GENERATION OF HAPLOID PLANTS

Abstract

The present invention relates to non-transgenic and transgenic plants, preferably crop plants, comprising a mutation causing an alteration of the amino acid sequence in the CATD domain of the centromere histone H3 (CENH3), preferably within the loop1 or the α2-helix of the CATD domain, which have the biological activity of a haploid inducer. Further, the present invention provides methods of generating the plants of the present invention and haploid and double haploid plants obtainable by crossing the plants of the present invention with wildtype plants as well as methods of facilitating cytoplasm exchange.

Claims

1. A plant having biological activity of a haploid inducer and comprising a nucleotide sequence encoding a centromer histone H3 (CENH3) protein comprising a CATD domain, wherein the nucleotide sequence comprises a mutation causing in the CATD domain an alteration of the amino acid sequence of the CENH3 protein, wherein said alteration confers the biological activity of a haploid inducer.

2. The plant according to claim 1, wherein the mutation causes an alternation in a loop1 corresponding to nucleotides from position 340 to position 378 set forth in SEQ ID No. 37 of the CENH3 protein derived from Arabidopsis thaliana set forth in SEQ ID No. 38 and being positioned within the CATD domain, wherein the alteration confers the biological activity of a haploid inducer, or the mutation causes an alternation in an α2-helix corresponding to nucleotides from position 379 to position 465 set forth in SEQ ID No. 37 of the CENH3 protein derived from Arabidopsis thaliana set forth in SEQ ID No. 38 and being positioned within the CATD domain, wherein the alteration confers the biological activity of a haploid inducer.

3. The plant according to claim 1, wherein the mutation causes one or more of the following substitutions or deletions of a specified amino acid as defined: TABLE-US-00012 Amino acid(s) Position within the loop1 1 T, S or A 2 H, Q, N, A, Y, F, G, D or E 3 M, Q, I, F, Y, A, E, N, R, L, H or G 4 L, F, V, I or Y 5 A, T, S, C or M 6 P, N, D, R, A, T, F, R, H, S or K 7 X 8 Q, Y, D, K, R, E, G, S, P, H, N or A 9 I, V or P 10 N, G, T, E, or S 11 R or P 12 W or Y 13 T, Q or S Position within the α2-helix 1 A, P, V or L 2 E, D, Q, H or L 3 A 4 L or V 5 V, L, M, I, R, Y or T 6 S or A 7 I or L 8 Q 9 E 10 A or S 11 A or T 12 E 13 D, N, F, I or Y 14 Y, F or H 15 L, I or V 16 V or I 17 G, R, E, H, N, T, E, D or Q 18 L, M or I 19 F, M or L 20 S, E, D or G 21 D, M, V, N, E, A, R or K 22 S, G, A or T 23 M, W, N or H 24 L or H 25 C or L 26 A or T 27 L or I 28 H 29 A or S

4. The plant according to claim 1, wherein the mutation causes a substitution or deletion of a specified amino acid of SEQ ID No. 1 or 49.

5. The plant according to claim 1, wherein crossing between the plant and a wildtype plant or a plant expressing wildtype CENH3 protein yields at least 0.1% haploid progeny.

6. The plant according to claim 1, wherein the nucleotide sequence comprising the mutation is an endogenous gene or a transgene.

7. The plant according to claim 6, wherein the nucleotide sequence comprising the mutation is a transgene and at least one endogenous gene encoding a CENH3 protein is inactivated or knocked out.

8. The plant according to claim 1, wherein the amino acid asparagine at position 2 of SEQ ID No. 49 is substituted, or the amino acid alanine at position 95 of SEQ ID No. 55 is substituted, or the amino acid proline at position 6 of SEQ ID No. 49 is substituted, or the amino acid proline at position 121 of SEQ ID No. 52 is substituted, or the amino acid tryptophan at position 12 of SEQ ID No. 49 is substituted, or the amino acid tryptophan at position 127 of SEQ ID No. 52 is substituted, or the amino acid alanine at position 1 of SEQ ID No. 1 is substituted, or the amino acid alanine at position 107 of SEQ ID No. 58 is substituted, or the amino acid leucine at position 4 of SEQ ID No. 1 is substituted, or the amino acid leucine at position 132 of SEQ ID No. 52 or position 92 of SEQ ID No. 34 or position 130 of SEQ ID No. 38 or position 106 of SEQ ID No. 61 is substituted, or the amino acid leucine at position 7 of SEQ ID No. 1 is substituted, or the amino acid leucine at position 109 of SEQ ID No. 61 is substituted, or the amino acid glutamine at position 8 of SEQ ID No. 1 is substituted, or the amino acid glutamine at position 114 of SEQ ID No. 58 or position 110 of SEQ ID No. 61 is substituted, or the amino acid alanine at position 10 of SEQ ID No. 1 is substituted, or the amino acid alanine at position 138 of SEQ ID No. 52 is substituted, or the amino acid cysteine at position 25 of SEQ ID No. 1 is substituted, or the amino acid cysteine at position 153 of SEQ ID No. 52 is substituted, or the amino acid alanine at position 26 of SEQ ID No. 1 is substituted, or the amino acid alanine at position 154 of SEQ ID No. 52 is substituted.

9. A part of the plant according to claim 1 comprising a mutation causing in the CATD domain an alteration of the amino acid sequence of the CENH3 protein and said alteration confers the biological activity of a haploid inducer.

10. A haploid plant obtainable by crossing a plant according to claim 1 with a plant expressing wildtype CENH3 protein.

11. A double haploid plant obtainable by converting the haploid plant according to claim 10 into a double haploid plant.

12. A method of generating a haploid plant, comprising the steps of: a) crossing a plant according to claim 1 to a plant expressing wildtype CENH3 protein, and b) identifying the haploid progeny plant generated from the crossing step.

13. A method of generating a double haploid plant, comprising the steps of: a) crossing a plant according to claim 1 to a plant expressing wildtype CENH3 protein, b) identifying a haploid progeny plant generated from the crossing step, and c) converting the haploid progeny plant into a double haploid plant.

14. A method of facilitating a cytoplasm exchange, comprising the steps of: x) crossing a plant according to claims 1 to 8 claim 1 as ovule parent with a plant expressing wildtype CENH3 protein as pollen parent, and y) obtaining a haploid progeny plant comprising the chromosomes of the pollen parent and the cytoplasm of ovule parent.

15. A method of generating a plant according to claim 1, comprising the steps of: i) subjecting seeds of a plant to a sufficient amount of the mutagen ethylmethane sulfonate to obtain M1 plants, ii) allowing sufficient production of fertile M2 plants, iii) isolating genomic DNA of M2 plants and iv) selecting individuals possessing an alteration of the amino acid sequence in the CATD domain of CENH3.

16. A nucleotide sequence encoding at least the CATD domain of a CENH3 protein comprising a mutation causing in the CATD domain an alteration of the amino acid sequence of the CENH3 protein.

17. A vector comprising the nucleotide sequence of claim 16.

18. A plant cell or a host cell comprising the nucleotide sequence of claim 16, or a vector comprising said nucleotide sequence, as a transgene.

19. A method of generating a transgenic plant comprising the steps of: yy) transforming a plant cell with the nucleotide sequence of claim 16, or a vector comprising said nucleotide sequence, and zz) regenerating a plant having the biological activity of a haploid inducer from the plant cell.

20. The part of the plant according to claim 9, wherein the part is a shoot vegetative organ, root, flower or floral organ, seed, fruit, ovule, embryo, plant tissue or cell.

Description

[0270] FIG. 1 shows schematically a mechanistic model relating to methods of the present invention.

[0271] FIG. 2 shows an alignment of the amino acid sequences of Arabidopsis thaliana (first row), Beta vulgaris (second row), Brassica napus (third row), Zea mays (forth row), Sorghum bicolor (fifth row) as well as a diagram showing the level of conservation over these five plant species.

EXAMPLES

Example 1: Mutagenesis of Barley a and βCENH3 by Targeting Local Lesions IN Genomes (TILLING)

[0272] To identify whether a single point mutation of endogenous CENH3 could result in a haploid inducer an EMS-induced TILLING population of diploid barley (Hordeum vulgare) (Gottwald et al., 2 (2009), BMC Res Notes, 258), a species encoding two functional variants of CENH3 (α and βCENH3) (Sanei et al., 108 (2011), Proc Natl Acad Sci USA, E498-505) was screened. Assuming the complementation of either CENH3 variant a functional mutation of αCENH3 or βCENH3 would still allow the generation of offspring.

[0273] To do this, a TILLING population of 10,279 EMS treated diploid barley (Hordeum vulgare) plants of cv. Barke to identify mutant alleles of a and βCENH3 was screened. Four and three primer combinations

TABLE-US-00004 Hv_aCENH3_EX1 + 2 + 3_F: (SEQ ID No. 2) AGGCAGGGTCTCAATTCCTT, Hv_aCENH3_EX1 + 2 + 3_R: (SEQ ID No. 3) GTCCCATCATCCATCGTCTT, Hv_aCENH3_EX4 + 5_F: (SEQ ID No. 4) CCCACTTCCTTGTTGTGGAC, Hv_aCENH3_EX4 + 5_R: (SEQ ID No. 5) GGCGATAAATGTATCTTGCATTC, Hv_aCENH3_EX6_F: (SEQ ID No. 6) TGGTAGCAACCAGAGCTACG, Hv_aCENH3_EX6_R: (SEQ ID No. 7) ACTGGCATGTTTCCTTCTGC, Hv_aCENH3_EX7_F: (SEQ ID No. 8) CGGACGGAGGGAGTATTTCT, Hv_aCENH3_EX7_R: (SEQ ID No. 9) GGACATGCCCAAAGAAAGTG, Hv_bCENH3_EX1 + 2_F: (SEQ ID No. 10) GCCAGCGAGTACTCCTACAAG, Hv_bCENH3_EX1_R: (SEQ ID No. 11) TTGAGTTACCAGCCACCACTC, Hv_bCENH3_EX3_F: (SEQ ID No. 12) GTCATGCACTGTGTCTTGCA, Hv_bCENH3_EX3_R: (SEQ ID No. 13) TGCTAAGATCGGATAACTGTGG, Hv_bCENH3_EX4_F: (SEQ ID No. 14) TGCTCCTGAACAAACTGAACC, Hv_bCENH3_EX4_R: (SEQ ID No. 15) GTGGCCGTCAGTACAATCG

[0274] were used to amplify all exons of the α and βCENH3 variants and parts of the corresponding introns, respectively, by using PCR with a heteroduplex step as described earlier (Gottwald et al., (2009), BMC Res Notes, 258). PCR products were digested with dsDNA Cleavage Kit and analysed using Mutation Discovery Kit and Gel-dsDNA reagent kit on the AdvanCETM FS96 system according to manufacturer's guidelines (Advanced Analytical, IA, USA).

[0275] RNA extraction, PCR and quantitative real time RT-PCR

[0276] Total RNA was isolated from roots, leaves using the Trizol method (Chomczynski and Sacchi, 162 (1987), Anal Biochem, 156-159) from anthers (microscopically staged between meiosis and development of mature pollen), carpel, endosperm and embryo by Picopure RNA isolation kit (Arcturus) according to manufacturer. The absence of genomic DNA contamination was confirmed by PCR using GAPDH primers (see Table 3). 10 μI of PCR mixture contained 1 μI of cDNA template, 5 μI of 2×Power SYBR

[0277] Green PCR Master Mix (Applied Biosystems), 0.33 mM of the forward and reverse primers for each gene (see Table 3). Reactions were run in an Applied Biosystems 7900HT Fast Real-Time PCR System. The PCR was performed using the following conditions: 95° C. for 10 min, followed by 40 cycles at 95° C. for 15 s, at the annealing temperature of 60° C. for 60 s. Three technical replicates were performed for each cDNA sample. Fast Real-Time PCR System and data were analyzed with SDS software v 2.2.2. Transcript levels of each gene were normalized to GAPDH by the following formula: R=2.sup.̂(-(CtGOI-CtH))*100, where R=relative changes, GOI=gene of interest, and H=housekeeping (GAPDH). The specificity and efficiency of both primers were determined by qRT-PCR using a dilution series of plasmids of cloned full length barley a and βCENH3 genes. A similar Ct value (the PCR cycle at which the fluorescent signal of reporter dye exceeds background level) for equal amount of plasmid indicates that both primers can amplify specific transcripts with the same efficiency.

TABLE-US-00005 TABLE 3 Primer name Sequence (5′ to 3′) GAPDH-F CAATGATAGCTGCACCACCAACTG (SEQ ID No. 21) GAPDH-R CTAGCTGCCCTTCCACCTCTCCA (SEQ ID No. 22) Hvα-F AGTCGGTCAATTTTCTCATCCC (SEQ ID No. 23) Hvα-R CTCTGTAGCCTCTTGAACTGC (SEQ ID No. 24) Hvβ-F GCCATTGTCGAACAAGAAGG (SEQ ID No. 25) Hvβ-R TAACACGGTGCGAATGAATG (SEQ ID No. 26) CH3A + L130_F_for phos-GACAGCTGAAGCATTTGTTGCTCTTC (SEQ ID No. 27) CENH3L130_I_for phos_GACAGCTGAAGCTATTGTTGCTCTTC (SEQ ID No. 28) CENH3L130_F + phos-CAACGATTGATTTGGGGAGGG I_rev (SEQ ID No. 29) cenh3-1_mut_for GGTGCGATTTCTCCAGCAGTAAAAATC (SEQ ID No. 30) cenh3-1_mut_rev CTGAGAAGATGAAGCACCGGCGATAT (SEQ ID No. 31) cenh3-1_mut2429r AACTTTTGCCATCCTCGTTTCTGTT (SEQ ID No. 32)

[0278] Only missense point mutations were identified for both barley CENH3 variants.

[0279] The non-functionality of mutated CENH3s of homozygous M2 mutants was tested by immunostaining of the centromeres with CENH3 variant-specific antibodies. Mitotic and meiotic chromosomes of H. vulgare wildtype and homozygous TILLING line 4528 (plant according to the present invention) have been subjected to immunostaining with antibodies specific for αCENH3 and βCENH3. αCENH3 and βCENH3 signals at centromeres were revealed in all mutants, while only the homozygous TILLING line 4528 which contains a leucine to phenylalanine substitution at amino acid 92 (SEQ ID No. 36), i.e. amino acid position 4 of the consensus sequence SEQ ID No. 1, displayed no centromeric βCENH3 signals in mitotic, meiotic and interphase cells. The leucine to phenylalanine substitution at amino acid 92 of SEQ ID No. 36 of the H. vulgare β-CENH3 sequence corresponds to a single nucleotide substitution from C to

[0280] T at position 274 of the H. vulgare β-CENH3 cDNA sequence (SEQ ID No. 35).

[0281] Only weak dispersed βCENH3 signals outside centromeres were found in this line. Transcription levels of a and βCENH3 in wildtype (cv Barke) (SEQ ID no. 33 and 34) and TILLING line 4528 with mutated βCENH3 have been measured. The relative expression level of a and βCENH3 was measured in different tissues using specific primers (Table 3). cDNA was prepared from total RNA and gene expression levels were normalized to the expression level of glyceryl phosphate dehydrogenase (GRPTA). Obviously, the centromeric loading of the mutated βCENH3 variant seems to be impaired, while no different transcription level between wild type and mutated βCENH3 was found. Hence, centromeres exclusively composed of αCENH3 are sufficient for mitotic centromere function in barley as no obvious chromosome segregation defects, such as anaphase bridges, as well as changes of ploidy or cycle vales was found. In addition, no obvious changes of the plant habitus were observed in mutant plants. In particular, no significant differences in phenotype, ploidy levels, cycle values and growth phenotype between homozygous plants of TILLING line 4528 and barley wildtype could be detected.

[0282] The issue was addressed whether missing βCENH3 is compensated by additional αCENH3 to maintain the centromere function in the mutant. Therefore, αCENH3 immunostaining signals of wildtype (126 centromeres measured) and of line 4528 (56 centromeres measured) were comparatively quantified by pixel intensity measurements. An increase of 19.8% αCENH3 in the mutant indicates that the missing βCENH3 is partly compensated by additionally incorporated αCENH3. The βCENH3 mutation is located in an evolutionarily highly conserved targeting domain (CATD) defined by parts of α1helix, loop 1 and α2helix of the histone fold. This domain is required for centromere loading of CENH3 by chaperons.

[0283] Indirect Immunostaining

[0284] Indirect immunostaining of nuclei and chromosomes was carried out as described previously (Sanei et al., 108 (2011), Proc Natl Acad Sci USA, E498-505). CENH3 of barley was detected with guenia pig anti-αCENH3-specific and rabbit anti-βCENH3-specific antibodies. A rabbit HTR12-specific antibody (abcam, ab72001) was used for the detection of A. thaliana CENH3 (AtCENH3). Epifluorescence signals were recorded with a cooled CCD-camera (ORCA-ER, Hamamatsu). Imaging was performed by using an Olympus BX61 microscope and an ORCA-ER CCD camera (Hamamatsu). To analyse the structures of immunosignals and chromatin at an optical resolution of ˜100 nm (super-resolution) Structured Illumination Microscopy (SIM) was applied using a C-Apo 63×/1.2 W Korr objective of an Elyra microscope system and the software ZEN (Zeiss, Germany). Images were captured separately for each fluorochrome using appropriate excitation and emission filters. The images were merged using the Adobe Photoshop 6.0 software. To determine the amount of a and βCENH3 in nuclei fluorescence intensities were measured using the TINA 2.0 software in maximum intensity projections generated from stacks of optical SIM sections through the specimens by the ZEN software. An intensity threshold was set to computationally subtract the background pixels from the image. The corrected sum of grey values of all signals within the nucleus was used to determine the CENH3 content. 3D-rendering based on SIM image stacks was done using the ZEN software.

Example 2: Arabidopsis thaliana

[0285] To proof whether the mutation in the CATD domain caused the observed impaired centromere loading, eYFP was N-terminally fused to the coding sequence (CDS) of A. thaliana CENH3 (SEQ ID No. 37, protein: SEQ ID No. 38) with an L/I (leucine/isoleucine) (CDS: SEQ ID No. 39, protein SEQ ID No. 40) or L/F (leucine/phenylalanine) (CDS: SEQ ID No. 41, protein SEQ ID No. 42) exchange of the corresponding positions (L130I or L130F, corresponding to amino acid position 92 in βCENH3 of barley, i.e. amino acid position 4 of the consensus sequence SEQ ID No. 1) in A. thaliana CENH3. The leucine to isoleucin substitution at position 130 of A. thaliana corresponds to a single nucleotide substitution from C to A at position 388 of SEQ ID no. 37.

[0286] The amino acid substitution from leucine to phenylalanine at position 130 is caused by two nucleotide substitutions, namely TC to AT at positions 387 and 388 of SEQ ID No. 37.

[0287] Double labelling of transgenic A. thaliana with corresponding anti-wild type CENH3 and anti-GFP revealed a significantly reduced centromere targeting of the mutated CENH3s.

[0288] Next, to test for haploid inducer ability A. thaliana genomic CENH3 constructs with a L130I or L130F exchange were used to transform heterozygous CENH3 knock-out A. thaliana plants (Ravi and Chan, Nature, 464 (2010), 615-618). Genotyping identified homozygous CENH3 null mutants which were complemented with either genomic CENH3 wild type, L130I or L130F constructs. As viable diploid plants containing either of the constructs were obtained, it is likely that this mutation does not impair the centromere function in homozygous A.thaliana plants. When CENH3 null mutants expressing a point mutated L130F CENH3 protein were crossed to wild type, chromosomes from the mutant are eliminated, producing haploid progeny. Flow cytometric analysis revealed that 10.7% of the F1 plants were haploid.

[0289] Cloning and Generation of CENH3 Transgenes

[0290] To generate CENH3 genomic fragments carrying mutations, resulting in phenylalanine 130 (F130) and isoleucine 130 (1130) instead of wild-type leucine 130 (L130), a genomic CENH3 fragment in pCAMBIA1300 vector used to complement cenh3-1/cenh3-1 (cenh3 null mutant) (Ravi and Chan, Nature, 464 (2010), 615-618; Ravi et al., Genetics, 186 (2010), 461-471) was subcloned via the unique HindIII and BamHI sites into pBlueScript II KS (Strategene, www.stratagene.com). Mutations of CENH3, L130I or L130F, were generated in pBlueScript II KS using a Phusion® Site-Directed Mutagenesis Kit (Finnzymes, www.finnzymes.com) according to manufacturer's instructions with minor changes as described. Following 5′-phosphorylated primers were used for mutagenesis: CH3A+L130_F_for, CENH3L130_I_for and CENH3L130_F+I_rev. Mutated CENH3 genomic fragments were subcloned via the unique HindIII and BamHI sites into the initial pCAMBIA1300 containing a hygromycin resistance marker. All constructs were verified by sequencing. For primers see Table 3, above.

[0291] To generate p35S::eYFP-CENH3 fusion constructs containing mutations within the CENH3 CDS, resulting in L130I or L130F, a plasmid (p35S-BM; Schmidt, www.dna-cloning.com) containing a p35S::eYFP-CENH3 expression (Lermontova, 18 (2006), Plant Cell, 615-618) was used as template for the Phusion® Site-Directed Mutagenesis Kit (Finnzymes, www.finnzymes.com). Primers and strategies to introduce desired mutations were the same as above. Resulting expression cassettes (35Spro, eYFP-(mutated) CENH3 and NOS terminator) were subcloned via unique Sfi1 restriction sites into the pLH7000 vector containing a phosphinotricine resistance marker (Schmidt, www.dna-cloning.com) and verified by sequencing.

[0292] Plant Transformation, Culture Conditions and Cross-Pollination

[0293] A. thaliana wild-type (SEQ ID No. 37 and 38) and cenh3-1/CENH3 heterozygotes plants (both accession Columbia-0) were transformed by the floral dip method (Clough and Bent, 16 (1998), Plant J, 735-743). Transgenic progenies were selected on Murashige and Skoog solid medium containing the corresponding antibiotic. Plants were germinated on Petri dishes under long-day conditions (20° C. 1611 light/18° C. 8 h dark), grown for 4 weeks under short-day conditions (20° C. 8 h light/18° C. 16 h dark) and then shifted to long-day conditions again. For crossing, the closed buds of mutant cenh3 A. thaliana were emasculated by removing the immature anthers with the help of forceps. The stigmas of emasculated buds were fertilized with the yellowish pollen from mature anthers of freshly opened wild type A. thaliana flowers.

[0294] DNA Extraction and Genotyping of A. thaliana

[0295] Genomic DNA preparations and PCR-based genotyping were performed using standard methods. DNA was extracted according to Edwards et al. (1991), Nucleic Acids Res 19, 1349.

[0296] Plants were genotyped for cenh3-1 in a dCAPS genotyping reaction. The dCAPS primers, cenh3-1_mut_for and cenh3-1_mut_rev, were used to amplify CENH3. Amplicons were digested with EcoRV and resolved on a gel. cenh3-1 mutant allele is not cut (215 bp) while the WT CENH3 allele is cut (191 and 24 bp). For primers see Table 3. To genotype the endogenous CENH3 locus for cenh3-1 in the offspring of cenh3-1/CENH3 plants transformed with the CENH3 genomic locus (untagged CENH3 transgene with L130, L130I or L130F), an initial PCR reaction was performed with one primer outside of the transgene CENH3 locus, allowing specific amplification of the endogenous and not the transgenic CENH3 locus. Primers used were cenh3-1mut_for and cenh3-1_mut2320r/cenh3-1_mut2429r. Amplicons were purified and used as template for a second dCAPS PCR genotyping reaction as described above for cenh3-1 plants. For Primers see Table 3. Presence of transgene was verified by PCR.

[0297] Flow Cytometric Analysis of Plants and Seeds

[0298] For flow cytometric ploidy analyses of plants equal amounts of leaf material of 5 to 10 individuals were chopped simultaneously in nuclei isolation buffer (Galbraith et al. (1983), Science 220, 1049-1051) supplemented with DNas-free RNase (50 μg/ml) and propidium iodide (50 μg/ml) with a sharp razor blade. The nuclei suspensions were filtered through 35 μm cell strainer cap into 5 ml polystyrene tubes (BD Biosciences) and measured on a FACStar.sup.PLUS cell sorter (BD Biosciences) equipped with an argon ion laser INNOVA 90C (Coherent). Approximately 10,000 nuclei were measured and analysed using the software CELL Quest ver. 3.3 (BD Biosciences). The resulting histograms were compared to a reference histogram representing a diploid wild type plant. In cases where an additional peak at the haploid position was detected, the plants were individually measured again to identify the haploid individuals.

[0299] Nuclei isolation of seeds was performed as described above using the nuclei isolation buffer. MA VI (100 mM Tris-HCl, 5.3 mM MgCl.sub.2, 86 mM NaCl, 30.6 mM sodium citrate, 1.45 mM Triton X-100, pH 7.0; supplemented with 50 DNas-free RNase and 50 μg/ml propidium iodide). Nuclei suspensions were measured on a FACSAria cell sorter (BD Biosciences) and analysed using the FACS Diva software ver.

[0300] 6.1.3 (BD Biosciences). Similarly as above, first 10 to 20 seeds were pooled to identify lines with haploid embryos and in a second step single seeds were co-chopped together with leaf material from Raphanus sativus (Genebank Gatersleben, accession number: RA 34) as internal reference to confirm the occurrence of haploid seeds.

Example 3: Beta vulgaris

[0301] Further, the functional significance of the identified mutation was assayed also in the sugar beet Beta vulgaris. RFP reporter constructs containing the cDNA of Beta vulgaris CENH3 (SEQ ID No. 43, protein SEQ ID No. 44) with an L106F (SEQ ID No. 45, protein SEQ ID No. 46) or L1061 (SEQ ID No. 47, protein SEQ ID No. 48) exchange (corresponding to amino acid position 92 of barley, amino acid position 4 of the consensus sequence SEQ ID No. 1) were generated and used for stable transformation of sugar beet and a reduced centromere targeting of the mutated CENH3s was detected.

[0302] The amino acid substitution from leucine to phenylalanine at position 106 is caused by two nucleotide substitutions, namely C to Tat position 316 and G to Tat position 318 of SEQ ID no. 43.

[0303] The amino acid substitution from leucine to isoleucine at position 106 is caused by two nucleotide substitutions, namely C to A at position 316 and G to T at position 318 of SEQ ID no. 43.

[0304] Plant Transformation and Culture Conditions

[0305] Beta vulgaris wild-type leaves of 6-8 week old plants (grown under semi-controlled greenhouse conditions) were transiently transformed by particle bombardment (300 μg gold coated with 0.5 μg plasmid DNA). Bombarded leaves were incubated for 48-72 h (25° C. 16 h light (350 μmolm.sup.−2s.sup.−1)/8 h dark) before microscopic analysis. Stable transformation of B. vulgaris callus was performed as described in Lindsey & Gallois, 1990 (Journal of experimental botany, 41(5), 529-536) (selection via kanamycin). After approx. 2 month (24° C. 16 h light (55 μmolm.sup.−2s.sup.−1)/8 h dark) callus cells were microscopically analysed.

[0306] Cloning and Generation of CENH3 Transgenes

[0307] To generate the 35S::RFP-CENH3 fusion construct, CENH3 was amplified from sugar beet cDNA with the following primers:

TABLE-US-00006 (SEQ ID No. 16) BvCENH3-cds1: GGATCCATGAGAGTTAAACACACTGC, (SEQ ID No. 17) BvCENH3-cds2: GGATCCTGTTCAGTTACCATCCCCTC,

[0308] cloned into a vector containing a 35Spro, RFP and 35S-terminator expression cassette For constructs containing mutations within the CENH3 coding sequence, resulting in F106 and 1106 instead of L106, the above mentioned plasmid containing the 35S::RFP-CENH3 expression cassette was used as template for primer based mutagenesis. The PstI site close to the position of the desired mutation was used to split CENH3 into two parts. Via mutations in the Primers the desired mutations were integrated:

TABLE-US-00007 (SEQ ID No. 18) BvCENH3_mut_Fw: ATGGATCCATGAGAGTTAAACACACTGC, (SEQ ID No. 19) BvCENH3_L -> F_Rv: CTCTGCAGCCTCTTGAAGGGCCATAAAAGC, (SEQ ID No. 20) BvCENH3_L -> I_Rv: CTCTGCAGCCTCTTGAAGGGCCATAATAGC.

[0309] Resulting expression cassettes (35Spro, RFP-(mutated) CENH3 and 35S-terminator) were verified by sequencing.

[0310] Analysis of CENH3 binding in B. vulgaris

[0311] To analyse the binding of CENH3 and the mutated CENH3 either leaf or callus material was analysed using a C-Apo 63×/1.2 W Korr objective of an Axio Imager M2 microscope system and the software ZEN (Zeiss, Germany).

Example 4: Identification and Testing of Other CENH3 Mutants

[0312] For the identification of other single point mutations within the endogenous gene of CENH3 which cause an amino acid substitution or a deletion of one or more amino acids of the sequence of the translated. CENH3. Even if Ravi und Chan 2010 highlighted only the particular importance of the N terminal domain, above described studies on mutants in another part of CENH3 like α2-helix (Example 1 to 3) gave indications that the modification of CATD domain of CENH3 can result in a destabilization of the CENH3 binding capacities to DNA. Therefore the focus was on identification of other mutations within the CATD, in particular in the loop1 and α2-helix. Additionally, it should be demonstrated that due to the high level of conservation of the CATD domain between the species, an identified mutation has the potential to confer the biological activity of a haploid inducer to different plant species.

[0313] For that TILLING populations having high mutation rates have been generated for two other monocot plants namely for corn (Zea mays) and sorghum (Sorghum bicolor), and for two dicot plants namely for rape seed (Brassica napus) and sugar beet (Beta vulgaris). In order to screen for mutations in the endogenous CENH3 gene which result in at least one amino acid substitution or a deletion of at least one amino acid in the CATD domain of the translated CENH3 protein, amplicons covering all exons of the CENH3 genes as well as parts of the corresponding introns, respectively, have been developed as exemplary described above for barley (Example 1) and between 1000 and 10000 individuals per plant species have been analyzed by means of Sanger's sequencing method. In addition, M2 sugar beet plants have been tested for mutations using specific PCR.

[0314] Furthermore, the affect of the identified mutation within the CENH3 gene on the primary and secondary structure of the encoded protein have been evaluated using inter alia the software Prof (Rost, B. and Sander, C. (1994a). Combining evolutionary information and neural networks to predict protein secondary structure. Proteins, 19(1), 55-72. Rost, B. and Sander, C. (1994b). Conservation and prediction of solvent accessibility in protein families. Proteins, 20(3), 216-26. Rost, B., Casadio, R., Fariselli, P., and Sander, C. (1995). Transmembrane helices predicted at 95% accuracy. Protein Sci, 4(3), 521-33.).

[0315] The non-functionality of mutated CENH3s of homozygous mutants has been tested for example by immunostaining of centromeres with CENH3 specific antibodies as described above (Examples 1 and 2). Identified TILLING lines showed significantly reduced or impaired centromeric loading by the mutated CENH3. Plants having a genome which was heterozygous for such mutation(s) were viable and no obvious changes of the plant habitus were observed, i.e. phenotype, poidy levels, cycle values and growth were comparable to corresponding wildtype plants with regard to statistic accuracy.

[0316] The biological activity of a haploid inducer in the identified mutants has been evaluated by crossing the mutant plants with a tester plant of the same species: The tester plant carries the wildtype form of CENH3. The maternal as well as the paternal performance of haploid induction have been tested. For that, the mutant plants have been used either as ovule parent or as pollen parent in the cross with the tester plant. Putative haploid progeny from this cross can be determined quickly if the used tester lines carry a recessive non-CENH3 mutation. So, the haploid plants show the recessive phenotype. For example, in corn the manifestation of the mutation glossy (Mutants of maize, Neuffer, M G et al. 1997. Cold Spring Harbor Laboratory, New York) can be used. Haploid progeny from these crosses can be determined quickly if the used tester lines carry a recessive non-CENH3 mutation. So, the haploid plants show the recessive phenotype. For example, in corn the manifestation of the mutation glossy (Mutants of maize, Neuffer, M G et al. 1997. Cold Spring Harbor Laboratory, New York) can be used.

[0317] Additionally, cytogenetic analyses of mitose and meiose with the inductors indicates also for suitability of mutants as haploid inducers and homozygosity has been determined by use of molecular markers, polymorph for tester and potential inductor. Haploidy as such could be tested cytogenetically.

[0318] The following Tables shows the missense and deletion mutations which confer the biological activity of a haploid inducer to investigated plant species:

TABLE-US-00008 TABLE 4 mutation of the CENH3 derived from Brassica napus (aa: amino acid; nd: not determined, y: yes, n: no). Amino acid substitution is given as X#Y, i.e. amino acid X (one letter code) is substituted for amino acid Y at position #. mutation identifier (SEQ ID Nos of genomic DNA; chance in cDNA; amino codon codon secondary acid) wildtype mutant mutation structure BN_CenH3_26 cct tct P121S n (62; 63; 64) BN_CenH3_27 tgg tga W127stop n (65; 66; 67) BN_CenH3_28 ctt ttt L132F y (68; 69; 70) BN_CenH3_29 gcg acg A138T n (71; 72; 73) BN_CenH3_30 tgc tac C153Y y (74; 75; 76) BN_CenH3_31 gct gtt A154V y (77; 78; 79)

TABLE-US-00009 TABLE 5 mutation of the CENH3 derived from Zea mays (aa: amino acid; nd: not determined, y: yes, n: no). Amino acid substitution is given as X#Y, i.e. amino acid X (one letter code) is substituted for amino acid Y at position #. mutation identifier (SEQ ID Nos of genomic DNA; chance in cDNA; amino codon codon secondary acid) wildtype mutant mutation structure ZM_CenH3_07 gca aca A107T nd (80; 81; 82) ZM_CenH3_08 caa taa Q114stop nd (83; 84; 85)

TABLE-US-00010 TABLE 6 mutation of the CENH3 derived from Sorghum bicolor (aa: amino acid; nd: not determined, y: yes, n: no). Amino acid substitution is given as X#Y, i.e. amino acid X (one letter code) is substituted for amino acid Y at position #. mutation identifier (SEQ ID Nos of genomic DNA; chance in cDNA; amino codon codon secondary acid) wildtype mutant mutation structure SB_CenH3_04 gca gta A95V nd (86; 87; 88)

TABLE-US-00011 TABLE 7 mutation of the CENH3 derived from Beta vulgaris (nd: not determined, y: yes, n: no). Amino acid substitution is given as X#Y, i.e. amino acid X (one letter code) is substituted for amino acid Y at position #. mutation identifier (SEQ ID Nos of genomic DNA; chance in cDNA; amino codon codon secondary acid) wildtype mutant mutation structure Bv_CENH3_04 ctg cag L106Q nd (89; 90; 91) Bv_CENH3_05 ctt cct L109P nd (92; 93; 94) Bv_CENH3_06 caa cta Q110L nd (95; 96; 97)

[0319] The crossings with the tester plants the TILLING plants with mutated endogenous CENH3 yielded at least 0.5% haploid progeny. For example, in Brassica napus the mutations C153Y and A154V showed induction rates between 0.5% and 1%. In a few cases induction rates of 2% or more could be reached. Frequently the induction rate was higher if the tester was used as male parent in the cross.

[0320] Moreover, the result of crossing demonstrated that identified mutations could be functional also in other plant species. Thus, mutation at amino acid position 4 of the consensus sequence SEQ ID No. 1, whereby leucine has been substituted for phenylalanine created induction activity in Hordeum vulgare (L92F) as shown in Examples 1 to 3 but also in Brassica napus (L132F). Therefore mutations could be introduced into other plant species by techniques like TILLING, Mutagenesis or genome editing (e.g. CRISPR/Cas, TALENs, Zinc Finger nucleases etc.). Moreover, the biological activity and efficiency of a haploid inducer could be further improved by combining different identified mutations in one plant and/or modifying the genetic background of the haploid inducer. The combination of different mutations could be achieved efficiently by genome editing, or the mutant haploid inducer is mutagenized for a second time.