IP Library › Granted Patent US 12,203,080
Granted Patent B2
US 12,203,080 · App. 17/142,063 · Granted Jan 21, 2025

Method for removing genetic linkage in a plant

Inventors: Paul Bundock (Wageningen, NL); Jeroen Stuurman (Wageningen, NL)
Assignee: KEYGENE N.V.
C12N15/8213A01H1/045C12N9/22C12N15/8241C07K2319/81
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Quick Facts
Patent No.
US 12,203,080
App. No.
17/142,063
Granted
Jan 21, 2025
Kind
B2
Abstract

The current disclosure relates to the field of plants, in particular to the fields of plant breeding and plant genetics. More particular, the disclosure concerns inventive methodology that may be useful in improving plant properties. In particular the invention may be useful in removing linkage drag. Also provided are plant and plant parts obtained with the method disclosed herein.

Claims (30)

1. A method of inducing a targeted translocation between a first locus A and a second locus B present on a first plant chromosome in a plant or plant cell, the method comprising:

(a) providing at least one somatic plant cell comprising:

(i) the first chromosome comprising an introgression comprising the first locus A and the second locus B, wherein the first locus A and the second locus B exhibit linkage drag with one another; and

(ii) at least a second chromosome, wherein the chromosomes are homologous or homeologous chromosomes of each other;

(b) introducing a double strand break at a target sequence in the first chromosome in the at least one somatic plant cell, wherein the double strand break in the first chromosome is introduced between the first locus A and the second locus B thereby providing a first part of the first chromosome comprising the first locus A and a second part of the first chromosome comprising the second locus B;

(c) introducing a double strand break in the second chromosome in the at least one somatic plant cell, thereby providing a first part of the second chromosome and a second part of the second chromosome; and

(d) identifying, using the at least one somatic plant cell obtained under (c), at least one modified plant cell comprising a translocation between the first locus A and the second locus B on the first chromosome, and wherein the first part of the first chromosome comprising the first locus A is ligated to the second part of the second chromosome by non-homologous end joining and not by meiotic recombination,

wherein the double strand break in the first chromosome and the double strand break in the second chromosome is introduced by at least one site-specific nuclease selected from the group consisting of a zinc finger nuclease, a meganuclease, a TAL-effector nuclease or a nuclease of CRISPR system introduced into the at least one somatic plant cell of (a).

2. The method of claim 1 , wherein in (d), the second part of the first chromosome is ligated to the first part of the second chromosome.

3. The method of claim 1 , wherein the second chromosome does not comprise a locus that is identical to the first locus A and/or does not comprise a locus that is identical to the second locus B.

4. The method according to claim 1 , wherein multiple site-specific nucleases are used for introducing the double strand break in the first chromosome and/or the double strand break in the second chromosome.

5. The method of claim 1 , wherein the double strand break in the first chromosome and the double strand break in the second chromosome are introduced by the same site-specific nuclease.

6. The method according to claim 1 , wherein locus A and/or locus B are part of a gene.

7. The method according to claim 6 , wherein locus A and/or locus B are part of a promoter.

8. The method of claim 1 , wherein no more than one double strand break is introduced in the first chromosome and no more than one double strand break is introduced in the second chromosome.

9. The method according to claim 1 , wherein the identifying of (d) comprises any one of sequencing, selective amplification of ligated parts of the first chromosome, phenotyping the offspring and fluorescent in-situ hybridization (FISH).

10. The method of claim 1 , further comprising:

(e) regenerating a plant from a somatic plant cell obtained after step (c) of claim 1 ;

(f) generating seed from the regenerated plant by selfing or crossing with another plant;

(g) growing a plant from seed obtained in step (f), and;

(h) optionally, screening the plant obtained in step (g) for targeted translocation.

11. The method of claim 1 , wherein the somatic plant cell is selected from the group consisting of a protoplast, and/or a plant cell obtained from a hybrid.

12. The method of claim 1 , wherein the plant is a diploid, triploid, tetraploid, pentaploid, hexaploid, octaploid, decaploid, dodecaploid or a amphidiploid.

13. A method for providing a plant P1 obtained from a plant P2, wherein plant P2 is a F1 hybrid produced by crossing two parent plants, wherein the plant P2 is characterized by the presence of a first locus A and a second locus B on a first chromosome, and wherein the plant P1 is characterized by the presence of a targeted translocation between the first locus A and the second locus B, the method comprising:

(a) providing at least one somatic plant cell from the plant P2 comprising the first chromosome comprising an introgression comprising the first locus A and the second locus B, wherein the first locus A and the second locus B exhibit linkage drag with one another, and further comprising at least a second chromosome, wherein the chromosomes are homologous or homeologous chromosomes of each other;

(b) introducing a double strand break at a target sequence in the first chromosome in the at least one somatic plant cell, wherein the double strand break in the first chromosome is introduced between the first locus A and the second locus B, thereby providing a first part of the first chromosome comprising the first locus A and a second part of the first chromosome comprising the second locus B;

(c) introducing a double strand break in the second chromosome in the at least one somatic plant cell, thereby providing a first part of the second chromosome and a second part of the second chromosome;

(d) identifying, using the at least one somatic plant cell obtained under step (c), at least one modified plant cell comprising a translocation between the first locus A and the second locus B on the first chromosome, and further, wherein the first part of the first chromosome comprising the first locus A is ligated to the second part of the second chromosome by non-homologous end joining and not by meiotic recombination; and

(e) regenerating the plant P1 from the at least one modified plant cell identified in (d);

wherein the double strand break in the first chromosome and the double strand break in the second chromosome is introduced by at least one site-specific nuclease selected from the group consisting of a zinc finger nuclease, a meganuclease, a TAL-effector nuclease or a nuclease of CRISPR system introduced into the at least somatic one plant cell of (a).

Continuity (3)
Continuation 14655633
Provisional Application 61746399 · Dec 27, 2012
Related Publication 20210123068A1 · Apr 29, 2021
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