Abstract:
The invention relates to the use of a gene of the Argonaute family, a transcript of said gene or the ORF of said gene in order to produce plants that are completely or partially apomictic. The invention can be used to control apomixis in cultivated species having sexual reproduction.

Description:
This application is the U.S. national phase of International Application No. PCT/IB2010/054769, filed 21 Oct. 2010, which designated the U.S. and claims priority to France Application No. 09/05046, filed 21 Oct. 2010, the entire contents of each of which are hereby incorporated by reference. 
     FIELD OF THE INVENTION 
     The subject of the invention is means for regulating reproductive development in cultivated plants. More particularly, the subject of the invention is the development of plants which reproduce completely or partially by gametophytic apomixis, i.e. asexually by means of seeds. 
     BACKGROUND OF THE INVENTION 
     Gametophytic apomixis is a form of asexual reproduction through seeds. It exists in numerous angiosperms, and close to 400 apomictic species have been listed. However, no apomictic plants are found among the main cultivated cereals (corn, wheat or rice), but only in wild plants, a few cultivated fodder species, and certain fruit species. Apomixis is a genetically controlled mechanism. Apomictic plants develop female gametes without prior meiosis. The gametes thus formed contain a genome identical to that of the somatic tissue from which they are derived. The development of the embryo from these gametes takes place without fertilization by a male gamete, i.e. by parthenogenesis. The genome of the embryo thus formed is therefore strictly identical to that of its mother plant, without paternal contribution. Apomixis is therefore a mode of cloning through seeds which ensures that genotypes are perpetuated identically through the generations. 
     The use of apomixis in a controlled manner in cultivated species offers many potential applications. These applications relate to the propagation of unstable genotypes, the control of pollen contaminations, methods for improving plants, and methods for the commercial production of seeds. 
     None of these applications can be envisioned in the main cultivated species such as wheat, corn, rice and the like, on the basis of current technologies. No apomictic forms are in fact known in these various species, and no genetic system which makes it possible to induce apomixis in sexual plants is known. 
     Many laboratories have, over the past years, endeavored to develop apomictic plants, either by attempting to transfer the determinants of apomixis from wild plants to cultivated plants, or by inducing apomictic phenotypes in sexual plants by mutagenesis. Neither of these two approaches has produced an apomictic genotype in a species in which this mode of reproduction did not exist beforehand. 
     Results recently published in the journal Nature (Ravi, M., Marimuthu, M. P., and Siddiqi, I. (2008). Gamete formation without meiosis in  Arabidopsis . Nature 451: 1121-1124.), show, in  Arabidopsis , that the inactivation of a gene involved in meiosis, called DYAD, the function of which is to regulate chromatid cohesion during meiosis, makes it possible to produce approximately 0.1% of gametes which escape meiosis. The remainder of the gametes, and therefore 99.9% of the descendants, are sterile. It is even probable that the frequency of non-meiotic gametes is not significantly different, in these mutant plants, than that in sexual plants, and that these gametes in fact become apparent only because the mutation kills, moreover, all the gametes normally derived from sexuality. 
     Another recent study (Erfurth, I., Jolivet, S., Froger, N., Catrice, O., Novatchkova, M., and Mercier, R. (2009). Turning meiosis into mitosis. PLoS Biol. 7: e1000124), shows that it is possible, in  Arabidopsis , to change meiotic division into mitotic division through the simultaneous activation of three genes involved in meiosis (osd1/Atspo11-1/Atrec8). The triple mutant produces functional diploid gametes. However, these gametes are fertilized, the descendants are not therefore apomictic, and it is not known whether this result is transposable to species other than  Arabidopsis.    
     The work by the inventors in the field have shown that it is possible to induce a completely or partially apomictic phenotype in corn by manipulating the expression of particular genes. The seeds produced evade meiotic reproduction and are fertile. These results advantageously apply to other cultivated plants such as rice or wheat. 
     SUMMARY OF THE INVENTION 
     The invention is therefore directed toward the use of specific nucleotide sequences, the manipulation of which makes it possible to develop cultivated plants, such as corn, rice and wheat, which reproduce completely or partially by gametophytic apomixis. 
     The objective of the invention is also to provide a method for producing apomictic plants. 
     According to yet another aspect, the invention aims to use apomixis in sexually reproducing cultivated species, in a controlled manner, for developing numerous applications, as will be set out hereinafter. 
     The invention is thus directed toward the use of a gene of the Argonaute family, of a transcript of this gene or of the ORF thereof for producing partially or completely apomictic plants. 
     This involves more especially a gene of the Argonaute family encoding a protein of sequence SEQ ID No. 1. 
     More particularly, the invention is directed toward the use of a gene of the Argonaute family corresponding to the sequence SEQ ID No. 2 or of the transcript of such a gene corresponding to the sequence SEQ ID No. 3, or of the ORF of sequence SEQ ID No. 4. 
     In the use according to the invention, the gene defined above is inactivated by mutagenesis. 
     Such an inactivation allows the formation of gametes of apomeiotic origin. The plants of which the protein is inactivated reproduce apomeiotically. 
     The invention is also directed toward a method for inducing a completely or partially apomictic phenotype in cultivated species such as corn, rice or wheat, characterized in that it comprises the targeted inactivation, by means of a transposable element, for example of Mutator type, of a gene, of a transcript of this gene, or of the ORF thereof, as defined above, and the identification of the mutated locus. 
     The use of apomixis in a controlled manner in cultivated species offers numerous potential applications. They relate to the propagation of unstable genotypes, the control of pollen contaminations, methods for improving plants, and methods for the commercial production of seeds. 
     The first application relates to the clonal propagation, through seeds, of genetically unstable genotypes. This is in particular the case for all hybrid plants; these hybrid plants produce, through genetic mixing during meiosis and fertilization, descendants which are different than one another, and different from their mother plant. This is also the case for cultivated species which have levels of ploidy that are unstable in meiosis, such as triploid forms. 
     In certain cultivated species, in order to maintain a high level of genetic purity, it is necessary to rigorously control pollination, in order to avoid contamination with pollen derived from more or less distant neighboring fields, it being possible for the pollen to move over quite variable distances, depending on the species, the climatic conditions, or the dissemination factors such as insects. In the case of apomictic plants, however, the genome resulting from the male gametes does not contribute to the next generation. The use of apomictic plants would therefore make it possible to dispense with the risks of contamination. Apomixis is therefore a totally unique method for controlling genetic purity. It is also potentially an effective method for avoiding undesirable transgene flow in the case of the growing of genetically modified organisms. 
     Apomixis also offers new perspectives in terms of plant improvement. It would in fact make it possible to use, as a new variety, any genotype selected as advantageous, provided that it involves a genetically determined criterion, regardless of the genetic structure, since, provided that it is apomictic, it becomes genetically stable. It is therefore possible to envision developing varieties directly from hybrid, optionally interspecific, forms, while dispensing with the stabilization steps currently necessary, such as successive self-fertilization steps, or the production of double haploids. This method therefore allows a considerable amount of time to be saved, but also opens the door to the introduction of completely new genetic materials into selection programs, and in particular of genetic materials which, in sexual plants, induce strong sterility. This is the case, for example, for most interspecific crosses. 
     A very important application relates to the production of hybrid seeds. As it is carried out today, this involves the controlled large-scale hybridization of genetically stable parental ecotypes. These are generally homozygous lines, obtained by various methods (production of doubled haploids, self-fertilizations, etc.). One of the two parents is used as the male, the other as the female. Only the females produce the commercial seeds. The yield from the seed production parcels is generally low compared with the hybrids, for three reasons: (1) the male lines are necessary but use a large part of the space without producing seeds; (2) the parental lines generally have a much lower yield than the hybrids owing to inbreeding depression; (3) pollination control involves physical or genetic castration of the lines used as females, a process which leads to a considerable loss of yield. However, in the case of apomictic plants, it could be envisioned to produce the seeds directly from hybrids, and therefore with much higher yields, using 100% of the available area, without the need to control pollination, and without a castration step. The advantage of using apomixis for seed production is very significant in species such as corn, where hybrid forms are already produced, for reasons of reduced costs, but also in autogamous species, for instance wheat or rice, where large-scale controlled hybridizations are difficult. The production of a few apomictic hybrid plants would be sufficient to initiate the large-scale production of genetically stable, hybrid seeds. 
     The partially or completely apomictic plants or plant seeds of cultivated species such as corn, rice and wheat, characterized in that they comprise inactivated alleles of a gene as defined above, also fall within the field of the invention. 
     The plants or plant seeds of the invention are advantageously as obtained by inactivation of the gene by mutagenesis or according to the method as described above, in order to induce in the cultivated plants a completely or partially apomictic phenotype. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Other features and advantages of the invention are given by way of illustration in the examples which follow, in which reference is made to  FIGS. 1 to 5 , which represent, respectively, 
         FIGS. 1A to 1C , the results relating to identification of apomeiotic plants in corn by means of a genetic screen; 
         FIG. 2 , the response of the cytological analysis of the FNR4 mutant; 
         FIG. 3 , the phylogenetic tree of the multigene family comprising the FNR4 mutant; 
         FIG. 4 , the structure of the FNR4 gene and the sites for transposable-element insertions in the various alleles that are available; and 
         FIG. 5 , the FNR4 expression profile. 
     
    
    
     DEFINITIONS 
     The term “gametophytic apomixis” refers to a form of asexual reproduction through seeds, in which the gametes produced in the female gametophytes have not undergone meiotic reduction, and therefore have the same ploidy and the same genetic makeup as the mother plant. Gametophytic apomixis involves two successive stages: apomeiosis and parthenogenesis. 
     “Apomeiosis” corresponds to the mechanisms via which apomictic plants evade meiosis. 
     “Parthenogenesis” corresponds to the development of embryos without fertilization and without paternal genetic contribution. 
     DETAILED DESCRIPTION OF THE INVENTION 
     Example 1 
     Identification of Apomeiotic Plants by Means of a Targeted Genetic Screen 
     The results obtained are shown in  FIGS. 1A to 1C : 
     In order to identify apomeiotic plants, a population of mutagenesis based on transposable elements of Mutator type, in a diploid genetic background, was constructed ( FIG. 1A ). The screen is based on the response of the albumen to a divergence in the respective ploidy levels of the male and female gametes. 
     When, in a wild-type corn plant, a diploid female is crossed with a tetraploid male, this results in an early abortion of the development of the albumen and therefore of the seed. 
     The results obtained are shown in  FIG. 1B . When the gametes are at the same ploidy level (both haploids, or both diploids), the seed develops normally. In this screen, a tetraploid plant was used as male, and 15 000 mutagenized plants were used as females. The investigation related to the plants which, owing to the absence of meiotic reduction induced by mutation, produce gametes which are themselves diploid, and therefore in equilibrium with the genomic contribution of the diploid male gametes produced by the pollinator. In this screen, the vast majority of the plants exhibited the expected response, i.e. abortion of seed development. However, in the case of the fnr4 (Female Non Reduction 4) mutant, crossing with a tetraploid tester results in the formation of normally developed seeds. When analyzing these normally developed seeds, by means of a conventional flow cytometry analysis ( FIG. 1C ), it is verified that the embryo of these seeds exhibits a tetraploid genome, which is therefore the result of the fusion of two diploid gametes. 
     The mutant plants therefore indeed produce apomeiotic gametes. The cytological analysis of the fnr4 mutant, reported on  FIG. 2 , indicates that the failure of meiosis is the result of defective chromosome condensation during meiosis and the disorganization, which results therefrom, of the mycotubule network constituting the meiotic spindle. The image is obtained by immunolocalization, firstly, of a specific antibody against beta-tubulin, visualized with a secondary antibody coupled to a fluorochrome (visible in the green range), and by labeling of the DNA with propidium iodide (visible in the red range). W23 is a wild-type line. The sequences of 3 mutant alleles, sequence SEQ ID Nos 7-9, comprising the Mutator insertion sequence, are given at the end of the description. 
     Example 2 
     Identification of the Modified Function in the Mutant Plants 
     Since the apomeiotic phenotype of the FNR4 mutant results from the random insertion of a transposable element of Mutator type into the genome, the sequence, which is known, of the Mutator elements was used for identifying the mutated locus by means of a conventional analysis of co-segregation between the phenotype and the sites of insertion of the various Mutator elements. 
     A candidate sequence could thus be identified and cloned. Analysis of three independent mutations in the same gene, all resulting from insertions of transposable elements, shows an identical phenotype. The locus identified therefore indeed corresponds to that responsible for the apomeiotic phenotype. Comparison of the sequence of the locus in the public database makes it possible to associate a biological function with this gene. It unambiguously appears to belong to a multigene family, comprising about twenty loci, all identified as encoding proteins of Argonaute type. These sequences of the gene (genomic, transcript and reading frame) correspond, respectively, to the sequences SEQ ID Nos 2, 3 and 4, and that of the corresponding protein to SEQ ID No. 1. 
     A phylogenetic tree comparing the protein sequence of FNR4 and that of the other known Argonautes in corn and in  Arabidopsis  is presented in  FIG. 3 . It includes the known members in  Arabidopsis  (AGO00X) and corn (AGO10X). The position, in this tree, of the MEL1 gene, which is another Argonaute gene, described in rice as being involved in the regulation of meiosis, is also indicated. The phenotype of a mel1 mutant is very different than fnr4, but it is important to note that it is, moreover, a clearly distinct Argonaute. 
       FIG. 4  gives the structure of the FNR4 gene and the sites for transposable element insertions in the various alleles that are available. 
     Example 3 
     Expression Profile of the Gene and of the Protein in the Wild-Type Forms, the Apomeiotic Mutants and the Apomictic Plants 
     The RT-PCR method was used to analyze the expression profile of the gene. The results show that the gene is expressed constitutively in all the tissues analyzed (leaves, mature pollen, pre-meiotic ovules, ovules during meiosis, ovules during gametogenesis, mature ovules before fertilization). 
     Using synthetic peptides designed on the basis of the sequence of the protein, antibodies specific for sequences SEQ ID No. 5 and SEQ ID No. 6, which make it possible to visualize by Western blotting the presence of the protein on extracts sampled at various stages of development, were generated. The detection of the antibody by immunodetection is shown in  FIG. 5 , A: Expression of the protein transcripts in corn. The specificity of the antibody used is validated through the use of mutant lines. B: tissue localization of the protein, limited to the basal part of the mature embryo sac; detection with a secondary antibody coupled to a fluorochrome (in green). DNA visualized in blue. The examination of  FIG. 5A  shows a band of expected size on the basis of the protein sequence, approximately 100 kD. The protein detected, unlike the RNA, is found only in the ovules, before and during meiosis. A residual presence is visible in the post-meiosis ovules, but no protein is detected in the other tissues of the plant. 
     The specificity of the antibody for the protein of the FNR4 locus was verified by testing, in parallel, the presence of the protein on ovules of the mutant plants. The signal of the antibody completely disappears in the mutants, thereby indicating excellent specificity. This shows, moreover, that the mutant plants indeed correspond to a loss of function. 
     Immunolocalization techniques were then used to study the localization of the protein at the tissue level. The results, shown in  FIG. 5B , indicate that the protein is exclusively localized in the reproductive cells of the ovule. The gene is therefore expressed constitutively in the plant, but regulated post-transcriptionally, the corresponding protein having an expression profile exclusive to the reproductive cells. 
     The expression profile of the gene in apomictic ecotypes that are hybrid between corn and  Tripsacum  was also studied. These apomictic hybrid plants, obtained by crossing sexual corn plants and apomictic  Tripsacum  plants, exhibit the genome of the two species in the same cytoplasm. Using primers capable of detecting both the corn alleles and the  Tripsacum  alleles, the expression profile of the gene in the somatic and reproductive tissues of these plants is verified. It is noted that the transcripts detected by RT-PCR are absent from the apomictic forms.