Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—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
  • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
  • C12N15/8289—Male sterility

Definitions

• PPR peptide sequences capable of restoring the male fertility of plants carrying a male sterility inducing cytoplasm
• the invention relates in particular to PPR peptide sequences capable of restoring the male fertility of plants carrying a cytoplasm inducing male sterility, nucleotide sequences coding for these peptides, a process for obtaining fertile male plants, plants obtained by a such process.
• Large-scale production of Brassica hybrids requires control of the pollination system and efficient transfer of pollen between the parent plants. For many hermaphrodite plants, in fact, to obtain hybrids on a commercial scale, it is necessary to prevent self-pollination. This is relatively simple in the case of corn, for example insofar as the pollen-producing organs are separated from the female organs of the plant and manual or mechanical castration is sufficient.
• Cytoplasmic male sterility which is present in several higher plants is a trait inherited by the maternal route and which prevents the production of a functional pollen, while maintaining female fertility (Nedel et al, 1994).
• the sterility mechanisms notably involve the expression of particular mitochondrial proteins.
• Different systems inducing cytoplasmic male sterility are for example recalled in the document US 20020100078.
• Various techniques are known from the prior art for introducing a fertility restorer gene from a radish plant to a Brassica plant.
• US Patent 5,644,066 for example describes a method of introducing a restorer gene using a protoplast fusion technique.
• US Patent 5,973,233 and US Application 2002/49998 describe methods of introducing a radish restorer gene, while avoiding the introduction of an unwanted gene part inducing the production of gluconisolates. Fertility restorative systems are thus known for the production of hybrid seeds.
• the invention aims to provide the nucleotide sequences and the peptide sequences encoded by these nucleotide sequences, the expression of which is essential for the process of restoring male fertility in plants.
• the invention also aims to provide a method using the nucleotide sequences described to modulate male fertility in plants carrying a sterility-inducing cytoplasm.
• O ⁇ fl38 gene which induces male cytoplasmic sterility
• Ogura codes for a polypeptide of 137 amino acids and is co-transcribed with the OrfB gene.
• the Ofrb gene is the homolog of 1 ⁇ TP8 in the mitochondria of plants (Gray et al).
• the physiological function of the cms-inducing gene product is still unknown. This product is present in all tissues and found in membrane fractions of mitochondria (Grêlon et al, 1994).
• cytoplasmic sterility (orf 138) is transmitted only by the female parent.
• the sterility conferred by this system offers the advantage of being total, that is to say that there is no production of pollen.
• the production of only hybrid seeds is carried out using natural vectors (insects, pollinating wind).
• Cms Ogura is used for the production of commercial hybrid rapeseed seeds FI.
• plants such as rapeseed, for which the seeds constitute the harvest, it is necessary, even essential, to have nuclear genes restoring fertility making it possible to suppress cms in hybrids.
• nuclear restorers exist naturally in the radish species and have been introduced into sterile male Ogura rapeseed plants, allowing the restoration of male fertility (Ffeyn, 1976). This restoration has been shown to be accompanied by a decrease in the amount of the FORF138 protein, but not in the corresponding mRNA (Krishnasamy & Makaroff, 1994; Bellaoui et al., 1999).
• the restorer locus was introduced by crossing into the rapeseed genome, along with an unknown amount of associated genetic information from the radish, and conventional breeding methods were used to improve the restorative fertility genotypes.
• a significant amount of radish genetic material has been introduced in place of around 50 cm of rapeseed genome (Foisset et al., 1998), and is responsible for the difficulties encountered in improving the restored lines.
• Rfo the fertility restorer gene
• the inventors thus succeeded in identifying and cloning the Rfo locus, using an appropriate cloning strategy, associated with comparative genetic and physical maps between Arabidopsis and the radish, more specifically concerning the Rfo locus.
• Comparative genetic mapping studies have demonstrated the collinearity between two chromosomal segments between closely related species.
• Various studies have shown that large regions, including chromosomes, can be collinear, while in other studies, only certain small regions, limited to a few centimeters, exhibit collinearity (Synthesis Schmidt, 2000; Schmidt 2002) .
• the inventors have succeeded in obtaining significant results in the identification and cloning of the Rfo locus involved in the restoration of fertility, by using a strategy of genetic and physical mapping and of positional cloning and by studying microsyntenia between Arabidopsis. and radish.
• the inventors have thus succeeded in identifying the genes encoding proteins from the family of proteins PPR (class of proteins having several repeats of a motif called pentatricopeptide), capable of restoring fertility in Brassica plants carrying the cytoplasm Ogura and / or Kosena.
• the subject of the invention is, according to a first aspect, a PPR peptide sequence (pentatricopeptide) capable of restoring the male fertility of plants, comprising at least one sequence chosen from: a) SEQ ID No. 1 (PPR protein designated PPR-A), or SEQ ID No. 2 (PPR protein designated PPR-B), or SEQ LD No.
• PPR-C protein b) a homologous peptide sequence comprising at least 80% identity with a sequence from a), c) a biologically active peptide fragment representative of a sequence of a) or b).
• restore fertility is meant that the male fertility obtained in a plant in which the expression of PPR-A and / or PPR-B and / or PPR-C has been induced (or the expression of variants of these proteins indicated in b) or c)) is at least 20, preferably at least 50, 70, 80, 90% of the level of fertility of a plant not carrying the sterility-inducing cytoplasm.
• homologous peptide sequence any peptide sequence which differs from the sequence SEQ ID N ° 1 or SEQ ID N ° 2 and SEQ ID N ° 3 by substitution, deletion, and / or insertion of an amino acid or a reduced number of amino acids, at positions such that these homologous peptide sequences constitute a PPR protein having a fertility restoring activity as described above.
• such a homologous peptide sequence is identical to at least 80% of the sequence SEQ LD No. 1 or SEQ ID No. 2 or SEQ ID No. 3, preferably at least 85, 90, 92, 95, 98, 99% of the sequence originating from the template nucleotide sequence presented in the sequences SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3.
• amino acids typically have at least 100 amino acids, preferably at least 300, 400, 500 consecutive amino acids of the sequence from which they are derived.
• These fragments have a fertility restoring activity, preferably at least 20, 30, 50, 80, 90%, or even more than 100%, of the restoring activity obtained with SEQ ID No. 1, or SEQ H) No. 2 OR SEQ LD N ° 3.
• the skilled person has known methods, such as the manufacture of plants transformed according to the method described below.
• the invention thus relates to "natural variants", that is to say the fertility restoring PPR peptides or their fragments which may exist naturally, corresponding in particular to truncations, substitutions, deletions and / or additions of amino acid residues.
• These natural variants generally result from the genetic polymorphism of the population, and have an activity which is not substantially modified compared to the wild peptide.
• the invention also relates to PPR peptides comprising at least one mutation.
• the term “mutation” is intended to denote any change which has occurred in the sequence of PPR peptides, other than those present in its natural variants, and / or in its counterparts, while retaining a fertility restoring activity.
• the methods for typing polymorphisms of PPR genes are variable depending on the type of polymorphism using: - the PCR-RFLP technique (amplification followed by enzymatic digestion of the PCR product) when the polymorphism was located on an enzymatic restriction site.
• oligonucleotide ligation tests differential ligation
• SNP single nucleotid polymorphism
• the invention also relates to an isolated nucleotide sequence, coding for a peptide sequence above.
• this sequence can be chosen from: a) a nucleotide sequence chosen from SEQ LD No. 4 (nucleotide sequence coding for SEQ ID No. 1 and designated ppr-A), or SEQ ID No.
• nucleotide sequence coding for SEQ LD N ° 2 and designated ppr-B amino acid sequence coding for SEQ ID N ° 3 and designated ppr-C
• b) a nucleotide sequence complementary to a sequence of a b) a nucleotide sequence complementary to a sequence of a
• genomic nucleotide sequences in their natural chromosomal environment that is to say in their natural state, these are sequences which have been isolated, that is to say -to say that they were taken directly or indirectly, for example by copy (cDNA), their environment having been at least partially modified.
• nucleic sequence is meant a fragment of DNA and / or natural RNA isolated or synthetic, designating a precise sequence of nucleotides, modified or not, making it possible to define a fragment, a segment or a region of a nucleic acid.
• homologous nucleotide sequence is meant any nucleotide sequence which differs from the sequence SEQ ID No 4 or SEQ ID No 5 or SEQ ID No 6 by substitution, deletion, and / or insertion of a nucleotide or of a reduced number of nucleotides, at positions such that these homologous nucleotide sequences encode a PPR protein having a fertility restoring activity as described above.
• such a homologous nucleotide sequence is identical to at least 80% of the sequence SEQ E) No 4 or SEQ ID No 5 or SEQ ID No 6, preferably at least 85, 90, 92, 95, 98 , 99%.
• the invention covers nucleotide sequences which due to the degeneracy of the genetic code encode PPR proteins having the amino acid sequence of proteins of sequence a) to c) above.
• the invention covers allelic variant nucleotide sequences, in particular SNP sequences.
• percentage of identity between two nucleic acid (or peptide) sequences is meant a percentage of identical nucleotides (or amino acids) between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistics and the differences between the two sequences being distributed randomly and over their entire length.
• Optimal alignment of sequences for comparison can be achieved using mathematical algorithms. In a preferred, non-limiting manner, we can cite various algorithms recalled in Altschul et al (1998), in particular the algorithms presented in Altschul et al (1990), Karlin and Altschul (1993). We could use for example the programs:
• -BLAST in particular BLASTN, BLAST2 (Tatusova et al., 1999), gapped BLAST (Altschul et al, 1997), or -FASTA (Altschul et al., 1990).
• BLAST2 Teatusova et al., 1999
• gapped BLAST Altschul et al, 1997)
• -FASTA Altschul et al., 1990.
• such a homologous nucleotide sequence hybrid specifically to the sequence complementary to the sequence SEQ ID No. 4 or SEQ LD No. 5 or SEQ ID No. 6, under stringent conditions.
• the parameters defining the appropriate stringency conditions are known to those skilled in the art, in particular described in Ausubel et al. (1995).
• the specific hybridization conditions or high stringency will be such as to ensure the detection of at least 90%, preferably 95% or preferably 99% of identity between one of the two sequences and the complementary sequence of the other.
• the invention also relates to the mutated nucleotide sequences insofar as they comprise at least one point mutation and preferably at most 10% of mutation, without significantly altering the PPR activity at the origin of the fertility restoration.
• the invention also relates to probes or primers, which can be used in methods of detecting, identifying, assaying or amplifying the above nucleic sequences. These methods may involve techniques known to those skilled in the art, including PCR type amplification techniques using nucleotide primers corresponding to portions of the sequence SEQ ID No. 4 or SEQ ID No. 5 or SEQ LD # 6 ,.
• a primer is defined, within the meaning of the invention, as being a fragment of single-stranded nucleic acid or a denatured double-stranded fragment comprising at least 10 bases, preferably at least 15, 30 bases, which can be used to amplify a nucleotide sequence ci -above.
• a probe is defined, within the meaning of the invention, as being a fragment of single-stranded nucleic acid or a denatured double-stranded fragment comprising at least 10 bases, preferably at least 15, 30 bases, and having a specificity of hybridization under conditions determined to form a hybridization complex with a target nucleic acid.
• Such probes or primers will in particular be used in marker-assisted selection programs, for example for monitoring the introgression in a plant of a gene coding for a PPR protein according to the invention.
• these probes or primers are specific for the sequences corresponding to the male fertility restoring allele in the sense that they do not hybridize to the sequences corresponding to the sterility maintaining allele in conditions of high stringency.
• the temperature for the hybridization reaction is between approximately 25 and 65 ° C, in particular between 35 and 65 ° C in a saline solution at a concentration of approximately 0 8 at 1 M.
• the pairing with the specific probes of the invention can be carried out at temperatures above 50 ° C. for 1 to 2 mM MgCl 2 .
• the hybridization temperature of an oligonucleotide being calculated on the basis of 2 ° C by A or T and 4 ° C by G or C, an oligonucleotide consisting for example of a combination of 7 A and 5 C hybridizes at 34 ° C. This rule, well known to those skilled in the art, makes it possible to calculate the hybridization temperature that can be envisaged for all the probes or primers according to the invention.
• the probes in accordance with the invention can be used for the analysis of samples of genetic material extracted from plants to be tested, this can for example be analyzes of the Southern or Northern type. They can also be used for the in situ detection of the allele sought, for example techniques of the FISH (Fluorescence in situ hybridization) or BAC FISFf (In situ hybridization on BAC clone) type, etc.
• the invention relates to detection kits comprising at least one probe and / or primer as defined above and means for detecting the presence or absence of the specific hybridization. These kits can be used in detection methods based on PCR amplification.
• the amplification may relate, from the genetic material of the plant to be tested, to the sequence responsible for restoring the male fertility of plants carrying the sterility-inducing cytoplasm.
• the amplification conditions will be defined according to the pair of primers chosen so that said pair of primers hybridizes only with its complementary sequence present in the sought allele. Amplification with the primers included in the kit makes it possible to detect the presence of the sequence of interest in the plant tested.
• the invention also relates to a nucleotide construct, intended to be introduced into a plant whose fertility is to be restored, comprising at least one nucleotide sequence described above coding for a PPR protein with fertility restoring activity and a promoter sequence capable of controlling expression of this sequence in sufficient quantity to restore fertility.
• PPR protein with fertility restoring activity is meant at least one peptide sequence a) to c) as defined above.
• constructions nucleotides combining at least two PPR sequences according to the invention can be used for the transformation of sterile plants, and preferably, a construct containing both the three PPR sequences according to the invention linked to the endogenous promoter of PPRB or else to the LEA promoter are used for said transformations.
• nucleotide sequences encoding at least one of the proteins PPR-A and PPR-B and PPR-C may be introduced into a plant. ).
• a promoter is a nucleotide sequence which is capable of modulating or controlling the level of transcription of a gene under the control of this promoter, and which allows (or provides a site for) the binding of RNA polymerase during of the transcript.
• the position of a promoter is defined relative to the site of the start of transcription.
• consensus promoter sequences found in plant promoters, in particular a TATA box located approximately 19 to 27 bases upstream of this site and a CAAT box located approximately 70 to 80 bases upstream of this site.
• RNA stabilization and effect on translation for example the leader EMCN (Elroy-Stein et al, 1989) and the leader TMN (Gallie et al, 1989).
• regulatory boxes operating at a transcriptional level, capable of at least partially controlling the activity of a promoter; these latter regulation boxes can be inducible by a physiological factor or by the environment of the plant.
• the promoter used may be a constitutive promoter (this promoter controls the expression of the Rfo gene in all plant tissues), or specific for anthers and sufficiently active to control the expression of the Rfo gene specifically in anthers (such a promoter is capable of controlling the expression of RNA or of PPR polypeptides in the anthers at a level sufficient to obtain the production of functional pollen and a restoration of fertility; for example, the LEA promoter may be used (Hughes and Galau, 1989 and 1991) or the specific Brassica promoter described in US Patent 5,356,799).
• the promoter is chosen from promoters of PPR genes from cloned and sequenced radishes.
• the preparation of such a DNA construct can be carried out in various ways suitable for those skilled in the art. Briefly, at least part of the Rfo gene capable of restoring fertility (preferably a sequence encoding PPR-A or PPR-B or PPR-C) can be cloned downstream of the promoter using restriction enzymes to ensure its insertion in an orientation appropriate to the promoter so that it is expressed. Once this DNA of interest is operably linked to the promoter, the construct thus formed can be cloned into a plasmid or another vector.
• the promoter sequence controls the transcription of the gene of interest into a functional messenger RNA.
• the prepared nucleotide construct typically also includes a transcription termination region.
• nucleotide construct comprising not only a promoter operably linked to a gene of interest, but also at least one sequence capable of modifying the activity of the promoter, in particular an enhancer sequence and / or a leading streak.
• the invention relates to a cloning and / or expression vector comprising a nucleotide construct as described above. According to another aspect, the invention relates to a host cell transformed with the nucleotide sequences described above.
• the invention also relates to antibodies capable of specifically recognizing the polypeptides according to the invention or any fragment thereof.
• the antibodies are produced by methods well known to those skilled in the art based on the natural production of antibodies in reaction against the introduction of a polypeptide foreign to the organism, in this case the polypeptide defined by the sequence SEQ ID No. 1,2 or 3, any fragment thereof or any derived polypeptide having the same physiological function in the plant.
• said antibodies are capable of specifically recognizing a polypeptide corresponding to the fertility restoring allele.
• Said antibodies can be used in immunochemistry tests to detect in a population of plants the presence of the polypeptides defined by the sequences SEQ ID No. 1, 2 or 3 or derived polypeptides.
• the invention therefore also relates to a kit for detecting the fertility restorative allele in plants comprising an antibody defined above.
• the invention relates to plant cells transformed by a vector as defined above, using a cellular host capable of infecting said plant cells by allowing the integration into the genome of the latter, of sequences nucleotides encoding PPR proteins, in particular A and B and C initially contained in the vector used.
• transformation refers to a genetic manipulation of plant cells capable of being transformed, such as, for example, callus cells, embryos, cells in suspension in cultures (cultures derived for example from calluses, embryos, leaf tissue, young inflorescences, anthers).
• a Brassica transgenic plant can be obtained by various dicotyledon transformation techniques known to those skilled in the art, including, without limitation, the transfer of DNA mediated by Agrobacterium tumefaciens, using a vector carrying a disarmed T-DNA, or direct transfer methods such as protoplast transformation methods or biolistic methods.
• the techniques for cultivating and regenerating whole plants from the tissues thus transformed are known to those skilled in the art, for example described in the references Hansen and Wright (1999), Komari et al. (1998).
• Genetic engineering techniques for the genetic transformation of plant cells or tissues will be used for the transfer of a nucleotide construct comprising a PPR peptide sequence, a promoter, if appropriate transcription termination sequences.
• the present invention includes plants genetically transformed with a vector according to the invention. These plants comprise the nucleotide construct or the vector as defined above, which comprise all or part of one of the sequences defined by the sequences SEQ LD ⁇ ° 4 to SEQ JJD N ° 6.
• the transformed plants which are the subject of the present invention are derived from plants which are male-sterile before transformation.
• the present invention can be implemented in the context of marker-assisted selection (SAM).
• SAM marker-assisted selection
• This selection method is much more effective than a simple phenotypic evaluation since all the loci corresponding to different characters of interest can be analyzed together on a single DNA sample, regardless of the culture conditions of the plant sampled.
• Selection assisted by markers makes it possible to implement accelerated backcrossing techniques consisting in following the introgression of the restorative sequence which is the subject of the invention by visualizing hybridization with a probe or amplification with a pair of primers.
• the invention thus also relates to a method for detecting the restorative sequences in plants, preferably in crucifers and in a preferred manner in plants of the genus Brassica.
• the present invention also relates to a method of using the Rfo gene in a scheme for producing hybrids from a restorative plant obtained according to the invention.
• the production of hybrids requires the crossing between an improved maintenance line "A” and an improved restorative line "B” obtained according to the present invention. This makes it possible to quickly obtain fertile hybrids containing the interesting agronomic characteristics of parent "A” and parent "B".
• the method which is the subject of the invention has the advantage of introgressing only the Rfo gene by freeing itself from the adjacent sequences present in the restorative locus originating from radish and thus eliminating in the improved restorative line B and in hybrids the effects pleiotropics of these sequences causing problems of increased lodging, high levels of glucosinolates in seeds, female fertility and reduced yield.
• the techniques and agents for selecting plant cells and / or plant tissues incorporating marker nucleotide sequences associated with the gene of interest are also well known to those of skill in the art, and include, but are not limited to, the use of marker genes such as genes conferring resistance to an antibiotic or to herbicides, or other positive selection systems, cited for example in Gelvin 1998, in particular the system based on selection on mannose, in the presence of the gene for MPI (Mannose-6-phosphate isomerase) (Hansen and Wright, 1999), or selection systems coupled with the elimination of marker genes after selection (Ebinuma et al., 1997).
• the transformed plants can also be selected by PCR screening in the absence of selection marker genes (McGarvey and Kaper, 1991).
• the invention also relates to the tissues or parts of plants, plants, or seeds containing the Rfo nucleic acid sequences according to the invention.
• plant tissue refers to any tissue of a plant, in a plant or in a crop. This term includes whole plants, plant cells, plant organs, plant seeds, protoplasts, calluses, cell cultures and all other plant cells organized as a functional and / or structural unit. Parts of regenerated plants such as flowers, seeds, leaves, stems, fruit, pollen, tubers and the like are also within the scope of the invention.
• the invention includes fertile transgenic plants obtained as well as their progeny and the product of this progeny.
• Transgenic hybrid plants obtained by crossing at least one plant according to the invention with another, also form part of the invention.
• the transgenic plants according to the invention comprise in particular a T0 or RO transgenic plant, that is to say the first plant regenerated from transformed cells, the transgenic plant Tl or RI, that is to say the first generation of descendants, and the plants of the descendants of subsequent generations obtained which understand and sufficiently express the Rfo DNA to restore fertility.
• a large number of suitable techniques can be used, for example PCR analysis or Southern blot hybridization techniques, to determine the structure of the Recombinant DNA, the detection of RNA transcribed from the DNA of the gene of interest expressed in the cells of anthers of transformed plants, using Northern blot techniques or RT-PCR amplification, identification of the production of the protein encoded by the gene of interest, such as gel electrophoresis of proteins, Western blot techniques.
• the invention relates to a process for obtaining a plant expressing at least a part of the Rfo gene and having a fertile male phenotype, resulting from the introduction of a construction as described above, in at least one plant cell, then the culture of the cell thus transformed so as to regenerate a plant containing in its genome said expression cassette.
• the invention thus also relates to a method for restoring the fertility of a Brassica plant, characterized in that it comprises the introduction into a sterile male plant of a nucleotide sequence coding for a PPR protein.
• the PPR protein is a protein of sequence chosen from the peptide sequences a) to c) defined above, preferably PPR-A, PPR-B or PPR-C. It is understood that the invention covers such a method using at least one nucleotide sequence obtained by a method of screening for sequences originating from the Rfo gene and capable of restoring fertility. They are in particular nucleotide sequences defined in a) to c) above. Those skilled in the art have in the present application appropriate screening techniques. It is also possible to construct crossover plans such as those described in application US 0220083483 involving a male sterility inducing gene then a fertility restorer gene.
• the plant is Brassica napus.
• the invention also relates to a method of introducing a fertility restorer gene from a radish plant, into a Brassica plant, characterized in that it comprises the crossing or cell fusion between the Brassica plant expressing a gene.
• cytoplasmic male sterility Ogura and / or Kosena which induces the sterility of this Brassica plant and a plant expressing at least part of the Rfo gene and having a fertile male phenotype, so as to introduce the Rfo gene restoring fertility into the plant Brassica genus and to obtain a hybrid plant with restored fertility, the part of the Rfo gene introduced preferably comprising a sequence coding for PPR-A or PPR-B or PPR-C.
• the invention also relates to the use of at least one nucleotide sequence coding for PPR-A or PPR-B or PPR-C (or a functional peptide variant defined above), for restoring fertility in Brassica.
• the invention also relates to a process for obtaining hybrid seeds by crossing between a fertility restoring plant obtained by a above process and a sterile male plant.
• the invention also relates to a method for screening polymorphic nucleotide sequences capable of restoring fertility, characterized in that it comprises:
• the invention also relates to a screening method for detecting the presence of a nucleotide sequence coding for a PPR protein in a plant, characterized in that it comprises an amplification step using at least minus a leader sequence as defined above and / or a hybridization step using at least one probe sequence as defined above.
• the invention also relates to a process for the production of hybrid rapeseed varieties whose male fertility is restored, comprising the introduction of a nucleotide sequence according to the invention into a sterile male rapeseed plant used as a male parent and expressing the PPR gene and the crossing between said plant with a sterile maize rapeseed line used as female, and production of the hybrid variety resulting from crossing.
• the invention covers hybrid seeds whose fertility is restored by the PPR gene obtained from plants obtained by the method described above and also the use of a plant expressing the nucleotide sequences according to the invention for restoring fertility. of a rapeseed line used as a female in a cross or in the production of seeds of a hybrid variety.
• the invention in another aspect, relates to a cloning and / or expression vector comprising an isolated nucleotide sequence as described above.
• the invention also relates to a recombinant host cell characterized in that it is transformed with this recombinant vector.
• the recombinant host cell can be chosen from eukaryotic or prokaryotic cells, for example bacteria, in particular E. coli or Agrobacterium, for example Agrobacterium tumefaciens, yeasts, animal cells and preferably plant cells, more specifically brassicacea cells.
• the invention relates to a recombinant plant multicellular organism comprising a recombinant cell mentioned above.
• the invention also relates to a transgenic plant transformed with a vector described above.
• This transgenic plant is for example a carrier of the cytoplasm inducing cytoplasmic mass sterility transformed by the vector mentioned above.
• the invention also relates to a nucleotide construct, characterized in that it consists of the sequences SEQ ID No. 4, SEQ ID No. 5 and SEQ LD No. 6 operatively linked to a promoter sequence.
• promoter sequence is meant a sequence comprising one or more elements for regulating transcription.
• This promoter sequence can also include one or more insulators and / or enhancers.
• the promoter chosen is the endogenous promoter of PPR or other promoters specific for endogenous or exogenous plants.
• plant-specific promoters is meant, for example, any promoter allowing optimal expression of the sequences of the invention in plants.
• the CaMN35S or GRE promoter can be used.
• Figure 1A represents a map of markers closely linked to the Rfo locus in radishes.
• Figure 1B represents the results of BLAST tests with AFLP markers compared to the A.thaliana genome.
• FIG. 2 represents the analysis of microsyntenia around the Rfo locus between the radish and Arabidopsis.
• the genetic map is based on the analysis of 6900 plants segregated with Rfo.
• the physical Arabidopsis card is deducted from the AGI database.
• the dark lines physically locate the markers on the DAC : rabidopsis and the arrows locate them on the genetic map.
• FIG. 3 represents a high resolution genetic and physical map near the Rfo locus.
• A contigs from BAC to 'A.thaliana corresponding to the synthetic region of the Rfo locus (position of the markers indicated by the triangles).
• B genetic map of the Rfo locus in radishes.
• C BAC radish contigs representing the physical map of the Rfo locus. The positions of the markers are determined by genetic mapping using the recombinants.
• the term “recombinant” is intended to mean a plant whose genotype presents a recombination of the markers characteristic of the parents of the population around Rfo. The number of recombinants is indicated on the physical map between the markers.
• FIG. 1 The position of the markers on the BAC contigs is indicated by vertical dotted lines.
• the Rfo locus is indicated by a black band.
• BAC 64-16-7 positive with the markers flanking Rfo, physically delimits the Rfo locus.
• - Figure 4 shows a physical map of BAC 64-16-7 and a prediction of genes from its sequence using the GENSCAN program. The position, name and orientation of genes are indicated by oriented bands. The genes were obtained by comparison with the Arabidopsis genome. The position of the markers closely linked to the Rfo gene is indicated by the vertical arrows.
• Figure 5 shows flowers from the Ogura cms Tanto transgenic lines and non-transgenic control. In Figure 5A, the flowers are from a restored transgenic plant showing normal anthers and pollen production.
• the flowers originate from a non-transgenic plant serving as a control (non-transgenic control) showing short filaments and the cavities of the anther ("locules") empty without pollen.
• the petals were removed from the two flowers for a better analysis of the results.
• sequence SEQ ID No. 7 is a sequence of 22,770 base pairs comprised between the markers flanking the PPR cluster (the markers M-F24-D7.9 and M-F24-D7.13 ).
• the nucleotide sequence of the 3 PPR is extracted, in the order PPR C, PPR B, PPR
• SEQ ID No. 8 to SEQ ID No. 31 correspond to the sequences of the primers used to identify the markers in FIG. 3 in the reading direction 5 '->3'. ATG, stop, or introns have been predicted with computer software on the reference sequence of 22,770 base pairs.
• Example 1 Identification of the PPR (pentatricopeptide) peptide sequence capable of restoring the male fertility of plants.
• the radish Rfo fertility restorer gene has been mapped and AFLP markers associated with the Rfo gene have been identified.
• the inventors have shown that, near the Rfo gene, the sequence collinearity between the radish and Arabidopsis is limited to a reduced interval.
• AFLP analysis was done in combination with a segregation analysis.
• the DNA studied is obtained from the offspring from a cross between a sterile male European radish line, 7ms, and a homozygous radish line for the Rfo gene.
• 800 combinations of PstJ-MséJ primers were studied to determine the polymorphism, and three new AFLP markers, designated R3, RI 5 and R5, were identified as directly associated with the Rfo gene.
• R3 and RI 5 are perfectly linked to the Rfo gene in a population of more than 900 segregated plants, and R5 is located at 0.1 cM from Rfo (Fig. 1).
• the markers R3 and RI 5 are homologous to sequences carried by the BAC F24D7 ⁇ 'Arabidopsis by BLAST analysis, in a genomic region of 20 kb, and the marker R5 is found in the same region 150 kb further on. It follows that the genomic DNA of the radish near the Rfo locus is probably collinear with the DNA & Arabidopsis of BAC F24D7.
• the markers derived from chromosome 1 of Arabidopsis are located near the Rto gene in radishes.
• PCR markers derived from six BAC clones of Arabidopsis, covering a physical distance of 1100 kb.
• the markers were chosen from coding sequences (CDS) predicted AGI.
• CDS coding sequences
• the primers were chosen from exons, to increase the chances of amplification of the radish DNA.
• the primers are also on the edge of a suspected intron, so as to increase the chances of identifying a polymorphism between fertile male and sterile male radishes.
• the primers are from 60 CDS and the PCR was carried out on Arabidopsis and on the fertile and sterile radish (Table 1). These markers have been mapped with respect to the Rfo gene in the radish.
• 11 polymorphic markers between the fertile and sterile radish were identified.
• the markers M-T12P18.15, M-F24D7.4, M-F24D7.9, M-F24D7.13, M-F24D7.16, M-F24D7.17, M-F2K11.1 are strictly genetically linked with the gene Rfo.
• the marker M-F2K11.19 is located 0.6 cM from the Rfo gene.
• the markers M-F22C12.1 and M-T12P18.9 are located at 0.2 cM and 0.1 cM from the Rfo gene respectively.
• the sequences of the different primers used SEQ ID No. 8 to SEQ ID No. 31 are indicated in the attached list of sequences.
• the inventors have thus designed two pairs of oligonucleotides capable of amplifying specifically the locus M-T12P18.9 and M-F2K11.19 of the fertile line (respectively SEQ ID N ° 8 and SEQ ID N ° 9 and SEQ ID N ° 28 and SEQ ID N ° 29). Plants with recombination events closely associated with the Rfo gene have been identified as lines carrying only the marker M-T12P18.9 or the marker M-F2K11.19 linked in cis to the Rfo gene.
• a library of BAC was constructed from nuclear DNA derived from a line of radish D81 homozygous for the Rfo gene.
• the bank consists of 120,000 clones.
• the size of the inserts of 100 randomly sampled BAC clones was determined by digestion with Notl and PFGE. The size of the inserts varies between 100 kb and 200 kb.
• the bank represents at least 23 times the haploid genome of radishes.
• the library was screened with markers closely linked to the Rfo gene (M-F16M19.21, M-F2K11.19, M-F2K11.1, R15, M-T12P18.9 and M-F22C12.1), and positive BACs have been identified and isolated.
• the BAC clones were aligned and ordered in a single contig. The order of the BAC clones in the contig is compatible with the genetic mapping of markers around the Rfo gene.
• the Rfo locus was physically located on a BAC clone, BAC 64-16-7, positive with the flanking markers of the Rfo gene (Fig. 3).
• BAC 64-16-7 (referenced SEQ LD No. 32) was sequenced using a "shotgun" sequencing technique, and the sequence was assembled in a single 127 kb contig. The sequence redundancy was at least 10 times the length of the BAC sequence. Two additional tests were performed to verify the quality of the consensus sequence. First, the predicted restriction map was compared with the footprint of BAC 64-16-7 and the results were consistent. In addition, the sequences of the markers associated with the Rfo gene were aligned with the sequence of BAC 64-16-7, and it was possible to verify that the genetic order of the markers corresponded to the physical order on the sequence.
• the Rfo gene is located in a genetic interval of 0.029 cM between the markers F24D7.9 and F24D7.13. These two markers delimit the Rfo gene physically in 22 kb on the sequence of BAC 64-16-7.
• the sequence interval F24D7.9 / F24D7.13 includes the genes coding for three proteins belonging to the PPR family of proteins (Fig. 4), designated PPR-A, PPR-B and PPR-C. It follows that the restorative activity is coded either by the A gene or by the B gene or by the C gene or by the combination of at least two of these genes.
• Plant material Two progenies were produced in segregation for the Rfo gene.
• F4 families have been produced from heterozygous radish (D radish) genotypes from Asia.
• An F2 progeny of 20,000 seeds was obtained from FI hybrids between a sterile European radish line (7ms) and a radoz genotype homozygous for the Rfo gene (D81.8) chosen from the F3 family of radish D .
• AFLP analysis Nos et al., 1995 was carried out on DNA samples from sterile male and fertile male plants (Giovanni et al. 1991; Michelmore et al., 1991 ), according to the Keygene NV protocol (Wageningen, Netherlands) using restriction enzymes Pstl and MseJ, and a set of primers corresponding to the adapters Pstl and yel with two or three selective nucleotides at the 3 'end respectively.
• the ALFP products were separated from the acrylarnide gels in which they were initially detected, then re-amplified by PCR, using the same primers and conditions as for the AFLP amplification, then clones using the vector pGEM-T easy (Promega). The sequences of the cloned fragments were compared with the genome of Arabidopsis thaliana using the BLAST program.
• Specific primers were designed from this first AFLP sequence and were used to amplify the DNA of fertile male or sterile male parents. The two different amplification products were sequenced and compared with each other. The specific primers were designed around the region showing a polymorphism between the fertile male radish and the sterile male, so as to detect this polymorphism by PCR amplification or by digestion after PCR. When no sequence polymorphism was found between the two parents, the AFLP sequence was amplified by PCR using the Clontech kit (Universal Genome Walker Kit). Different genomic DNA fragments linked to an adapter were constructed according to the manufacturer's protocol ((Universal Genome Walker, Clontech, USA).
• Young leaves were collected in 96-well plates (2-ml well, Costar, Corning) containing glass beads (diameter 3 mm, Polylabo), 3 beads per well, and lyophilized. The plates were sealed using Easy Peel seal (ABgene), and the lyophilized sheets were placed with the beads in a mixer.
• DNA was extracted using 600 ⁇ L of extraction buffer containing 0.5 M NaCl, 0.1 M Tris, 50 mM EDTA, and 20 mM sodium metabisulfite (added before use) .
• the plates were sealed using appropriate seals (Corning Inc., NY, USA), and incubated in a water bath at 95 ° C for one hour. The plates were centrifuged at 3000 rpm for 20 minutes. The supernatant (280 ⁇ L) is transferred into 96-well plates (0.65 ml per well, ABgene, Advanced Biotechnbologies), containing 35 ⁇ L of 10 M ammonium acetate, 280 ⁇ L of isopropanol, and mixed .
• the plates are centrifuged at 3000 rpm for 30 minutes. The supernatant is decanted by inverting the plate, the DNA precipitates are washed with 70% ethanol and air-dried by incubation at 60 ° C for about 20 minutes. The DNA precipitates are resuspended in 50 ⁇ L of TE buffer at pH 8.0 containing 25 ⁇ g / ml of RNase, with gentle stirring at 37 ° C for one hour.
• BAC radish library Construction of the BAC radish library (Zhang et al, 1995)
• the nuclei are isolated from radish leaves 10 days old and included in an agarose gel.
• the DNA is then partially digested with Ec RI and HindJJl.
• the agarose pieces are separated into four or eight pieces and then incubated on ice for one hour with only restriction digestion buffer (enzyme buffer + spermidine + BSA), then with the enzyme restriction which is added at different concentrations for an additional 30 minutes on ice and at 37 ° C for approximately 30 minutes.
• the reaction is stopped by adding 0.5M ⁇ DTA.
• the pieces are loaded onto a 1% low melting point agarose gel (Seakem agarose, etc.) then subjected to CHEF electrophoresis in a CHEF DRU (BioRad), in an IX TAE buffer (10 h at 4.4N / cm with 3.5s of initial and final interruption time, and 5 hours at 4.5N / cm with 2s for initial and final interruption time, at 12 ° C).
• the compression zones containing the AD ⁇ of approximately 100 to 200kb are separated from the gel.
• a second size selection is made: the separate strip is subjected to a second migration under the same electrophoresis conditions.
• the DNA is eluted from the agarose by electroelution in a TAE IX buffer (4 h 30 at 6 V / cm with 50 s for initial and final interruption time). After dialysis using T 10 E1, the DNA is quantified on a 0.6% agarose gel.
• the radish DNA selected by the size (xy ng) pending is linked to a pGUCI vector desphosphorylated and digested with HindJU or EcoRN (10 to 50 ng) with 10U of T4 ligase, at 16 ° for 16 hours, in a volume total of 50 ⁇ L.
• the ligation is dialyzed again with T KJ ⁇ Q.I, using a 0.025 ⁇ m Millipore membrane filter, for 2 hours at room temperature in a Petri dish.
• White colonies are removed (Robot Q-Bot, Genetix) and individually transferred into 384-well micro titration plates (Genetix) containing conservation medium (36mM K 2 HPO 4 , 13.2mM KH 2 PO, l , 7mM ratea citrate, 6.8mm ⁇ H4 2 SO 4 , 0.4mM MgSO 4 , 4.4% V / N glycerol, 12.5 ⁇ g / mL chloramphenicol, in LB medium), then cultivated overnight at 37 ° C and stored at - 80 ° C.
• conservation medium 36mM K 2 HPO 4 , 13.2mM KH 2 PO, l , 7mM ratea citrate, 6.8mm ⁇ H4 2 SO 4 , 0.4mM MgSO 4 , 4.4% V / N glycerol, 12.5 ⁇ g / mL chloramphenicol, in LB medium
• 117,120 clones were obtained, 63,100 from restriction by EcoRI and 81,020 from restriction by HindJJJ.
• the plasmid AD ⁇ of 70 randomly selected clones is isolated ( ⁇ .D. Young and
• the average size of the average inserts is more than 100kb, with the following distribution: 13.2% with a size between 150 and 200kb, 51.5% between 100 and 150kb, 33.8% between 50 and 100kb, and 1.5% less than 50kb.
• the DNA isolated from the 384 colonies on a plate represents a pool.
• the pools are screened. Once an individual plate is identified, two duplicates of that plate are prepared on LB agar; all the clones corresponding to each of the 24 columns are taken together from the first duplicate and the PCR is carried out on these 24 mixtures of clones. After identification of a positive column, a PCR is carried out on each of the 16 corresponding colonies of this column, so as to identify a positive BAC clone.
• the inventors have also developed a protocol for subcloning the genes of the PPR cluster from the clone BAC 64-16-7.
• the sub-cloning of each PPR gene (candidate for Rfo) is carried out in the binary vector pEC2, in order to carry out transgenesis experiments on sterile male rapeseed plants and to demonstrate that it is the candidate gene which effectively restores male fertility.
• PPR A the BAC 64-16-7 clone is digested with the restriction enzymes NdeI and SpeJ and the 6.5 kb fragment is made “blunt end” and cloned in the BamHJ site (blunt end rendering) of the vector binary. This fragment contains the coding part of PPR A, 2.6 kb upstream and 1.8 kb downstream.
• PPR B the BAC 64-16-7 clone is digested with the restriction enzyme Spel and the 7.1 kb fragment is made “blunt end” and cloned in the BamHI site (blunt end) of the binary vector . This fragment contains the coding part of PPR B, 3.1 kb upstream and 1.9 kb downstream.
• PPR C the clone BAC 64-16-7 is digested with the restriction enzymes Bam ⁇ I and Spel (rendered “blunt end”) and the 7.5 kb fragment is cloned in the BamHI-Hindlll site (rendered blunt end) of the binary vector. This fragment contains the coding part of PPR C, 2 kb upstream and 3.6 kb downstream 4.
• PPRA and PPRB the clone BAC 64-16-7 is digested with the restriction enzyme BstEU and BamHI and the fragment of 21 kb is rendered "blunt end” and cloned into the BamHI site (rendered blunt end) of the binary vector. This fragment contains PPR B and PPRA.
• PPR C and PPR B the clone BAC 64-16-7 is digested with the restriction enzymes EcoRI and Apal (rendered “blunt end”) and the 16.8 kb fragment is cloned in the BamHI site (rendered “ blunt end ”) of the binary vector. This fragment contains PPR C and PPR B as well as 2.4 kb upstream and 3.6 kb downstream
• PPR C and PPR A Clone 1 BAC 64-16-7 is digested with NotI, (rendered “blunt end”) is ligated to the 7.5 kb fragment BamHI and Spel, carrying PPRC (rendered “blunt end” ") And described in 4. This chimeric clone will express PPRA and PPR C
• PPR A, PPR B and PPR C the clone BAC 64-16-7 is digested independently by the restriction enzymes ⁇ coRI ("rendered” blunt end ") and Kpn and by Kpn ⁇ and Sali (" rendered blunt end ”) .
• the 2 EcoRI-Kp ⁇ l and Kp ⁇ -Sall fragments of 9.8 kb and 13.5 kb respectively will be cloned simultaneously in the BamHI site (rendered "blunt end") of the binary vector.
• This fragment contains PPR C, PPR B and PPR A as well as 2.4 kb upstream and 2.4 kb downstream
• the inventors develop molecular markers for genetic selection and the characterization of germplasm.
• the nucleic sequence presented in the following example corresponds to the complete sequence of BAC64 which includes the fertility restoring sequences.
• Probes can be designated on the nucleic acid sequence adjacent to the fertility restorer sequences. These probes can be used in a program to select fertility restorative lines or follow the evolution of the Rfo locus in a germplasm collection for the identification of new restorative sequences. These probe sequences designated on the BAC64 have the same importance as the use of the restorative sequences in the selection assisted by markers or in the programs of characterization of germplasm. This use includes searching the rapeseed deletion mutant banks of restored lines which have lost, for example, the radish sequences linked in cis to the fertility restoring sequences. This work can be done in order to reduce, for example, the size of the radish DNA introduced into rapeseed.
• Example 2 Functional restoration of an Ogura cms rapeseed (sterile) with a nucleotide construct containing PPR B:
• pSCN PPRB was integrated into the C58pMP90 strain of Agrobacterium tumefaciens (C Koncz & J Schell, MGG 204: 383-396 (1986):
• the promoter of T (L) -D ⁇ A gene 5 confrols the tissue-specific expression of chimaeric genes carried by a novel type of Agrobacterium binary vector.) to be used for plant transformation.
• the putative transgenic shoots were identified by PCR by using primers in the gene coding for the selection marker Npt ⁇ (Bevan et al.
• NptU confers resistance to kanamycin
• the primers confirm the presence of the two ends of the T-DNAs used for the transformation.
• the transgenic plants were then transferred to the greenhouse and isolated to avoid any risk of cross-pollination.
• Table 3 Fertility of Ogura cms plants transformed with the nucleotide construct containing the PPRB transgene demonstrated by PCR analysis.

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