DOMINANT MUTATION IN THE TDM GENE LEADING TO DIPLOGAMETES PRODUCTION IN PLANTS

Abstract

The invention relates to a dominant mutation in the TDM gene leading to the production of 2n gametes in plants, to the plants comprising said mutation, and to their use in plant breeding. The invention relates also to plants in which the dominant mutation in the TDM gene is combined with the inactivation of a gene involved in meiotic recombination in plants and a gene involved in the monopolar orientation of the kinetochores during meiosis. These plants which produce apomeiotic gametes are also useful in plant breeding.

Claims

1. A method for obtaining a plant producing Second Division Restitution 2n gametes, wherein said method comprises providing, by random or targeted mutagenesis or by genetic transformation, a plant comprising a dominant mutation within a gene, herein designated as TDM gene, coding for a protein designated herein as TDM protein, wherein said protein has at least 75% sequence identity with amino acid residues 1 to 286 of the TDM protein of SEQ ID NO: 1 when said plant is Brassica spp. or at least 30% identity with said residues when said plant is different from Brassica spp, and the 60 first amino acids of said protein comprise a motif X.sub.1X.sub.2X.sub.3, wherein X.sub.1 is a Threonine (T), X.sub.2 is a Proline (P), and X.sub.3 is a Proline (P) or a Glutamine (Q), designated herein as TPP/Q motif, and wherein said dominant mutation comprises the mutation of at least one residue of the motif X.sub.1X.sub.2X.sub.3 of said TDM protein and results in the ability of the plant to produce Second Division Restitution 2n gametes.

2. The method according to claim 1, wherein said motif is situated in a region of said protein which is that situated from positions 16-18 of SEQ ID NO: 1.

3. The method according to claim 1, wherein said residue is the T residue or its adjacent P residue.

4. The method according to claim 3, wherein said mutation is selected from the group consisting of: the substitution of said T and/or P residues with a different residue and the deletion of said T and/or P residues, alone or with 1 or 2 residues flanking said T and/or P residues.

5. The method according to claim 1, wherein said mutation abrogates phosphorylation at the T residue of said motif.

6. The method according to claim 1, which comprises: providing by random or targeted mutagenesis, a plant having said dominant mutation within an allele of a TDM gene, said plant being heterozygous for this mutation.

7. The method according to claim 1, wherein said plant is a transgenic plant, and said method comprises: a) transforming at least one plant cell with a vector containing a DNA construct comprising a TDM gene having said dominant mutation; b) cultivating said transformed plant cell in order to regenerate a plant having in its genome a transgene containing said DNA construct.

8. A method for obtaining a plant producing apomeiotic gametes, wherein said method comprises: a) providing a plant comprising said dominant mutation in a TDM gene as defined in claim 1; b) inhibiting in said plant a first protein involved in initiation of meiotic recombination in plants, said protein being selected among: a protein designated as spo11-1 protein, wherein said protein has at least 40%, and by order of increasing preference, at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence identity, or at least 60%, and by order of increasing preference, at least, 65, 70, 75, 80, 85, 90, 95 or 98% sequence similarity with the spo11-1 protein of SEQ ID NO: 29; a protein designated as spo11-2 protein, wherein said protein has at least 40%, and by order of increasing preference, at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence identity, or at least 60%, and by order of increasing preference, at least, 65, 70, 75, 80, 85, 90, 95 or 98% sequence similarity with the spo11-2 protein of SEQ ID NO: 30; a protein designated as prd1 protein, wherein said protein has at least 25%, and by order of increasing preference, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence identity, or at least 35%, and by order of increasing preference, at least, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence similarity with the prd1 protein of SEQ ID NO: 31; a protein designated as prd2 protein, wherein said protein has at least 25%, and by order of increasing preference, at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence identity, or at least 35%, and by order of increasing preference, at least, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence similarity with the prd2 protein of SEQ ID NO: 32; a protein designated as PAIR1 protein, wherein said protein has at least 30%, and by order of increasing preference, at least 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence identity, or at least 40%, and by order of increasing preference, at least, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence similarity with the PAIR1 protein of SEQ ID NO: 33; a protein designated as DFO protein, wherein said protein has at least 30%, and by order of increasing preference, at least 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence identity, or at least 40%, and by order of increasing preference, at least, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence similarity with the DFO protein of SEQ ID NO: 34, and c) inhibiting in said plant a second protein designated as REC8 protein, wherein said protein has at least 40%, and by order of increasing preference, at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence identity, or at least 45%, and by order of increasing preference, at least, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence similarity with the REC8 protein of SEQ ID NO: 35.

9. The method according to claim 8, wherein inhibition of at least one of the SPO11-1, SPO11-2, PRD1, PRD2, DFO, PAIR1, or REC8 proteins is obtained by mutagenesis of the gene encoding said protein of or of its promoter, and selection of the mutants having partially or totally lost the activity of said protein.

10. The method according to claim 8, wherein inhibition of at least one of the SPO11-1, SPO11-2, PRD1, PRD2, DFO, PAIR1, or REC8 proteins is obtained by expressing in said plant of a silencing RNA targeting the gene encoding said protein.

11. A DNA construct comprising the TDM gene having a dominant mutation as defined in claim 1.

12. A plant producing Second Division Restitution 2n gametes, obtainable by the method of claim 1, wherein the plant comprises said dominant mutation in the motif X.sub.1X.sub.2X.sub.3 of the TDM protein which results in the ability of the plant to produce Second Division Restitution 2n gametes.

13. A plant producing Second Division Restitution 2n gametes, which is a transgenic plant containing a transgene comprising the DNA construct of claim 11.

14. A plant producing apomeiotic gametes, obtainable by a method of claim 8, wherein the plant comprises said dominant mutation in the motif X.sub.1X.sub.2X.sub.3 of the TDM protein which results in the ability of the plant to produce Second Division Restitution 2n gametes.

15. A method for producing Second Division Restitution 2n gametes, wherein said method comprises cultivating a plant obtainable by a method of claim 1, wherein the plant comprises said dominant mutation in the motif X.sub.1X.sub.2X.sub.3 of the TDM protein which results in the ability of the plant to produce Second Division Restitution 2n gametes, and recovering the gametes produced by said plant.

16. A method for producing apomeiotic gametes, wherein said method comprises cultivating a plant obtainable by a method of claim 8, wherein the plant comprises said dominant mutation in the motif X.sub.1X.sub.2X.sub.3 of the TDM protein which results in the ability of the plant to produce Second Division Restitution 2n gametes, and recovering the gametes produced by said plant.

Description

[0089] In addition to the above arrangements, the invention also comprises other arrangements, which will emerge from the description which follows, which refers to exemplary embodiments of the subject of the present invention, with reference to the attached drawings in which:

[0090] FIG. 1 represents alignment of TDM proteins from various angiosperm species. Sequences were aligned with T-Coffee (v6.85) with default parameters (http://toolkit.tuebingen.mpg.de/t_coffee). The sequence alignment was edited with BioEdit. Only the first half of the sequences which is conserved in TDM proteins is shown. The residues showing more than 80% identity in the TDM proteins which are aligned are shaded. The conserved region comprising the TPP/Q motif is boxed.

[0091] FIG. 2 represents the phylogenetic tree of TDM proteins from various angiosperms, TDM_like1 proteins from Brassicales and TDM-like proteins from Arabidopsis thaliana and Brachypodium distachyon. The analysis was performed on the Phylogeny.fr platform and comprised the following steps. Sequences were aligned with T-Coffee (v6.85) using the following pair-wise alignment methods: the 10 best local alignments (Lalign_pair), an accurate global alignment (slow_pair). After alignment, positions with gap were removed from the alignment. The phylogenetic tree was reconstructed using the maximum likelihood method implemented in the PhyML program (v3.0 aLRT). Proteins of the TDM clade are shown for all species (including TDM-like1 in Brassicales). More distant TDM paralogues are shown only for Arabidopsis thaliana and Brachypodium distachyon.

At: Arabidopsis thaliana. Al: Arabidopsis lyrata. Bra: Brassica rapa. Sly: Solanum lycopersicum. St: Solanum tuberosum. Csa Cucumis sativus. Eucgr: Eucalyptus grandis. Cp: Carica papaya. ME: Manihot esculenta. TC: Theobroma cacao. Goraii Gossypium raimondii. FV: Fragaria vesca. Pp: Prunus persica. LI: Lotus japonicus. MT: medicago truncatula. GM: Glycine max. Pv Phaseolus vulgaris. VV: Vitis vinifera. Aq: Aquilegia caerulea. OS: Oryza sativa japonica. OSINDICA: Oryza sativa indica. BD: Brachypodium distachyon. SB: Sorghum bicolor. ZM: Zea mays. Si: Setaria italica.

[0092] FIG. 3 shows that spo11-1 rec8 (s)-40 mutant produces dyads and is tetraploid. (A to C). Male meiotic products stained by toluidine blue. (A) Wild type produces tetrads of spores. (B) spo11-1 rec8 produces unbalanced polyads of spores. (C) spo11-1 rec8 (s)-40 produces dyads of spores. (D to F) Mitotic caryotype. (D) Wild type is diploid, having ten chromosomes aligned on mitotic metaphase plates. (E) spo11-1 rec8 is diploid. (F) spo11-1 rec8 (s)-40 is tetraploid, having 20 chromosomes aligned on mitotic metaphase plates. Scale bar=10 μM.

[0093] FIG. 4 illustrates meiotic products of TDM-P17L, TDM-T16A and TDM-Δ14-19. (A) spo11-1 rec8 mutants transformed with TDM-P17L. Wild type plants transformed by (B) TDM-P17L, (C) TDM-T16A or (D) TDM-Δ14-19. Dyads of spores are observed, compared to tetrads in wild type (FIG. 3A) and polyads in spo11-1 rec8 (FIG. 3B).

[0094] FIG. 5 illustrates meiotic chromosome spreads in wild type, spo11-1 rec8, TDM-P17 and spo11-1 rec8 TDM-P17 plants. (A to D) Meiosis in wild type. (A) Five bivalents align at metaphase I and (B) pairs of homologous chromosome are distributed into two nuclei at telophase I. (C) Five pairs of sister chromatids align on the two metaphase plates. (D) Four balanced nuclei are formed at telophase II. (E to H) Meiosis in spo11-1 rec8. The first division resembles a mitotic division with (E) alignment at 10 pairs of chromatids on the metaphase plates and (F) segregation into two groups of 10 chromatids. (G) single chromatids fail to align properly at metaphase II, resulting into (H) a variable number of unbalanced nuclei at telophase II. (I to J) Meiosis in wild type plant transformed with TDM-P17. A single, meiosis I-like division is observed. (K to L) Meiosis in spo11-1 rec8 plants transformed with TDM-P17. A single, mitotic-like division is observed.

EXAMPLES

Experimental Procedures

1. Growth Conditions and Genotyping

[0095] Arabidopsis plants were cultivated in greenhouse as previously described (Vignard et al., PLoS Genet., 2007, 3, 1894-1906) or in vitro on Arabidopsis medium, as previously described (Estelle and Somerville, Mol. Genet., 1987, 206, 200-206) at 21° C., under a 16-h to 18-h photoperiod and 70% relative humidity.

[0096] spo11-1-3 rec8-2 plants were genotyped as previously described (d'Erfurth et al., PLoS Biol., 2009, 7, e1000124). tdm-3 plants were genotyped as described in Cromer et al., PLoS Genet., 2012, 8, e1002865.

2. EMS Mutagenesis and Mutation Identification

[0097] EMS mutagenesis was performed as previously described (Crismani et al., Science, 2012, 336, 1588-1590). Whole genome sequencing was done by HigSeg™ 2000 (Illumina). A list of SNPs was generated compared to the reference genome of Arabidopsis thaliana TAIR10 (cultivar Columbia).

3. Cytology and Ploidy Analysis

[0098] Male meiotic products observation, chromosomes spreads, and ploidy measurement were carried out using the techniques described by d'Erfurth et al. (PLoS Genet., 2008, 4, e1000274).

4. Directed Mutagenesis Constructs and Plant Tranformation

[0099] TDM genomic fragment was amplified by PCR using TDM U (5′-GACATCGGCACTTGCTTAGAG-3′; SEQ ID NO: 36) and TDM L (5′-GCGATATAGCTCCCACTGGTT-3′; SEQ ID NO: 37). The amplification covered 986 nucleotides before the ATG and 537 nucleotides after the stop codon. The PCR product was cloned, by Gateway™ technology (Invitrogen), into the pDONR207™ vector (Invitrogen), to create pENTR-TDM, on which directed mutagenesis was performed using the Stratagene QuickChange™ Site-Directed Mutagenesis Kit, according to the manufacturer's instructions. The mutagenic primers used to generate mutated version of TDM were SEQ ID NO: 38 to 41:

TABLE-US-00002 TDM-P17L: 5′-GAGTTTACTATACTCTGCCGCCGGCGAGAAC-3′; TDM-T16A: 5′-CTCCACCTGGAGTTTACTATGCCCCGCCGCCGGCGAGA-3′; TDM-Y14A: 5′-CCACCTGGAGTTGCGTATACTCCGCCGCGGCG-3′; TDM-Δ14-19: 5′-CCACCTGGAGTTGCGAGAACAAGTGATCATGTGGC-3′;
and their respective reverse complementary primers. To generate binary vectors for plant transformation, an LR recombination reaction was performed with the binary vector for the Gateway™ system, pGWB1 (Nakagawa et al., Journal of Bioscience and Bioengineering. 2007, 104, 34-41). The resulting binary vectors, pTDM, pTDM-P17L, pT16A, and pTDM-Y14A, were transformed using the Agrobacterium-mediated floral dip method (Clough, S. J. and Bent A. F., Plant J., 1998, 16, 735-743) on wild type plants and plant populations segregating for the spo11-1 or rec8 or tdm-3 mutation. Transformed plants were selected on agar plates containing 20 mg/L hygromycin.

Example 1: A Dominant Mutation in TDM Leads to Premature Meiotic Exit

[0100] To identify new genes controlling meiotic progression, a genetic screen was designed based on the idea that mutations that lead to the skipping of the second meiotic division such as osd1 and cyca1;2/tam will restore the fertility of mutants that have unbalanced chromosome segregation defect only at the second meiotic division (d'Erfurth et al., PLoS Biol., 2009, 7, e1000124; d'Erfurth et al., PLoS Genet., 2010, 6, e1000989 and WO 2010/079432). This is the case of spo11rec8 double mutants, in which the first meiotic division resembles a mitosis (balanced segregation of sister chromatids to opposite poles) but the second division is unbalanced and leads to aneuploid gametes and hence very limited fertility (FIG. 5) (Chelysheva et al., J. Cell. Sci., 2005, 118, 4621-32). Mutations in OSD1 (d'Erfurth et al., PLoS Biol., 2009, 7, e1000124 2009) or CYCA1;2/TAM (d'Erfurth et al., PLoS Genet., 2010, 6, e1000989), that lead to meiotic exit before meiosis II, are indeed able to restore fertility of spo11-1 rec8. Thus, a genetic screen was ran based on the restoration of fertility of spo11-1 rec8, aiming at identifying mutants conferring similar defects than osd1 or tam. Despite their meiotic segregation defect, spo11-1 rec8 plants produced enough residual seeds that were mutagenized with ethylmethane sulfonate (EMS). The M1 plants that are presumably heterozygous for EMS mutations were self-fertilized and harvested in bulks of ˜5 to produce M2 families. About 2000 M2 families (400 bulks) were screened for increased fertility compared to spo11-1 rec8 non-mutagenized control.

[0101] Three bulks segregated plants with increased fertility. Genotyping confirmed that they were spo11-1 rec8 mutants which indicated that were genuine suppressors. Analysis of male meiotic products stained by toluidine blue showed that in all three cases, fertile plants produced dyads of spores, as observed in osd1 or cyca1;2/tam, instead of tetrads, as observed in wild type, suggesting that the second meiotic division did not occur in those plants (FIG. 3). Sequencing of candidate genes (CYCA1;2/TAM and OSD1) identified recessive mutations in CYCA1;2/TAM in two of the three families. The identified mutations were a splicing site in exon 7 (TAIR10 chr1:29082522 C>T) and a mutation in the 5′UTR region which introduced an upstream out of frame start codon (TAIR10 chr1:29084174 G>A). A complementation test showed that they were allelic, confirming that the mutations in CYCA1;2 caused the dyad phenotype and the restoration of fertility. The third family (spo11rec8(s)-40) had no mutation in OSD1 and CYCA1;2 and is the focus of this study.

[0102] Chromosome spreads unexpectedly showed that the four plants were tetraploids (FIG. 3). This suggested that the causal mutation was dominant and caused the production of diploid gametes in both male and female organs of the M1 plant. Whole genome sequencing of the bulk of two sister plants with ˜100× coverage revealed the presence of 1144 SNPs compared to wild type. However, only 15 SNPs appeared as homozygote. These few homozygote SNPs were dispersed in the genome suggesting that they were present in the spo11-1 rec8 line before mutagenesis, rather than resulting from fixation of EMS induced mutations. The fact that almost all detected mutations were heterozygote further suggested that the causal mutation was dominant. This mutation would have been phenotypically expressed in the M1 plant leading, in combination with spo11-1 rec8 mutation, to the production of diploid clonal gamete as observed in a spo11-1 rec8 osd1 triple mutant (MiMe, d'Erfurth et al., PLoS Biol., 2009, 7, e1000124 and WO 2010/079432), hence maintaining heterozygosity of EMS induced mutations from the M1 plant in the tetraploid M2 plants.

[0103] Candidate causal mutations were then looked for among the heterozygote SNPs. Among these 1129 mutations, 341 were predicted to affect a coding sequence (non-sense, missense or splicing site). Among them, a mutation in TDM resulting in an amino acid change (TDM-P17L), appeared as a good candidate as the potential causal dominant mutation. TDM was previously shown to be essential for meiotic exit at the end of meiosis II. Even if the meiotic defect observed in tdm knockout mutants (an extra round of division) differs drastically from the (spo11rec8(s)-40) defect, a dominant mutation in TDM appeared as a potential candidate to be the causal mutation in (spo11rec8(s)-40).

[0104] To test this hypothesis, a genomic clone containing the TDM gene (including promoter and terminator) that is able to complement tdm-3 mutant (n=8 transformants, 100% tetrads) was produced and mutated to recreate the mutation identified in the screen (TDM-P17L). When introduced in spo11-1rec8 plants, the TDM-P17L clone restored fertility of primary transformants (n=2/3. spo11-1 rec8: 0.1 seeds per fruit (n=197), spo11-1 rec8 TDM-P17L#15: 25 seeds per fruit (n=15), spo11-1 rec8 TDM-P17L#67: 48 seeds per fruit (n=10)) and led to the production of dyads (FIG. 4, table II). This demonstrates that the mutation in TDM is indeed the causal dominant mutation in spo11rec8(s)-40. Analysis of meiotic chromosome spreads in spo11-1rec8 TDM-P17L transformants showed a mitotic-like first division, with 10 univalents aligned at metaphase-I and sister chromatids segregated at anaphase I, and absence of second division (FIG. 5). Next, the ploidy level of spo11-1 rec8 TDM-P17L offspring was explored. Among selfed progeny, only tetraploids (4n) were found (Table III). When spo11-1rec8 TDM-P17L pollen was used to fertilise a wild-type plant, all the resulting progeny were triploid (Table III). When spo11-1 rec8 TDM-P17L ovules were fertilised with wild-type pollen grains, only triploid plants were found (Table III). This demonstrated that spo11-1 rec8 mutants transformed by TDM-P17L produce high levels of male and female (100%) mitosis-like derived spores, which result in functional diploid gametes.

[0105] When introduced in wild type plants and tdm-3 mutants, the TDM-P17L genomic clone modified the meiotic phenotype of both genotypes by the production of dyads (FIG. 4, Table II).

TABLE-US-00003 TABLE II Meiotic product of primary transformants Number of Transformed independent Male meiotic Construct genotype transformants products — wild type — Tetrads — tdm-3 — lobed monads — osd1 or tam — Dyads — spo11-1 rec8 — Polyads TDM tdm-3 8 8 tetrads TDM-P17L spo11-1 rec8 3 2 dyads 1 lobed monads wild type 20  14 dyads 2 dyads and tetrads 4 lobed monads tdm-3 2 2 dyads TDM-T16A wild type 2 2 dyads TDM- wild type 6 3 dyads Δ14_19 1 dyads and tetrads 2 tetrads tdm-3 4 4 dyads TDM-Y14A wild type 5 5 tetrads tdm-3 3 2 tetrads 1 lobed monads

[0106] TDM-P17L plants that produced dyads showed a wild type first division and an absence of meiosis II (FIG. 5) which caused the formation of 2n gametes, a phenotype reminiscent of the one from osd1 and cyca1;2/tam (d'Erfurth et al., PLoS Biol., 2009, 7, e1000124 and WO 2010/079432; d'Erfurth et al., PLoS Genet., 2010, 6, e1000989). Ploidy levels were measured among the offspring of TDM-P17L plants (Table III). Among selfed progeny, tetraploids and triploids were found. When TDM-P17L ovules were fertilised with wild-type pollen grains, diploid and triploid plants were isolated (Table III).

TABLE-US-00004 TABLE III Ploidy of spo11-1 rec8 TDM-P17L and TDM-P17L offsprings Crossed as Crossed as Trans- male with female with formant Selfed wild type wild type spo11-1 #15  100% 4n 100% 3n 100% 3n rec8 (n = 25) (n = 5)  (n = 24) TDM- #67  100% 4n 100% 3n 100% 3n P17L (n = 24) (n = 10) (n = 18) TDM- #1 100% 4n nd 43% 3n, 57% 2n P17L (n = 4)  (n = 7)  #2 60% 4n, 40% 3n nd  5% 3n, 95% 2n (n = 30) (n = 18) #3 73% 4n, 27% 3n nd nd (n = 11) #4 nd nd 27% 3n, 73% 2n (n = 15) #8 nd nd 22% 3n, 78% 2n (n = 18)

[0107] In summary, the tdm-p17L dominant mutation confers a similar meiotic defect than the recessive osd1 or tam mutations, leading to the premature exit from meiosis before the second division and consequently to the production of diploid male and female gametes.

[0108] TDM belongs to a small family of protein conserved in plants. For instance, the Arabidopsis genome contains five other genes showing significant sequence similarity with TDM (FIG. 2). These TDM-like genes are of unknown function. The analysis of the protein sequences showed that the causal mutation was in a small domain conserved only in the TDM protein subfamily that contains typically one or two genes per plant species (FIG. 1). The Pro 17 amino acid is absolutely conserved as well as the adjacent Thr16 amino acid (FIG. 1). This defines a minimum consensus phosphorylation site on the T16. To test this two other potential loss-of-phosphorylation versions of the genomic TDM gene were created at that site by substituting the phosphorylable amino acid by a non phosphorylable one (TDM-T16A), and by deleting the entire conserved domain (TDM-Δ14_19). Both TDM-T16A and TDM-Δ14_19 gave the dyad phenotype in a dominant manner when introduced into wild type plants, recapitulating the effect of TDM-P17L (Table II; FIG. 4). Further, when introduced into tdm-3 mutants, TDM-Δ14_19 also showed the dyad phenotype (Table II). However mutation of the TDM tyrosine 14 (TDM-Y14A), a slightly less conserved amino acid of the domain, was unable to confer the dyad phenotype when introduced in wild type and was able to complement the tdm-3 mutation (Table II). In summary, expression of TDM-P17L, -T16A and -Δ14-19 mutations are equally able to dominantly confer premature meiosis exit. As TP is a potential phosphorylation sites, this results suggest that TDM may be regulated by phosphorylation to ensure the meiosis I to meiosis II transition.