Methods and compositions for generating complex trait loci

Patent metadata

Field	Value
Publication number	US-11312969-B2
Application number	US-201916551195-A
Country	US
Kind code	B2
Filing date	Aug 26, 2019
Priority date	Jan 27, 2012
Publication date	Apr 26, 2022
Grant date	Apr 26, 2022

Abstract

Compositions and methods are provided for stacking multiple independent transgenic loci into the genome of a plant. Compositions include plants, seeds or plant cells comprising at least one transgenic target site and at least one genomic locus of interest integrated at different genomic sites within a genomic window. Plant breeding techniques can be employed such that the transgenic target site and the genomic locus of interest can be bred together. In this way, multiple independent transgene integrations can be generated within a genomic window to create a complex trait locus. The complex trait locus is designed such that the transgenic target sites and/or genomic loci of interest can segregate independently of each other, thus providing the benefit of altering a complex trait locus by breeding-in and breeding-away specific elements. Various methods can also be employed to modify the target sites such that they contain a variety of polynucleotides of interest.

First claim

That which is claimed:

1. A maize plant or seed having in its genome a genomic window comprising at least a first transgenic target site comprising at least two non-identical FRT recombination sites, a second transgenic target site comprising at least two non-identical FRT recombination sites, and a genomic locus of interest; wherein each of said genomic locus of interest, said first transgenic target site and said second transgenic target site have different genomic sites; and wherein the genomic window is flanked by a first marker and a second marker; wherein the first marker is UMC2196, and the second marker is UMC1006.

2. The plant or seed of claim 1 , wherein the non-identical FRT recombination sites of the first transgenic target site comprise a FRT1 site, a FRT5 site, a FRT6 site, a FRT7 site, a FRT12 site, or a FRT87 site.

3. The plant or seed of claim 1 , wherein the non-identical FRT recombination sites of the second transgenic target site comprise a FRT1 site, a FRT5 site, a FRT6 site, a FRT7 site, a FRT12 site, or a FRT87 site.

4. The plant or seed of claim 1 , wherein the non-identical FRT recombination sites of the first transgenic target site comprise a FRT1 site and a FRT87 site.

5. The plant or seed of claim 1 , wherein the non-identical FRT recombination sites of second first transgenic target site comprise a FRT1 site and a FRT87 site.

6. The plant or seed of claim 1 , wherein the non-identical FRT recombination sites of the first transgenic target site comprise a FRT1 site and a FRT6 site.

7. The plant or seed of claim 1 , wherein the non-identical FRT recombination sites of second transgenic target site comprise a FRT1 site and a FRT6 site.

8. The plant or seed of claim 1 , wherein said genomic window further comprises a third transgenic target site comprising a fourth FRT recombination site, a fifth FRT recombination site and a sixth FRT recombination site, wherein
(i) said fourth, fifth and said sixth FRT recombination sites are dissimilar with respect to one another; or
(ii) said fourth, fifth and said sixth FRT recombination sites are dissimilar and have a decreased compatibility with respect to one another;
and said third transgenic target site has a different genomic insertion site than said first transgenic target site, said second transgenic target site and said genomic locus of interest.

9. The maize plant or seed of claim 1 , wherein
(a) said genomic locus of interest confers a trait comprising male sterility, site-specific recombination, abiotic stress tolerance, altered phosphorus, altered antioxidants, altered fatty acids, altered essential amino acids, altered carbohydrates, herbicide tolerance, insect resistance or disease resistance; or
(b) said genomic locus of interest comprises a transgene or a native trait.

10. The plant or seed of claim 1 , wherein said first transgenic target site or said second transgenic target site comprises at least one polynucleotide of interest.

11. A method for producing a second plant, the method comprising applying plant breeding techniques to a first plant, or parts thereof, wherein said first plant is the plant of claim 1 , and wherein application of said techniques results in the production of said second plant, wherein said second plant comprises at least one additional transgenic target site or at least one additional genomic locus of interest within said genomic window when compared to said first plant; wherein each of said additional transgenic target site and said additional genomic locus of interest have a different genomic insertion site with respect to each other and with respect to said first transgenic target site, said second transgenic target site and said genomic locus of interest.

12. The method of claim 11 , wherein the at least one additional transgenic target site comprises a polynucleotide of interest.

13. The method of claim 11 , wherein said second plant comprises at least one less transgenic target site or at least one less genomic locus of interest within said genomic window when compared to said first plant.

14. A method of producing a complex trait locus in the genome of a plant, the method comprising:
(a) providing a first plant having in its genome a genomic window comprising at least a first transgenic target site comprising at least two non-identical FRT recombination sites, and wherein said first plant does not comprise a first genomic locus of interest; wherein the genomic window is flanked by a first marker and a second marker; wherein the first marker is UMC2196, and the second marker is UMC1006;
(b) breeding to said first plant a second plant comprising at least two non-identical FRT recombination sites, wherein said second plant comprises in its genome the first genomic locus of interest and said second plant does not comprise said first transgenic target site; and,
(c) selecting a progeny plant from step (b) comprising said first transgenic target site and said genomic locus of interest;
wherein said first transgenic target site and said first genomic locus of interest have different genomic insertion sites in said progeny plant.

15. A method of producing a complex trait locus in the genome of a plant, the method comprising:
(a) providing a first plant having in its genome a genomic window at least a first transgenic target site and a second transgenic target site having different genomic insertion sites, wherein said first transgenic target site comprises at least two non-identical FRT recombination sites and said second transgenic target site comprises at least two non-identical FRT recombination sites, wherein said first plant does not comprise a first genomic locus of interest; wherein the genomic window is flanked by a first marker and a second marker; wherein the first marker is UMC2196, and the second marker is UMC1006;
(b) breeding to said first plant a second plant, wherein said second plant comprises in its genome the first genomic locus of interest, wherein said second plant does not comprise said first transgenic target site or said second transgenic target site; and,
(c) selecting a progeny plant from step (b) comprising said first transgenic target site, said second transgenic target site and said first genomic locus of interest;
wherein each of said first transgenic target site, said second transgenic target site and said first genomic locus of interest have a different genomic insertion site in said progeny plant.

16. The method of claim 15 , wherein said method further comprises (a) breeding to said progeny plant a third plant comprising a second genomic locus of interest, wherein said third plant comprises in said genomic window said second genomic locus of interest, wherein said third plant does not comprise said first transgenic target site, said second transgenic target site or said first genomic locus of interest in said genomic window; and,
(b) selecting a second progeny plant from step (a) comprising said first transgenic target site, said second transgenic target site, said first genomic locus of interest, and said second genomic locus of interest; wherein each of said first transgenic target site, said second transgenic target site, said first genomic locus of interest and said second genomic locus of interest have a different genomic insertion site in said second progeny plant.

17. The method of claim 15 , wherein
(a) the first genomic locus of interest confers a trait comprising male sterility, site-specific recombination, abiotic stress tolerance, altered phosphorus, altered antioxidants, altered fatty acids, altered essential amino acids,

Assignees

• Pioneer Hi Bred Int
• Du Pont

Inventors

• Falco Saverio Carl
• Lassner Michael
• Li Zhongsen
• Scelonge Christopher J

Classifications

• C12N9/16

  acting on ester bonds (3.1) · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N15/8213 Primary

  Targeted insertion of genes into the plant genome by homologous recombination · CPC title