Methods and compositions for generating complex trait loci

Patent metadata

Field	Value
Publication number	US-12146144-B2
Application number	US-202318459484-A
Country	US
Kind code	B2
Filing date	Sep 1, 2023
Priority date	Jan 27, 2012
Publication date	Nov 19, 2024
Grant date	Nov 19, 2024

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 nonidentical 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 MMP76, and the second marker is RZ444E.

2. The maize 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 maize 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 maize 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 maize 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 maize 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 maize 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 maize 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 maize 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. The maize plant or seed of claim 1 , wherein said second transgenic target site comprises
(a) the same non-identical FRT recombination sites as said first transgenic target site; or
(b) different non-identical FRT recombination sites as said first transgenic target site.

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

13. A method for producing a second maize plant, the method comprising applying plant breeding techniques to a first maize plant, or parts thereof, wherein said first maize plant is the maize plant of claim 1 , and wherein application of said techniques results in the production of said second maize plant, wherein said second maize 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 maize 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.

14. The method of claim 13 , wherein the at least one additional transgenic target site comprises a polynucleotide of interest.

15. The method of claim 13 , wherein said second maize 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 maize plant.

16. A method of producing a complex trait locus in the genome of a maize plant, the method comprising:
(a) providing a first maize 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 maize 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 MMP76, and the second marker is RZ444E;
(b) breeding to said first maize plant a second maize plant comprising at least two non-identical FRT recombination sites, wherein said second maize plant comprises in its genome the first genomic locus of interest and said second maize plant does not comprise said first transgenic target site; and,
(c) selecting a progeny maize 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 maize plant.

17. A method of producing a complex trait locus in the genome of a maize plant, the method comprising:
(a) providing a first maize 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 nonidentical FRT recombination sites and said second transgenic target site comprises at least two non-identical FRT recombination sites, wherein said first maize 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 MMP76, and the second marker is RZ444E;
(b) breeding to said first maize plant a second maize plant, wherein said second maize plant comprises in its genome the first genomic locus of interest, wherein said second maize plant does not comprise said first transgenic target site or said second transgenic target site; and,
(c) selecting a progeny maize 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 maize plant.

18. The method of claim 17 , wherein said method further comprises
(a) breeding to said progeny maize plant a third maize plant comprising a second genomic locus of interest, wherein said third maize plant comprises in said genomic window said second genomic locus of interest, wherein said third maize plant does not comprise said first transgenic target site, said second transgenic target site or said first genomic locus of interest in said genomic wind

Assignees

• Pioneer Hi Bred Int
• Corteva Agriscience Llc

Inventors

• Falco Saverio Carl
• Lassnser 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