Methods for Inducing Apomixis in Plants

Abstract

The present invention relates to methods for inducing apomixis in a plant, methods for the production of apomictic plants and the plants and plant seeds obtained thereby.

Claims

1-15. (canceled)

16. A method for the production of a transgenic apomictic plant, comprising the following steps: m) providing a plant cell of a sexually propagating plant, which comprises a nucleotide sequence coding for a protein with DEDDh exonuclease activity under control of a cis-acting regulatory element, n) modifying the cis-acting regulatory element controlling the nucleotide sequence coding for a protein with DEDDh exonuclease activity by creating at least one ATHB-5 and at least one SORLIP2AT transcription factor binding site therein, and o) regenerating the plant cell obtained in step n), which contains the newly created at least one regulatory nucleotide core sequence into a transgenic plant exhibiting apomixis.

17. The method according to claim 16, wherein the plant cell provided in step m) is transformed with a plant vector containing an exogenous nucleotide sequence element comprising a nucleotide sequence encoding a trans-acting apomixis effector.

18. The method according to claim 17, wherein the trans-acting apomixis effector is an over expressed trans acting apomixis effector.

19. The method according to claim 18, wherein the trans-acting apomixis effector is a transcription factor, in particular ATHB-5, SORLIP2AT, or POLASIG.

20. The method according to claim 16, wherein the nucleotide sequence coding for a protein with the activity of a DEDDh exonuclease comprises a nucleotide sequence selected from the group consisting of a1) the polynucleotide defined in any one of SEQ ID NO: 22 to 54, or a fully complementary strand thereof, b1) a polynucleotide encoding a polypeptide with the amino acid sequence defined in any one of SEQ ID NO: 1 to 21 or a fully complementary strand thereof and c1) a polynucleotide variant having a degree of sequence identity of more than 70% to the nucleic acid sequence defined in a1) or b1) of a fully complementary strand thereof.

21. The method according to claim 16, wherein the nucleotide sequence coding for a protein with the activity of a DEDDh exonuclease comprises a nucleotide sequence selected from the group consisting of a2) the polynucleotide defined in any one of SEQ ID NO: 22, 23, 27, 28, 32, 33 or a fully complementary strand thereof, b2) a polynucleotide encoding a polypeptide with the amino acid sequence defined in any one of SEQ ID NO: 4, 5, 6 or a fully complementary strand thereof, and c2) a polynucleotide variant having a degree of sequence identity of more than 70% to the nucleic acid sequence defined in a2) or b2) or a fully complementary strand thereof.

22. A transgenic apomictic plant produced according to the method of claim 16.

23. A transgenic plant material from a plant according to claim 22.

24. The method of claim 16, wherein the ATHB-5 binding site is any one of SEQ ID NO: 66 or 67 and the SORLIP2AT binding site is any one of SEQ ID NO: 76 or 77.

25. The method of claim 16, wherein the method further comprises interrupting or deleting at least one regulatory nucleotide target sequence in said cis-acting regulatory element that is a Dof2, a Dof3, or a PBF transcription factor binding site.

26. The method of claim 25, wherein the Dof2, Dof3 or PBF transcription factor binding site is selected from the group consisting of SEQ ID NO: 80, 81, 82, 83, 84, and 85.

Description

[0156] The figures show:

[0157] FIG. 1 apo-specific TBS in the positive strands that appear in all and only apo alleles. (SEQ ID NO.: 86, nucleotide residues 1-112; SEQ ID NO.: 87, nucleotide residues 1-112; SEQ ID NO.: 88, nucleotide residues 1-113; SEQ ID NO.: 89, nucleotide residues 1-112; SEQ ID NO.: 90, nucleotide residues 1-112; SEQ ID NO.: 91, nucleotide residues 1-112; SEQ ID NO.: 92, nucleotide residues 1-113; SEQ ID NO.: 93, nucleotide residues 1-112; SEQ ID NO.: 94, nucleotide residues 1-112; SEQ ID NO.: 95, nucleotide residues 1-112; SEQ ID NO.: 96, nucleotide residues 1-112; SEQ ID NO.: 97, nucleotide residues 1-112; SEQ ID NO.: 98, nucleotide residues 1-112; SEQ ID NO.: 99, nucleotide residues 1-112; SEQ ID NO.: 100, nucleotide residues 1-112; SEQ ID NO.: 101, nucleotide residues 1-112; SEQ ID NO.: 102, nucleotide residues 1-113; SEQ ID NO.: 103, nucleotide residues 1-112; SEQ ID NO.: 104, nucleotide residues 1-112; SEQ ID NO.: 105, nucleotide residues 1-111; SEQ ID NO.: 106, nucleotide residues 1-112; SEQ ID NO.: 107, nucleotide residues 1-123; SEQ ID NO.: 108, nucleotide residues 1-123; SEQ ID NO.: 109, nucleotide residues 1-123; SEQ ID NO.: 110, nucleotide residues 1-123; SEQ ID NO.: 111, nucleotide residues 1-123; SEQ ID NO.: 112, nucleotide residues 1-123; SEQ ID NO.: 113, nucleotide residues 1-123; SEQ ID NO.: 114, nucleotide residues 1-123; SEQ ID NO.: 115, nucleotide residues 1-123; SEQ ID NO.: 116, nucleotide residues 1-123; SEQ ID NO.: 117, nucleotide residues 1-123; SEQ ID NO.: 118, nucleotide residues 1-123; SEQ ID NO.: 119, nucleotide residues 1-123; ATHB-5 (agtTTATTc), nucleotide residues 62-70 of SEQ ID NO.: 107).

[0158] FIG. 2 apo-specific TBS in the negative strands that appear in all and only apo alleles. (SEQ ID NO.: 86, nucleotide residues 1-112; SEQ ID NO.: 87, nucleotide residues 1-112; SEQ ID NO.: 88, nucleotide residues 1-113; SEQ ID NO.: 89, nucleotide residues 1-112; SEQ ID NO.: 90, nucleotide residues 1-112; SEQ ID NO.: 91, nucleotide residues 1-112; SEQ ID NO.: 92, nucleotide residues 1-113; SEQ ID NO.: 93, nucleotide residues 1-112; SEQ ID NO.: 94, nucleotide residues 1-112; SEQ ID NO.: 95, nucleotide residues 1-112; SEQ ID NO.: 96, nucleotide residues 1-112; SEQ ID NO.: 97, nucleotide residues 1-112; SEQ ID NO.: 98, nucleotide residues 1-112; SEQ ID NO.: 99, nucleotide residues 1-112; SEQ ID NO.: 100, nucleotide residues 1-112; SEQ ID NO.: 101, nucleotide residues 1-112; SEQ ID NO.: 102, nucleotide residues 1-113; SEQ ID NO.: 103, nucleotide residues 1-112; SEQ ID NO.: 104, nucleotide residues 1-112; SEQ ID NO.: 105, nucleotide residues 1-111; SEQ ID NO.: 106, nucleotide residues 1-112; SEQ ID NO.: 107, nucleotide residues 1-123; SEQ ID NO.: 108, nucleotide residues 1-123; SEQ ID NO.: 109, nucleotide residues 1-123; SEQ ID NO.: 110, nucleotide residues 1-123; SEQ ID NO.: 111, nucleotide residues 1-123; SEQ ID NO.: 112, nucleotide residues 1-123; SEQ ID NO.: 113, nucleotide residues 1-123; SEQ ID NO.: 114, nucleotide residues 1-123; SEQ ID NO.: 115, nucleotide residues 1-123; SEQ ID NO.: 116, nucleotide residues 1-123; SEQ ID NO.: 117, nucleotide residues 1-123; SEQ ID NO.: 118, nucleotide residues 1-123; SEQ ID NO.: 119, nucleotide residues 1-123; SORLIP1AT (GTGGC), nucleotide residues 51-55 of SEQ ID NO.: 107; SORLIP2AT (GGCCC), nucleotide residues 53-57 of SEQ ID NO.: 107; POLASIG1 (TTTATT), nucleotide residues 64-69 of SEQ ID NO.: 107)

[0159] FIG. 3 apo-specific TBS in the negative strands that appear in all and only apo alleles. (SEQ ID NO.: 86, nucleotide residues 1-112; SEQ ID NO.: 87, nucleotide residues 1-112; SEQ ID NO.: 88, nucleotide residues 1-113; SEQ ID NO.: 89, nucleotide residues 1-112; SEQ ID NO.: 90, nucleotide residues 1-112; SEQ ID NO.: 91, nucleotide residues 1-112; SEQ ID NO.: 92, nucleotide residues 1-113; SEQ ID NO.: 93, nucleotide residues 1-112; SEQ ID NO.: 94, nucleotide residues 1-112; SEQ ID NO.: 95, nucleotide residues 1-112; SEQ ID NO.: 96, nucleotide residues 1-112; SEQ ID NO.: 97, nucleotide residues 1-112; SEQ ID NO.: 98, nucleotide residues 1-112; SEQ ID NO.: 99, nucleotide residues 1-112; SEQ ID NO.: 100, nucleotide residues 1-112; SEQ ID NO.: 101, nucleotide residues 1-112; SEQ ID NO.: 102, nucleotide residues 1-113; SEQ ID NO.: 103, nucleotide residues 1-112; SEQ ID NO.: 104, nucleotide residues 1-112; SEQ ID NO.: 105, nucleotide residues 1-111; SEQ ID NO.: 106, nucleotide residues 1-112; SEQ ID NO.: 107, nucleotide residues 1-123; SEQ ID NO.: 108, nucleotide residues 1-123; SEQ ID NO.: 109, nucleotide residues 1-123; SEQ ID NO.: 110, nucleotide residues 1-123; SEQ ID NO.: 111, nucleotide residues 1-123; SEQ ID NO.: 112, nucleotide residues 1-123; SEQ ID NO.: 113, nucleotide residues 1-123; SEQ ID NO.: 114, nucleotide residues 1-123; SEQ ID NO.: 115, nucleotide residues 1-123; SEQ ID NO.: 116, nucleotide residues 1-123; SEQ ID NO.: 117, nucleotide residues 1-123; SEQ ID NO.: 118, nucleotide residues 1-123; SEQ ID NO.: 119, nucleotide residues 1-123; LIM1 (aagaggaGGTGG), nucleotide residues 43-54 of SEQ ID NO.: 107).

[0160] FIG. 4 sex-specific TBS in the negative strands that appear in all and only sex alleles._(SEQ ID NO.: 86, nucleotide residues 1-112; SEQ ID NO.: 87, nucleotide residues 1-112; SEQ ID NO.: 88, nucleotide residues 1-113; SEQ ID NO.: 89, nucleotide residues 1-112; SEQ ID NO.: 90, nucleotide residues 1-112; SEQ ID NO.: 91, nucleotide residues 1-112; SEQ ID NO.: 92, nucleotide residues 1-113; SEQ ID NO.: 93, nucleotide residues 1-112; SEQ ID NO.: 94, nucleotide residues 1-112; SEQ ID NO.: 95, nucleotide residues 1-112; SEQ ID NO.: 96, nucleotide residues 1-112; SEQ ID NO.: 97, nucleotide residues 1-112; SEQ ID NO.: 98, nucleotide residues 1-112; SEQ ID NO.: 99, nucleotide residues 1-112; SEQ ID NO.: 100, nucleotide residues 1-112; SEQ ID NO.: 101, nucleotide residues 1-112; SEQ ID NO.: 102, nucleotide residues 1-113; SEQ ID NO.: 103, nucleotide residues 1-112; SEQ ID NO.: 104, nucleotide residues 1-112; SEQ ID NO.: 105, nucleotide residues 1-111; SEQ ID NO.: 106, nucleotide residues 1-112; SEQ ID NO.: 107, nucleotide residues 1-123; SEQ ID NO.: 108, nucleotide residues 1-123; SEQ ID NO.: 109, nucleotide residues 1-123; SEQ ID NO.: 110, nucleotide residues 1-123; SEQ ID NO.: 111, nucleotide residues 1-123; SEQ ID NO.: 112, nucleotide residues 1-123; SEQ ID NO.: 113, nucleotide residues 1-123; SEQ ID NO.: 114, nucleotide residues 1-123; SEQ ID NO.: 115, nucleotide residues 1-123; SEQ ID NO.: 116, nucleotide residues 1-123; SEQ ID NO.: 117, nucleotide residues 1-123; SEQ ID NO.: 118, nucleotide residues 1-123; SEQ ID NO.: 119, nucleotide residues 1-123; Dof2/Dof3 (ttGCTTTaaaa), nucleotide residues 58-68 of SEQ ID NO.: 86; Dof2/Dof3 (also) (TGCTTT), nucleotide residues 59-64 of SEQ ID NO.: 86; PBF (GCTTT), nucleotide residues 60-64 of SEQ ID NO.: 86).

[0161] The invention will now be illustrated by way of example.

EXAMPLE 1

Screening and Isolation of Apomixis-Inducing Gene (Apollo Gene)

[0162] 1.a) Plant Material and Seed Screen Analysis

[0163] Plants were grown from seedlings onwards in a phytotron under controlled environmental conditions. The flow cytometric seed screen was used to analyse reproductive variability in 18 Boechera accessions (Table IV).

TABLE-US-00004 TABLE IV Boechera accessions used in Microarrays and RT-PCR analyses. Table IV - Boechera accessions used in Microarrays and RT-PCR analyses. Apomeiosis Accession frequency Collection locality B08-1 1 Birch Creek, Montana B08-11 1 Sliderock, Ranch Creek, Granite, Montana B08-33 1 Mule Ranch, Montana B08-111 1 Morgan Switch Back, Idaho B08-81 1 Vipond Park, Beaverhead, Montana B08-168 1 Vipond Park, Beaverhead, Montana B08-43 1 Mule Ranch, Montana B08-66 1 Highwood Mtns, Montana B08-104 1 Lost Trail Meadow B08-215 1 Blue Lakes road, California B08-369 0 Twin Saddle, Idaho B08-376 0 Sagebrush Meadow, Montana B08-380 0 Buffalo Pass, Colorado B08-355 0 Gold Creek, Colorado B08-329 0 Big Hole Pass, Montana B08-385 0 Parker Meadow, Idaho B08-344 0 Bandy Ranch, Montana B08-390 0 Panther Creek

[0164] Single seeds were ground individually with three 2.3 mm stainless steel beads in each well of 96-well plate (PP-Master-block 128.0/85 MM, 1.0 ml 96 well plate by Greiner bio-one, http World Wide Web internet site gbo.com) containing 50 l extraction-nuclei isolation buffer (see below) using a Geno-Grinder 2000 (SPEX Certi-Prep) at rate of 150 strokes/minute for 90 seconds.

[0165] A two-step procedure consisting of an isolation and staining buffer was used: (a) isolation buffer I0.1M Citric acid monohydrate and 0.5% v/v Tween 20 dissolved in H.sub.2O and adjusted to pH 2.5); and (b) staining buffer II0.4M Na.sub.2HPO.sub.4.12H.sub.2O dissolved in H.sub.2O plus 4 g/ml 4,6-Diamidinophenyl-indole (DAPI) and adjusted to pH 8.5. 50 l of isolation buffer I was added to each seed per well in a 96-well plate before grinding, and a further 160 l buffer I was added after grinding to recover enough volume through filtration (using Partec 30 m mesh-width nylon filters). 100 l of staining buffer II was then added to 50 l of the resultant suspension (isolated nuclei), and incubated on ice for 10 minutes before flow cytometric analysis. To avoid sample degradation over the 2-hour period required for the analysis of 96 samples, the sample plate was sealed with aluminum sealing tape.

[0166] All sample plates were analysed on a 4 C. cooled Robby-Well auto-sampler hooked up to a Partec PAII flow Cytometer (Partec GmbH, Mnster, Germany). Two single seeds from SAD 12, a known sexual self-fertile Boechera were always included as an external reference at well positions 1 and 96 in order to normalize other peaks and correct peak shifts over the analysis period. SAD 12 seeds were composed exclusively of 2C embryo to 3C endosperm ratio, which reflected an embryo composition of C (C denotes monoploid DNA content) maternal (Cm) genomes+C paternal (Cp)=2C genomes, and an endosperm composition of 2Cm+Cp=3C.

[0167] Based upon the present high-throughput flow-cytometric seed screen data, all apomictic accessions were shown to be characterized by 100% apomictic seed production.

[0168] 1.b) Ovule Micro-Dissection

[0169] Ovules at megasporogenesis between stages 2-II to 2-IV were selected where megaspore mother cell is differentiated, inner and outer integument initiated in order to examine changes in gene expression associated with meiosis and apomeiosis. The gynoecia of sexual and apomictic Boechera were dissected out from non-pollinated flowers at the stage of megasporogenesis in 0.55 M sterile mannitol solution, at a standardized time (between 8 and 9 a.m.) over multiple days. Microdissections were done in a sterile laminar air flow cabinet using a stereoscopic Microscope (1000 Stemi, Carl Zeiss, Jena, Germany) under 2 magnification. The gynoecium was held with forceps while a sterile scalpel was used to cut longitudinally such that the halves of the silique along with the ovules were immediately exposed to the mannitol. Individual live ovules were subsequently collected under an inverted Microscope (Axiovert 200M, Carl Zeiss) in sterile conditions, using sterile glass needles (self-made using a Narishige PC-10 puller, and bent to an angle of about)100 to isolate the ovules from placental tissue. Using a glass capillary (with an opening of 150 m interior diameter) interfaced to an Eppendorf Cell Tram Vario, the ovules were collected in sterile Eppendorf tubes containing 100 l of RNA stabilizing buffer (RNA later, Sigma). Between 20 and 40 ovules per accession were collected in this way, frozen directly in liquid nitrogen and stored at 80 C.

[0170] 1.c) Ovule RNA Isolation

[0171] Total RNA extractions were carried out using PicoPure RNA isolation kit (Arcturus Bioscience, CA). RNA integrity and quantity was verified on an Agilent 2100 Bioanalyzer using the RNA Pico chips (Agilent Technologies, Palo Alto, Calif.).

[0172] 1.d) Microarray

[0173] 1.d.i) Microarray Design

[0174] The 454 (FLX) technology was used to sequence the complete transcriptomes of 3 sexual and 3 apomictic Boechera accessions, as a first step in the design of high-density Boechera-specific microarrays for use in comparisons of gene expression and copy number variation. The goal of transcriptome sequencing was thus to identify all genes which can be expressed during flower development, followed by the spotting of all identified genes onto an (Agilent) microarray.

[0175] This was accomplished by pooling flowers at multiple developmental stages separately for sexual and apomictic plants, followed by a cDNA normalization procedure in order to balance out transcript levels to increase the chance that all observable mRNA species are sequenced. Furthermore, a 3-UTR (untranslated region) anchored 454 procedure was employed such that mRNA sequences were biased towards their 3-UTRs, regions which demonstrate relatively high (but not random) levels of variability, to enable the identification of allelic variation.

[0176] The 454 sequences were assembled using the CLC Genomics workbench using standard assembly parameters for long-read high-throughput sequences, after trimming of all reads using internal sequence quality scores. In doing so, 36 289 contig sequences and 154 468 non-assembled singleton sequences were obtained. This data was provided to ImaGenes (GmbH, Germany) for microarray development using their Pre-selection strategy (PSS) service.

[0177] The PSS service worked as follows: 14 different oligonucleotides (each 60 bp in length) per contig and 8 oligonucleotides per singleton, including the anti-sense sequence of each oligo, were bioin-formatically designed and spotted onto two 1 million-spot test arrays. These test-arrays were probed using (1) a complex cRNA mixture (obtained by pooling tissues and harvesting all RNA from them), and (2) genomic DNA extracted from leaf tissue pooled from a sexual and an apomictic individual. Based upon the separate hybridization results from the cRNA and genomic DNA samples, and after all quality tests, a final 2105 000 spot array was designed. This array should contain multiple oligonucleotides (i.e. technical replicates) of every gene expressed during Boechera flower development.

[0178] 1.d.ii) Hybridization

[0179] cRNA was prepared and labelled using the Quick-Amp One-Color Labeling Kit (Agilent Technologies, CA) and hybridized to the Agilent custom Boechera arrays (8 and 10 biological replicates were hybridized for sexual and apomictic genotypes respectively).

[0180] 1.d.iii) Statistical Analysis

[0181] Analyses were performed using GeneSpring GX Software (version 10) and candidate probes significantly differentially expressed (p0.05) between apomictic and sexual plants were selected based on the following parameters: (a) percentile shift 75 normalization, median as baseline, reproductive mode (apomictic or sexual) as interpretation (1st level), T-test unpaired as statistical analysis and Bonferroni FWER multiple test corrections. Using the highest level of significance cutoff led to the identification of 4 different spots on the microarray (p<0.01 for the first three and p<0.05 for the fourth). Importantly, when the oligonucleotide sequences of these 4 spots were BLASTed to a 454 cDNA sequence database, all 4 blasted to the same Boechera transcript. Thus, not only has the present experiment been corrected for biological noise, furthermore a single differentially-expressed transcript between the microdissected ovules of all sexual and apomictic genotypes, with 4 technical replicates for the specific gene on the microarray was detected. This gene is expressed to a similar fashion when comparing both diploid and triploid apomictic ovules to those of sexuals, and hence its expression behavior is apparently not influenced by ploidy. Finally, a search for homologues to this Boechera transcript demonstrated that it is involved with the cell cycle in other species, thus supporting evidence regarding deregulation of the sexual pathway as a means to produce apomixis.

EXAMPLE 2

Characterisation of Apomixis-Inducing Gene

[0182] 2.a) Candidate Gene Characterization

[0183] 2.a.i) Genome Level

[0184] 2.a.i.1) Cloning

[0185] The full-length transcript from all 18 accessions was cloned and sequenced (TOPO-TA Cloning kit, Invitrogen) using proofreading polymerase (Accuprime). The transcript is highly polymorphic, and is characterized by comparable levels of single nucleotide polymorphisms between sexual and apomicts. Nevertheless, a single apomixis polymorphism is found in all 10 apomictic accessions, but not in any sexual accession. SEQ ID No. 46 to 54 show the genomic and the coding sequence of three sexual alleles, namely S011a, S355a and S390a. SEQ ID No. 37 to 45 show the genomic and the coding sequence of three apomictic alleles, namely A011a, A043a and A081a. Considering that the geographic collection points of all accessions range from California to the American mid-west (i.e. 1000's of kilometers), the sharing of this polymorphism in all apomicts is highly significant. Finally, the SNP polymorphism spectrum surrounding the apomixis polymorphism reflects that found in all other alleles in both sexual and apomictic accessions. Hence the apomixis polymorphism appears to have undergone recombination during the evolution of Boechera, but which is nonetheless shared by all apomicts, regardless of different genetic, ploidy or geographic backgrounds.

[0186] 2.a.i.2) BAC

[0187] Pooled DNA of all tissues accessions was used as a template for hybridization probes generation. Two probes of different size (1.6 and 2.3 kb) were prepared by PCR amplification using two pairs of specific primers of the candidate gene genomic sequence. Both probes were labeled and used for hybridization on a apomictic Boechera BAC library. There were 8 positive hybridizations. The respective isolated BACs (PureLink Plasmid DNA Purification kit) were named 1, 2a, 2b, 3, 4, 5, 6 and 7. Selected BACs were retested using specific primers for the candidate gene. All BACs were confirmed except the BAC-3. The other seven BACs were fingerprinted by restriction enzyme digestion. BAC-1 and BAC-2a seemed to be redundant with the other BACs. The BACs: 2b, 4, 5, 6 and 7 were sequenced.

[0188] BAC sequences could be assembled together for the pairs 2b_4 and 5_7, whereas BAC-6 remained alone.

[0189] BAC sequences were characterized by comparison with other plant sequences.

[0190] 2.a.ii) Transcriptome Level

[0191] RACE experiments (SMARTer RACE cDNA Amplification Kit) were performed.

[0192] The results revealed that mRNA corresponding to apomictic accessions has a truncated 5 extreme upstream the apomixis polymorphism whereas sexual accessions have 200 bp of additional length.

[0193] Once 5 and 3 mRNA extremes were known, further PCRs over all tissues cDNA were performed for complete splicing profile characterization.

[0194] 2.b) Validation

[0195] 2.b.i) QRT-PCR

[0196] An allele-specific qRT-PCR analysis of the candidate gene on the microdissected live ovules (megaspore mother cell stage) from 6 sexual and 10 diploid apomictic Boechera accessions (3 technical replicates per accession) was completed. Using two different forward PCR primers which spanned the apomixis-specific polymorphism which was identified from the gene sequences, it was possible to measure transcript abundance for both the sexual and apomictic alleles separately.

[0197] cDNA was prepared using RevertAid H Minus reverse transcriptase.

[0198] For the real-time PCR reactions the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, Calif.) was used. QRT-PCR amplifications were carried out in a 7900HT Fast RT-PCR System machine (Applied Biosystems) with the following temperature profile for SYBRgreen assays: initial denaturation at 90 C. for 10 min, followed by 40 cycles of 95 C. for 15 sec. and 60 C. for 1 min. For checking amplicon quality, a melting curve gradient was obtained from the product at the end of the amplification. The Ct, defined as the PCR cycle at which a statistically significant increase of reporter fluorescence is first detected, was used as a measure for the starting copy numbers of the target gene. The mean expression level and standard deviation for each set of three technical replicates for each cDNA was calculated. Relative quantitation and normalization of the amplifled targets were performed by the comparative AACt method using a calibrator sample in reference to the expression levels of the housekeeping gene UBQ10.

[0199] The results are conclusive: the apomictic allele is exclusively expressed in the microdissected ovules of all apomictic accessions, while the sexual allele is never expressed in any, which means sexual or apomictic, ovule. Both alleles are expressed in other tissues, namely somatic tissue. Hence, it appears very reasonable to assume that the sexual allele is inactive/silenced during normal sexual ovule development, while the expression of the apomictic allele is correlated with apomeiotic ovule development.

EXAMPLE 3

Transformation of Arabidopsis thaliana with Apomixis-Inducing Gene

[0200] 3.a) Plant Transformation

[0201] Transformations of Arabidopsis thaliana (sex) (hybrids F1) and Boechera (sex) with the gene of the present invention are able to show a change of their reproductive mode into apomictic seed production. For this, the complete genomic allele (including complete promoter) has been cloned in pNOS-ABM.

[0202] In addition, different constructs are used to characterize the role of the present regulatory elements, in particular the promoter of the present invention, in its expression. For this, both apo and sex promoters have been exactly connected to the ATG in front of gus in pGUS-ABM.

[0203] Complete BAC-4 is as well used for transformations.

EXAMPLE 4

[0204] For promoter analysis of the present regulatory elements the plant PAN software (release 1.0.2007) (http World Wide Web internet site plantpan.mbc.nctu.edu.tw/gene_group/index.php; Chang et al., (2008) PlantPAN: Plant Promoter Analysis Navigator, for identifying combinatorial cis-regulatory elements with distance constraint in plant gene group, BMC Genomics, 9:561) has been used. What is claimed is: