Generation of haploid plants
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.
1. A plant having biological activity of a haploid inducer and comprising a nucleotide sequence encoding a centromere histone H3 (CENH3) protein comprising a CATD domain, wherein the nucleotide sequence comprises a mutation encoding a polypeptide having one or more amino acid substitutions at positions 2, 6, and 12 of a loop1 consensus sequence, wherein said alteration confers the biological activity of a haploid inducer, wherein the plant is homozygous with respect to the mutation and is not able to express a native CENH3 protein,
wherein the loop1 consensus sequence comprises: T, S, or A at position 1: H, Q, N, A, Y, F, G, D or E at position 2; M, Q, I, F, Y, A, E, N, R, L, H or G at position 3; L, F, V, I or Y at position 4; A, T, S, C or M at position 5; P, N, D, R, A, T, F, R, H, S or K at position 6; Q, Y, D, K, R, E, G, S, P, H, N or A at position 8; I, V or P at position 9; N, G, T, E or S at position 10; R or P at position 11; W or Y at position 12; and T, Q or S at position 13, and
wherein the plant is selected from the group consisting of Sorghum bicolor, Saccharum officinarium, Zea mays, Arabidopsis thaliana, Brassica rapa , and Brassica juncea.
2. 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.
3. The plant according to claim 1 , wherein the nucleotide sequence comprising the mutation is an endogenous gene or a transgene.
4. The plant according to claim 3 , wherein the nucleotide sequence comprising the mutation is a transgene.
5. The plant according to claim 1 , wherein the one or more amino acid substitutions comprise:
a substitution for the amino acid H, Q, N, A, Y, F, G, D or E at position 2 of the loop1 consensus sequence;
a substitution for the amino acid P, N, D, R, A, T, F, R, H, S or K at position 6 of the loop1 consensus sequence; and
a substitution for the amino acid W or Y at position 12 of the loop1 consensus sequence.
6. A part of the plant according to claim 1 comprising a mutation causing in the CATD domain an alteration of the amino acid sequence at positions 2, 6, and 12 of the loop1 sequence of the loop1 consensus sequence.
7. 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.
8. 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.
9. A method of facilitating a cytoplasm exchange, comprising the steps of:
x) crossing a plant according to 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.
10. 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 comprising said nucleotide sequence comprising said one or more mutations of the loop1 consensus sequence, wherein said substitution confers the biological activity of a haploid inducer, and wherein the plant is homozygous with respect to the mutation and is not able to express a native CENH3 protein.
11. An isolated nucleotide sequence encoding a centromere histone H3 (CENH3) protein comprising a CATD domain, wherein the nucleotide sequence encodes a polypeptide having substitutions at one or more of positions 2, 6, and 12 of a loop1 consensus sequence of SEQ ID NO: 49,
wherein the substitution at position 2 is not H, Q, N, A, Y, F, G, D or E,
wherein the substitution at position 6 is not P, N, D, R, A, T, F, R, H, S or K,
wherein the substitution at position 12 is not W or Y, and
wherein the polypeptide has the biological activity of a haploid inducer.
12. A vector comprising the nucleotide sequence of claim 11 .
13. A plant cell or a host cell comprising the nucleotide sequence of claim 11 , or a vector comprising said nucleotide sequence, as a transgene.
14. A method of generating a transgenic plant comprising the steps of:
yy) transforming a plant cell with the nucleotide sequence of claim 11 , or a vector comprising said nucleotide sequence, and
zz) regenerating a plant having the biological activity of a haploid inducer from the plant cell.
15. The part of the plant according to claim 6 , wherein the part is a shoot vegetative organ, root, flower or floral organ, seed, fruit, ovule, embryo, plant tissue or cell.
16. The plant according to claim 1 , wherein the one or more mutations comprise:
a V at position 2 of the loop1 sequence of the loop1 consensus sequence.