CN1248296A - Means for identifying nucleotide sequences involved in apomixis - Google Patents

Abstract

The present invention relates to a method for identifying nucleotide sequences involved in apomixis in apomictic plants in Poaceae, especially maize. It is characterized in that the method comprises: identifying the meiotic mutation whose corresponding gene shows orthologous to the gene involved in apomixis expression in Poaceae genome through phenotype analysis, genetic mapping and transposon markers. The invention also relates to the use of the cloned genes for the identification and isolation of orthologous sequences in apomictic plants. The invention also relates to the use and modification of sequences isolated in apomictic forms for inducing apomictic development in sexual plants.

Description

Method for identifying apomixis-related nucleotide sequences

The object of the present invention is a method for identifying, isolating and characterizing nucleotide sequences involved in apomixis.

The invention more particularly relates to methods and tools enabling the identification of these sequences in the genome of apomictic plants, their subsequent isolation and characterization.

The invention also relates to the transgenic applications and products obtained using these sequences.

In its modern conception, apomixis or apogamy includes all phenomena of asexual reproduction by seed. Apomictic plants are found among 300 species of angiosperms in 35 families. The different forms of apomixis (generally used only in female reproduction) are characterized by the absence of meiosis, fertilization of the ovum and parthenogenetic development of the embryo. Apomixis thus results in the production of offspring that are genetically identical to their parent plants.

The natural counterpart of apomixis is sexual reproduction, or fusion of the sexes. In contrast to apomixis, sexual reproduction involves processes that involve both meiosis and gametogamy. Meiosis randomly distributes homologous chromosomes derived from the parents into different gametes. Meiosis also allows recombination between homologous chromosomes by crossing over. Gametogamy is the fusion between gametes. Gamete mating reunites in a single individual a specific combination of genetic information from two parents. Sexual fusion thus produces genetically unique progeny through recombination of the parental genomes.

During the cycle of plant development, the alternation of two successive generations, separated by meiosis and fertilization, is also observed. The first generation corresponds to the asexual generation. One or more cells of the sporophyte undergo meiosis, producing meiotic spores. The meiospores develop into gametophytes, which represent gametophytic generations (starting from gametes). Fusion of the gametes produces a zygote, which represents a return to the asexual generation.

In sexual angiosperms, the female gametophyte (embryo sporium) develops into a multicellular structure (ovule) that is very different from the sporophyte. During ovule development, a special cell, the protosporium, undergoes two successive stages: megasporogenesis (formation of reduced megaspores, or meiospores, from the Forms the female gametophyte), producing a multicellular gametophyte (ovule) containing a single gamete. Nearly 80% of angiosperms involve this type of development (Polygonum type). The different forms of apomixis correspond to a range of variations on this topic.

The first classification of apomixis according to the origin of the embryo is two basic forms. For adventitious embryogenesis, embryos are somatically differentiated directly from the nucellus or integument of the ovule. Thus there is no alternation of generations of sporophytes and gametophytes. Gametophytic apomixis, on the other hand, is characterized by the formation of unreduced female gametophytes, and the parthenogenetic development of embryos from the ovum. In the following, any reference to apomixis refers to gametophyte apomixis.

According to the source of female gametophytes, there are two types of gametophyte apomixis. For the asporotic form, the unreduced embryonic sac is derived from the soma of the ovule, usually nucellus. For the multiple sporulation form, the embryonic ascus is derived from the germ cell, the sporogenous cell. Ameiotic reproduction includes both asporulation and multiple sporulation. There are actually a large number of different processes leading to the formation of unreduced gametophytes.

In sexual angiosperms, the male gametophyte (pollen grain) contains two types of reproductive cells. One fertilizes the ovule to form an embryo, and the other combines with the nucleus of the central cell derived from the endosperm. Both the embryo and the endosperm are thus sexual. This is called double fertilization and is a characteristic feature of angiosperms. In most apomictic plants, the embryo develops without fertilization, while the endosperm remains sexual. When central cell fertilization is required for endosperm development, it is called pseudozygous or pseudozygous apomixis; when both the embryo and endosperm develop without fertilization, it is called independent apomixis.

At the embryonic level, apomixis is actually equivalent to asexual reproduction via seeds. This results from a combination of the following clearly identifiable factors: absence of meiosis or formation of embryonic sacs without meiosis, parthenogenesis or formation of embryos in the absence of fertilization of the egg ball. The absence of meiosis and parthenogenesis ensures the alternation of sporophyte and gametophyte generations, but not the alternation of different nuclear stages: sporophyte and gametophyte remain at the ploidy level.

But at the plant level, apomixis is actually a form of mixed reproduction that includes sexual fusion and asexual reproduction. In fact, apomixis in general is an optional phenomenon: it occurs in the progeny of apomictic plants in "alternative" individuals, ie genetically different from the parent plant. For apomictic development in the strict sense, a combination of apomixis and azorosity is required. Alternatives may arise if one or both conditions are not met. Depending on the success or failure of meiosis and fertilization, the possible types of progeny in non-obligatory apomixis plants are as follows: Fertilize no fertilization Meiosis n+n n+0 numerous divisions 2n+n 2n+0

In the definition of a heterozygote of the "n+n" or "2n+n" isotype, the first term refers to the state of the gametophyte, ie, subtraumatic (n) or non-subtractive (2n). The second term reflects the presence or absence of fertilization of the ovum. The "2n+0" category represents strictly apomixis, while the "n+n" category represents sexual fusion. The "2n+n" class leads to genome accumulation, while the "n+0" class leads to haploidization of the parent plant. The proportions of each of these different classes vary from species to species, and even from plant to plant within a given species. In the following part of the description, references to apomixis relate to these non-obligatory forms of apomixis and also to mandatory forms of apomixis which produce only 2n+0 offspring.

The genetic determination mechanisms for apomixis are poorly understood. It is now recognized that asexually reproducing angiosperms are derived from sexually reproducing ancestors, and that the transition from one form of reproduction to another will manifest genetic determination mechanisms. Thus, apomixis is the result of the expression of an apomictic gene or allele that is present and expressed in apomictic plants but absent or non-functional in sexual plants.

The vast majority of work on the genetic control of apomixis involves apomyosis and more often aposporosis. There is now sufficient consensus on the principles of Mendelian inheritance of asporulation (see references (1) and (2), references are listed at the end of the description). Ploidospore formation is poorly understood, but existing results (3, 4) show that accepted hypotheses in asporogenous plants can undoubtedly be applied to ploidospore formation plants. How the genes involved in these different models act has remained a mystery. But these analyzes tend to suggest that the genes responsible for apomixis can alone initiate the entire apomictic program.

Apomixis has attracted much interest because of its potential to improve plants. Applying apomixis to major crops would be an easy way to stabilize heterosis. This would be a potential revolution in the way reproductive systems are manipulated.

Numerous programs have been developed with the aim of identifying "apomictic genes" and introducing them into crops.

The earliest and probably most advanced work was using natural apomictic plants. The corresponding allele is present and functional. Transfer to important crops is either through interspecific crosses between crops and apomictic mating plants, or by segregation of the corresponding genes for subsequent transgenic introduction into target species ((5); (6); (7) and (8 )).

Another approach that is rapidly developing is the generation of apomixis in sexual plants by different mutagenesis methods. Arabidopsis A. thalina is the most commonly used model ((9), (10) and (8)). Similar studies have been found for Petunia hybrida (10) and Hieracium (8). In all these cases, the underlying hypothesis is that this involves only simple genetic control, and that a few alleles with little transfer or modification may provide sufficient conditions for apomixis expression. Apomixis is understood here as the consequence of the preceding events: failure of meiosis and parthenogenesis.

The present inventors' work in this field led them to search for genes orthologous in maize to genes involved in the expression of apomixis in species of Tripsacum (a genus that is married to maize). In this specification and claims, "orthologous genes" refer to different genes derived from a common gene, or symbiotic homologous genes, and also refer to species containing these genes. The genes have the same function in apomixis.

Triplegrass belongs to the bluestem family. This is the only known marriage of the genus Maize on the American continent. (4) and (11) have been the most complete studies on the reproductive mode of Triscystis. These efforts have identified the following:

- all polyploid accessions reproduce by means of apomixis,

- Apromixis in apomictic forms is mainly Antennaria type and rarely Taraxacum type,

- the embryonic sac in the form of multiple sporulation derived directly from the megasporophyte by three consecutive mitotic divisions,

- meiotic failure in the form of multiple sporulation associated with the absence of corpus sacrum around the megasporophyte,

-Analysis of F1 populations between the ploidospore forming forms of maize and Tripsacus reveals a simple inheritance of unreduced sporulation,

- Genetic mapping of various alleles detected by molecular maize probes close to loci responsible for apomeiosis; these probes are umc28, csu68 and umc62,

- These probes allowed the identification of a partial homology relationship between the chromosome responsible for the ameiosis of Tripsia spp. and the distal end of the long arm of chromosome 6 in maize.

The work of the present inventors led them to propose and demonstrate that the genes responsible for apomixis in Tripsacum have one or more orthologs in the genomes of sexual grasses, particularly maize. This concept has allowed the development of a general strategy for identifying and then cloning the nucleotide sequences responsible for apomixis and new tools for its implementation.

It is therefore an object of the present invention to provide a method for identifying genes orthologous to genes involved in apomixis in Poaceae, especially maize.

The purpose of the present invention is also to provide a method for isolating this gene sequence.

The object of the present invention is also the use of this sequence for isolating the sequences of the corresponding orthologous genes in apomictic plants, and the use of these sequences in transgenesis.

The object of the present invention is also a method for demonstrating the functional relationship between the isolated sequences and the expression of the apomictic phenotype in apomictic plants.

The method according to the invention for the identification of nucleotide sequences orthologous to genes responsible for all or part of the apomictic development in one form of apomixis in Poaceae, in particular maize, characterized in that the phenotype is expressed close to Mutations with or similar to the phenotypes observed in apomictic forms were mapped in the Poaceae, particularly maize genomes, in order to identify those mutations showing orthologs to genes involved in apomictic reproduction.

The associated phenotypes are identified here based on four features of the plural sporulation form, which can be observed individually or together: (a) the mutations are specific for megasporogenesis and do not affect male reproductive function, (b) they result in sporogenesis Protocells form unreduced gametes, (c) they are characterized by the absence of corpus sacrum around the megaspore mother cell, (d) a control point that normally functions during embryo sac formation shows inactivation, although the process of megasporogenesis One of the stages fails but the embryonic sac forms normally.

The identification of the nucleotide sequences referred to in the methods defined above comprises in one embodiment of the invention the identification of loci or genes, or the mapping of meiotic mutations in the genome of Poaceae, particularly maize, to identify Those that are orthologous to genes involved in apomixis.

The invention relates in particular to a method for identifying gene sequences orthologous to genes controlling apomixis in apomixis, in Poaceae, especially maize, characterized by studying phenotypic expression and by means of Molecular markers capable of mapping the loci responsible for apomixis in said apomictic forms determined the position of various meiotic mutations in the genome of Poaceae, especially maize.

Typically, mutations mapped according to the invention are cloned and sequenced.

The present inventors specifically demonstrated that the genes involved in the expression of apomixis in Tripsia have one or more orthologous genes in maize.

The present application of the series of markers in maize and Tripsacum has led to the identification of candidate genes (1) having the same genomic location as the genes controlling the formation of multiple spores, i.e. they are located in maize and Tripsacum Diploid sporulation in the same chromosomal region of the segment of homologous control genes, and (2) associated phenotypes determined according to the above criteria. Preferably, said position relates to the elongate and afd loci in the maize genome.

The method of the invention is also characterized in that it comprises the use of transposon markers to map the meiotic mutations. The invention particularly relates to the tagging of elongate loci with transposons.

Transposons of known sequence can be used to create mutations in genes that are only expressed with a known phenotype. Insertion of transposons in genes to be isolated is often characterized by loss of function. For recessive alleles, this is often characterized by the appearance of the recessive phenotype in heterozygous plants. Transposons of particular interest include Mutator or Ac/Ds type transposable elements.

Preferably, cloning and sequencing of the targeted mutations is performed.

Mutated genes can be isolated by marking the insertion site of the transposon, and the various loci into which the transposon has been inserted are cloned, if desired, and sequenced by conventional techniques of Mendelian analysis and molecular biology (12) and (13).

For loci that eg correspond more specifically to the elongate locus, the locus expressed by the allele el1 is first phenotyped. It was then located by genetic mapping, and the position was compared to the position of the locus controlling multiple sporulation in Tripsys spp. This locus is then tagged with the aid of a transposon, isolated, and then sequenced.

The present invention also relates to the use of at least a part of the sequence defined above for identifying and then isolating such sequences or orthologous genes in apomictic forms.

The present invention relates to isolated nucleotide sequences. These sequences are characterized in that they are orthologous to sequences responsible for all or part of development in apomictic forms. The invention also relates to sequences that are functionally homologous to the above identified sequences.

The present invention particularly relates to nucleotide sequences of this type corresponding to mutant elongate genes.

The invention also relates to nucleic acids comprising one or more of the sequences as defined above and the regulatory sequences required for expression in plant material.

The invention also relates to cloning and expression vectors containing such nucleic acids, and to cellular hosts, such as Agrobacterium tumefaciens, containing these vectors.

The invention also relates to the use of such sequences, if appropriate together with other alleles characteristic of the apomictic form, for the transformation of plant material, plant cells, plants of various developmental stages and seeds for their apomictic development . The alleles correspond to other genes than the orthologous genes provided by the present invention.

The invention relates in particular to a method for producing apomictic plants, characterized in that the sequence of the mutant elongate gene as defined above is used.

Transformed plant material per se is included within the scope of the present invention, characterized in that it contains in its genome at least the part of the sequence involved in apomictic development, where appropriate with other characteristic alleles of the apomictic form Together.

It is envisaged that the cells, plants and seeds are of the family Poaceae. They are especially corn based.

Transformation of plant material, cells, plants and seeds is best carried out using conventional techniques for genetic modification.

Now take the example of obtaining a transgenic maize plant.

A. Obtaining corn callus and using it as a target for genetic transformation

Regardless of the method employed (electroporation, Agrobacterium, microfilaments, particle gun), genetic transformation of maize generally requires the use of rapidly dividing undifferentiated cells that retain the ability to regenerate whole plants. Cells of this type make up the fragile embryogenic callus of maize (so-called type II).

These calli were obtained from immature embryos of genotype H1 II or (A188xB73) using the method and medium described by Armstrong (1994). The calli thus obtained were proliferated and maintained by successive subcultures every 15 days on the starting medium.

Plantlets were then regenerated from these calluses by adjusting the hormonal and osmotic balance of the cells by the method described by Vain et al. (1989). The plants are domesticated in a greenhouse where they can cross or self-fertilize.

B. Genetic Transformation of Maize Using Pellet Guns

The previous paragraph describes the acquisition and regeneration of the cell line required for transformation; a method for genetic transformation resulting in stable integration of the modified gene in the plant genome is described here. The method relies on the use of a particle gun; the target cells are fragments of the callus described in paragraph 1 above. These fragments with a surface area of 10 to 20 mm2 were placed 16 pieces/dish in the center of a Petri dish containing the same medium as the starting medium but supplemented with 0.2M manna 4 hours before bombardment Sugar alcohol + 0.2M sorbitol. Plasmids carrying the genes to be introduced were purified on Qiagen columns according to the manufacturer's instructions. It was then deposited on tungsten particles (M10) according to the procedure described by Klein et al., Nature, 1987, 327, pp. 70-73. The particles thus coated are fired at the target cells with a gun as described by J. Finer (1992).

The thus bombarded callus plates were then sealed with Scellofrais(R) and incubated at 27[deg.] C. in the dark. The first subculture was performed 24 hours thereafter, and then every 15 days for 3 months on the same medium as the starting medium (in which the selection agent had been added). Selective agents that can be used are generally active ingredients of certain insecticides (Basta®, Round up®) or certain antibiotics (hygromycin, kanamycin, etc.).

After 3 months or less, callus is obtained whose growth is not inhibited by the selection agent, usually and in most cases by cell division that has integrated one or more copies of the selection gene in its genetic inheritance The resulting cell composition. The probability of obtaining such a callus is about 0.8 calli/bombard plate.

These calli are identified, singly isolated, expanded, and cultured to regenerate plantlets. All these manipulations were performed in media containing selection agents in order to avoid interference of non-transformed cells.

Such regenerated plants are acclimated and then grown in a greenhouse, at which point they can be crossed or self-fertilized.

C. Genetic Transformation of Maize Using Agrobacterium tumefaciens

The technique used is reported in Ishida et al. (Nature Biotechnology, 1996, 14: 745-750) or in Horsch et al., Science, 1984, 223, pp. 496-498.

The present invention thus provides methods for producing apomictic plant populations with transposable elements.

In particular, the present invention provides methods for inducing apomictic development in sexual plants, particularly maize.

In another application of the invention, at least a part of the above defined sequences are used to identify and isolate orthologous locus sequences in apomictic forms.

The present invention thus relates to hybridization probes prepared from said sequences and primers useful in PCR techniques.

Such probes and primers specifically correspond to those prepared from elongate sequences.

Hybridization or PCR techniques are best performed conventionally.

The present invention also relates to a method for identifying and isolating the genes responsible for the formation of multiple sporulation in Tripsia apomicticum, characterized in that at least a part of the sequence of the elongate locus is used.

The method of the invention is also characterized in that the sequence isolated in Trip. apomicticum is used for functional analysis of the relationship between the sequence and apomictic expression. As illustrated in the Examples, the mutagenesis method was particularly used to demonstrate the relationship between the apomictic Trip.

According to the present invention, the relationship between said sequence and apomyosporosis was specifically demonstrated.

Other characteristics and advantages of the invention are given in the following examples. In these examples reference will be made to Figures 1-5:

- Figure 1: Genetic mapping of the chromosomal segments controlling the formation of multiple spores in the apomictic tetraploid Trip.

- Figure 2: Construction of the mapping population of the el1 mutation (elongate),

- Figure 3: Construction of mutagenized populations for marker elongate loci,

- Figure 4: Phenotypic characterization of megaspore mother cells in plants homozygous for the allele el1 and maize plants with the wild allele at the elongate locus, and comparison with sexual and apomictic forms of Tripsia ,

- Figure 5: Construction of a mutagenized population of maize-Tripsa hybrids for the functional analysis of the relationship between the isolated sequences and apomictic expression in apomictic plants.

Example 1: Genetic Mapping of Apomeiosis in Tripsia

The following describes the generation of genetic maps controlling the chromosomal segments that control the apomictic and sexual forms of Tripsysia apomixis.

1) Material

- Mapping of the diploid Tripsia: the two parents used were sexual diploid plants (2n=36) of the ORSTOM-CIMMYT collection, kept at the Tlaltizapan Experimental Station, State of Morelos, Mexico. These are Tripsacum maizar Hern. and Randolph with accession number CIMMYT #99-1114, and Tripsacum dactyloides var. meridionale de Wet and Timothy with accession number CIMMYT #575-5136. The population contained 175 F1 plants, 56 of which were used for mapping.

- Mapping of apomeiosis: The mapping population contained 232 F1 maize-Tripsa plants. Parent maize (H1 ) is a maize hybrid (2n=2x=20) between two CIMMYT lines (CML135-CML139). The other is tetraploid apomictic T.dactyloides (2n=4x=72), the preservation number is CIMMYT #65-1234. Apomictic plants are used as males.

2) method

. Analysis of the mode of reproduction: This was performed using the method of Leblanc et al. (4).

. Detection of RFLP-type molecular markers associated with apomeiosis:

The strategy described below is generally comparable to the method described by Michelmore et al. (14) for the detection of molecular markers associated with specific phenotypic responses.

Probes were obtained from the University of Missouri (Columbia).

About 100 RFLP probes were selected on the existing maize genetic map to achieve a density of about 20-30 cM between two markers (see (4), Appendix 4). Various reference maps were used: UMC map (University of Missouri, Columbia; Maize Data Bank, UMC95 map), a map from Cornell University (15) and various maps provided by CIMMYT (16). The chi2 test was used to determine potential linkage and the method of Allard (17) was used to evaluate recombination values. Because the donor parent of the segment controlling apomeiotic reproduction is a heterozygous tetraploid plant, three conditions are required to detect the linkage between the multiple sporulation and the RFLP alleles: (1) the gene is present between the two parents There is an RFLP polymorphism at the locus, (2) the allele is heterozygous in Tripsia, (3) the allele must be a monodominant combination in the tetraploid, which can be distinguished from the other three open.

- Mapping of diploids

F1 mapping populations between two heterozygous parents belonging to two different species were used. The plotting method was as described by Ritter et al. (18).

3) Results

- Identification of RFLP markers associated with apomeiosis

Figure 1 shows the genetic mapping of the chromosomal segments controlling apomeiotic reproduction, and a comparison with Tripsia and maize sexual diploid plants. "Apo" corresponds to the locus responsible for apomeiotic reproduction. The location of umc71 on maize chromosome 6 is roughly marked, and the allele on chromosome 6 was obtained from the latest version of the UMC map. The map was obtained from 52 plants of the F1 population between maize and Tripsia. In this population, meiosis and ploidosporation segregated 1:1 (24 apomictic plants, 28 sexual plants, chi2 = 0.31, p = 0.6). Eighty-four probes were tested and probes were selected on the UMC map to cover the maize genome as broadly as possible. 90% of them detected at least one polymorphism between maize and Tripsascus. Three probes representing polymorphic and apomict-specific alleles were tested on the entire F1 population. One allele detected by probe umc28 was shown to be associated with apomeiosis. In the second step, 14 probes close to the umc28 locus on the UMC map were tested. Four of these, umc71, umc62, csu68 and cdo202, detected multiple RFLP alleles that were both associated with diplosporulation and completely co-segregated between them.

- Comparative mapping between sexual diploids and apomictic tetraploids

Five markers associated with diplosporulation were mapped on diploid populations. They can all be on the same parent (575-5136). Note that these five markers are all strictly linked in tetraploid plants, but segregate with significant recombination values in both diploid parents and in maize.

-Comparative mapping of maize-tripsa:

Probes that detect alleles associated with chromosomal segments that control polyspore formation in Tripsia spp. detected all alleles belonging to the same linkage group in the map of maize. This is the long arm of chromosome 6. Some of them also detected alleles in other regions of the genome, notably chromosomes 3 and 8. The positions of various probes associated with apoiosis on the map of maize are shown in the table below:

clone location

UMC38* 6L; 8L; 3L;

UMC62 6L

UMC71 6L; 8L;

UMC28 6L

CSU68 6L; 8L; 3L

CD0202 6L; 8L; 3L

*: Not related to multiple sporulation, but belonging to the same linkage group.

Example 2: Identification of Orthologous Genes in Maize

The genes responsible for the expression of apomeiosis in Tripsia spp. were studied in maize. The goal was to identify candidate genes in maize that have (1) the same genomic location as apoinomycetes and (2) phenotypes associated with apoinomyces.

There are many meiotic mutants in maize. The phenotypic criteria for selection of candidate genes were as follows: (1) presence of distinctly differentiated sporogenous cells, (2) complete absence of meiotic induction in these cells, or failure of such induction at an early stage, (3) comparison with meiosis The ability to generate functional gametophytes unrelated to the failure of mitosis, (4) the absence of corpus sacrum around the megaspore parental cells or at least a very marked decrease. Among the potential candidates, i.e., those with all of the above characteristics or parts, those whose positions in the maize genome were unknown or imprecise were mapped using reference loci, which were derived from the unreduced Those detected by the probes used for sporulation mapping.

-Materials and methods

The results reported in the work below were performed on the recessive meiotic mutant elongate (el1) (19). In plants homozygous for the el1 locus, chromosomes remain depolymerized during the metaphase and late stages of the first division, giving rise to various chromosomal abnormalities, a significant fraction of which are unreduced gametophytes (30 to 70%, depending on the mutation in particular genetic background). Pollen from normal plants is fertilized to form triploid embryos and defective pentaploid endosperms.

The precise location of the elongate locus was unknown until the present invention. But it was previously known to belong to the long arm of chromosome 8 in maize. It therefore did not map directly to the arm of the maize chromosome identified by Leblanc et al. as homologous to genes controlling apomeiosis. Although located on chromosome 8, it likely belongs to a segment with duplicates between the distal long arm of chromosome 6 and some part of chromosome 8 (15).

Figure 2 describes the construction of the el1 (elongate) mutant mapping population. Three F1 plants with heterozygous genotype El1/e1 were backcrossed with three homozygous e11/e1 plants. For each individual species, 50 plants were grown, self-fertilized and their phenotypes were evaluated. El1/el1 seeds were expected to be normal, whereas el1/el1 seeds had malformed endosperms. In order to confirm the elongate phenotype, 10-20 embryos from seeds with defective endosperm were taken as samples and analyzed by flow cytometer according to the method proposed by Galbraith et al. (20). The el1 mutant was obtained from the Maize Genetic Stock Center, Urbana, Illinois in the form of seeds produced by self-fertilization of plants homozygous for the el1 allele in the genetic background (further information unclear). A homozygous line W23 with a wild phenotype was used to construct the population. Linkage was detected and recombination values were evaluated using Mapmaker 2.0 software (for Mackintosh machines).

-Comparison of phenotypic expression in apomeiotic plants and elongate

The phenotypic expression of the elongate mutation in homozygous ell/el1 plant sporogenous cells was analyzed using cytoembryological techniques as described by Leblanc et al. (11). Immature inflorescences were collected on four types of material: (1) sexual diploid Tripsia, (2) apomictic tetraploid Tripsia, the line described by Leblanc et al. (11), (3) wild phenotype A homozygous El1/Ell maize line (W23), and (4) a homozygous el1/el1 line.

.Use transposable elements to mark and isolate elongate locus sequences

Tagging with transposons is the use of transposons of known sequence to create mutations in genes whose expression is only known phenotype. Insertion of transposons in genes to be isolated is often characterized by loss of function. In the case of recessive alleles, it is often characterized by the appearance of the recessive phenotype in heterozygous plants. By marking the insertion site of the transposon, the mutant gene itself can be cloned. Thus, the mutant gene itself is isolated by marking the insertion site of the transposon, and the various loci into which the transposon has been inserted are cloned by conventional techniques of Mendelian analysis and molecular biology. The following experiments were performed with the Mutator system (21).

The populations used to mark the elongate locus with transposons are shown graphically in Figure 3 (f: frequency of occurrence; [EL] and [el]: dominant and recessive phenotypes; El*: marked etc. allele. Plants homozygous for the recessive mutation were crossed with plants homozygous for the wild allele El1. In the gamete population produced by the El1/E11 parents, one or more mutations were found at the elongate locus. The insertion resulted in The loss of function of this allele resulted in the discovery of some F1 plants of the el1/Ell genotype but the el1 phenotype. The gene was thus marked.

result:

Mapping of -elongate loci:

In the populations studied in isolation, the el1 and El1 phenotypes segregated in a 1:1 population (Chi2 = 0.5; p = 0.6). Various RFLP probes belonging to chromosomes 3, 6, and mostly chromosome 8 were tested on 50 plants of this population with the three restriction enzymes EcoRI, BamHI and HindIII. Probes that detect meaningful polymorphisms are then analyzed on another 100 strains. Umc28, umc62, and umc71 did not display elongate-associated polymorphic alleles using the three enzymes tested. While Csu68 and cdo202 detected alleles associated with elongate. The linkage between the three loci assessed by percent recombination is as follows: cdo 202 csu68 elongatecsu68 9.3 7.41.9

- Phenotype identification:

Fig. 4 shows a comparison of the development of protospores in various types of materials (A: mother cells of megaspores in W23, which is the El1/El1 genotype of maize; B: mother cells of megaspores in homozygous el1/e1 maize ; C: mother cells of megaspores in sexual diploid Trip. The various developmental stages observed were identified and matched between the various forms observed on the basis of ovule integument size and morphology (11). For the sexual forms of maize and Tripsia: completely similar developmental features were observed: identical morphology of cells and nuclei (in particular: mother cells of rectangular megaspores, thick prominent walls, corpus sacrum very prominent on cell walls emergence, from the mother cell of the megaspore to the formation of the megaspore). The same similarity was observed when comparing the triple sporulation form of Trip. The nuclei of the sexual forms, the ploidospore forms of Trip.

Transposon markers for -elongate loci:

A population of 12,500 F1 plants generated according to Figure 3 was grown at the CIMMYT experimental station in Tatizapan, Mexico. These 12,500 plants were fertilized with maize hybrids (CML135*CML62 hybrids) that had lost the active Mutator. The ears of these 12,500 plants were monitored to maturity to find those that were heterozygous for this locus but expressed the elongate mutation. For plants with defective endosperms, the corresponding embryos were analyzed by flow cytometry. Two plants, designated TTE1-5 and TTE1-7, were identified to contain defective endosperms associated with triploid embryos. These plants express the elongate phenotype in heterozygous ell/Ell plants. In these plants, the wild allele of this locus is marked with one of the Mutator-type transposons. Seeds from crosses of these two plants with the CML135*CML62 hybrid formed a population segregating for this marker allele at the elongate locus: half of the plants produced from this cross had the el1/El genotype (el1 from TTEl plants, El from CML135 *CML62), the other half is the El1*/El genotype, where El1* is the transposon-marked allele and comes from TTEl plants. Such genes at the elongate locus can be identified and cloned by analyzing the co-segregation of various copies of transposable elements in these plants and the elongate loci found with the RFLP probes mentioned below.

- Example 3: Generation and use of populations for demonstrating the relationship between candidate alleles and the expression of total or partial apomictic developmental phenotypes

The overall scheme and materials used are shown in Figure 5. The strain containing the Mutator element was obtained from Mr. Freeling, University of California (Berkeley). The double haploid BC2-28 plants used here were those previously reported by Leblanc et al. (6). They are apomictic plants with haploid genomes from the respective parents of maize and tripsa. We used these plants to introduce Mutator elements into apomictic material.

A population of 1000 apomictic BC2-28 plants was first constructed from a single apomictic double haploid plant, and 2n+0 type plants were selected from its progeny. In this way we created 1000 copies of the same apomictic polyhaploid genotype. These 1000 plants were crossed with Mutator clones. Among the progeny obtained, we selected alternative 2n+n plants, ie those that had incorporated the genome from the Mutator clone. These clones have about 200 copies of each type of Mutator. It is thus desirable to harvest an average of 100 copies of apomictic plants. Selection of BC2-28 plants and alternative apomictic BC3-38 plants was performed using a simple morphological criterion. Double haploids actually have a very recognizable phenotype that is very different from that caused by the accumulation of extra maize genomes (6). In total about 35,000 seeds (BC2-28 xMutators) were acquired. They were both grown in the summer of 1996 at the CIMMYT experimental station in Tlaltizapan, Morelos state. Choose an alternative 2n+n one month after germination. About 7,500 2n+n plants were obtained, ie almost 20% of the alternative plants. This population was recrossed with maize hybrids (CML135*CML62) without active Mutator elements, resulting in a population of approximately 150,000 seeds, representative of the reverse genetic population. This population represents ideal material for analyzing the effect of a given sequence on apomictic expression. In fact, the Tripsia allele responsible for the expression of this trait is here in a monodominant combination (single copy in the genome). Thus to confirm the allele-function relationship it is only necessary to examine the phenotypic effect of the transposon inserted into this sequence. This allele-function relationship is asserted if the mutation causes a loss of function. Since the sequences of both the transposon and the gene of interest are known, plants in which alleles are mutated can be identified by conventional PCR techniques.

references

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Claims (24)

1. A method for identifying in Poaceae, especially maize, nucleotide sequences orthologous to sequences responsible for all or part of apomictic development in apomixis forms, characterized in that the phenotype is close to or similar to that of apomixis Mutations in the phenotypes observed in the apomixis form were mapped in the Poaceae genome, particularly the maize genome, to identify those showing orthologs to genes involved in apomixis. 2. A method according to claim 1, characterized in that the meiotic mutations are mapped in the Poaceae genome, in particular the Maize genome, to identify those showing orthologs to genes involved in apomixis. 3. The method according to claim 2, characterized in that the various meiotic mutations are placed in the Poaceae genome, in particular in maize located in the genome. 4. Method according to claim 3, characterized by using molecular markers capable of mapping the locus of diplosporulation in Tripsysia spp. 5. Method according to claim 4, characterized in that said mapping involves the elongate and afd loci. 6. A method according to any one of claims 1-5, characterized in that it further comprises marking the localized meiotic mutations with transposons. 7. The method according to claim 4, characterized in that the marking with transposons is carried out at the elongate locus using Mutator or Ac/Ds type transposable elements. 8. Method according to any one of the preceding claims, characterized in that the located mutations are cloned and sequenced. 9. The method according to claim 6 or 7, characterized in that the mutated gene is cloned after marking the transposon insertion site by segregation analysis and, if necessary, is sequenced. 10. Nucleotide sequences, characterized in that they are orthologous to the sequences responsible for all or part of the apomictic development in apomictic forms, and homologous sequences. 11. Nucleotide sequence according to claim 10, characterized in that it corresponds to a mutated elongate gene. 12. Nucleic acid comprising one or more sequences as defined in claim 10 or 11 and the regulatory sequences required for expression in plant material. 13. Cloning and expression vector comprising the nucleic acid of claim 12. 14. A cellular host comprising the vector of claim 13. 15. The sequence according to claim 10 or 11, where appropriate together with other alleles characteristic of the apomictic form, for introduction into the genome of plant material, plant cells, plants of various developmental stages and seeds in order to render them apomixis Applications in reproductive development. 16. Poaceae, especially maize plant cell, characterized in that it contains in its genome at least the part involved in the apomictic development of the sequence according to claim 10 or 11. 17. Poaceae plant, especially maize, characterized in that it contains in its genome at least the part involved in the apomictic development of the sequence according to claim 10 or 11. 18. The seed of Poaceae, especially maize, characterized in that it contains in its genome at least the part involved in the apomictic development of the sequence according to claim 10 or 11. 19. Method for producing apomictic plants, characterized in that the nucleotide sequence according to claim 11 is used. 20. Use of at least a part of the sequence of claim 10 or 11 for identifying and isolating orthologous sequences of loci in apomixis forms. 21. Hybridization probes and molecular primers, characterized in that they are prepared from the sequences of claims 10 or 11. 22. Hybridization probes and molecular primers according to claim 18, characterized in that they are prepared from sequences of elongates. 23. A method for identifying and isolating the genes responsible for apomeiotic reproduction in apomictic Trip. 24. The use of a mutagenic population to demonstrate the relationship between the isolated sequence and apomictic expression in Tripsia sativa of claim 20.

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