Method for removing genetic linkage in a plant
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.
1. A method for removing genetic linkage 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 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, 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 at a corresponding locus or location in the second chromosome, thereby providing a first part of the second chromosome and a second part of the second chromosome, wherein the double strand break in the first chromosome and the double strand break in the second chromosome are introduced at the same or corresponding genomic position on both of the homologous or homeologous chromosomes; and
(d) identifying, using the at least one plant cell obtained under step (c), at least one plant cell wherein the genetic linkage between the first locus A and the second locus B on the first chromosome has been removed, 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,
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; and wherein the at least one site-specific nuclease is a zinc finger nuclease, a meganuclease, a TAL-effector nuclease or a nuclease of CRISPR system introduced into the at least one plant cell of step (a).
2. The method of claim 1 , wherein in step (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 of claim 1 , wherein the double strand break in the first chromosome and the double strand break in the second chromosome is introduced by the same site-specific nuclease.
5. 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.
6. The method of claim 1 , wherein:
(i) the first chromosome comprises the first locus A and the second locus B, wherein the first locus A is linked to a desirable trait of a first character and the second locus B is linked to a undesirable trait of the first character or a second character;
(ii) wherein the second chromosome does not comprise a locus that is identical to the second locus B linked to a undesirable trait of the first character or a second character, and/or;
(iii) one double strand break is introduced between the first locus A and the second locus B on the first chromosome and at a corresponding locus or location at the second chromosome.
7. The method of claim 6 , wherein the first or second character is selected from the group consisting of yield, resistance to disease or pests, growth, size, amount of seeds, or resistance against salt, heat, cold, water or drought stress.
8. The method of claim 1 , further comprising:
(e) regenerating a plant from a plant cell obtained after step (b) 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 removal of genetic linkage.
9. The method of claim 1 , wherein the provided plant cell is a somatic plant cell.
10. The method of claim 9 , wherein the somatic plant cell is selected from the group consisting of a protoplast, and/or a plant cell obtained from a hybrid.
11. The method of claim 1 , wherein the plant is a diploid, triploid, tetraploid, pentaploid, hexaploid, octaploid, decaploid, dodecaploid or a amphidiploid.
12. A method for providing a plant P1 obtained from a plant P2, wherein the plant P2 is characterized by the presence of genetic linkage between a first locus A and a second locus B on a first chromosome, and wherein the plant P1 is characterized by the absence of the genetic linkage, the method comprising:
(a) providing at least one plant cell from the plant P2 comprising the first chromosome comprising an introgression comprising the first locus A and the second locus B and further comprising at least a second chromosome, wherein the first locus A and the second locus B exhibit linkage drag with one another, and wherein the chromosomes are homologous or homeologous chromosomes of each other;
(b) introducing a double strand break at an target sequence in the first chromosome, 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 at a corresponding locus or location in the second chromosome, thereby providing a first part of the second chromosome and a second part of the second chromosome, wherein the double strand break in the first chromosome and the double strand break in the second chromosome are introduced at the same or corresponding genomic position on both of the homologous or homeologous chromosomes;
(d) identifying, using the at least one plant cell obtained under step (c), at least one plant cell wherein the genetic linkage between the first locus A and the second locus B on the first chromosome has been removed, 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; and
(e) regenerating the plant P 1 from the at least one 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, and wherein the at least one site-specific nuclease is a zinc finger nuclease, a meganuclease, a TAL-effector nuclease or a nuclease of CRISPR system introduced into the at least one plant cell of step (a).