Optimal soybean loci
As disclosed herein, optimal native genomic loci have been identified in dicot plants, such as soybean plants, that represent best sites for targeted insertion of exogenous sequences.
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 soybean genomic sequence of at least 1 Kb determined to comprise the following characteristics:
a. the level of methylation of said nongenic sequence is 1% or less;
b. said nongenic sequence shares less than 40% sequence identity with any other sequence contained in the Glycine max genome;
c. said nongenic sequence is located within a 40 Kb region of a known or predicted expressive soybean coding sequence; and
d. said nongenic sequence exhibits a recombination frequency within the soybean genome of greater than 0.01574 cM/Mb; wherein said nongenic sequence comprises a 1 Kb sequence having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 2639, SEQ ID NO: 3682, SEQ ID NO: 4050, SEQ ID NO: 4593, SEQ ID NO: 4622, SEQ ID NO: 4879, SEQ ID NO: 4932, SEQ ID NO: 5102, SEQ ID NO: 5122, SEQ ID NO: 5520, SEQ ID NO: 5698, SEQ ID NO: 6087, SEQ ID NO: 6515, SEQ ID NO: 6571, SEQ ID NO: 6586, SEQ ID NO: 6775 and SEQ ID NO: 6935; and
a DNA of interest, wherein the DNA of interest is inserted into said nongenic sequence.
2. The soybean plant, soybean plant part, or soybean plant cell of claim 1 , wherein said nongenic sequence is 1 Kb to 5.73 Kb in length and contains no methylated cytosine residues.
3. The soybean plant, soybean plant part, or soybean plant cell of claim 1 , wherein a 40 Kb region of native soybean genome comprising said nongenic sequence also comprises at least one known or predicted soybean coding sequence, or a sequence comprising a 2 Kb upstream and/or 1 Kb downstream sequence of a known soybean gene.
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 a gene expression cassette comprising an insecticidal resistance gene or herbicide tolerance gene.
6. A method of making a transgenic plant cell comprising a DNA of interest targeted to one nongenic soybean genomic sequence, the method comprising:
a. selecting an optimal nongenic soybean genomic sequence of at least 1 Kb, wherein
said nongenic soybean genomic sequence comprises the following characteristics:
ii). the level of methylation of said nongenic sequence is 1% or less;
ii). said nongenic sequence shares less than 40% sequence identity with any other sequence contained in the Glycine max genome;
iii). said nongenic sequence is located within a 40 Kb region of a known or predicted expressive soybean coding sequence; and
iv). said nongenic sequence exhibits a recombination frequency within the soybean genome of greater than 0.01574 cM/Mb; further wherein said nongenic sequence comprises a 1 Kb sequence having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 2639, SEQ ID NO: 3682, SEQ ID NO: 4050, SEQ ID NO: 4593, SEQ ID NO: 4622, SEQ ID NO: 4879, SEQ ID NO: 4932, SEQ ID NO: 5102, SEQ ID NO: 5122, SEQ ID NO: 5520, SEQ ID NO: 5698, SEQ ID NO: 6087, SEQ ID NO: 6515, SEQ ID NO: 6571, SEQ ID NO: 6586, SEQ ID NO: 6775 and SEQ ID NO: 6935;
b. introducing a site specific nuclease into a plant cell, wherein the site specific nuclease cleaves said nongenic soybean genomic sequence;
c. introducing the DNA of interest into the plant cell;
d. targeting the DNA of interest into said nongenic locus, wherein the cleavage of said nongenic sequence facilitates integration of the polynucleotide sequence into said nongenic locus; and
e. selecting transgenic plant cells comprising the DNA of interest targeted to said nongenic locus.
7. The method of making a transgenic plant cell of claim 6 , 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.
8. The method of making a transgenic plant cell of claim 6 , wherein said DNA of interest is integrated within said nongenic locus via a homology directed repair integration method.
9. The method of making a transgenic plant cell of claim 6 , wherein said DNA of interest is integrated within said nongenic locus via a non-homologous end joining integration method.