOP3A protein mutated at the C-terminus. This host cell can be a prokaryotic cell, for example, a cell of Agrobacterium, or a eukaryotic cell, for example a plant cell genetically transformed by at least one 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.

[0065] The present invention also relates to a mutant plant characterized in that it contains a mutation in the gene RECQ4encoding a RECQ4 protein having at least 40% sequence identity with the RECQ4 protein of sequence SEQ ID NO: 1 and further comprising a region having at least 60% sequence identity with the region extending from positions 407 to 959 of sequence SEQ ID NO: 1, said mutation inducing inhibition of the expression or function of the RECQ4 protein in said plant and being selected from point mutations or deletions of all or part of the coding sequence or promoter of said gene.

[0066] In one embodiment, the invention relates to a mutant plant as described above, characterized in that it further contains: a mutation in the gene TOP3A encoding a TOP3A protein having at least 50% sequence identity with the TOP3A protein of sequence SEQ ID NO: 2, the gene FIDGencoding a FIDG protein having at least 45% sequence identity, or at least 60% sequence similarity with the AtFIDG protein of sequence SEQ ID NO: 46 and containing an AAA-ATPase domain and a VSP4 domain, and / or the gene FANCM encoding a FANCM protein having at least 30% sequence identity, or at least 45% sequence similarity with the AtFANCM protein of sequence SEQ ID NO: 45, and containing a DEXDc helicase domain and a HELICc helicase domain, said mutation inducing inhibition of the expression or function of the TOP3A, FIDG and / or FANCM protein in said plant or at least a recombinant DNA sequence whose transcript is an interfering RNA targeting the gene TOP3A, FIDG Or FANCM,placed under transcriptional control of a functional promoter in said plant; and / or a recombinant DNA sequence encoding a TOP3A protein having at least 50% sequence identity with the TOP3A protein of sequence SEQ ID NO: 2, comprising an unaltered TOPRIM domain and Topoisomerase IA domain and at least one zinc finger domain which is inactivated, placed under transcriptional control of a functional promoter in said plant.

[0067] The present invention also relates to a transgenic plant containing a transgene comprising a recombinant DNA sequence whose transcript is an interfering RNA targeting the RECQ4 gene, placed under transcriptional control of a functional promoter in said plant, said gene RECQ4encoding a RECQ4 protein having at least 40% sequence identity with the RECQ4 protein of sequence SEQ ID NO: 1 and further comprising a region having at least 60% sequence identity with the region extending from positions 407 to 959 of sequence SEQ ID NO: 1.

[0068] In one embodiment, the invention relates to a transgenic plant as defined above, characterized in that it further contains: a recombinant DNA sequence whose transcript is an interfering RNA targeting the gene TOP3A encoding the TOP3A protein having at least 50% sequence identity with the TOP3A protein of sequence SEQ ID NO: 2, the gene FIDG encoding a FIDG protein having at least 45% sequence identity, or at least 60% sequence similarity with the AtFIDG protein of sequence SEQ ID NO: 46 and containing an AAA-ATPase domain and a VSP4 domain, and / or the gene FANCMencoding the FANCM protein having at least 30% sequence identity, or at least 45% sequence similarity with the AtFANCM protein of sequence SEQ ID NO: 45, and containing a DEXDc helicase domain and a HELICc helicase domain, said recombinant DNA sequence being placed under transcriptional control of a functional promoter in said plant or a mutation in the gene TOP3A, FIDG and / or FANCM, said mutation inducing inhibition of the TOP3A, FIDG and / or FANCM protein in said plant; and / or a recombinant DNA sequence encoding a TOP3A protein having at least 50% sequence identity with the TOP3A protein of sequence SEQ ID NO: 2, comprising an unaltered TOPRIM domain and Topoisomerase IA domain and at least one zinc finger domain which is inactivated, placed under transcriptional control of a functional promoter in said plant.

[0069] The invention also encompasses plants genetically transformed by at least one DNA construct of the invention. Preferably, said plants are transgenic plants, in which said construct(s) are contained within a transgene integrated into the plant's genome, such that it is transmitted to successive generations of plants. Expression of the DNA construct of the invention results in negative regulation of gene expression. RECQ4 and / or TOP3A, negative regulation of gene expression TOP3A being combined with the expression of a C-terminally mutated TOP3A protein, which confers to said transgenic plants a higher level of meiotic COs than that of the wild-type plants (not containing the DNA construct of the invention) from which they are derived. Generally, this level of meiotic COs is at least 50% higher, preferably at least twice as high, as that of the wild-type plants from which they are derived.

[0070] Particularly advantageously, the meiotic COs level can be further increased by combining, in the same plant, the inhibition of the RECQ4 and / or TOP3A protein with that of the FANCM and / or FIDG protein. In this case, the meiotic COs level is at least 3 times higher, preferably at least 5 times higher, than that of the wild-type plants from which they are derived.

[0071] The increase in meiotic COs levels by inhibition of the FANCM protein is described in PCT Application WO2013038376.

[0072] The FANCM protein is defined as a protein having at least 30% and, in increasing order of 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 increasing order of preference, at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence similarity with the AtFANCM protein of Arabidopsis thaliana(GenBank: NP_001185141; UniProtKB: F4HYE4), and containing a helicase domain DEXDc (cd00046) and a helicase domain HELICc (cd00079). Preferably, the DEXDc helicase domain has at least 65%, and in increasing order of preference, at least 70, 75, 80, 85, 90, 95, or 98% sequence identity, or at least 75%, and in increasing order of preference, at least 80, 85, 90, 95, or 98% sequence similarity with the DEXDc domain (amino acids 129–272) of the AtFANCM protein, and the HELICc helicase domain has at least 60%, and in increasing order of preference, at least 65, 70, 75, 80, 85, 90, 95, or 98% sequence identity, or at least 70%, and in increasing order of preference, at least 75, 80, 85, 90, 95, or 98% sequence similarity with the domain HELICc (amino acids 445-570) of the AtFANCM protein. The polypeptide sequence of the AtFANCM protein is represented in the sequence list in the appendix under SEQ ID NO: 45.

[0073] The identity and sequence similarity values ​​shown for the FANCM protein are calculated using the BLASTP or Needle program with default parameters. Similarity calculations are performed using the BLOSUM62 matrix.

[0074] The sequence of the FIDGETIN-LIKE 1 protein of Arabidopsis thaliana is represented in the sequence list in the appendix under SEQ ID NO: 46. A search of sequence databases identified orthologs of AtFIDG in a wide range of eukaryotes, and it is very likely that this protein is conserved across all land plants. Non-limiting examples of orthologs of AtFIDG are listed in the table below. Table III AtFIDG Ortholog Sequences : Plant Access number Brachypodium distachyon Genbank: XM_003576467 Carica papaya Phytozome: evm.model.supercontig_171.22 PLAZA: CP00171G00260 Fragaria vesca Phytozome: mrna25885.1 PLAZA: FV6G37220 Glycine max Phytozome: Glyma19g18350.2 and Glyma05g14440.2 PLAZA: GM19G18350 and GM05G14440 Oryza sativa GenBank: ABA97741 Populus tricocarpa GenBank: POPTR_0001s33870 Ricinus communis Genbank: XM_002509479 Solanum lycopersicum Genbank: XM_004233540.1 Bicolor sorghum Genbank: XM_002442067.1 Theobroma cacao Phytozome: Thecc1EG017182t1 PLAZA: TC04G003320 Vitis vinifera GenBank: CBI21358 Zea mays GenBank: DAA54951 Hordeum vulgare Genbank: BAK02801. Triticum urartu Genbank: EMS65393.1.

[0075] The FIDG protein has at least 35%, and in ascending order of preference, at least 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence identity, or at least 50%, and in ascending order of preference, at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence similarity with the AtFIDG protein of sequence SEQ ID NO: 46, and containing an AAA-ATPase domain (PF00004) and a VSP4 domain (PF09336).

[0076] Preferably, said FIDG protein has at least 70%, and in increasing order of preference, at least 75, 80, 85, 90, 95 or 98% sequence identity, or at least 80%, and in increasing order of preference, at least 85, 90, 95 or 98% sequence similarity with any of the orthologs of AtFIDG the list of which is shown in Table III above.

[0077] According to a preferred embodiment of the present invention, the AAA-ATPase domain of said FIDG protein has at least 60%, and in increasing order of preference, at least 65, 70, 75, 80, 85, 90, 95 or 98% sequence identity, or at least 70%, and in increasing order of preference, at least 75, 80, 85, 90, 95 or 98% sequence similarity with the AAA-ATPase domain of the AtFIDG protein (amino acids 431-561 of SEQ ID NO: 46).

[0078] According to another preferred embodiment of the present invention, the VSP4 domain of said FIDG protein has at least 60%, and in increasing order of preference, at least 65, 70, 75, 80, 85, 90, 95 or 98% sequence identity, or at least 70%, and in increasing order of preference, at least 75, 80, 85, 90, 95 or 98% sequence similarity with the VSP4 domain of the AtFIDG protein (amino acids 623-672 of SEQ ID NO: 46).

[0079] Advantageously, said FIDG protein further contains a RAD51-binding domain, having at least 50%, and in ascending order of preference, at least 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence identity, or at least 70%, and in ascending order of preference, at least 75, 80, 85, 90, 95 or 98% sequence similarity with amino acids 268-346 of SEQ ID NO: 46.

[0080] The identity and sequence similarity values ​​shown for the FIDG protein are calculated over the entire length of the compared sequences using the Needleman-Wunsch global alignment algorithm (Needle EMBOSS program with default parameters). Similarity calculations are performed using the BLOSUM62 matrix.

[0081] The present invention can be applied particularly in the field of plant breeding, in order to accelerate the development of new varieties. It also facilitates recombination between related species, and therefore the introgression of traits of interest. Furthermore, it accelerates the creation of genetic maps and positional cloning.

[0082] The present invention applies to a wide range of monocotyledonous or dicotyledonous plants of agronomic interest. By way of non-limiting examples, one may mention rapeseed, sunflower, potato, maize, wheat, barley, rye, sorghum, rice, soybean, bean, carrot, tomato, courgette, pepper, aubergine, turnip, onion, pea, cucumber, leek, artichoke, beetroot, cabbage, cauliflower, lettuce, endive, melon, watermelon, strawberry, apple tree, pear tree, plum tree, poplar, grapevine, cotton, rose, tulip, etc.

[0083] The present invention will be better understood with the aid of the following supplementary description, which refers to non-limiting examples illustrating the effects of gene mutations ATERCQ4 Or AtTOP3A, alone or in combination with gene mutations FANCM,on meiotic recombination and the Cosine rate, with references to the attached drawings in which: figure 1 Phylogenetic tree of RECQ4 proteins from flowering plants. The analysis was performed on the Phylogeny.fr platform according to the following steps: the sequences were aligned with T-Coffee (v6.85) using the following pairwise alignment methods: the 10 best local alignments (Lalign_pair), and a global exact alignment (slow_pair). After alignment, positions with gaps were removed from the alignment. The phylogenetic tree was reconstructed using the maximum likelihood method, implemented in the PhyML v3.0 aLRT program. The protein sequences were collected from the PLAZA database ( http: / / bioinformatics.psb.ugent.be / plaza / ) and that of the phytozome ( http: / / www.phytozome.net. ): At : Arabidopsis thaliana. Al Arabidopsis lyrata. Bra: Brassica rapa. Esa: Thellungiella halophila. Cp: Carica papaya. Tcacao: Theobroma cacao. Graimndii: Gossypium raimondii. VV: Vitis vinifera. Ppersica: Prunus persica. RC: Ricinus communis. ME: Manihot esculenta. Solyc: Solanum lycopersicum. Eucgr: Eucalypsus grandis. Phvul: Phaseolus vulgaris. Glyma: Glycine max. OS: Oryza sativa. BD: Brachypodium distachyon. Si: Setaria italica. Zmays: Zea mays. Sbicolor: Sorghum bicolor. figure 2 . Alignment of eukaryotic 3a TOPOISOMERASES using T-Coffee. The areas that have been preserved are indicated by a vertical or horizontal line. At : arabidopsis thaliana. Sc. Saccharomyces cerevisiea. Os Oryza sativa. Hs Homo sapiens. Ds Drosophila melanogaster. Coenorabditis elegans. Sp Schizosaccharomyces pombe.The alignment was performed using T-Coffee ( http: / / toolkit.tuebingen.mpg.de / tcoffee ) with the default settings. figure 3 . The double mutation of RECQ4A And RECQ4B restores the formation of bile acids in mutants zmm. Average number of bivalents per male meiocyte. Ring bivalents (black). Straight bivalents (grey). Univalent pairs (black dots). The number of metaphase I cells analyzed is indicated in parentheses. figure 4 . The RECQ4A and RECQ4B helicases redundantly limit CO2 formation during meiosis. Genetic distances in four intervals, measured using lines carrying fluorescent markers, were calculated with the Perkins equation and are expressed in centiMorgans. I2a and I2b are adjacent intervals on chromosome 2, and I5c and I5d on chromosome 5. figure 5 . The mutation top3a-R640X restores the training of bivalents in mutants zmm. Average number of bivalents per male meiocyte. Ring bivalents (black). Straight bivalents (grey). Univalent pairs (black dots). The number of metaphase I cells analyzed is indicated in parentheses. figure 6 . TOP3A and FANCM independently limit CO formation during meiosis. Genetic distances in four intervals, measured using lines carrying fluorescent markers, were calculated with the Perkins equation and are expressed in centiMorgans. I2a and I2b are adjacent intervals on chromosome 2, and I5c and I5d on chromosome 5. figure 7 . RECQ4 and FANCM independently limit CO formation during meiosis. Genetic distances in two intervals, measured using lines carrying fluorescent markers, were calculated with the Perkins equation and are expressed in centiMorgans. I2a and I2b are adjacent intervals on chromosome 2. EXAMPLE 1: OBTAINING GENE MUTANTS RECQ4 GENE MUTATION DELETERS ZMM

[0084] Seeds of the mutant msh4 d Arabidopsis thalianain the Landsberg eracta genetic background (cshl_GT14269; Drouaud et al., PLoS Genet., 2013, 9, e1003922; Higgins et al., Genes Dev., 2004, 18, 2557-2570) were mutated by EMS (ethylmethane sulfonate). Plants grown from the mutated seeds (population M1, heterozygous for the EMS-induced mutations, and homozygous for the gene mutation) ZMM ) have an identical phenotype, resulting from the inactivation of the gene ZMM,This results in a sharp decrease in the frequency of co-occurring chromosomes (COs), leading to a significant decrease in the number of bivalents and a substantial drop in fertility (resulting in "semi-sterile" plants). This leads to the formation of short siliques that are easily distinguishable from those of wild-type plants. The M1 plants were self-pollinated to produce a population of offspring (population M2; approximately 1000 families) potentially containing plants homozygous for the EMS-induced mutations. Plants in population M2 have longer siliques than the wild-type plants. zmm homozygous individuals from the M1 generation were selected and genotyped to verify their homozygous status for the mutation msh4. These are suppressors.

[0085] Two lines of mutant suppressors of the gene mutation AtMSH4, called msh4(s)84 and msh4(s)101,are the subject of this study. Whole-genome sequencing of these mutants showed that the corresponding mutations were located in the gene At1g10930 encoding the RECQ4A helicase. The mutant msh4(s)84 includes the change C>T at position TAIR 10: chrl:3652474 which introduces a stop codon at codon W387. The mutant msh4(s)101 includes the C>T change at position TAIR 10: chr1:3650343 which introduces the G762D substitution. These two suppressors are allelic, demonstrating that mutations in RECQ4A are the causal mutations of fertility restoration.

[0086] Comparison of the Landsberg ecotype sequence (Gan et al., Nature, 2011, 477, 7365, 419-23) with that of the Columbia ecotype genome showed that their loci RECQ4Bdiffered by a series of single nucleotide and insertion / deletion polymorphisms, including two that introduce a premature stop codon (Q430>STOP) and a read frame shift (Aa 501) in Lansdberg. This strongly suggests that the gene RECQ4B The gene is not functional in Lansdberg.

[0087] In light of these results, it was hypothesized that the mutation recq4a Columbia was unable to compensate for the effects of that of msh4 due to redundancy with RECQ4B during meiosis. This would explain why the mutation of RECQ4A alone, would be capable of offsetting the effects of that of msh4 at Landsberg but not Columbia.

[0088] Therefore, the mutations msh4, recq4a, And recq4b were combined in the Columbia genetic background. The number of bivalents in metaphase I of the triple mutant msh4 recq4a-4 recq4b-2 was compared with that of wild plants (Col), of plants zmm ( shock1 / Atzip2 Or msh4 ) and double mutants msh4 recq4a-4 And msh4 recq4b-2.

[0089] The results are illustrated by the Figure 3 While wild plants consistently show 5 bivalents per meiosis, mutants Atmsh4 show a sharp decrease in the number of bivalents, and therefore the appearance of univalents. In wild plants, normal chromosome segregation is observed, leading to the formation of balanced gametes. In contrast, in mutants zmm, We observe poor chromosome segregation resulting from a decrease in the number of bivalents, induced by the mutation zmm, and which leads to unbalanced, non-viable gametes.

[0090] Furthermore, as previously shown (Higgins et al., The Plant Journal, 2011, 65, 492-502), double mutants msh4 recq4a-4 And msh4 recq4b-2 are almost sterile like the mutant msh4 and have a number of bivalents in metaphase I, similar to that of msh4 ( Figure 3 ).

[0091] On the contrary, the triple mutant msh4 recq4a-4 recq4b-2 is fertile and its number of bivalents is restored to the level of that of the wild type ( Figure 3 Similarly, the double mutation recq4a recq4b is also capable of restoring fertility and bivalent formation in another mutant zmm, shoc1 / Atzip2 ( Figure 3 ).

[0092] These results show that at Columbia, RECQ4A And RECQ4B have redundant functions that prevent the formation of meiotic COs in mutants zmm. EXEMPLE 2: INFLUENCE OF GENE INACTIVATION RECQ4 ON THE FREQUENCY OF MEIOTIC RECOMMENDATION

[0093] The effects of mutations recq4 then measured meiotic recombination in a wild plant (Columbia), i.e, in the presence of ZMMs functional. Meiotic recombination frequency was measured by tetrad analysis using fluorescently labeled cell lines, as described by Berchowitz & Copenhagen, Nat. Protoc., 2008, 3, 41-50). Genetic distance was measured at four different genetic intervals: two adjacent intervals on chromosome 2 (I2a and I2b) and two adjacent intervals on chromosome 5 (I5c and I5d). The results are illustrated by the Figure 4 .

[0094] Among simple mutants recq4a And recq4b, Genetic distances were not significantly different from the wild type for any of the tested intervals (p>5%). In contrast, genetic distances were considerably increased in the double mutant recq4a recq4b (p<10 -9< ). This increase was by a mean factor of 6.2 with a range of 4.6 to 8.6.

[0095] These results show that RECQ4A and RECQ4B play a major, redundant role in limiting CO formation during meiosis and are the most potent meiotic anti-CO genetic factors identified to date. Furthermore, this demonstrates that having more than six times the CO levels of the wild type does not have deleterious effects on chromosome integrity, balanced chromosome segregation, meiotic completion, or fertility. EXEMPLE 3 : OBTAINING GENE MUTANTS TOP3A GENE MUTATION DELETERS ZMM

[0096] A screening process similar to that carried out for msh4 (Example 1) was performed to search for mutant suppressors zmm hei10 (Chelysheva et al., PLoS Genet., 2012,8, e1002799). Among 1000 mutagenized lines derived from the mutant hei10-2, a suppressor, hei10(s)61, exhibiting higher fertility and number of bivalents than the mutant hei10, has been isolated ( Figure 5 Cloning the mutation from the genetic map defined a 2.4 Mb gap on chromosome 5, between positions Tair10_chr5:23.819.915 and Tair10_chr5:26.497.664. Whole-genome sequencing of hei10(s)61 identified a nonsense mutation within this interval, in the gene At5g63920 which codes for TOPOISOMERASE 3 alpha (TOP3A).

[0097] This gene appears to be a good candidate given that TOP3A and Sgs1 belong to the RTR complex, a conserved complex that is essential for maintaining genome integrity (Mankouri and Hickson, Trends Biochem Sci., 2007, 32, 538-46).

[0098] In order to test whether this mutation was the cause of the phenotype, hei10(s)61 was transformed with a 10kb genomic clone containing TOP3A. All the transformants exhibited the phenotype d'hei10 (24 independent lines), were sterile and had 1.8 bivalents ± 0.75 in metaphase I (average of 3 independent lines; Figure 5 This demonstrates that the mutation in TOP3A was in fact the causal mutation that restored fertility and the formation of bivalents in the suppressor hei10(s)61. This shows that AtTOP3A prevents the formation of additional CO in the context hei10. The mutation hei10(s)61 change Arg640 into a stop codon (hereinafter referred to as top3a-R640X ) . Therefore, top3a-R640X code for a protein that has intact TOPRIM and Topoisomerase domains but is truncated of the latter 286 amino acids that contain the two Zinc finger domains, predicted ( Figure 2 ).

[0099] The increase in the number of bivalents and the restoration of fertility observed in the mutant hei10 top3a-R640X were also observed in a double mutant msh5 top3a-R640X (4,2 ± 0.9 bivalents per meiosis; Figure 5 showing that the observed suppression was not specific to the genetic background hei10.

[0100] Plants hei10-2 top3a-2 And hei10-2 top3a-R640X differ in both their somatic and meiotic phenotype. Like a simple mutant top3a-2, hei10-2 top3a-2 shows stunted growth and complete sterility, contrasting with the normal growth and fertility of hei10-2 top3a-R640X. During meiosis, the double mutant hei10 top3a-2 was indistinguishable from a simple mutant top3a-2, with aberrant structures in metaphase I and massive fragmentation in anaphase I. In hei10-2 top3a-R640X, the observed bivalents (4.2 per cell, compared to 1.7 in hei10-2, figure 6 ), segregate in anaphase I without chromosome fragmentation. The phenotype top3a-2 has shown that TOP3A is essential to prevent meiotic catastrophe, while top3a-R640X appears to be a separation of function leading to the restoration of COs in the genetic background hei10, but retaining its effective repair activity. This suggests that TOP3A has a dual function during meiosis, being essential both for resolving meiotic recombination intermediates and for limiting CO formation.

[0101] In plants hei10-2 top3a-R640X / top3a-2, No fragmentation was observed, showing that a copy of TOP3AR640X is sufficient to repair the recombination intermediates. The number of bivalents in this genetic background is even higher than in the double mutant hei10 top3a-R640X (4.9±0.1 vs 4.2±1.1 bivalents ±, T-test, figure 5 ), suggesting that the TOP3A-R640X protein could retain anti-CO activity.

[0102] Next, the effect of the mutation top3a-R640X in plants possessing a HEI10 functional, was analyzed. In accordance with the results obtained in the context hei10, plants top3a-R640X show no somatic abnormalities and are fertile [seeds per fruit: wild type=61 ±4 (n=40), top3a-R640X = 61±3 (n = 40)]. Meiosis of the mutant top3a-R640X is almost indistinguishable from that of the wild, with no observation of fragmentation.

[0103] However, a low frequency of univalents was observed in the single mutant top3a-R640X (0.3 per cell, Figure 5 ), whereas univalents are not observed in the wild type. This suggests that obligatory CO is slightly affected in the mutant top3a-R640X. Plants top3a-R640X / top3a-2 show no developmental abnormalities, are fertile, and no univalents have been observed ( Figure 5 ). EXEMPLE 4 : INFLUENCE OF GENE MUTATION TOP3A ON THE FREQUENCY OF MEIOTIC RECOMMENDATION

[0104] The meiotic recombination frequency of the mutant top3a-R640X a then was analyzed by tetrad analysis using lines labeled with a fluorescent marker, in 4 different genetic intervals, as described in example 2 ( Figure 6 In all tested intervals, genetic distances were increased compared to wild-type (P < 10⁻⁵). This increase averaged by a factor of 1.5, with a range of 1.3 to 1.8. This shows that TOP3A has anti-CO activity not only in a context hei10, but also in wild plants for ZMMs. EXEMPLE 5 : INFLUENCE OF COMBINED GENE INACTIVATION TOP3A AND FANCM ON THE FREQUENCY OF MEIOTIC RECOMMENDATION

[0105] The FANCM helicase was the first meiotic anti-CO gene described in Arabidopsis (PCT Request WO 2013 / 038376; Crismani et al., Science, 336, 6088, 1588-90). To test whether TOP3A and FANCM act in the same pathway, the recombination frequency was measured in the double mutant top3a-R640Xfancm-1 ( Figure 6 In the four intervals tested, it was observed that the genetic distances were significantly greater in the double mutant top3a-R640X fancm-1 than in the two single mutants (p<10⁻⁵). The increase in CO in the double mutant, compared to the wild type, was on average a factor of 4.9, with a range of 3.5 to 5.6. The effects of the two mutations on CO formation are cumulative, demonstrating that FANCM and TOP3A act on two parallel pathways to limit CO formation during meiosis. Interestingly, the double mutant top3a-R640X fancm-1 has normal growth, wild-type meiosis and is completely fertile [seeds per fruit: wild type = 61 ±4 (n=40), top3a-R640X fancm-1 =65 ±2 (n = 40)]. This confirms that having five times more COs than the wild type does not have immediate deleterious effects on chromosome integrity, balanced chromosome segregation, completion of meiosis, and fertility. EXEMPLE 6 : INFLUENCE OF COMBINED GENE INACTIVATION FANCM AND RECQ4 ON THE FREQUENCY OF MEIOTIC RECOMMENDATION

[0106] The FANCM helicase was the first meiotic anti-CO gene described in Arabidopsis (PCT Request WO 2013 / 038376; Crismani et al., Science, 336, 6088, 1588-90). To test whether RECQ4 and FANCM act in the same pathway, the recombination frequency was measured in the triple mutant recq4a recq4b fancm-1 ( Figure 7 In both intervals tested, it was observed that the genetic distances were significantly greater in the triple mutant recq4a recq4b fancm-1 than in the case of the double mutant recq4a recq4b (p< 0.01) and the simple mutant fancm-1 (p<10⁻⁶). The increase in CO₂ in the triple mutant, compared to the wild type, was on average a factor of 9. The effects of gene mutations recq4 And fancm The effects on CO formation are cumulative, demonstrating that FANCM and RECQ4 act in parallel to limit CO formation during meiosis. Interestingly, the triple mutant recq4a recq4b fancm-1 has normal growth, wild-type meiosis (n=450) and is completely fertile [seeds per fruit: wild type = 53.7 ±4.8 (n=40), recq4a recq4b fancm-1 =49.2 ±5.3 (n = 40)]. This confirms that having nine times more COs than the wild type does not have immediate deleterious effects on chromosome integrity, balanced chromosome segregation, completion of meiosis, and fertility.

Claims

1. A method for increasing the frequency of meiotic crossovers in a plant, characterized in that it comprises inhibiting in said plant the expression or function of at least one protein of the RTR complex called RECQ4, by mutagenesis of the gene encoding said protein or its promoter, by extinction of the gene encoding said protein, or by an inhibitor specifically binding to a functional domain of said protein, said RECQ4 protein having at least 40% sequence identity with the RECQ4 protein of sequence SEQ ID NO: 1 and further comprising a region having at least 60% sequence identity with the region extending from positions 407 to 959 of the sequence SEQ ID NO: 1.

2. The method according to claim 1, characterized in that it further comprises inhibiting in said plant the expression or function of a protein of the RTR complex called TOP3A, by mutagenesis of the gene encoding said protein or its promoter, by extinction of the gene encoding said protein, or by an inhibitor specifically binding to a functional domain of said protein, said TOP3A protein having at least 50% sequence identity with the TOP3A protein of sequence SEQ ID NO: 2.

3. The method according to claim 1 or claim 2, <b>characterized in that it further comprises inhibiting in said plant the expression or function of at least one protein selected from: - a protein hereinafter referred to as FIDG, said protein having at least 45% sequence identity, or at least 60% sequence similarity, with the AtFIDG protein of sequence SEQ ID NO: 46 and containing an AAA-ATPase domain and a VSP4 domain, and - a protein hereinafter referred to as FANCM, said protein having at least 30% sequence identity, or at least 45% sequence similarity with the AtFANCM protein of sequence SEQ ID NO: 45, and containing a DEXDc helicase domain and a HELICc helicase domain, by mutagenesis of the gene encoding said protein or its promoter, by extinction of the gene encoding said protein, or by an inhibitor specifically binding to a functional domain of said protein.

4. The method according to any one of claims 1 to 3, characterized in that the inhibition of the RECQ4 and / or TOP3A protein and, where applicable, the FIDG and / or FANCM protein, is obtained by silencing the gene encoding said protein by expressing in said plant an interfering RNA targeting said gene.

5. An expression cassette, characterized in that it comprises a recombinant DNA sequence whose transcript is an interfering RNA targeting the RECQ4 gene, placed under the transcriptional control of a functional promoter in a plant cell, said RECQ4 gene encoding a RECQ4 protein having at least 40% sequence identity with the RECQ4 protein of sequence SEQ ID NO: 1 and further comprising a region having at least 60% sequence identity with the region extending from positions 407 to 959 of the sequence SEQ ID NO: 1.

6. The expression cassette according to claim 5, characterized in that it further comprises a recombinant DNA sequence encoding a TOP3A protein comprising an unaltered TOPRIM domain and a Topoisomerase IA domain and at least one zinc finger domain that is inactivated, placed under the transcriptional control of a functional promoter in a plant cell, said TOP3A protein having at least 50% sequence identity with the TOP3A protein of sequence SEQ ID NO: 2.

7. The expression cassette according to claim 5 or 6, characterized in that it further comprises a recombinant DNA sequence whose transcript is an interfering RNA targeting the FANCM or FIDG gene, placed under the transcriptional control of a functional promoter in a plant cell, said FANCM gene encoding a FANCM protein having at least 30% sequence identity, or at least 45% sequence similarity, with the AtFANCM protein of sequence SEQ ID NO: 45, and containing a DEXDc helicase domain and a HELICc helicase domain, and said FIDG gene encoding a FIDG protein having at least 45% sequence identity, or at least 60% sequence similarity, with the AtFIDG protein of sequence SEQ ID NO: 46 and containing an AAA-ATPase domain and a VSP4 domain.

8. A recombinant vector containing an expression cassette according to any one of claims 5 to 7.

9. A host cell transformed with a recombinant vector according to claim 8.

10. A mutant plant characterized in that it contains a mutation in the RECQ4 gene encoding a RECQ4 protein having at least 40% sequence identity with the RECQ4 protein of sequence SEQ ID NO: 1 and further comprising a region having at least 60% sequence identity with the region extending from positions 407 to 959 of the sequence SEQ ID NO: 1, said mutation inducing the inhibition of the expression or function of the RECQ4 protein in said plant and being selected from the point mutations or deletion of all or part of the coding sequence or the promoter of said gene.

11. The mutant plant according to claim 10, characterized in that it further contains: - a mutation in the TOP3A gene encoding a TOP3A protein having at least 50% sequence identity with the TOP3A protein of sequence SEQ ID NO: 2, the FIDG gene encoding an FIDG protein having at least 45% sequence identity, or at least 60% sequence similarity, with the AtFIDG protein of sequence SEQ ID NO: 46 and containing an AAA-ATPase domain and a VSP4 domain, and / or the FANCM gene encoding a FANCM protein having at least 30% sequence identity, or at least 45% sequence similarity, with the AtFANCM protein of sequence SEQ ID NO: 45, and containing a DEXDc helicase domain and a HELICc helicase domain, said mutation inducing the inhibition of the expression or function of the TOP3A, FIDG and / or FANCM protein in said plant, or at least one recombinant DNA sequence whose transcript is an interfering RNA targeting the TOP3A, FIDG, or FANCM gene, placed under the transcriptional control of a functional promoter in said plant; and / or - a recombinant DNA sequence encoding a TOP3A protein having at least 50% sequence identity with the TOP3A protein of sequence SEQ ID NO: 2, comprising an unaltered TOPRIM domain and a non-altered Topoisomerase IA domain and at least one zinc finger domain that is inactivated, placed under the transcriptional control of a functional promoter in said plant.

12. A transgenic plant containing a transgene comprising a recombinant DNA sequence whose transcript is an interfering RNA targeting the RECQ4 gene, placed under the transcriptional control of a functional promoter in said plant, said RECQ4 gene encoding a RECQ4 protein having at least 40% sequence identity with the RECQ4 protein of sequence SEQ ID NO: 1 and further comprising a region having at least 60% sequence identity with the region extending from positions 407 to 959 of sequence SEQ ID NO: 1.

13. The transgenic plant according to claim 12, characterized in that it further contains: - a recombinant DNA sequence whose transcript is an interfering RNA targeting the TOP3A gene encoding the TOP3A protein having at least 50% sequence identity with the TOP3A protein of sequence SEQ ID NO: 2, the FIDG gene encoding an FIDG protein having at least 45% sequence identity, or at least 60% sequence similarity, with the AtFIDG protein of sequence SEQ ID NO: 46 and containing an AAA-ATPase domain and a VSP4 domain, and / or the FANCM gene encoding the FANCM protein having at least 30% sequence identity, or at least 45% sequence similarity with the AtFANCM protein of sequence SEQ ID NO: 45, and containing a DEXDc helicase domain and a HELICc helicase domain, said recombinant DNA sequence being placed under the transcriptional control of a functional promoter in said plant, or a mutation in the TOP3A, FIDG and / or FANCM gene, said mutation inducing the inhibition of the TOP3A, FIDG and / or FANCM protein in said plant; and / or - a recombinant DNA sequence encoding a TOP3A protein having at least 50% sequence identity to the TOP3A protein of sequence SEQ ID NO: 2, comprising an unaltered TOPRIM domain and a non-altered Topoisomerase IA domain and at least one zinc finger domain that is inactivated, placed under the transcriptional control of a functional promoter in said plant.