Optimal soybean loci

• US 9,909,131 B2
• Filed: 11/03/2014
• Issued: 03/06/2018
• Est. Priority Date: 11/04/2013
• Status: Active Grant

• Current Assignee: Corteva Agriscience LLC (Corteva, Inc.)
• Original Assignee: Dow Agrosciences LLC (Corteva, Inc.)
• Inventors: Sastry-Dent, Lakshmi, Cao, Zehui, Sriram, Shreedharan, Webb, Steven R., Camper, Debra L., Ainley, W. Michael
• Primary Examiner(s): Keogh, Matthew R
• Application Number:
  US14/531,748
• Publication Number:
  US 20150128308A1
• Time in Patent Office: 1,219 Days
• Field of Search: None
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/8243 involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
• CPC Class Codes: C12N 15/8273 for drought, cold, salt resistance
• CPC Class Codes: C12N 15/8274 for herbicide resistance
• CPC Class Codes: C12N 15/8286 for insect resistance
• CPC Class Codes: Y02A 40/146 Genetically Modified [GMO] plants, e.g. transgenic plants

Abstract

As disclosed herein, optimal native genomic loci of soybean plants have been identified that represent best sites for targeted insertion of exogenous sequences.

Claims

1. A soybean plant, soybean plant part, or soybean plant cell comprising a recombinant nucleic acid molecule, said recombinant nucleic acid molecule comprising:

a nongenic nucleic acid molecule of at least 1 Kb, wherein

a. the level of methylation of said nongenic nucleic acid molecule is 1% or less;

b. the nongenic nucleic acid molecule shares less than 40% sequence identity with any other nucleic acid molecules contained in the soybean genome;

c. the nongenic nucleic acid molecule is located within a 40 Kb region of a known or predicted expressive soybean coding nucleic acid molecule; and

d. the nongenic nucleic acid molecule exhibits a recombination frequency within the soybean genome of greater than 0.01574 cM/Mb, wherein said nongenic nucleic acid molecule has at least 95% sequence identity with a nongenic nucleic acid molecule selected from the group consisting of

SEQ ID NO;

639, identified as soy_OGL_1423,

SEQ ID NO;

137, identified as soy_OGL_1434,

SEQ ID NO;

76, identified as soy_OGL_4625,

SEQ ID NO;

440, identified as soy_OGL_6362,

SEQ ID NO;

43, identified as soy_OGL_308,

SEQ ID NO;

566, identified as soy_OGL_307,

SEQ ID NO;

4236, identified as soy_OGL_310,

SEQ ID NO;

47, identified as soy_OGL_684,

SEQ ID NO;

2101, identified as soy_OGL_682, and

SEQ ID NO;

48, identified as soy_OGL_685; and

a DNA of interest, wherein the DNA of interest is inserted into said nongenic nucleic acid molecule to produce said recombinant nucleic acid molecule.

2. The soybean plant, soybean plant part, or soybean plant cell of claim 1 wherein said DNA of interest is inserted within 2 Kb, 1.75 Kb, 1.5 Kb, 1.25 Kb, 1.0 Kb, 0.75 Kb, 0.5 Kb, or 0.25 Kb of a zinc finger target site.

3. The soybean plant, soybean plant part, or soybean plant cell of claim 1, wherein said DNA of interest is inserted between a pair of zinc finger target sites.

4. The soybean plant, soybean plant part, or soybean plant cell of claim 1, wherein said DNA of interest comprises a gene expression cassette comprising an insecticidal resistance gene, herbicide tolerance gene, nitrogen use efficiency gene, water use efficiency gene, nutritional quality gene, DNA binding gene, and selectable marker gene.

5. The soybean plant, soybean plant part, or soybean plant cell of claim 1, wherein said DNA of interest comprises two or more gene expression cassettes.

6. The soybean plant, soybean plant part, or soybean plant cell of claim 1, wherein two or more of said nongenic nucleic acid molecules each comprise an inserted DNA of interest to produce two or more recombinant nucleic acid molecules wherein the two or more recombinant nucleic acid molecules are located on a same chromosome.

7. The soybean plant, soybean plant part, or soybean plant cell of claim 1, wherein said DNA of interest is modified during insertion of said DNA of interest into said nongenic nucleic acid molecule.

8. The soybean plant, soybean plant part, or soybean plant cell of claim 1 wherein the nongenic soybean genomic nucleic acid molecule has at least 95% sequence identity with a nucleic acid molecule selected from the group consisting of

SEQ ID NO:

639, identified as soy_OGL_1423,

SEQ ID NO;

137, identified as soy_OGL_1434,

SEQ ID NO;

76, identified as soy_OGL_4625,

SEQ ID NO;

440, identified as soy_OGL_6362,

SEQ ID NO;

43, identified as soy_OGL_308,

SEQ ID NO;

566, identified as soy_OGL_307, and

SEQ ID NO;

4236, identified as soy_OGL_310.

9. The soybean plant, soybean plant part, or soybean plant cell of claim 1 wherein the nongenic soybean genomic nucleic acid molecule has at least 95% sequence identity with a nucleic acid molecule selected from the group consisting of

SEQ ID NO:

43, identified as soy_OGL_308,

SEQ ID NO;

566, identified as soy_OGL_307, and

SEQ ID NO;

4236, identified as soy_OGL_310.

10. A method of making a transgenic plant cell comprising a DNA of interest, the method comprising:

a. selecting a target nongenic soybean genomic nucleic acid molecule of at least 1 Kb, wherein

i. the level of methylation of said nongenic nucleic acid molecule is 1% or less;

ii. the nongenic nucleic acid molecule shares less than 40% sequence identity with any other nucleic acid molecule contained in the soybean genome;

iii. the nongenic nucleic acid molecule is located within a 40 Kb region of a known or predicted expressive soybean coding nucleic acid molecule; and

iv. the nongenic nucleic acid molecule exhibits a recombination frequency within the soybean genome of greater than 0.01574 cM/Mb, wherein said nongenic nucleic acid molecule has at least 95% sequence identity to a nucleic acid molecule selected from the group consisting of

SEQ ID NO;

639, identified as soy_OGL_1423,

SEQ ID NO;

137, identified as soy_OGL_1434,

SEQ ID NO;

76, identified as soy_OGL_4625,

SEQ ID NO;

440, identified as soy_OGL_6362,

SEQ ID NO;

43, identified as soy_OGL_308,

SEQ ID NO;

566, identified as soy_OGL_307,

SEQ ID NO;

4236, identified as soy_OGL_310,

SEQ ID NO;

47, identified as soy_OGL_684,

SEQ ID NO;

2101, identified as soy_OGL_682, and

SEQ ID NO;

48, identified as soy_OGL_685;

b. selecting a site specific nuclease that specifically binds and cleaves said target nongenic soybean genomic nucleic acid molecule;

c. introducing said site specific nuclease into a soybean plant cell;

d. introducing the DNA of interest into the plant cell;

e. inserting the DNA of interest into said target nongenic soybean genomic nucleic acid molecule; and

,

f. selecting transgenic plant cells comprising the DNA of interest targeted to said nongenic nucleic acid molecule.

11. The method of making a transgenic plant cell of claim 10, wherein said site specific nuclease is selected from the group consisting of a zinc finger nuclease, a CRISPR nuclease, a TALEN, a homing endonuclease or a meganuclease.

12. The method of making a transgenic plant cell of claim 10, wherein said DNA of interest is integrated within said nongenic nucleic acid molecule via a homology directed repair integration method.

13. The method of making a transgenic plant cell of claim 10, wherein said DNA of interest is integrated within said nongenic nucleic acid molecule via a non-homologous end joining integration method.

14. The method of making a transgenic plant cell of claim 10, wherein two or more of said DNA of interest are inserted into two or more of said target nongenic soybean genomic nucleic acid molecules, optionally on a same chromosome.

15. The method of making a transgenic plant cell of claim 10, wherein said DNA of interest is modified during insertion of said DNA of interest into said target nongenic soybean genomic nucleic acid molecules.

16. The method of making a transgenic plant cell of claim 10, wherein the nongenic soybean genomic nucleic acid molecule has at least 95% sequence identity with a nucleic acid molecule selected from the group consisting of

SEQ ID NO:

639, identified as soy_OGL_1423,

SEQ ID NO;

137, identified as soy_OGL_1434,

SEQ ID NO;

76, identified as soy_OGL_4625,

SEQ ID NO;

440, identified as soy_OGL_6362,

SEQ ID NO;

43, identified as soy_OGL_308,

SEQ ID NO;

566, identified as soy_OGL_307, and

SEQ ID NO;

4236, identified as soy_OGL_310.

17. The method of making a transgenic plant cell of claim 10, wherein the nongenic soybean genomic nucleic acid molecule has at least 95% sequence identity with a nucleic acid molecule selected from the group consisting of

SEQ ID NO:

43, identified as soy_OGL_308,

SEQ ID NO;

566, identified as soy_OGL_307, and

SEQ ID NO;

4236, identified as soy_OGL_310.

18. A soybean plant, soybean plant part, or soybean plant cell comprising a recombinant nucleic acid molecule, said recombinant nucleic acid molecule comprising:

a nongenic nucleic acid molecule sequence selected from the group consisting of

SEQ ID NO;

639, identified as soy_OGL_1423,

SEQ ID NO;

137, identified as soy_OGL_1434,

SEQ ID NO;

76, identified as soy_OGL_4625,

SEQ ID NO;

440, identified as soy_OGL_6362,

SEQ ID NO;

43, identified as soy_OGL_308,

SEQ ID NO;

566, identified as soy_OGL_307,

SEQ ID NO;

4236, identified as soy_OGL_310,

SEQ ID NO;

47, identified as soy_OGL_684,

SEQ ID NO;

2101, identified as soy_OGL_682,

SEQ ID NO;

48, identified as soy_OGL_685, and

a DNA of interest, wherein the DNA of interest is inserted into said nongenic nucleic acid molecule.

19. The soybean plant, soybean plant part, or soybean plant cell of claim 18, wherein said nongenic nucleic acid molecule is selected from the group consisting of

SEQ ID NO:

639, identified as soy_OGL_1423,

SEQ ID NO;

43, identified as soy_OGL_308,

SEQ ID NO;

566, identified as soy_OGL_307, and

SEQ ID NO;

4236, identified as soy_OGL_310.

20. The soybean plant, soybean plant part, or soybean plant cell of claim 18, wherein said nongenic nucleic acid molecule is selected from the group consisting of

SEQ ID NO:

43, identified as soy_OGL_308,

SEQ ID NO;

566, identified as soy_OGL_307, and

SEQ ID NO;

4236, identified as soy_OGL_310.