Optimal maize loci

• US 10,093,940 B2
• Filed: 11/03/2014
• Issued: 10/09/2018
• 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.
• Primary Examiner(s): Keogh, Matthew R
• Application Number:
  US14/531,739
• Publication Number:
  US 20150128310A1
• Time in Patent Office: 1,436 Days
• Field of Search: None
• CPC Class Codes: A01H 5/10 Seeds
• 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

Abstract

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

Claims

1. A maize plant, maize plant part, or maize 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 maize genome;

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

d. the nongenic nucleic acid molecule exhibits a recombination frequency within the maize genome of greater than 0.00041 cM/Mb, wherein said nongenic nucleic acid molecule has at least 90% sequence identity with a nongenic nucleic acid molecule selected from the group consisting of

SEQ ID NO;

387, identified as loci_137693_G1,

SEQ ID NO;

463, identified as loci_265551_G1,

SEQ ID NO;

560, identified as loci_128078_G1,

SEQ ID NO;

573, identified as loci_168286_G1,

SEQ ID NO;

1268, identified as loci_3733_G1,

SEQ ID NO;

2030, identified as loci_203075_G1,

SEQ ID NO;

2053, identified as loci_232484_G1,

SEQ ID NO;

4425, identified as loci_136086_G1,

SEQ ID NO;

2033, identified as loci_203704_G1,

SEQ ID NO;

2709, identified as loci_127268_G1,

SEQ ID NO;

2731, identified as loci_204637_G1,

SEQ ID NO;

3230, identified as loci_291068_G1,

SEQ ID NO;

3357, identified as loci_232222_G1,

SEQ ID NO;

3428, identified as loci_43577_G1,

SEQ ID NO;

424, identified as loci_204726_G1, and

SEQ ID NO;

4529, identified as loci_232228_G1; and

a DNA of interest, wherein the DNA of interest comprises a non-native exogenous sequence, and said DNA of interest is inserted into said nongenic nucleic acid molecule to produce said recombinant nucleic acid molecule.

2. The maize plant, maize plant part, or maize plant cell of claim 1 wherein said DNA of interest is inserted within 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 specific for said nongenic sequence.

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

4. The maize plant, maize plant part, or maize plant cell of claim 1, wherein said DNA of interest comprises an analytical domain.

5. The maize plant, maize plant part, or maize plant cell of claim 1, wherein said DNA of interest does not encode a peptide.

6. The maize plant, maize plant part, or maize plant cell of claim 1, wherein said DNA of interest encodes a peptide.

7. The maize plant, maize plant part, or maize 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.

9. The maize plant, maize plant part, or maize 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.

11. The maize plant, maize plant part, or maize plant cell of claim 1 wherein said nongenic maize nucleic acid molecule is selected from the group consisting of SEQ ID NO:

2731, identified as optimal loci_204637, SEQ ID NO;

4425, identified as optimal_loci_136086, SEQ ID NO;

2053, identified as optimal_loci_232484, SEQ ID NO;

2030, identified as optimal_loci_203075, SEQ ID NO;

1268, identified as optimal_loci_3733, SEQ ID NO;

573, identified as optimal_loci_168286, SEQ ID NO;

560, identified as optimal_loci_128078, SEQ ID NO;

463, identified as optimal_loci_265551, SEQ ID NO;

2709, identified as optimal_loci_127268, SEQ ID NO;

424, identified as optimal_loci_204726, and SEQ ID NO;

3357, identified as optimal_loci_232222.

12. The maize plant, maize plant part, or maize plant cell of claim 1 wherein said nongenic sequence is selected from the group consisting of SEQ ID NO:

2731, identified as optimal_loci_204637, SEQ ID NO;

4425, identified as optimal_loci_136086, SEQ ID NO;

2053, identified as optimal_loci_232484, SEQ ID NO;

2030, identified as optimal_loci_203075, SEQ ID NO;

1268, identified as optimal_loci_3733, SEQ ID NO;

573, identified as optimal_loci_168286, SEQ ID NO;

560, identified as optimal_loci_128078 and SEQ ID NO;

463, identified as optimal_loci_265551.

13. The maize plant, maize plant part, or maize plant cell of claim 1 wherein said nongenic sequence is selected from the group consisting of SEQ ID NO:

2731, identified as optimal loci_204637, SEQ ID NO;

2030, identified as optimal_loci_203075 and SEQ ID NO;

560, identified as optimal_loci_128078.

14. The maize plant, maize plant part, or maize plant cell of claim 1 wherein said nongenic sequence is SEQ ID NO:

387, identified as optimal_loci_137693_G1.

19. The maize plant, maize plant part, or maize plant cell of claim 1, wherein said DNA of interest comprises a gene expression cassette comprising an insecticidal resistance gene, or herbicide tolerance gene.

8. A maize plant, maize plant part, or maize 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 maize genome;

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

d. the nongenic nucleic acid molecule exhibits a recombination frequency within the maize genome of greater than 0.00041 cM/Mb, wherein said nongenic nucleic acid molecule has at least 90% sequence identity with a nongenic nucleic acid molecule selected from the group consisting of

SEQ ID NO;

387, identified as loci_137693_G1,

SEQ ID NO;

463, identified as loci_265551_G1,

SEQ ID NO;

560, identified as loci_128078_G1,

SEQ ID NO;

573, identified as loci_168286_G1,

SEQ ID NO;

1268, identified as loci_3733_G1,

SEQ ID NO;

2030, identified as loci_203075_G1,

SEQ ID NO;

2053, identified as loci_232484_G1,

SEQ ID NO;

4425, identified as loci_136086_G1,

SEQ ID NO;

2033, identified as loci_203704_G1,

SEQ ID NO;

2709, identified as loci_127268_G1,

SEQ ID NO;

2731, identified as loci_204637_G1,

SEQ ID NO;

3230, identified as loci_291068_G1,

SEQ ID NO;

3357, identified as loci_232222_G1,

SEQ ID NO;

3428, identified as loci_43577_G1,

SEQ ID NO;

424, identified as loci_204726_G1, and

SEQ ID NO;

4529, identified as loci_232228_G1; and

a DNA of interest, wherein said DNA of interest comprises two or more gene expression cassettes and said DNA of interest is inserted into said nongenic nucleic acid molecule to produce said recombinant nucleic acid molecule.

10. The maize plant, maize plant part, or maize plant cell of claim 8, wherein at least one of said two or more gene expression cassettes comprises an insecticidal resistance gene or herbicide tolerance gene.

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

a. selecting a target nongenic maize 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 maize genome;

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

iv. the nongenic nucleic acid molecule exhibits a recombination frequency within the maize genome of greater than 0.00041 cM/Mb, wherein said nongenic nucleic acid molecule has at least 90% sequence identity to a nucleic acid molecule selected from the group consisting of

SEQ ID NO;

387, identified as loci_137693_G1,

SEQ ID NO;

463, identified as loci_265551_G1,

SEQ ID NO;

560, identified as loci_128078_G1,

SEQ ID NO;

573, identified as loci_168286_G1,

SEQ ID NO;

1268, identified as loci_3733_G1,

SEQ ID NO;

2030, identified as loci_203075_G1,

SEQ ID NO;

2053, identified as loci_232484_G1,

SEQ ID NO;

4425, identified as loci_136086_G1,

SEQ ID NO;

2033, identified as loci_203704_G1,

SEQ ID NO;

2709, identified as loci_127268_G1,

SEQ ID NO;

2731, identified as loci_204637_G1,

SEQ ID NO;

3230, identified as loci_291068_G1,

SEQ ID NO;

3357, identified as loci_232222_G1,

SEQ ID NO;

3428, identified as loci_43577_G1,

SEQ ID NO;

424, identified as loci_204726_G1, and

SEQ ID NO;

4529, identified as loci_232228_G1;

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

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

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

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

,

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

16. The method of making a transgenic plant cell of claim 15, wherein said DNA of interest is integrated within said nongenic locus via a homology directed repair integration method.

17. The method of making a transgenic plant cell of claim 15, wherein said DNA of interest is integrated within said nongenic locus via a non-homologous end joining integration method.

18. The method of making a transgenic plant cell of claim 15, wherein two or more of said DNA of interest are inserted into two or more of said target nongenic maize genomic loci that are located on the same chromosome.