Means and Methods to Induce Apomixis in Plants

Abstract

The present invention relates to nucleic acid molecules for use in inducing apomixis in a plant, transgenic cells, in particular transgenic plant cells, comprising said nucleic acid molecule, transgenic plants, in particular plant seeds, comprising said nucleic acid molecule, methods for inducing apomixis in a plant, methods for the production of apomictic plants and uses thereof.

Claims

1. An isolated nucleic acid molecule for use in inducing apomixis in a plant comprising a polynucleotide, which is selected from the group consisting of a) the polynucleotide defined in any one of SEQ ID No. 22 to 62, or a fully complementary strand thereof, b) 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 c) a polynucleotide variant having a degree of sequence identity of more than 70% to the nucleic acid sequence defined in a) or b), or a fully complementary strand thereof.

2. The isolated nucleic acid molecule of claim 1, which comprises a polynucleotide coding for a protein with exonuclease activity, which polynucleotide is 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), or a fully complementary strand thereof.

3. The isolated nucleic acid molecule of claim 2, wherein the polynucleotide comprises a nucleic acid 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.

4. The isolated nucleic acid molecule of claim 1, which comprises a polynucleotide which polynucleotide is able to act as a regulatory element and is selected from the group consisting of a3) the polynucleotide defined in any one of SEQ ID No. 55 to 62 or 65 or a fully complementary strand thereof and b3) a polynucleotide variant having a degree of sequence identity of more than 70% to the nucleic acid sequence defined in a3), or a fully complementary strand thereof.

5. A vector comprising the nucleic acid molecule of any one of claims 1 to 4.

6. The vector of claim 5, wherein the nucleic acid molecule of any one of claims 1 to 3 is operably linked to at least one regulatory element.

7. The vector of claim 5 or 6, wherein the nucleic acid molecule of any one of claim 1 or 4 is operably linked to at least one protein coding nucleotide sequence.

8. A host cell containing the vector of claims 5 to 7.

9. A protein encoded by a nucleotide acid sequence according to any one of claims 1 to 3.

10. A transgenic plant, plant cell or plant material comprising at least one transgenic nucleic acid molecule of any one of claims 1 to 4 or the vector of any one of claims 5 to 7.

11. A cell culture, preferably a plant cell culture comprising a cell according to claim 8.

12. A method for inducing apomixis in a plant, wherein the expression of a nucleotide sequence encoding a protein capable of inducing apomixis in a plant according to any one of claims 1 to 3 is induced in the ovule of said plant.

13. The method for inducing apomixis in a plant according to claim 12, wherein the expression is induced by transforming a plant cell with the isolated nucleic acid molecule of any one of claims 1 to 4 or the vector according to any one of claims 5 to 7 and regenerating the transformed plant cell into a transformed plant that contains the at least one nucleic acid sequence according to claims 1 to 4 so as to induce apomixis in the plant.

14. A method for the production of an apomictic plant, wherein a plant cell is transformed with a nucleic acid molecule capable of inducing the expression of nucleotide sequence encoding a protein capable of inducing apomixis in a plant according to any one of claims 1 to 4 and regenerating the transformed plant cell into a transformed plant that contains the nucleic acid molecule so as to induce apomixis in the plant.

15. The method for the production of an apomictic plant by transforming according to claim 14, wherein the plant cell is transformed with the isolated nucleic acid molecule of any one of claims 1 to 4 or the vector of claims 5 to 7 and the transformed plant cell is regenerated into a transformed plant that contains the at least one nucleic acid sequence according to claims 1 to 4 so as to induce apomixis in the plant.

16. A method for isolating an apomixis inducing nucleic acid molecule from a plant, wherein the isolated nucleic acid molecule of any one of claims 1 to 4 is used to screen and isolate nucleic acid sequences derived from the plant.

17. A transgenic plant, plant cell or plant material comprising a cell according to any one of claim 8, 10 or 11 or produced according to a method according to any one of claim 14 or 15 or progeny thereof.

18. The transgenic plant, plant cell or plant material according to claim 17 transgenically expressing the nucleotide acid sequence of any one of claims 1 to 4.

19. A method for identifying an effector for apomixis in a plant, wherein the transgenic plant, plant cell or plant material according to any one of claim 17 or 18 is cultivated.

Description

EXAMPLE 1

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

1.a) Plant Material and Seed Screen Analysis

[0180] 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. 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

[0181] 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, on the world wide web at “gbo.com”) containing 50 μl extraction-nuclei isolation buffer (see below) using a Geno-Grinder 2000 (SPEX Certi-Prep) at rate of 150 strokes/minute for 90 seconds.

[0182] A two-step procedure consisting of an isolation and staining buffer was used: (a) isolation buffer I—0.1M 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 II—0.4M Na.sub.2HPO.sub.4.12H.sub.2O dissolved in H.sub.2O plus 4 μg/ml 4′,6-Diamidinophenyl-indole (DAPI) and adjusted to pH 8.5. 50 μl of isolation buffer I was added to each seed per well in a 96-well plate before grinding, and a further 160 μl buffer I was added after grinding to recover enough volume through filtration (using Partec 30 μm mesh-width nylon filters). 100 μl of staining buffer II was then added to 50 μl 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.

[0183] All sample plates were analysed on a 4° C. cooled Robby-Well auto-sampler hooked up to a Partec PAII flow Cytometer (Partec GmbH, Münster, 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)=2 C genomes, and an endosperm composition of 2 Cm+Cp=3 C.

[0184] Based upon the present high-throughput flow-cytometric seed screen data, all apomictic accessions were shown to be characterized by 100% apomictic seed production.

1.b) Ovule Micro-Dissection

[0185] 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 μm interior diameter) interfaced to an Eppendorf Cell Tram Vario, the ovules were collected in sterile Eppendorf tubes containing 100 μl 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.

1.c) Ovule RNA Isolation

[0186] 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.).

1.d) Microarray

1.d.i) Microarray Design

[0187] 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.

[0188] 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.

[0189] 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.

[0190] 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 bioinformatically 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 2×105 000 spot array was designed. This array should contain multiple oligonucleotides (i.e. technical replicates) of every gene expressed during Boechera flower development.

1.d.ii) Hybridization

[0191] 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).

1.d.iii) Statistical Analysis

[0192] Analyses were performed using GeneSpring GX Software (version 10) and candidate probes significantly differentially expressed (p≤0.05) 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

2.a) Candidate Gene Characterization

2.a.i) Genome Level

2.a.i.1) Cloning

[0193] 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.

2.a.i.2) BAC

[0194] 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.

[0195] BAC sequences could be assembled together for the pairs 2b_4 and 5_7, whereas BAC-6 remained alone.

[0196] BAC sequences were characterized by comparison with other plant sequences.

2.a.ii) Transcriptome Level

[0197] RACE experiments (SMARTer RACE cDNA Amplification Kit) were performed.

[0198] 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.

[0199] Once 5′ and 3′ mRNA extremes were known, further PCRs over all tissues cDNA were performed for complete splicing profile characterization.

2.b) Validation

2.b.i) QRT-PCR

[0200] 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.

[0201] cDNA was prepared using RevertAid H Minus reverse transcriptase.

[0202] 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 amplified targets were performed by the comparative ΔΔCt method using a calibrator sample in reference to the expression levels of the house-keeping gene UBQ10.

[0203] 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

Construction of Transformation Vectors and Transformation of Arabidopsis thaliana with Apomixis-Inducing Gene

Plant Transformation

[0204] 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.

[0205] 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.

[0206] Complete BAC-4 is as well used for transformations.