# US 2017/0280645 A1 · Dominant mutation in the tdm gene leading to diplogametes production in plants

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- Number: US 2017/0280645 A1
- Document ID: US20170280645A1
- Status: Published application
- Kind: A1
- Application number: 15/308,807
- Filed: 2015-06-01
- Published: 2017-10-05
- Earliest claimed: 2014-06-02
- Assignees: Institut National de la Recherche Agronomique
- Applicants: Institut National De La Recherche Agronomique
- Inventors: Raphaël Mercier; Marta Cifuentes; Laurence Cromer
- Web page: https://patentgrep.com/patent/US20170280645A1

## CPC

- A01H 1/08: Methods for producing changes in chromosome number
- A01H 1/022: {Genic fertility modification, e.g. apomixis}
- A01H 4/008: {Methods for regeneration to complete plants}
- C07K 14/415: from plants
- C12N 15/8218: {Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]}
- C12N 15/8241: {Phenotypically and genetically modified plants via recombinant DNA technology}
- C12N 15/8287: {for fertility modification, e.g. apomixis}
- A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

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

## Claims

### Claim 1

A method for obtaining a plant producing Second Division Restitution 2n gametes,
wherein said method comprises providing, by random or targeted mutagenesis or by genetic transformation, a plant comprising a dominant mutation within a gene, herein designated as TDM gene, coding for a protein designated herein as TDM protein,
wherein said protein has at least 75% sequence identity with amino acid residues 1 to 286 of the TDM protein of SEQ ID NO: 1 when said plant is Brassica spp. or at least 30% identity with said residues when said plant is different from Brassica spp, and the 60 first amino acids of said protein comprise a motif X1X2X3, wherein X1 is a Threonine (T), X2 is a Proline (P), and X3 is a Proline (P) or a Glutamine (Q), designated herein as TPP/Q motif,
and wherein said dominant mutation comprises the mutation of at least one residue of the motif X1X2X3 of said TDM protein and results in the ability of the plant to produce Second Division Restitution 2n gametes.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1)

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

### Claim 4 (depends on 3)

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

### Claim 5 (depends on 1)

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

### Claim 6 (depends on 1)

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

### Claim 7 (depends on 1)

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

### Claim 8 (depends on 1)

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

### Claim 9 (depends on 8)

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

### Claim 10 (depends on 8)

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

### Claim 11 (depends on 1)

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

### Claim 12 (depends on 1)

A plant producing Second Division Restitution 2n gametes, obtainable by the method of claim 1, wherein the plant comprises said dominant mutation in the motif X1X2X3 of the TDM protein which results in the ability of the plant to produce Second Division Restitution 2n gametes.

### Claim 13 (depends on 11)

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

### Claim 14 (depends on 8)

A plant producing apomeiotic gametes, obtainable by a method of claim 8, wherein the plant comprises said dominant mutation in the motif X1X2X3 of the TDM protein which results in the ability of the plant to produce Second Division Restitution 2n gametes.

### Claim 15 (depends on 1)

A method for producing Second Division Restitution 2n gametes, wherein said method comprises cultivating a plant obtainable by a method of claim 1, wherein the plant comprises said dominant mutation in the motif X1X2X3 of the TDM protein which results in the ability of the plant to produce Second Division Restitution 2n gametes, and recovering the gametes produced by said plant.

### Claim 16 (depends on 8)

A method for producing apomeiotic gametes, wherein said method comprises cultivating a plant obtainable by a method of claim 8, wherein the plant comprises said dominant mutation in the motif X1X2X3 of the TDM protein which results in the ability of the plant to produce Second Division Restitution 2n gametes, and recovering the gametes produced by said plant.