Methods and compositions for increasing harvestable yield via editing GA20 oxidase genes to generate short stature plants
The present disclosure provides compositions and methods for the editing or mutating of specific subtypes of GA20 oxidase genes and specific zygosity combinations of those edits or mutations. Modified plants, and plant parts and cells thereof, having mutations reducing the expression or activity of GA20 oxidase genes are further provided with improved characteristics, such as reduced plant height and increased lodging resistance, but without off-types. Methods are further provided for making modified plants, and plant parts and cells thereof, having one or more mutations in specific subtypes of GA20 oxidase genes.
1 . A modified corn plant, or plant part thereof, comprising a mutant allele of a GA20 oxidase_3 gene at the GA20 oxidase_3 locus, wherein a wild-type allele of the GA20 oxidase_3 gene encodes a GA20 oxidase_3 protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 9, and a mutant allele of a GA20 oxidase_5 gene at the GA20 oxidase_5 locus, wherein a wild-type allele of the GA20 oxidase_5 gene encodes a GA20 oxidase_5 protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 15, and wherein
(i) said modified corn plant is homozygous for the mutant allele at said GA20 oxidase_3 locus and is heterozygous for the mutant allele at said GA20 oxidase_5 locus, or
(ii) said modified corn plant is heterozygous for the mutant allele at said GA20 oxidase_3 locus and is homozygous for the mutant allele at said GA20 oxidase_5 locus, and
wherein said modified corn plant has a shorter plant height relative to a wild-type control plant.
2 . The modified corn plant, or plant part thereof, of claim 1 , wherein said modified corn plant is homozygous for the mutant allele at said GA20 oxidase_3 locus and is heterozygous for the mutant allele at said GA20 oxidase_5 locus, and wherein said GA20 oxidase_3 locus comprises a heteroallelic combination of mutant alleles or two identical mutant alleles.
3 . The modified corn plant, or plant part thereof, of claim 1 , wherein said modified corn plant is homozygous for the mutant allele at said GA20 oxidase_5 locus and is heterozygous for the mutant allele at said GA20 oxidase_3 locus, and wherein said GA20 oxidase_5 locus comprises a heteroallelic combination of mutant alleles or two identical mutant alleles.
4 . The modified corn plant, or plant part thereof, of claim 1 , wherein said mutant allele of the GA20 oxidase_3 gene or said mutant allele of said GA20 oxidase_5 gene exhibits an at least 50% reduction of expression or enzymatic activity relative to an unmodified, wild-type GA20 oxidase_3 or GA20 oxidase_5 gene allele.
5 . The modified corn plant, or plant part thereof, of claim 1 , wherein each mutant allele at the GA20 oxidase_3 locus comprises a mutation selected from the group consisting of a substitution, a deletion, an insertion, a duplication, and an inversion of one or more nucleotides relative to a wild-type GA20 oxidase_3 gene.
6 . The modified corn plant, or plant part thereof, of claim 1 , wherein each mutant allele at the GA20 oxidase_5 locus results in one or more of the following: a GA20 oxidase_5 protein truncation, a non-translatable GA20 oxidase_5 gene transcript, a non-functional GA20 oxidase_5 protein, a premature stop codon in the GA20 oxidase_5 gene, and any combination thereof.
7 . The modified corn plant, or plant part thereof, of claim 1 , wherein each mutant allele at the GA20 oxidase_5 locus comprises a mutation selected from the group consisting of a substitution, a deletion, an insertion, a duplication, and an inversion of one or more nucleotides relative to a wild-type GA20 oxidase_5 gene.
8 . The modified corn plant, or plant part thereof, of claim 1 , wherein said mutant allele of the GA20 oxidase_3 gene comprises a mutation selected from the group consisting of:
(a) a deletion of 13 bases starting at nucleotide base number 536;
(b) a deletion of nucleotide base number 542;
(c) an insertion of CC at nucleotide base number 542;
(d) a deletion of nucleotide base number 541;
(e) a deletion of 3 bases starting at nucleotide base number 540;
(f) a deletion of 2 bases starting at nucleotide base number 422;
(g) an insertion of an A at nucleotide base number 422;
(h) an insertion of a T at nucleotide base number 422;
(i) a deletion of nucleotide base number 564;
(j) an insertion of an A at nucleotide base number 564;
(k) an insertion of a C at nucleotide base number 565; and
(l) an insertion of a C at nucleotide base number 63;
wherein said nucleotide base numbering is based on SEQ ID No. 168 and counted from the first nucleotide of SEQ ID NO: 168 in the 5′ to 3′ direction.
9 . The modified corn plant, or plant part thereof, of claim 1 , wherein said mutant allele of the GA20 oxidase_3 gene comprises a mutation identified by one or more of SEQ ID Nos: 170 to 193 and 206 to 217 relative to the corresponding reference sequence in SEQ ID No: 168.
10 . The modified corn plant, or plant part thereof, of claim 1 , wherein said mutant allele of the GA20 oxidase_5 gene comprises a mutation selected from the group consisting of:
(a) a deletion of nucleotide base number 644;
(b) a deletion of 2 bases starting at nucleotide base number 644;
(c) an insertion of a T at nucleotide base number 644;
(d) a deletion of nucleotide base number 372;
(e) a deletion of nucleotide base number 786;
(f) a deletion of 5 bases starting at nucleotide base number 786;
(g) a deletion of 2 bases starting at nucleotide base number 101;
(h) an insertion of a T at nucleotide base number 102;
(i) a deletion of 3 bases starting at nucleotide base number 99;
(j) an insertion of an A at nucleotide base number 282; and
(k) an insertion of a C at nucleotide base number 282;
wherein said base numbering is based on SEQ ID No. 169 and counted from the first nucleotide of SEQ ID NO: 169 in the 5′ to 3′ direction.
11 . The modified corn plant, or plant part thereof, of claim 1 , wherein said mutant allele of the GA20 oxidase_5 gene comprises a mutation identified by one or more of SEQ ID Nos: 218 to 239 and 251 to 261 relative to the corresponding reference sequence in SEQ ID No: 169.
12 . The modified corn plant, or plant part thereof, of claim 1 , wherein said modified corn plant has an improved lodging resistance relative to an unmodified control plant.
13 . The modified corn plant, or plant part thereof, of claim 1 , wherein said modified corn plant does not have any significant off-types in at least one female organ or ear.
14 . A method of making a modified progeny corn plant, or plant part thereof, comprising:
(a) crossing a first corn plant comprising a mutant allele of a GA20 oxidase_3 gene at the GA20 oxidase_3 locus, wherein a wild-type allele of the GA20 oxidase_3 gene encodes a GA20 oxidase_3 protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 9, with a second corn plant comprising a mutant allele of a GA20 oxidase_5 gene at the GA20 oxidase_5 locus, wherein a wild-type allele of the GA20 oxidase_5 gene encodes a GA20 oxidase_5 protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 15; and
(b) selecting a progeny corn plant, or plant part thereof, from the cross in step (a) that is
(i) homozygous for one or more mutant alleles at the GA20 oxidase_3 locus and heterozygous for a mutant allele at the GA20 oxidase_5 locus, or
(ii) heterozygous for a mutant allele at the GA20 oxidase_3 locus and homozygous for one or more mutant alleles at the GA20 oxidase_5 locus,
wherein the progeny corn plant has a shorter plant height relative to a wild-type control plant.
15 . The method of claim 14 , wherein the first corn plant is:
(a1) homozygous for one or more mutant alleles at the GA20 oxidase_3 locus, or
(b1) heterozygous for a mutant allele at the GA20 oxidase_3 locus; and wherein the second corn plant is:
(a2) homozygous for one or more mutant alleles at the GA20 oxidase_5 locus, or
(b2) heterozygous for a mutant allele at the GA20 oxidase_5 locus.
16 . The modified corn plant, or plant part thereof, of claim 1 , wherein each mutant allele at the GA20 oxidase_3 locus results in one or more of the following: a GA20 oxidase_3 protein truncation, a non-translatable GA20 oxidase_3 gene transcript, a non-functional GA20 oxidase_3 protein, a premature stop codon in the GA20 oxidase_3 gene, and any combination thereof.
17 . The modified corn plant, or plant part thereof, of claim 1 , wherein said modified corn plant is at least 10% shorter than an unmodified control plant.
18 . The modified corn plant, or plant part thereof, of claim 1 , wherein said modified corn plant is at least 20% shorter than an unmodified control plant.
19 . The modified corn plant, or plant part thereof, of claim 1 , wherein said modified corn plant is at least 30% shorter than an unmodified control plant.
20 . The modified corn plant, or plant part thereof, of claim 1 , wherein the level of one or more active GAs in at least one internode tissue of the stem or stalk of said modified corn plant is lower than the same internode tissue of an unmodified control plant.
21 . The modified corn plant, or plant part thereof, of claim 1 , wherein said mutant allele of the GA20 oxidase_3 gene exhibits an at least 90% reduction of expression or enzymatic activity relative to an unmodified, wild-type GA20 oxidase_3 gene allele.
22 . The modified corn plant, or plant part thereof, of claim 1 , wherein said mutant allele of the GA20 oxidase_5 gene exhibits an at least 90% reduction of expression or enzymatic activity relative to an unmodified, wild-type GA20 oxidase_5 gene allele.
23 . The modified corn plant, or plant part thereof, of claim 1 , wherein the mutant allele of the GA20 oxidase_3 gene comprises a mutation relative to SEQ ID NO: 34.
24 . The modified corn plant, or plant part thereof, of claim 1 , wherein the mutant allele of the GA20 oxidase_3 gene comprises a mutation relative to SEQ ID NO: 168.
25 . The modified corn plant, or plant part thereof, of claim 1 , wherein the mutant allele of the GA20 oxidase_5 gene comprises a mutation relative to SEQ ID NO: 35.
26 . The modified corn plant, or plant part thereof, of claim 1 , wherein the mutant allele of the GA20 oxidase_5 gene comprises a mutation relative to SEQ ID NO: 169.
27 . The plant part of the modified corn plant of claim 1 , wherein the plant part is a corn plant organ or corn plant tissue of the modified corn plant.
28 . The plant part of the modified corn plant of claim 1 , wherein the plant part is a corn seed of the modified corn plant.
29 . The method of claim 14 , wherein the level of one or more active GAs in at least one internode tissue of the stem or stalk of said progeny corn plant is lower than the same internode tissue of an unmodified control plant.
30 . The method of claim 14 , wherein said mutant allele of the GA20 oxidase_3 gene exhibits an at least 90% reduction of expression or enzymatic activity relative to an unmodified, wild-type GA20 oxidase_3 gene allele.
31 . The method of claim 14 , wherein said mutant allele of the GA20 oxidase_5 gene exhibits an at least 90% reduction of expression or enzymatic activity relative to an unmodified, wild-type GA20 oxidase_5 gene allele.
32 . The method of claim 14 , wherein the mutant allele of the GA20 oxidase_3 gene comprises a mutation relative to SEQ ID NO: 34.
33 . The method of claim 14 , wherein the mutant allele of the GA20 oxidase_3 gene comprises a mutation relative to SEQ ID NO: 168.
34 . The method of claim 14 , wherein the mutant allele of the GA20 oxidase_5 gene comprises a mutation relative to SEQ ID NO: 35.
35 . The method of claim 14 , wherein the mutant allele of the GA20 oxidase_5 gene comprises a mutation relative to SEQ ID NO: 169.
36 . The method of claim 15 , wherein the first corn plant is homozygous for one or more mutant alleles of the GA20 oxidase_3 locus, and wherein said GA20 oxidase_3 locus comprises a heteroallelic combination of mutant alleles or two identical mutant alleles.
37 . The method of claim 15 , wherein the second corn plant is homozygous for one or more mutant alleles of the GA20 oxidase_5 locus, and wherein said GA20 oxidase_5 locus comprises a heteroallelic combination of mutant alleles or two identical mutant alleles.
38 . The method of claim 15 , wherein the first corn plant is:
(x1) homozygous for one or more mutant alleles of the GA20 oxidase_5 locus,
(y1) heterozygous for a mutant allele of the GA20 oxidase_5 locus, or
(z1) homozygous for a wild-type allele of the GA20 oxidase_5 locus; and wherein the second corn plant is:
(x2) homozygous for one or more mutant alleles of the GA20 oxidase_3 locus, or
(y2) heterozygous for a mutant allele of the GA20 oxidase_3 locus, or
(z2) homozygous for a wild-type allele of the GA20 oxidase_3 locus.