US20120042408A1 - Plants producing 2n gametes or apomeiotic gametes - Google Patents

Plants producing 2n gametes or apomeiotic gametes Download PDF

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US20120042408A1
US20120042408A1 US13/143,530 US201013143530A US2012042408A1 US 20120042408 A1 US20120042408 A1 US 20120042408A1 US 201013143530 A US201013143530 A US 201013143530A US 2012042408 A1 US2012042408 A1 US 2012042408A1
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protein
osd1
plant
spo11
gametes
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Raphael Mercier
Isabelle D'Erfurth
Nicole Froger
Sylvie Jolivet
Laurence Cromer
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Institut National de la Recherche Agronomique INRA
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
    • A01H1/022—Genic fertility modification, e.g. apomixis
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
    • A01H1/08—Methods for producing changes in chromosome number
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis

Definitions

  • the invention relates to plants that produce 2n Second Division Restitution (SDR) gametes, and to plants that produce apomeiotic gametes, and to their use in plant breeding.
  • SDR Second Division Restitution
  • 2n gametes are gametes having the somatic chromosome number rather than the gametophytic chromosome number. They have been shown to be useful for the genetic improvement of several crops (for review, cf. for instance RAMANNA & JACOBSEN, Euphytica 133, 3-18, 2003).
  • diplogametes allow crosses between plants of different ploidy level, for instance crosses between tetraploid crop plants and their diploid wild relatives, in order to use their genetic diversity in plant breeding programs.
  • meiosis I homologous chromosomes recombine and are separated into two cells, each of them comprising one entire haploid content of chromosomes.
  • meiosis II the two cells resulting from meiosis I further divide, and the sister chromatids segregate. The spores resulting from this division are thus haploid and carry recombined genetic information.
  • the abnormalities leading to 2n gametes formation include in particular abnormal cytokinesis, the skip of the first or second meiotic division, or abnormal spindle geometry (for review cf. VEILLEUX, Plant Breeding Reviews 3, 252-288, 1985, or BRETAGNOLLE & THOMPSON, New Phytologist 129, 1-22, 1995). These abnormalities lead to different classes of unreduced gametes. For instance, failure of the first meiotic division results in First Division Restitution (FDR) gametes, while failure of the second meiotic division results in Second Division Restitution (SDR) gametes.
  • FDR First Division Restitution
  • SDR Second Division Restitution
  • AtPS1 for Arabidopsis thaliana parallel spindles
  • This gene and its use for producing 2n pollen are disclosed in European Patent application 08490672, filed on Jul. 8, 2008, and in the publication of D′ERFURTH et al (PLoS Genet. 2008 November; 4(11):e1000274. Epub 2008 Nov. 28).
  • the inventors have now identified in the model plant Arabidopsis thaliana , another gene implicated in the formation of 2n gametes in plants. The inventors have found that inactivation of this gene results in the skipping of the second meiotic division. This generates diploid male and female spores, giving rise to viable diploid male and female gametes, which are SDR gametes.
  • This gene will be hereinafter designated OSD1, for omission of second division.
  • the sequence of the OSD1 gene of Arabidopsis thaliana is available in the TAIR database under the accession number At3g57860, or in the GenBank database under the accession number NM — 115648. This gene encodes a protein of 243 aa (GenBank NP — 191345), whose sequence is also represented in the enclosed sequence listing as SEQ ID NO: 1.
  • UVI4-Like gene (UVI4-L), in a publication of HASE et al. (Plant J, 46, 317-26, 2006), which describes its paralogue, named UVI4.
  • UVI4 acts as a suppressor of endo-reduplication and is necessary for maintaining the mitotic state whereas OSD1 (UVI4-L) does not appear to be required for this process.
  • OSD1 appears necessary for allowing the transition from meiosis Ito meiosis II.
  • the inventors have also identified in rice (Oryza sativa) an ortholog of the OSD1 gene of Arabidopsis thaliana .
  • the sequence of the OSD1 gene of Oryza sativa is available in the OryGenes or TAIR databases under the accession number Os02g37850. It encodes a protein of 234 aa, whose sequence is represented in the enclosed sequence listing as SEQ ID NO: 35.
  • the OSD1 proteins of Arabidopsis thaliana and Oryza sativa have 23.6% identity and 35% similarity over the whole length of their sequences
  • the invention thus provides a method for obtaining a plant producing Second Division Restitution 2n gametes, wherein said method comprises the inhibition in said plant of a protein hereinafter designated as OSD1 protein, wherein said protein has at least 20%, and by order of increasing preference, at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence identity, or at least 29%, and by order of increasing preference, at least 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence similarity with the AtOSD1 protein of SEQ ID NO: 1 or with the OsOSD1 protein of SEQ ID NO: 35.
  • OSD1 protein a protein hereinafter designated as OSD1 protein
  • said protein has at least 20%, and by order of increasing preference, at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 98% sequence similarity with the AtOSD1 protein of SEQ
  • protein sequence identity and similarity values provided herein are calculated over the whole length of the sequences, using the BLASTP program under default parameters, or the Needleman-Wunsch global alignment algorithm (EMBOSS pairwise alignment Needle tool under default parameters). Similarity calculations are performed using the scoring matrix BLOSUM62.
  • the SDR 2n gametes produced according to the invention are useful in all the usual applications of 2n gametes, for instance for producing polyploids plants, or to allow crosses between plants of different ploidy level. They can also be useful in methods of genetic mapping, for instance the method of “Reverse progeny mapping” disclosed in US Patent Application 20080057583.
  • the inventors have further found that by combining the inactivation of OSD1, with the inactivation of two other genes, one (SPO11-1) which encodes a protein necessary for efficient meiotic recombination in plants, and whose inhibition eliminates recombination and pairing (GRELON et al., Embo J, 20, 589-600, 2001), and another (REC8, At2g47980) which encodes a protein necessary for the monopolar orientation of the kinetochores during meiosis (CHELYSHEVA et al., J Cell Sci, 118, 4621-32, 2005), and whose inhibition modifies chromatid segregation, resulted in a genotype in which meiosis is totally replaced by mitosis without affecting subsequent sexual processes.
  • SPO11-1 which encodes a protein necessary for efficient meiotic recombination in plants, and whose inhibition eliminates recombination and pairing
  • REC8 At2g47980 encodes a protein necessary for
  • MiMe mitochondrial originating from meiosis.
  • This replacement of meiosis by mitosis results in apomeiotic gametes, retaining all the parent's genetic information (BICKNELL & KOLTUNOW, Plant Cell, 16 Suppl, S228-45, 2004).
  • FIG. 1 provides a schematic comparison between the mechanisms of mitosis, normal meiosis, meiosis in the osd1 mutant, meiosis in a mutant lacking SPO11-1 activity (Atspo11-1), meiosis in a double mutant lacking both SPO11-1 and REC8 activity (Atspo11-1/Atrec8), and meiosis in the MiMe mutant.
  • chromosomes During mitosis in diploid cells, chromosomes replicate and sister chromatids segregate to generate daughter cells that are diploid and genetically identical to the initial cell. During normal meiosis, two rounds of chromosome segregation follow a single round of replication. At division one, homologous chromosomes recombine and are separated. Meiosis II is more similar to mitosis resulting in equal distribution of sister chromatids. The obtained spores are thus haploid and carry recombined genetic information. In the osd1 mutant (this study) meiosis II is skipped giving rise to diploid spores and SDR gametes with recombined genetic information.
  • the Atspo11-1 mutant undergoes an unbalanced first division followed by a second division leading to unbalanced spores and sterility.
  • Atspo11-1/Atrec8 double mutant undergoes a mitotic-like division instead of a normal first meiotic division, followed by an unbalanced second division leading to unbalanced spores and sterility.
  • the presence of the Atspo11-1 and Atrec8 mutations leads to a mitotic-like first meiotic division and the presence of the osd1 mutation prevents the second meiotic division from occurring. Thus meiosis is replaced by a mitotic-like division.
  • the obtained spores and gametes are genetically identical to the initial cell.
  • the apomeiotic gametes produced by the MiMe mutant can be used, in the same way as the SDR 2n gametes, for producing polyploids plants, or for crossing plants of different ploidy level. They are also of interest for the production of apomictic plants, i.e plants which are able to form seeds from the maternal tissues of the ovule, resulting in progeny that are genetic clones of the maternal parent. Although it exists in over 400 species of angiosperms, very few crop species are apomictic and attempts to introduce this trait by crossing have failed (SAVIDAN, The Flowering of Apomixis: From Mechanisms to Genetic Engineering 2001; SPILLANE et al., Sexual Plant Reproduction, 14, 2001).
  • a further object of the present invention is thus a method for obtaining a plant producing apomeiotic gametes, wherein said method comprises the inhibition in said plant of the following proteins:
  • SEQ ID NO: 2 represents the sequence of the SPO11-1 protein of Arabidopsis thaliana . This sequence is also available in the Swissprot database under the accession number Q9M4A2.
  • SEQ ID NO: 3 represents the sequence of the SPO11-2 protein of Arabidopsis thaliana . This sequence is also available in the SwissProt database under the accession number Q9M4A1.
  • SEQ ID NO: 4 represents the sequence of the PRD1 protein of Arabidopsis thaliana . This sequence is also available in the GenBank database under the accession number ABQ12642.
  • SEQ ID NO: 5 represents the sequence of the PAIR1 protein of Arabidopsis thaliana . This sequence is also available in the GenBank database under the accession number NP — 171675.
  • SEQ ID NO: 6 represents the sequence of the Rec8 protein of Arabidopsis thaliana . This sequence is also available in the GenBank database under the accession number NP — 196168.
  • SPO11-1, SPO11-2, PRD1, PAIR1, and Rec8 proteins are conserved in higher plants, monocotyledons as well as dicotyledons.
  • orthologs of SPO11-1, SPO11-2, PRD1, PAIR1 and Rec8 proteins of Arabidopsis thaliana in monocotyledonous plants one can cite the Oryza sativa SPO11-1, SPO11-2, PRD1, PAIR1, and Rec8 proteins.
  • the sequence of the Oryza sativa SPO11-1 protein is available in GenBank under the accession number AAP68363; the sequence of the Oryza sativa SPO11-2 protein is available in GenBank under the accession number NP — 001061027; the sequence of the Oryza sativa PRD1 protein is available in GenBank under the accession number EAZ30311; the sequence of the Oryza sativa PAIR1 protein is available in SwissProt under the accession number Q75RY2; the sequence of the Oryza sativa Rec8 protein is available in GenBank under the accession number AAQ75095.
  • the inhibition of the above mentioned OSD1, SPO11-1, SPO11-2, PRD1, PAIR1, or Rec8 proteins can be obtained either by abolishing, blocking, or decreasing their function, or advantageously, by preventing or down-regulating the expression of the corresponding genes.
  • inhibition of said protein can be obtained by mutagenesis of the corresponding gene or of its promoter, and selection of the mutants having partially or totally lost the activity of said protein.
  • a mutation within the coding sequence can induce, depending on the nature of the mutation, the expression of an inactive protein, or of a protein with impaired activity; in the same way, a mutation within the promoter sequence can induce a lack of expression of said protein, or decrease thereof.
  • Mutagenesis can be performed for instance by targeted deletion of the coding sequence or of the promoter of the gene encoding said protein or of a portion thereof, or by targeted insertion of an exogenous sequence within said coding sequence or said promoter. It can also be performed by inducing random mutations, for instance through EMS mutagenesis or random insertional mutagenesis, followed by screening of the mutants within the desired gene. Methods for high throughput mutagenesis and screening are available in the art. By way of example, one can mention TILLING (Targeting Induced Local Lesions IN Genomes, described by McCallum et al., 2000).
  • those resulting in the ability to produce SDR 2n gametes can be identified on the basis of the phenotypic characteristics of the plants which are homozygous for this mutation: these plants can form at least 5%, preferably at least 10%, more preferably at least 20%, still more preferably at least 50%, and up to 100% of dyads as a product of meiosis.
  • those useful for obtaining a plant producing apomeiotic gametes can be identified on the basis of the phenotypic characteristics of the plants which are homozygous for this mutation, in particular the presence of univalents instead of bivalents at meiosis I, and the sterility of the plant.
  • mutants having a mutation within the REC8 gene those useful for obtaining a plant producing apomeiotic gametes can be identified on the basis of the phenotypic characteristics of the plants which are homozygous for this mutation, in particular chromosome fragmentation at meiosis, and sterility of the plant.
  • said method comprises:
  • step b) self fertilizing said plant of step a) in order to obtain a plant homozygous for said mutation.
  • said method comprises:
  • steps a) b) and c) crossing the plants of steps a) b) and c) in order to obtain a plant having a mutation within an allele of the OSD1 gene, a mutation within an allele of a gene selected among the SPO11-1, SPO11-2, PRD1, or PAIR1 gene, and a mutation within an allele of the REC8 gene, said plant being heterozygous for each mutation;
  • step f) self fertilizing the plant of step e) in order to obtain a plant homozygous for the mutation within the OSD1 gene, for the mutation within the gene selected among the SPO11-1, SPO11-2, PRD1, or PAIR1 gene, and for the mutation within the REC8 gene.
  • the inhibition of the target protein is obtained by silencing of the corresponding gene.
  • Methods for gene silencing in plants are known in themselves in the art. For instance, one can mention by antisense inhibition or co-suppression, as described by way of example in U.S. Pat. Nos. 5,190,065 and 5,283,323. It is also possible to use ribozymes targeting the mRNA of said protein.
  • RNA interference RNA interference
  • RNAi RNA interference
  • RNAi RNA interference
  • Various methods and DNA constructs for delivery of silencing RNAs are available in the art.
  • a “silencing RNA” is herein defined as a small RNA that can silence a target gene in a sequence-specific manner by base pairing to complementary mRNA molecules.
  • Silencing RNAs include in particular small interfering RNAs (siRNAs) and microRNAs (miRNAs).
  • DNA constructs for delivering a silencing RNA in a plant included a fragment of 300 bp or more (generally 300-800 bp, although shorter sequences may sometime induce efficient silencing) of the cDNA of the target gene, under transcriptional control of a promoter active in said plant.
  • silencing RNA constructs are those that can produce hairpin RNA (hpRNA) transcripts.
  • the fragment of the target gene is inversely repeated, with generally a spacer region between the repeats (for review, cf. WATSON et al., 2005).
  • amiRNAs artificial microRNAs directed against the gene to be silenced
  • silencing RNAs including in particular amiRNAs, in plants cf. for instance OSSOWSKI et al., (Plant J., 53, 674-90, 2008).
  • the present invention provides tools for silencing one or more target gene(s) selected among OSD1, SPO11-1, SPO11-2, PRD1, PAIR1, and REC8, including in particular expression cassettes for hpRNA or amiRNA targeting said gene (s).
  • An expression cassette of the invention may comprise for instance:
  • an expression cassette for hpRNA comprises:
  • said DNA construct(s) being placed under transcriptional control of said promoter.
  • said hairpin DNA construct comprises: i) a first DNA sequence of 200 to 1000 bp, preferably of 300 to 900 bp, consisting of a fragment of a cDNA of the target gene, or having at least 95% identity, and by order of increasing preference, at least 96%, 97%, 98%, or 99% identity with said fragment; ii) a second DNA sequence that is the complementary of said first DNA, said first and second sequences being in opposite orientations and ii) a spacer sequence separating said first and second sequence, such that these first and second DNA sequences are capable, when transcribed, of forming a single double-stranded RNA molecule.
  • the spacer can be a random fragment of DNA. However, preferably, one will use an intron which is spliceable by the target plant cell. Its size is generally 400 to 2000 nucleotides in length.
  • an expression cassette for an amiRNA comprises:
  • said DNA construct(s) being placed under transcriptional control of said promoter.
  • an expression cassette of the invention comprises a DNA construct targeting the OSD1 gene.
  • it comprises: a DNA construct targeting the OSD1 gene, a DNA construct targeting a gene selected among SPO11-1, SPO11-2, PRD1, and PAIR1, and a DNA construct targeting REC8.
  • promoters which are active in most tissues and cells and under most environmental conditions, as well as tissue-specific or cell-specific promoters which are active only or mainly in certain tissues or certain cell types, and inducible promoters that are activated by physical or chemical stimuli, such as those resulting from nematode infection.
  • Non-limitative examples of constitutive promoters that are commonly used in plant cells are the cauliflower mosaic virus (CaMV) 35S promoter, the Nos promoter, the rubisco promoter, the Cassaya vein Mosaic Virus (CsVMV) promoter.
  • Organ or tissue specific promoters that can be used in the present invention include in particular promoters able to confer meiosis-associated expression, such as the DMC1 promoter (KLIMYUK & JONES, Plant J, 11, 1-14, 1997); one can also use any of the endogenous promoters of the genes OSD1, SPO11-1, SPO11-2, PRD1, PAIR1, or REC8.
  • the DNA constructs of the invention generally also include a transcriptional terminator (for instance the 35S transcriptional terminator, or the nopaline synthase (Nos) transcriptional terminator).
  • a transcriptional terminator for instance the 35S transcriptional terminator, or the nopaline synthase (Nos) transcriptional terminator.
  • the invention also includes recombinant vectors containing a chimeric DNA construct of the invention.
  • said recombinant vectors also include one or more marker genes, which allow for selection of transformed hosts.
  • suitable vectors and the methods for inserting DNA constructs therein are well known to persons of ordinary skill in the art.
  • the choice of the vector depends on the intended host and on the intended method of transformation of said host.
  • a variety of methods for genetic transformation of plant cells or plants are available in the art for many plant species, dicotyledons or monocotyledons.
  • virus mediated transformation transformation by microinjection, by electroporation, microprojectile mediated transformation, Agrobacterium mediated transformation, and the like.
  • the invention also provides a host cell comprising a recombinant DNA construct of the invention.
  • Said host cell can be a prokaryotic cell, for instance an Agrobacterium cell, or a eukaryotic cell, for instance a plant cell genetically transformed by a DNA construct of the invention.
  • the construct may be transiently expressed; it can also be incorporated in a stable extrachromosomal replicon, or integrated in the chromosome.
  • said plant is a transgenic plant, and said method comprises:
  • said plant is a transgenic plant, and said method comprises:
  • said plant is a transgenic plant, and said method comprises:
  • the invention also encompasses plants able to produce SDR 2n gametes or apomeiotic gametes, obtainable by the methods of the invention.
  • said plants are transgenic plants, wherein said construct is contained in a transgene integrated in the plant genome, so that it is passed onto successive plant generations.
  • a chimeric DNA construct targeting the OSD1 gene resulting in a down regulation of the OSD1 protein, provides to said transgenic plant the ability to produce 2n SDR gametes.
  • the co-expression of a chimeric DNA construct targeting the OSD1 gene, a chimeric DNA construct targeting a gene selected among SPO11-1, SPO11-2, PRD1, and PAIR1, and a chimeric DNA construct targeting the REC8 gene results in a down regulation of the proteins encoded by these three genes and provides to said transgenic plant the ability to produce apomeiotic gametes.
  • the invention also encompasses a method for producing SDR 2n gametes, wherein said method comprises cultivating a plant obtainable by a method of the invention and recovering the gametes produced by said plant.
  • said gametes comprises at least 10%, more preferably at least 20%, and by order of increasing preference, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of viable 2n gametes.
  • the invention also encompasses a method for producing apomeiotic gametes, wherein said method comprises cultivating a plant obtainable by a method of the invention and recovering the gametes produced by said plant.
  • said gametes comprises at least 10%, more preferably at least 20%, and by order of increasing preference, at least 30%, 40%, 50%, or 60%, 70%, 80%, or 90% of viable apomeiotic gametes.
  • the present invention applies to a broad range of monocot- or dicotyledon plants of agronomical interest.
  • agronomical interest By way of non-limitative examples, one can mention potato, rice, wheat, maize, tomato, cucumbers, alfafa, sugar cane, sweet potato, manioc, clover, soybean, ray-grass, banana, melon, watermelon, cotton or ornamental plants such as roses, lilies, tulips, and narcissus.
  • Arabidopsis plants were cultivated as described in VIGNARD et al., (PLoS Genet, 3, 1894-906, 2007). For germination assays and cytometry experiments Arabidopsis were cultivated in vitro on Arabidopsis medium (ESTELLE & SOMERVILLE, Mol. Gen. Genet., 206, 200-06, 1987) at 21° C. with a 16 h day/8 h night photoperiod and 70% hygrometry.
  • ESTELLE & SOMERVILLE Mol. Gen. Genet., 206, 200-06, 1987
  • Plants were genotyped by PCR (30 cycles of 30s at 94° C., 30s at 56° C. and 1 min at 72° C.) using two primer pairs. For each genotype the primer pair is shown in Table I and the primer pair specific to the insertion is shown in Table II.
  • markers were amplified (40 cycles of 30s at 94° C., 30s at 58° C. and 30s at 72° C.) with the indicated primers and observed after migration on 3% agarose gel.
  • Meiotic spindles were observed according to the protocol described in MERCIER et al., (Genes Dev, 15, 1859-71, 2001) except that the DNA was counter-stained with DAPI. Observations were made using an SP2 Leica confocal microscope. Images were acquired with a 63 ⁇ water objective in xyz and 3D reconstructions were made using Leica software. Projections are shown. Cells were imaged at excitation 488 nm and 405 nm with AlexaFluor488 and DAPI respectively.
  • At3g57860 was selected as a good candidate due to its co-regulation with several known meiotic genes.
  • At3g57860 corresponds to the UVI4-Like gene (UVI4-L) which was briefly described in a study of its paralogue, the UVI4 gene (HASE et al., Plant J, 46, 317-26, 2006).
  • OSD1 Due to its role in meiosis (see below) we renamed the At3g57860 gene OSD1, for omission of second division.
  • OSD1 and UVI4 proteins are conserved throughout the plant kingdom but do not contain any obvious conserved known functional domains. No homologues were identified outside the plant kingdom.
  • the osd1-1 (pst15307) and the osd1-2 (GT21481) Ds insertional mutants are in the Nooseen (No-0) and Landsberg (Ler) backgrounds, respectively, and in both cases the insertion is in the second exon of the OSD1 gene.
  • the intron/exon structure of the OSD1 gene and the location of the two different Ds insertions are shown in FIG. 2 .
  • the OSD1 gene contains 3 exons and 2 introns and encodes a protein of 243 amino acids.
  • the positions of the two Ds insertions are indicated by triangles.
  • FIG. 3 represents meiosis in wild-type plants and FIG. 4 represents meiosis in osd1 mutants.
  • (H and I) Telophase II. Four haploid spores are formed (tetrad). Scale bar 10 ⁇ m
  • FIG. 4 (A and B) Male meiotic products stained with toluidine blue.
  • A A wild type tetrad.
  • B A dyad in the osd1-1 mutant.
  • C to D Male meiosis in osd1 is indistinguishable from wild type until telophase I (compare to FIG. 3 ), but no figures characteristic of a second division were observed.
  • C pachytene.
  • D diakinesis.
  • E metaphase I.
  • F Anaphase I.
  • G Telophase I.
  • H Metaphase I of female meiosis in osd1.
  • osd1 mutants Due to an absence of the second meiotic division, osd1 mutants produce high frequencies of viable diploid male and female gametophytes, which generate, after fecundation, viable tetraploid plants. However, this phenomenon differs from apomeiosis in that the produced gametes are genetically different from the mother plant.
  • Atspo11-1 and Atrec8 mutations lead to a mitotic-like first meiotic division and the osd1 mutation prevents the second meiotic division from taking place. This results in replacement of meiosis by a mitotic-like division, and in apomeiosis.
  • MiMe plants generate dyads (408/408) and are fertile (25 ⁇ 6 seeds per fruit).
  • the osd1 mutation therefore suppressed the sterility phenotype of the Atspo11-1/Atrec8 double mutant.
  • the Oriza sativa genome contains two OSD1/UVI4 homologue candidates (Os02g37850 and Os04g39670).
  • the two lines, AMBA12 and AMQF10 were genotyped by PCR to select homozygotes.
  • A Tetrad of spores in wild type
  • B Dyad of spores in AMB12.

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US20110179516A1 (en) * 2008-07-08 2011-07-21 Raphael Mercier Plants producing 2n pollen
US20120266324A1 (en) * 2011-04-15 2012-10-18 Pioneer Hi Bred International Inc. Self-Reproducing Hybrid Plants
US20140298507A1 (en) * 2010-12-01 2014-10-02 Mark D. Spiller Synthetic Clonal Reproduction Through Seeds
US9006515B2 (en) 2012-01-06 2015-04-14 Pioneer Hi Bred International Inc Pollen preferred promoters and methods of use
CN113396818A (zh) * 2021-06-23 2021-09-17 中国水稻研究所 一种利用多倍体杂种优势的植物育种方法
WO2022087616A1 (en) 2020-10-21 2022-04-28 Pioneer Hi-Bred International, Inc. Parthenogenesis factors and methods of using same
WO2024074888A2 (en) 2022-10-03 2024-04-11 The Regents Of The University Of California Circumventing barriers to hybrid crops from genetically distant crosses

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EP2510781A1 (de) 2011-04-15 2012-10-17 Institut National De La Recherche Agronomique Neue Verfahren zur Modifikation eines Pflanzenphänotyps
EP2574234A1 (de) 2011-09-29 2013-04-03 Rijk Zwaan Zaadteelt en Zaadhandel B.V. Quartettbrüten
CN106661589A (zh) 2014-06-02 2017-05-10 国家农艺研究所 在植物中产生二倍体配子的tdm基因中的显性突变
EP3292205B1 (de) * 2015-05-06 2023-08-02 Pioneer Hi-Bred International, Inc. Verfahren und zusammensetzungen zur herstellung von nichtreduzierten, nichtrekombinierten gameten und klonalen nachkommen
AU2016318051B2 (en) * 2015-09-04 2022-11-03 Keygene N.V. Diplospory gene
WO2017161264A1 (en) * 2016-03-18 2017-09-21 Pioneer Hi-Bred International, Inc. Methods and compositions for producing clonal, non-reduced, non-recombined gametes
EP3609315A4 (de) 2017-04-10 2021-01-06 The Regents of The University of California Erzeugung von haploiden pflanzen
CN109943585B (zh) * 2018-04-12 2020-05-15 中国水稻研究所 一种利用植物杂种优势的方法
CN117209577A (zh) * 2023-08-29 2023-12-12 中国科学院东北地理与农业生态研究所 一种植物减数分裂相关蛋白GmPRD1及其编码基因和应用
CN117965565A (zh) * 2024-03-28 2024-05-03 中国农业科学院生物技术研究所 蒺藜苜蓿MtPAIR1基因、基因编辑载体及其应用

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110179516A1 (en) * 2008-07-08 2011-07-21 Raphael Mercier Plants producing 2n pollen
US20140298507A1 (en) * 2010-12-01 2014-10-02 Mark D. Spiller Synthetic Clonal Reproduction Through Seeds
US20120266324A1 (en) * 2011-04-15 2012-10-18 Pioneer Hi Bred International Inc. Self-Reproducing Hybrid Plants
US9006515B2 (en) 2012-01-06 2015-04-14 Pioneer Hi Bred International Inc Pollen preferred promoters and methods of use
WO2022087616A1 (en) 2020-10-21 2022-04-28 Pioneer Hi-Bred International, Inc. Parthenogenesis factors and methods of using same
CN113396818A (zh) * 2021-06-23 2021-09-17 中国水稻研究所 一种利用多倍体杂种优势的植物育种方法
WO2024074888A2 (en) 2022-10-03 2024-04-11 The Regents Of The University Of California Circumventing barriers to hybrid crops from genetically distant crosses

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