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 comprising a mutant allele of a GA20 oxidase_3gene at an endogenous GA20 oxidase_3 locus, wherein a wild-type allele of the GA20 oxidase_3gene 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 an endogenous GA20 oxidase_5 locus, wherein a wild-type allele of the GA20 oxidase_5 gene encodes a GA20oxidase_5 protein comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 15, wherein said modified corn plant is homozygous for the mutant allele of the GA20oxidase_3 gene and homozygous for the mutant allele of the GA20 oxidase_5 gene, wherein said modified corn plant:
(a) has a 20% to 50% reduction in plant height relative to an unmodified control plant; and
(b) has an ear with no significant off-types, wherein said ear has:
(i) a number of kernels per ear that is at least 90% of the number of kernels per ear of the unmodified control plant; or
(ii) an average single kernel weight per ear that is at least 90% of the average single kernel weight per ear of the unmodified control plant.
2 . The modified corn plant of claim 1 , wherein said mutant allele of the GA20 oxidase_3 gene comprises a mutation in a sequence region selected from the group consisting of a promoter, 5′ UTR, first exon, first intron, second exon, second intron, third exon, 3′ UTR, terminator, and any combination thereof.
3 . The modified corn plant of claim 1 , wherein said mutant allele of the GA20 oxidase_3 gene 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 allele of the GA20 oxidase_3 gene.
4 . The modified corn plant of claim 1 , wherein said mutant allele of the GA20 oxidase_5 gene comprises a mutation in a sequence region selected from the group consisting of a promoter, 5′ UTR, first exon, first intron, second exon, second intron, third exon, 3′ UTR, terminator, and any combination thereof.
5 . The modified corn plant of claim 1 , wherein said mutant allele of the GA20 oxidase_5 gene 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 allele of the GA20 oxidase_5 gene.
6 . The modified corn plant of claim 1 , wherein said mutant allele of the GA20 oxidase_3 gene, said mutant allele of the GA20 oxidase_5 gene, or both comprises one or more mutations in the first exon of the corresponding gene.
7 . The modified corn plant of claim 1 , wherein said mutant allele of the GA20 oxidase_3 gene, said mutant allele of the GA20 oxidase_5 gene, or both comprises one or more mutations in the second exon of the corresponding gene.
8 . The modified corn plant of claim 1 , wherein said mutant allele of the GA20 oxidase_3 gene comprises a sequence selected from the group consisting of SEQ ID NOs: 170 to 193 or 206 to 217.
9 . The modified corn plant 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: 182 to 193 relative to the corresponding reference sequence in SEQ ID NOs: 194 to 205.
10 . The modified corn plant of claim 1 , wherein said mutant allele of the GA20 oxidase_5 gene comprises a sequence selected from the group consisting of SEQ ID NOs: 218 to 239 or 251 to 261.
11 . The modified corn plant 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: 229 to 239 relative to the corresponding reference sequence in SEQ ID NOs: 240 to 250.
12 . The modified corn plant of claim 1 , wherein said modified corn plant has improved lodging resistance relative to the unmodified control plant.
13 . The modified corn plant of claim 1 , wherein said mutant allele of the GA20 oxidase_3 gene comprises one or more mutation types selected from the group consisting of a nonsense mutation, a missense mutation, a frameshift mutation, a splice-site mutation, and any combination thereof.
14 . The modified corn plant of claim 1 , wherein said mutant allele of the GA20 oxidase_3 gene 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.
15 . The modified corn plant of claim 1 , wherein said mutant allele of the GA20 oxidase_5 gene comprises one or more mutation types selected from the group consisting of a nonsense mutation, a missense mutation, a frameshift mutation, a splice-site mutation, and any combination thereof.
16 . The modified corn plant of claim 1 , wherein said mutant allele of the GA20 oxidase_5 gene 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.
17 . The modified corn plant of claim 1 , wherein the stalk or stem diameter of said modified corn plant at one or more stem internodes is at least 5% greater than the stalk or stem diameter at the same one or more internodes of the unmodified control plant.
18 . The modified corn plant of claim 1 , wherein the stalk or stem diameter of said modified corn plant at one or more of the first, second, third, and/or fourth internode below the ear is at least 5% greater than the same internode of the unmodified control plant.
19 . The modified corn plant 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 at least 5% lower than the same internode tissue of the unmodified control plant.
20 . The modified corn plant 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 the unmodified control plant.
21 . The modified corn plant of claim 1 , wherein said modified corn plant comprises one or more traits, relative to the unmodified control plant, selected from the group consisting of improved lodging resistance, reduced green snap, deeper roots, earlier canopy closure, higher stomatal conductance, lower ear height, increased foliar water content, improved drought tolerance, improved nitrogen use efficiency, reduced anthocyanin content and area in leaves under normal or nitrogen-limiting or water-limiting stress conditions.
22 . The modified corn plant of claim 1 , wherein the mutant allele of the GA20 oxidase_3 gene and the mutant allele of the GA20 oxidase_5 gene are generated using an RNA-guided endonuclease and a guide RNA (gRNA) molecule, and wherein the gRNA molecule comprises a sequence that is at least 90% identical or complementary to one or more of SEQ ID NOs: 138 to 167.
23 . A plant part of the modified corn plant of claim 1 .
24 . The plant part of claim 23 , wherein said plant part is a meristem, a leaf, a stem, a node, a root, a flower, a floral organ structure, a seed, a fruit, a propagule, or any portion thereof.
25 . The plant part of claim 23 , wherein the said plant part is a seed.