WO2024098012A2 - Brassica ind gene mutations conferring improved pod shatter-resistance - Google Patents

Brassica ind gene mutations conferring improved pod shatter-resistance Download PDF

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WO2024098012A2
WO2024098012A2 PCT/US2023/078712 US2023078712W WO2024098012A2 WO 2024098012 A2 WO2024098012 A2 WO 2024098012A2 US 2023078712 W US2023078712 W US 2023078712W WO 2024098012 A2 WO2024098012 A2 WO 2024098012A2
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seq
plant
ind
mutation
brassica
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PCT/US2023/078712
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French (fr)
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Ratan Chopra
Mark Messmer
Rai M. KRISHAN
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Covercress Inc.
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses

Definitions

  • Loss-of-function mutations in an INDEHISCENT (IND) gene which encodes an atypical basic helix-loop-helix transcription factor can provide a reduced pod-shatter phenotype in Arabidopsis (Liljegren et al., 2004, Cell 116, 843-853).
  • Previously disclosed methods to achieve this trait include generation of point mutations in an INDEHISCENT (IND) gene through chemical mutagenesis, reducing expression of an IND gene using RNAi, or other geneediting based techniques for introducing mutations in an IND gene (US Patent Nos. 7,528,294 and 8809635; US Patent Applic. Pub. Nos.
  • Methods of obtaining Brassica plants with at least one mutant IND gene copy which can confer a pod shatter resistance trait comprising: (i) crossing a Brassica plant comprising at least one wild-type IND gene with a Brassica plant comprising at least one mutant ind gene encoding a mutant ind protein wherein one or more amino acid residues located C- terminal to the conserved basic Helix-Loop Helix (bHLH) domain of said mutant ind protein are substituted and/or deleted; and (ii) isolating Fl seed and/or Fl progeny plants comprising the mutant ind gene from the cross are provided.
  • bHLH conserved basic Helix-Loop Helix
  • Methods of obtaining Brassica plants with at least one mutant ind gene which can confer a pod shatter resistance trait comprising introducing a mutation in one or more nucleotides of at least one IND gene of a Brassica plant to obtain a Brassica plant comprising a mutant ind gene encoding a mutant ind protein wherein one or more amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of said mutant ind protein are substituted and/or deleted are provided.
  • bHLH conserved basic Helix-Loop Helix
  • Brassica plants and plant parts comprising at least one mutant ind gene encoding a mutant ind protein wherein one or more amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of said mutant ind protein are substituted and/or deleted, wherein the Brassica plant or plant part is not a Thlaspi arvense plant are provided.
  • bHLH conserved basic Helix-Loop Helix
  • Thlaspi arvense plants and plant parts comprising a mutant ind gene encoding a mutant ind protein wherein one or more amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of said mutant ind protein are substituted and/or deleted, wherein the Thlaspi arvense plants comprise two genomic DNA sequence polymorphisms which are not found together in any single naturally occurring Thlaspi arvense isolate are provided.
  • bHLH conserved basic Helix-Loop Helix
  • FIG 1 shows the results of field tests in Mt. Pulaski Illinois (“MPT’; left-most columns in each set of wild-type, indl-4 heterozygotes, and indl-4 homozygotes) and Havana, Illinois (“HVI”; right-most columns in each set of wild-type, indl-4 heterozygotes, and indl-4 homozygotes) that were evaluated in the spring of 2022.
  • MPT Pulaski Illinois
  • HVI Havana, Illinois
  • Figure 2 shows the result of evaluating pod shatter resistance in a greenhouse with wild-type, indl-4 heterozygotes, and indl-4 homozygotes.
  • Figure 3 A-E shows the alignment of genomic or coding sequences of the indicated Brassica IND genes.
  • the region highlighted in this alignment in boldface and underlining represents the bHLH domain and region with dashed underlining in the C-terminal protein coding region identifying a region which can be targeted with guide RNAs and Cas9 nucleases in gene editing methods.
  • Figure 4A-B shows the alignment of polypeptide sequences of the indicated Brassica IND proteins.
  • the region highlighted in this alignment in boldface and underlining represents the bHLH domain and the C-terminal region where amino acid residues are in lower case and italicized with dashed underlining represents the amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain.
  • a * symbol is located above the six C- terminal amino acids of the indicated Brassica IND proteins which comprise the consensus amino acid sequence YHNS(Q/D)(P/S/T) (SEQ ID NO: 32).
  • Figure 5 shows the T. arvense INDI DNA coding sequence (SEQ ID NO: 2) with the initiator methionine (Ml) codon through to the C -terminal threonine 182 (T182) codon and stop codon as well as the T. arvense INDI polypeptide (SEQ ID NO: 12).
  • Figure 6A shows the B. carinata INDla DNA coding sequence (SEQ ID NO: 4) with the initiator methionine (Ml) codon through to the C -terminal threonine 195 (T195) codon and stop codon as well as the IND polypeptide (SEQ ID NO: 14).
  • Figure 6B shows the B. carinata IND-b DNA coding sequence (SEQ ID NO: 5) with the initiator methionine (Ml) codon through to the C-terminal threonine 192 (T192) codon and stop codon as well as the IND polypeptide (SEQ ID NO: 15).
  • Figure 7A shows the C. sativa IND-a DNA coding sequence (SEQ ID NO: 6) with the initiator methionine (Ml) codon through to the C -terminal serine 167 (SI 67) codon and stop codon as well as the encoded IND polypeptide (SEQ ID NO: 16).
  • Figure 7B shows the C. sativa IND-b DNA coding sequence (SEQ ID NO: 7) with the initiator methionine (Ml) codon through to the C -terminal serine 167 (SI 67) codon and stop codon as well as the encoded IND polypeptide (SEQ ID NO: 17).
  • Figure 7C shows the C.
  • sativa IND-c DNA coding sequence (SEQ ID NO: 8) with the initiator methionine (Ml) codon through to the C -terminal serine 168 (SI 68) codon and stop codon as well as the encoded IND polypeptide (SEQ ID NO: 18).
  • Figure 8 shows the B. napus IND-a DNA coding sequence (SEQ ID NO: 9) with the initiator methionine (Ml) codon through to the C -terminal threoninel78 (T178) codon and stop codon as well as the encoded IND-a polypeptide (SEQ ID NO: 19).
  • Figure 9A and B show an alignment of DNA molecules for wild-type (TalNDI WT, SEQ ID NO: 2) and pARV55 (TaINDl_PSl)-Cas9 -generated alleles (B56924A1(+A) (SEQ ID NO: 33); B56914A5(+G) (SEQ ID NO: 34); B56930A5(+T) (SEQ ID NO: 35)).
  • Figure 10 shows an alignment of protein molecules for wild-type (TalNDI WT (SEQ ID NO: 12)) and pARV55 (TaINDl_PSl)-Cas9 -generated alleles (B56924A1(+A) (SEQ ID NO: 43); B56914A5(+G) (SEQ ID NO: 44); B56930A5(+T) (SEQ ID NO: 45)).
  • Figure 11 A and B show an alignment of DNA molecules for pennycress wild-type (TalNDI WT (SEQ ID NO: 2) and pARV56 (TaINDl_PS2)-Cas9 -generated alleles (B56929A1(+A) (SEQ ID NO: 36); B56922A5(+C) (SEQ ID NO: 37); B56941A1(+T) (SEQ ID NO: 38).
  • Figure 12 shows an alignment of protein molecules for penny cress wild-type (TalNDI WT (SEQ ID NO: 12) and pARV56 (TaINDl_PS2)-Cas9 -generated alleles (B56929A1(+A) (SEQ ID NO: 46); B56922A5(+C) (SEQ ID NO: 47); B56941A1(+T) (SEQ ID NO: 48)).
  • Figure 13 A and B show an alignment of DNA molecules for penny cress wild-type (TalNDI WT (SEQ ID NO: 2) and pARV58 (TaINDl_PS4)-Cas9 -generated alleles (B57581B1(+A) (SEQ ID NO: 39) and B57582A4(+T) (SEQ ID NO: 40).
  • Figure 14 shows an alignment of protein molecules for penny cress wild-type (TalNDI WT (SEQ ID NO: 12) and pARV58 (TaINDl_PS4)-Cas9 -generated alleles (B57581B1 (+A) (SEQ ID NO: 49) and B57582A4(+T) (SEQ ID NO: 50).
  • Figure 15A and B show an alignment of DNA molecules for penny cress wild-type (TalNDI WT (SEQ ID NO: 2) and pARV59 (TaINDl_PS5)-Cas9 -generated alleles B57663B2 (+A) (SEQ ID NO: 41) and B57449A1 (-19) (SEQ ID NO: 42).
  • Figure 16 shows an alignment of protein molecules for penny cress wild-type (TalNDI WT (SEQ ID NO: 12) and pARV59 (TaINDl_PS5)-Cas9 -generated B57663B2 (+A) (SEQ ID NO: 51) and B57449Al (-19) (SEQ ID NO: 52).
  • Figure 17A and B show penny cress reduced pod shattering reduction by the indl- 4 (SEQ ID NO: 73) or Cas9 edited INDI alleles depicted in Figures 9, 10, 11, and 12.
  • Fig. 17A shows measurement of force required to break the pennycress pods comprising the indl-4 mutation (2032:WG; SEQ ID NO: 73) or Cas9 edited INDI alleles compared to the wild-type genotypes B3:WG and B28:WG.
  • 17B shows a comparison of desirable cell separation layers observed in 2032-WG comprising the indl-4 mutation (2032:WG; SEQ ID NO: 73) and B56929A1 compared to the wild-type layers observed in the control genotype of B28:WG by toluidine blue staining of cross-sections.
  • Figure 18 A and B show an alignment of DNA molecules for Arabidopsis wild-type (AtINDl (SEQ ID NO: 1) and pARV56 (TaINDl_PS2)-Cas9 -generated alleles (At_indl_Pl(+A) (SEQ ID NO: 53); At_indl_P2(+T) (SEQ ID NO: 54); At_indl_P3(-34) (SEQ ID NO: 55)).
  • Figure 19 shows an alignment of protein molecules for Arabidopsis wild-type (AtINDl (SEQ ID NO: 11) and a pARV56 (TaINDl_PS2)-Cas9 -generated alleles (At_indl_Pl(+A) (SEQ ID NO: 58); At_indl_P2(+T) (SEQ ID NO: 59); (At_indl_P3(-34) (SEQ ID NO: 60)).
  • Figure 20 shows an alignment of DNA molecules for Arabidopsis wild-type (AtINDl (SEQ ID NO: 1) and a pARV72 (At_Cs_PS3)-Cas9 -generated allele (At_indl_P4(+A) (SEQ ID NO: 56).
  • Figure 21 shows an alignment of protein molecules for Arabidopsis wild-type (AtINDl (SEQ ID NO: 11) and a pARV72 (At_Cs_PS3)-Cas9 -generated allele (At_indl_P4(+A) (SEQ ID NO: 61).
  • Figure 22 shows an alignment of DNA molecules for Arabidopsis wild-type (AtINDl (SEQ ID NO: 1) and a pARV73 (At_Cs_PS4)-Cas9 -generated allele (At_indl_P5(+G) (SEQ ID NO: 57).
  • Figure 23 shows an alignment of protein molecules for Arabidopsis wild-type (AtINDl (SEQ ID NO: 11) and a pARV73 (At_Cs_PS4)-Cas9 -generated allele (At_indl_P5(+G) (SEQ ID NO: 62).
  • Figure 24A, B, and C show Arabidopsis reduced pod shattering optimization by a Cas9 edited INDI allele depicted in Figures 18 and 19.
  • Fig. 24A shows pods of a control and indl mutant At_indl_Pl(+A) (SEQ ID NO: 53 and 58) subjected to force.
  • Fig. 24B shows percentage of pods shattered for At_indl_Pl(+A) (SEQ ID NO: 53 and 58) Arabidopsis pods compared to the wild-type pods following subjection to force.
  • Fig. 24C shows a comparison of desirable cell separation layers observed in the At_indl_Pl(+A) (SEQ ID NO: 53 and 58) allele compared to the wild-type Col-0 control.
  • Figure 25 A, B, and C show an alignment of DNA molecules for Camelina sativa wild-type (Cs_INDl_Chr2_WT (SEQ ID NO: 6); CsINDl_Chr8_WT (SEQ ID NO: 7); and CsINDl_Chrl3_WT (SEQ ID NO: 8)) and pARV56 (TaINDl_PS2) generated alleles (C10012D7_Cs_Chr2(+A) (SEQ ID NO: 63); C10012D7_Cs_Chr8(+A) (SEQ ID NO: 64); C10012D7_Cs_Chrl3(+A) (SEQ ID NO: 65); C10012D10_Cs_Chr2(+T) (SEQ ID NO: 66); C10012D10_Cs_Chr2(+A) (SEQ ID NO: 63): C10012D10_Cs_Chr8(+T) (SEQ ID NO:
  • Figure 26 shows an alignment of protein molecules for Camelina sativa wild-type (Cs_INDl_Chr2_WT (SEQ ID NO: 16); CsINDl_Chr8_WT (SEQ ID NO: 17); and CsINDl_Chrl3_WT (SEQ ID NO: 18)) and pARV56 (TaINDl_PS2) generated alleles (C10012D7_Cs_Chr2(+A) (SEQ ID NO: 68); C10012D7_Cs_Chr8(+A) (SEQ ID NO: 69); C10012D7_Cs_Chrl3(+A) (SEQ ID NO: 70); C10012D10_Cs_Chr2(+T) (SEQ ID NO: 71); C10012D10_Cs_Chr2(+A) (SEQ ID NO: 68): C10012D10_Cs_Chr8(+T) (SEQ ID NO: 72); and
  • alleles refers to alternate forms of a DNA sequence at a genetic locus, that is, a position on a chromosome of a gene or other chromosome marker.
  • Brainssica plant refers to a plant in the family Brassicaceae.
  • a pairwise sequence alignment algorithm e.g, CLUSTAL O 1.2.4 with default parameters.
  • cultivar refers to a plant that has been cultivated.
  • a cultivar is generally developed using crossing, selfing, and/or selection and is maintained by any suitable method of propagation, through open pollination, selfing, or the like. Details of cultivar development can be found in “Principles of Cultivar Development” by Fehr, Macmillan Publishing Company (1993), which is herein incorporated by reference in its entirety.
  • elite and “elite line” refer to any line that has resulted from breeding and selection for desirable agronomic performance (typically commercial production). Generally, individuals in a line have similar parentage and one or more similar traits.
  • an "elite line” or “elite variety” can be an agronomically superior line or variety that has resulted from many cycles of breeding and selection for superior agronomic performance.
  • An "elite inbred line” is an elite line that is an inbred, and that has been shown to be useful for producing sufficiently high yielding and agronomically fit hybrid varieties (an “elite hybrid variety”).
  • elite germplasm is a germplasm resulting from breeding and selection for desirable agronomic performance (typically commercial production). Such germplasm may be agronomically superior germplasm, derived from and/or capable of giving rise to a plant with superior agronomic performance, such as an existing or newly developed elite line of a Brassica plant.
  • a “frameshift mutation” refers to a mutation in a nucleic acid sequence that results in the wild-type reading frame being shifted to different reading frame such that translation of an mRNA having a frameshift mutation results in a departure from the wildtype reading frame.
  • nonsense mutation refers to any mutation that results in the appearance of a stop codon where previously there was a codon specifying an amino acid. The presence of this premature stop codon results in the production of a truncated protein. Stop codons include TAA, TAG, and TGA codons.
  • amorphic allele refers to an allele of a gene having no gene activity in comparison to the wild-type allele of the gene. Amorphic alleles are also known as null or “knock-out” alleles.
  • isomorphic allele refers to an allele of a gene having wild-type gene activity.
  • hypomorphic allele refers to an allele of a gene having reduced or partially reduced but not null gene activity in comparison to the wild-type allele of the gene.
  • a hypomorphic allele can exhibit a subset of the phenotypes observed in an amorphic allele.
  • An example of an hypomorphic ind gene allele includes an ind allele which exhibits reduced pod shatter but does not also exhibit undesirable agronomic traits characteristic of amorphic ind alleles.
  • Gene activity comprises any measure of gene function.
  • Gene activity measures thus include measures of gene-mediated phenotypes e.g., pod shatter resistance and undesirable agronomic traits) and/or gene-encoded protein activity (e.g., transcriptional activation activity).
  • transcriptional activation activity can be assayed by monitoring expression of genes that are regulated by the gene with transcriptional activation activity.
  • IND refers to a wild-type INDEHISCENT gene or protein while the term “ind” refers to a mutant INDEHISCENT gene or protein.
  • the terms “include,” “includes,” and “including” are to be construed as at least having the features to which they refer while not excluding any additional unspecified features.
  • plant includes a whole plant and any descendant, cell, tissue, or part of a plant.
  • plant parts include any part(s) of a plant, including, for example and without limitation: seed (including mature seed and immature seed); a plant cutting; a plant cell; a plant cell culture; or a plant organ (e.g., pollen, embryos, flowers, fruits, shoots, leaves, roots, stems, and explants).
  • a plant tissue or plant organ may be a seed, protoplast, callus, or any other group of plant cells that is organized into a structural or functional unit.
  • a plant cell or tissue culture may be capable of regenerating a plant having the physiological and morphological characteristics of the plant from which the cell or tissue was obtained, and of regenerating a plant having substantially the same genotype as the plant.
  • Regenerable cells in a plant cell or tissue culture may be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, flowers, pods, or stalks.
  • some plant cells are not capable of being regenerated to produce plants and are referred to herein as “non-regenerable” plant cells.
  • variable means a group of similar plants that by structural features, genetic features, and/or performance which can be distinguished from other varieties within the same species.
  • the term variety refers to the botanical taxonomic designation whereby variety is ranked below species or subspecies, as well as the legal definition whereby the term “variety” refers to a commercial plant that is protected under the terms outlined in the International Convention for the Protection of New Varieties of Plants.
  • iPSR pod shatter resistant Brassica
  • iPSR Brassica plant cells, plant propagules, or plant parts which include seeds are also provided.
  • Such desirable ind alleles when in the homozygous state, can result in Brassica plants with useful pod shatter resistance traits while exhibiting improved agronomic performance in comparison to a control plant homozygous for an amorphic allele of the ind gene.
  • Such improved agronomic performance can include traits such as better threshability.
  • methods of obtaining iPSR Brassica by breeding, gene editing, and/or mutagenesis are also provided herein.
  • methods of using the iPSR Brassica including methods of harvesting seed from Brassica crops comprising the iPSR Brassica.
  • Desirable ind alleles provided herein which can confer an improved Pod Shatter Resistance (iPSR) phenotype include ind gene alleles comprising mutations encoding an ind protein where at least 5 or 6 of the amino acid residues located at the C-terminal end of a wild-type Brassica IND protein are deleted and/or substituted with different amino acid residues (e.g., including non-conservative amino acid substitutions).
  • the wild-type IND C-terminal six amino acids comprise the consensus amino acid sequence YHNS(Q/D)(P/S/T) (SEQ ID NO: 32).
  • iPSR mutations or “iPSR alleles” which can provide an iPSR Brassica plant
  • iPSR mutations or “iPSR alleles”
  • Table 1 Examples of the types of mutations which can provide an iPSR Brassica plant
  • Combinations of such iPSR mutations which include substitutions and deletions of at least the wild-type IND C-terminal five or six amino acids are also contemplated.
  • such mutations can be located or introduced at a target Brassica IND gene codon corresponding to the codon of the first N-terminal amino acid of the 18 amino acid C- terminal fragment located immediately C-terminal to the bHLH domain (e.g., the glycine codon in Figure 4) through all following C-terminal amino acid residues to the codon for the tyrosine or histidine residue of the SEQ ID NO: 19 consensus sequence.
  • Brassica plants and the corresponding IND genes which can contain or be made to contain such iPSR mutations include: (i) Brassica napus plants wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 9 or an allelic variant thereof or encodes a protein comprising the polypeptide of SEQ ID NO: 19 or an allelic variant thereof; (ii) Brassica carinata plants wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 4, SEQ ID NO: 5, or an allelic variant thereof or encodes a protein comprising the polypeptide of SEQ ID NO: 14, SEQ ID NO: 15, or an allelic variant thereof; (iii) a Camelina sativa plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or an allelic variant thereof or encodes a protein comprising the polypeptide of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:
  • such iPSR mutations encode an ind protein wherein at least 6 to 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 18 amino acid residues comprising the C-terminal consensus sequence of SEQ ID NO: 32 located at the C-terminus of a wild-type IND gene or an allelic variant thereof are deleted and/or are substituted with non-conservative amino acid residues.
  • Brassica plants and the corresponding ind genes which can encode such iPSR mutations include: (i) Brassica napus plants wherein the mutated ind gene encodes a mutant ind protein wherein the final 5 or 6 to 7, 8, 10, or 18 C-terminal amino acid residues of the wild-type IND protein comprising the polypeptide of SEQ ID NO: 19 or an allelic variant thereof are deleted and/or substituted (e.g., with a non-conservative amino acid residue) in the mutant ind protein; (ii) Brassica carinata plants wherein the mutated ind gene(s) encode a mutant ind protein wherein the final 5 or 6 to 7, 8, 10, or 18 C-terminal amino acid residues of the wild-type IND protein comprising the polypeptide of SEQ ID NO: 14, SEQ ID NO: 15, and/or an allelic variant thereof are deleted and/or substituted (e.g., with a non-conservative amino acid residue) in the mutant ind protein; (iii) a Came
  • certain Brassica IND coding sequences that can be targeted for introduction of iPSR mutations can include the wild-type IND coding sequences set forth in SEQ ID NO: 1-9 as well as other wild-type IND coding sequences with substantial sequence identity thereto (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1-9).
  • Allelic variants of wild-type IND genes found in distinct Brassica isolates or varieties that exhibit wild-type pod-shatter phenotypes can be targeted for introduction of iPSR mutations.
  • Introduction and fixation of the iPSR mutations in a homozygous state in such Brassica plants comprising wild-type IND coding sequences set forth in SEQ ID NO: 1-9, wildtype IND coding sequences with substantial sequence identity thereto, or allelic variants thereof that exhibit wild-type pod-shatter phenotypes can provide Brassica plants, seeds, seed lots, and plants exhibiting improved pod-shatter resistance in comparison to control Brassica plants comprising wild-type IND coding sequences set forth in SEQ ID NO: 1-9, wild-type IND coding sequences with substantial sequence identity thereto, or allelic variants thereof.
  • such wild-type IND allelic variants can comprise polynucleotide sequences that have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity across the entire length of the polynucleotide sequences of the wild-type IND coding regions of SEQ ID NO: 2, 4, 5, 6, 7, 8, or 9.
  • such allelic variants can also encode polypeptide sequences that have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity across the entire length of the polypeptide sequence of SEQ ID NO: 12, 14, 15, 16, 18, or 19.
  • iPSR mutations in the INDI gene of T. arvense are fixed in a homozygous state.
  • Such iPSR mutations include mutations in an ind gene encoding a mutant ind protein wherein one or more amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of said mutant ind protein are substituted and/or deleted (e.g,. any of the aforementioned mutations set forth in Table 1).
  • bHLH conserved basic Helix-Loop Helix
  • arvense include: (i) frameshift and/or nonsense mutations introduced in SEQ ID NO: 2 or an allelic variant thereof at a codon corresponding to G165 (GGA) through to H178 (CAC) of SEQ ID NO: 2 ( Figure 5); (ii) frameshift and/or nonsense mutations introduced in SEQ ID NO: 2 or an allelic variant thereof at a codon corresponding to D170 to anyone one of Y176, Y177, and/or H178 (CAC) of SEQ ID NO: 2; (iii) frame shift and/or nonsense mutations introduced in SEQ ID NO: 2 or an allelic variant thereof at a codon corresponding to SI 72, Cl 73, LI 74, or Cl 75 to anyone one of Y176, Y177, and/or H178 (CAC) of SEQ ID NO: 2; (iv) frame shift and/or nonsense mutations introduced in SEQ ID NO: 2 or an allelic variant thereof at a codon corresponding to Y176, Y177,
  • such iPSR mutations encode an ind protein wherein 1, 2, 3, 4, 5, or 6 to 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 18 amino acid residues located at the C-terminus of SEQ ID NO: 12 or an allelic variant thereof (e.g., amino acids encompassed by the C-terminal consensus sequence of SEQ ID NO: 32) are deleted and/or substituted with non-conservative amino acid residues (e.g,. any of the aforementioned mutations set forth in Table 1).
  • allelic variant thereof e.g., amino acids encompassed by the C-terminal consensus sequence of SEQ ID NO: 32
  • such iPSR mutations encode an ind protein wherein 6 to 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 18 amino acid residues comprising the C- terminal consensus sequence of SEQ ID NO: 32 located at the C-terminus of SEQ ID NO: 12 or an allelic variant thereof are deleted and/or substituted with non-conservative amino acid residues.
  • Such iPSR mutations can be introduced into a T. arvense plant by gene editing and/or breedingbased techniques.
  • iPSR mutations in the INDI gene of T. arvense include: (i) mutations encoding a T. arvense ind protein lacking 10 C-terminal amino acids of the wildtype INDI gene of T.
  • arvense e.g., the indl gene of SEQ ID NO: 33, the indl protein of SEQ ID NO: 43, and allelic variants thereof encoding or comprising indl proteins lacking the 10 C- terminal amino acids of the INDI protein wherein the allelic variants have at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 33 and/or SEQ ID NO: 43); (ii) mutations encoding a T. arvense ind protein lacking 8 C-terminal amino acids of the wild-type INDI gene of T.
  • arvense e.g., the indl gene of SEQ ID NO: 36, the indl protein of SEQ ID NO: 46, and allelic variants thereof encoding or comprising indl proteins lacking the 8 C-terminal amino acids of the INDI protein wherein the allelic variants have at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 36 and/or SEQ ID NO: 46); and (iii) mutations encoding a T. arvense ind protein lacking 6 C-terminal amino acids of the wild-type INDI gene of T.
  • the T. arvense plant comprising the iPSR mutation is: (i) a non-naturally occurring T. arvense plant; (ii) an elite T. arvense plant; (iii) a T.
  • T. arvense plant variety (iv) a T. arvense plant comprising two genomic DNA sequence polymorphisms which are not found together in any single naturally occurring T. arvense isolate; (v) a T. arvense plant which lacks a black seed trait (i.e., has yellow or brown seed in comparison to the black seed of Thlaspi arvense cultivar 2032), lacks a high fiber seed trait (i.e., has low fiber content seed in comparison to wild-type or cultivar 2032 T.
  • black seed trait i.e., has yellow or brown seed in comparison to the black seed of Thlaspi arvense cultivar 2032
  • high fiber seed trait i.e., has low fiber content seed in comparison to wild-type or cultivar 2032 T.
  • arvense lacks a reduced yield trait (i.e., exhibits increased seed yield in comparison to Thlaspi arvense cultivar 2032), and/or lacks an increased lodging trait (i.e., exhibits decreased lodging in comparison to Thlaspi arvense cultivar 2032); (vi) a T. arvense plant which lacks one or more genetic polymorphisms characteristic of the wild-type Thlaspi arvense cultivar 2032, representative seed of the cultivar having been deposited under NCMA Accession Number 202210002; (vii) a T.
  • the T. arvense plant which lacks one or more traits characteristic of the wild-type Thlaspi arvense cultivar 2032, representative seed of the cultivar having been deposited under NCMA Accession Number 202210002; and/or (viii) a T. arvense plant which is not Thlaspi arvense cultivar 2032, representative seed of the cultivar having been deposited under NCMA Accession Number 202210002.
  • Traits characteristic of Thlaspi arvense cultivar 2032 include black seed, high fiber seed trait, reduced yield, and/or increased lodging.
  • the T. arvense plant comprising any of the aforementioned the iPSR mutations is a non-naturally occurring T. arvense plant or an elite T.
  • arvense plant cultivar comprising a yellow/brown seed with low fiber seed trait (e.g., a yellow/brown seed coat color and low fiber content seed trait set forth in US Patent No. 10,709,151, incorporated herein by reference in its entirety) and/or comprising a low glucosinolate seed content trait (e.g., as set forth in US Patent No. 10,988,772, incorporated herein by reference in its entirety).
  • a yellow/brown seed with low fiber seed trait e.g., a yellow/brown seed coat color and low fiber content seed trait set forth in US Patent No. 10,709,151, incorporated herein by reference in its entirety
  • a low glucosinolate seed content trait e.g., as set forth in US Patent No. 10,988,772, incorporated herein by reference in its entirety.
  • Thlaspi arvense cultivar 2032 A deposit of at least 625 seeds of the Thlaspi arvense cultivar 2032 has been made with the Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA) at Bigelow Laboratory for Ocean Sciences at 60 Bigelow Drive, East Boothbay, Maine 04544.
  • NCMA Provasoli-Guillard National Center for Marine Algae and Microbiota
  • the Thlaspi arvense cultivar 2032 seeds have been given the Accession Number 202210002 by the NCMA as the International Depository Authority.
  • the seeds deposited with the NCMA on October 6, 2022, for Thlaspi arvense cultivar 2032 were harvested by hand in March 2020 from the CoverCress greenhouse at 1249 N. Warson Rd., St. Louis, Missouri 63132 prior to the filing date of this application.
  • Applicant has satisfied all the requirements of37 C.F.R. ⁇ 1.801-1.809, including providing an indication of the viability of the sample upon deposit. Applicant has no authority to waive any restrictions imposed by law on the transfer of biological material or its transportation in commerce. Applicant(s) do not waive any infringement of their rights granted under this patent or rights applicable to Thlaspi arvense cultivar 2032 under the Plant Variety Protection Act (7 USC 2321 et seq.).
  • Brassica carinata i.e., B. carinata
  • plants comprising an iPSR trait
  • iPSR mutations in the IND la and/or IND lb gene of B. carinata are fixed in a homozygous state. Wild-type B.
  • carinata plants typically comprise: (i) a wild-type IND la gene comprising the polynucleotide sequence of SEQ ID NO: 4 or an allelic variants thereof which encode the IND la protein of SEQ ID NO: 14 or an allelic variants thereof; and (ii) a wild-type IND lb gene comprising the polynucleotide sequence of SEQ ID NO: 5 or an allelic variant thereof which encode the IND-b protein of SEQ ID NO: 15 or an allelic variant thereof.
  • iPSR . carinata plants provided herein can comprise an iPSR mutation in the IND la and IND lb gene which are both fixed in the homozygous state.
  • iPSR B is a wild-type IND la gene comprising the polynucleotide sequence of SEQ ID NO: 4 or an allelic variants thereof which encode the IND la protein of SEQ ID NO: 14 or an allelic variants thereof.
  • carinata plants provided herein can comprise an iPSR mutation in the IND la gene while the IND-b gene comprises an amorphic or hypomorphic allele, and where both of the IND la and ind-b alleles are fixed in the homozygous state.
  • iPSR B. carinata plants provided herein can comprise an iPSR mutation in the IND-b gene while the IND la gene comprises an amorphic or hypomorphic allele, and where both of the IND la and ind-b alleles are fixed in the homozygous state.
  • Amorphic alleles of the IND la and IND lb gene include mutations in the bHLH domain or other mutations in those genes such as nonsense or frameshift mutations in codons for amino acid residues located N-terminal to the bHLH domain.
  • Hypomorphic alleles include those analogous to hypomorphic alleles of B. napus IND genes set forth in US Patent No. 8,809,635, which is incorporated herein by reference in its entirety.
  • the wild-type IND la and IND lb protein of B. carinata comprises a full length carboxy -terminal sequence of 18 amino acid residues which includes the C-terminal 6 amino acid consensus sequence of SEQ ID NO: 32 ( Figure 4B).
  • Such iPSR mutations include mutations in an indla and/or indlb gene encoding a mutant indla and/or indlb protein wherein one or more amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of the mutant indla and/or indlb protein are substituted and/or deleted (e.g., any of the aforementioned mutations set forth in Table 1).
  • bHLH conserved basic Helix-Loop Helix
  • carinata include: (i) frameshift and/or nonsense mutations introduced in SEQ ID NO: 4 or an allelic variant thereof at a codon corresponding to G178 (GGG) through to H191 (CAC) of SEQ ID NO: 4 ( Figure 6A) or frameshift and/or nonsense mutations introduced in SEQ ID NO: 5 or an allelic variant thereof at a codon corresponding to G175 (GGA) through to H188 (CAC) of SEQ ID NO: 5 ( Figure 6B); (ii) frameshift and/or nonsense mutations introduced in SEQ ID NO: 4 or an allelic variant thereof at a codon corresponding to D183 to anyone one of Y189, Y190, and/or H191 (CAC) of SEQ ID NO: 4 or frameshift and/or nonsense mutations in SEQ ID NO: 5 or an allelic variant thereof at a codon corresponding to DI 80 to anyone one of Y186, Y187, and/or Hl 88 (CAC) of SEQ ID NO:
  • such iPSR mutations encode an ind protein wherein 1, 2, 3, 4, 5, or 6 to 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 18 amino acid residues introduced at the C-terminus of SEQ ID NO: 14 or 15 or an allelic variant thereof (e.g., amino acids encompassed by the C-terminal consensus sequence of SEQ ID NO: 32) are deleted and/or substituted with non-conservative amino acid residues (e.g,. any of the aforementioned mutations set forth in Table 1).
  • allelic variant thereof e.g., amino acids encompassed by the C-terminal consensus sequence of SEQ ID NO: 32
  • such iPSR mutations encode an ind protein wherein 6 to 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 18 amino acid residues comprising the C-terminal consensus sequence of SEQ ID NO: 32 introduced at the C-terminus of SEQ ID NO: 14, 15, or an allelic variant thereof are deleted and/or substituted with non-conservative amino acid residues.
  • Such iPSR mutations can be introduced into a B. carinata plant by gene editing and/or breeding-based techniques.
  • the B. carinata plant comprising the iPSR mutation is: (i) a non-naturally occurring B. carinata plant; (ii) an elite B. carinata plant; and/or (iii) a 7>. carinata plant variety.
  • Cs-Gl hexapioid genome
  • Cs-G2 Cs-G2
  • Cs-G3 Kagale et al., Nat Commun 5, 3706 (2014). doi.org/10.1038/ncomms4706).
  • iPSR mutations in at least one of: (i) the IND-a gene of C.
  • sativa comprising the coding sequence of SEQ ID NO: 6 or an allelic variant thereof; (ii) the IND-b gene SEQ ID NO: 7 or an allelic variant thereof; and/or (iii) the IND-c gene comprising the coding sequence of SEQ ID NO: 8 are fixed in a homozygous state. Wild-type C.
  • sativa plants typically comprise: (i) a wild-type IND-a gene comprising the polynucleotide sequence of SEQ ID NO: 6 or an allelic variants thereof which encode the IND protein of SEQ ID NO: 16 or an allelic variants thereof; (ii) a wild-type IND-b gene comprising the polynucleotide sequence of SEQ ID NO: 7 or an allelic variant thereof, where SEQ ID NO: 7 encodes the IND protein of SEQ ID NO: 17; and (ii) a wild-type IND-c gene which encodes the IND-c protein of SEQ ID NO: 18 or an allelic variant thereof.
  • the wild-type IND proteins of C comprising the polynucleotide sequence of SEQ ID NO: 6 or an allelic variants thereof which encode the IND protein of SEQ ID NO: 16 or an allelic variants thereof.
  • a wild-type IND-b gene comprising the polynucleotide sequence of SEQ ID NO: 7 or an
  • iPSR C. sativa plants provided herein can comprise an iPSR mutation in the IND-a, IND-b, and IND-c gene which are all fixed in the homozygous state. In certain embodiments, iPSR C.
  • the sativa plants provided herein can comprise an iPSR mutation in at least one of the IND-a, IND-b, or IND-c genes while the remaining IND genes comprise an amorphic or hypomorphic allele, and where all of the ind-a, ind-b, and ind-c alleles are fixed in the homozygous state.
  • the iPSR C. sativa plants provided herein can comprise an iPSR mutation in at least two of the IND-a, IND-b, and IND-c genes, wherein any of the remaining IND-a, IND-b gene, or IND-c genes comprises an amorphic or hypomorphic mutation and wherein all mutations are fixed in the homozygous state.
  • iPSR C. sativa plants provided herein can comprise an iPSR mutation in the IND-a gene (e.g., in SEQ ID NO: 6 or an allelic variant thereof) which is fixed in the homozygous state, a mutation comprising an amorphic or hypomorphic allele of the IND-b gene (e.g., in SEQ ID NO: 7 or an allelic variant thereof) which is fixed in the homozygous state, and an amorphic or hypomorphic allele of the IND-c gene (e.g., in SEQ ID NO: 8 or an allelic variant thereof) which is fixed in a homozygous state.
  • sativa plants provided herein can comprise an iPSR mutation in the IND-b gene (e.g., in SEQ ID NO: 7 or an allelic variant thereof) which is fixed in the homozygous state, a mutation comprising an amorphic or hypomorphic allele of the IND-a gene e.g., in SEQ ID NO: 6 or an allelic variant thereof) which is fixed in the homozygous state, and an amorphic or hypomorphic allele of the IND-c gene (e.g., in SEQ ID NO: 8 or an allelic variant thereof) which is fixed in a homozygous state.
  • iPSR C e.g., in SEQ ID NO: 7 or an allelic variant thereof
  • sativa plants provided herein can comprise an iPSR mutation in the IND-c gene e.g., in SEQ ID NO: 8 or an allelic variant thereof) which is fixed in the homozygous state, a mutation comprising an amorphic or hypomorphic allele of the IND-a gene (e.g., in SEQ ID NO: 6 or an allelic variant thereof) which is fixed in the homozygous state, and an amorphic or hypomorphic allele of the IND-b gene (e.g., in SEQ ID NO: 7 or an allelic variant thereof) which is fixed in a homozygous state.
  • an iPSR mutation in the IND-c gene e.g., in SEQ ID NO: 8 or an allelic variant thereof
  • a mutation comprising an amorphic or hypomorphic allele of the IND-a gene e.g., in SEQ ID NO: 6 or an allelic variant thereof
  • Amorphic alleles of the IND-a, IND-b, and IND-c gene include mutations in the bHLH domain or other mutations in those genes such as nonsense or frameshift mutations in codons for amino acid residues located N-terminal to the bHLH domain.
  • Hypomorphic alleles include those analogous to hypomorphic alleles of B. napus IND genes set forth in US Patent No. 8,809,635, which is incorporated herein by reference in its entirety.
  • Such iPSR mutations include mutations in a C.
  • sativa ind-a, ind-b, and/or ind-c gene encoding a mutant ind protein wherein one or more amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of said mutant ind-a protein are substituted and/or deleted (e.g., any of the aforementioned mutations set forth in Table 1).
  • bHLH conserved basic Helix-Loop Helix
  • sativa include: (i) frameshift and/or nonsense mutations introduced in SEQ ID NO: 6, 7, 8, or an allelic variant thereof at a codon corresponding to G150 (GGA) through to H163 (CAC) of SEQ ID NO: 6 or 7 or at a codon corresponding to G151 (GGA) through to Hl 64 (CAC) of SEQ ID NO: 8 ( Figure 7A,B,C); (ii) frameshift and/or nonsense mutations introduced in SEQ ID NO: 6, 7, or an allelic variant thereof at a codon corresponding to Al 54 (GCT)to anyone one of Y161 (TAT), Y162 (TAC), and/or H163 (CAC) of SEQ ID NO: 6 or 7 or introduced in SEQ ID NO: 8 or an allelic variant thereof at a codon corresponding to Al 55 (GCT) to anyone one of Y162 (TAT), Y163 (TAC), and/or Hl 64 (CAC) of SEQ ID NO: 8;
  • such iPSR mutations encode an ind protein wherein 1, 2, 3, 4, 5, or 6 to 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 18 amino acid residues located at the C- terminus of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or an allelic variant thereof (e.g., amino acids encompassed by the C-terminal consensus sequence of SEQ ID NO: 32) are deleted and/or substituted with non-conservative amino acid residues (e.g,. any of the aforementioned mutations set forth in Table 1).
  • allelic variant thereof e.g., amino acids encompassed by the C-terminal consensus sequence of SEQ ID NO: 32
  • such iPSR mutations encode an ind protein wherein 6 to 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 18 amino acid residues comprising the C-terminal consensus sequence of SEQ ID NO: 32 located at the C-terminus of SEQ ID NO: 16, 17, 18, or an allelic variant thereof are deleted and/or substituted with nonconservative amino acid residues.
  • such iPSR mutations encode a C. sativa indla protein lacking 8 C-terminal amino acids of the wild-type INDla gene of C.
  • sativa e.g., the indl-a gene of SEQ ID NO: 63, the indl-a protein of SEQ ID NO: 68, and allelic variants thereof encoding or comprising an indla protein lacking the 8 C-terminal amino acids of the INDl-a protein wherein the allelic variants have at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 63 and/or SEQ ID NO: 68).
  • such iPSR mutations encode a C. sativa indl-b protein lacking 8 C-terminal amino acids of the wild-type INDlb gene of C.
  • sativa e.g., the indl-b gene of SEQ ID NO: 64, the indlb protein of SEQ ID NO: 69, and allelic variants thereof encoding or comprising an indl-b protein lacking the 8 C-terminal amino acids of the INDl-b protein wherein the allelic variants have at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 64 and/or SEQ ID NO: 69).
  • such iPSR mutations encode a C. -sativa indl-c protein lacking 8 C-terminal amino acids of the wild-type INDl-c gene of C.
  • sativa e.g., the indie gene of SEQ ID NO: 65, the indlb protein of SEQ ID NO: 70, and allelic variants thereof encoding or comprising an indie protein lacking the 8 C- terminal amino acids of the INDl-c protein wherein the allelic variants have at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 65 and/or SEQ ID NO: 70).
  • Such iPSR mutations can be introduced into a C. sativa plant by gene editing and/or breeding-based techniques.
  • the C. sativa plant comprising the iPSR mutation is: (i) a non- naturally occurring C. sativa plant; (ii) an elite C.
  • sativa plant (iii) a C. sativa plant variety; and/or (iv) a C. sativa plant comprising two genomic DNA sequence polymorphisms which are not found together in any single naturally occurring C. sativa isolate.
  • Wild-type B. napus plants typically comprise: (i) a wildtype IND-a gene comprising the polynucleotide sequence of SEQ ID NO: 9 or an allelic variants thereof which encode the IND-a protein of SEQ ID NO: 19 or an allelic variants thereof; and (ii) a wild-type IND-b gene comprising the polynucleotide sequence of SEQ ID NO: 10 or an allelic variant thereof.
  • the wild-type IND-a protein of B typically comprise: (i) a wildtype IND-a gene comprising the polynucleotide sequence of SEQ ID NO: 9 or an allelic variants thereof which encode the IND-a protein of SEQ ID NO: 19 or an allelic variants thereof; and (ii) a wild-type IND-b gene comprising the polynucleotide sequence of SEQ ID NO: 10 or an allelic variant thereof.
  • iPSR . napus plants provided herein can comprise an iPSR mutation in the IND- a gene which is fixed in the homozygous state and a wild-type IND-b gene which is fixed in a homozygous state.
  • napus plants provided herein can comprise an iPSR mutation in the IND-a gene which is fixed in the homozygous state and a mutation comprising an amorphic or hypomorphic allele of the IND-b gene which is fixed in a homozygous state.
  • Amorphic alleles of the IND-b gene include mutations in the bHLH domain or other mutations in those genes such as nonsense or frameshift mutations in codons for amino acid residues located N-terminal to the bHLH domain and include mutations in the IND-b gene set forth in US Patent No. 8,809,635, which is incorporated herein by reference in its entirety.
  • hypomorphic alleles of the IND-b gene include mutations in the bHLH domain or other mutations in the IND-b gene set forth in US Patent No. 8,809,635, which is incorporated herein by reference in its entirety.
  • Such iPSR mutations include mutations in an ind-a gene encoding a mutant ind-a protein wherein one or more amino acid residues located C-terminal to the conserved basic Helix- Loop Helix (bHLH) domain of said mutant ind-a protein are substituted and/or deleted (e.g., any of the aforementioned mutations set forth in Table 1).
  • iPSR mutations in the INDI -a gene of B are substituted and/or deleted (e.g., any of the aforementioned mutations set forth in Table 1).
  • napus include: (i) frameshift and/or nonsense mutations introduced in SEQ ID NO: 9 or an allelic variant thereof at a codon corresponding to G161 (GGG) through to H174 (CAC) of SEQ ID NO: 9 ( Figure 8); (ii) frameshift and/or nonsense mutations introduced in SEQ ID NO: 9 or an allelic variant thereof at a codon corresponding to S165 to anyone one of Y172 (TAT), Y173 (TAC), and/or H174 (CAC) of SEQ ID NO: 9; (iii) frame shift and/or nonsense mutations introduced in SEQ ID NO: 9 or an allelic variant thereof at a codon corresponding to D166, P167, S168, R169.
  • such iPSR mutations are characteristic in that they encode an ind protein wherein 1, 2, 3, 4, 5, or 6 to 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 18 amino acid residues introduced at the C- terminus of SEQ ID NO: 19 or an allelic variant thereof (e.g., amino acids encompassed by the C- terminal consensus sequence of SEQ ID NO: 32) are deleted and/or substituted with nonconservative amino acid residues (e.g,. any of the aforementioned mutations set forth in Table 1).
  • allelic variant thereof e.g., amino acids encompassed by the C- terminal consensus sequence of SEQ ID NO: 32
  • such iPSR mutations encode an ind protein wherein 6 to 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 18 amino acid residues comprising the C-terminal consensus sequence of SEQ ID NO: 32 introduced at the C-terminus of SEQ ID NO: 19 or an allelic variant thereof are deleted and/or substituted with non-conservative amino acid residues.
  • Such iPSR mutations can be introduced into a B. napus plant by gene editing and/or breeding-based techniques.
  • the B. napus plant comprising the iPSR mutation is: (i) a non- naturally occurring B. napus plant; (ii) an elite B. napus plant; (iii) a B.
  • B. napus plant variety (iv) a B. napus plant comprising two genomic DNA sequence polymorphisms which are not found together in any single naturally occurring B. napus isolate; and/or (iv) a B. napus plant which lacks a black seed trait (i.e., has yellow or brown seed) or lacks a high fiber seed trait.
  • B. napus plants include Brassica napus L. subsp. Napus, Brassica napus L. sub sp. napus forma annua, Brassica napus subsp. rapifera Metzg, and Brassica napus L. N ax . pabular ia plants.
  • Such aforementioned B. napus plants are also alternatively referred to as Brassica campe str is and Brassica oleracea plants.
  • Methods of obtaining Brassica plants with at least one mutant ind gene which can confer a pod shatter resistance trait by plant breeding methods are also provided herein.
  • the methods can comprise crossing a Brassica plant comprising at least one wildtype IND gene with a Brassica plant comprising at least one mutant ind gene containing an iPSR mutation; and (ii) isolating Fl seed and/or Fl progeny plants comprising the mutant ind gene from the cross.
  • Such Fl seed and/or Fl progeny can be identified and isolated by non-destructively assaying for the presence of the ind mutation in a nucleic acid detection assay (e.g., a nucleic acid hybridization-, amplification-, and/or sequencing-based assay for the presence of the iPSR mutation in the IND gene(s)).
  • a nucleic acid detection assay e.g., a nucleic acid hybridization-, amplification-, and/or sequencing-based assay for the presence of the iPSR mutation in the IND gene(s)
  • Non-destructive assays can be accomplished by sampling a portion of the Brassica plant or plant part while leaving plant parts necessary for seed production and/or seed viability intact.
  • the methods further comprise crossing the isolated Fl progeny plant comprising the mutant ind gene to a recurrent parent Brassica plant comprising a wild-type IND gene and isolating F2 seed and/or F2 progeny comprising the mutant ind gene and one or more genetic markers of the recurrent parent plant.
  • Examples of methods for obtaining new Brassica varieties or elite germplasm containing the iPSR mutations can be achieved by introgressing the iPSR mutation from a first Brassica line into the genetic background of a Brassica varieties or elite germplasm lacking the iPSR trait by using the Brassica varieties or elite germplasm as a recurrent parent in a series of backcrosses, where progeny which contain the iPSR mutation are selected (e.g., by a non-destructive nucleic acid assay) and carried forward into additional crosses to the recurrent parent.
  • the progeny plant containing the iPSR mutation can be selfed to fix the iPSR mutation in the homozygous state and obtain a new Brassica plant variety or elite germplasm exhibiting the iPSR trait.
  • linked and unlinked genetic markers which are characteristic of Brassica varieties and elite germplasm can include phenotypic markers: (i) yellow seed (e.g., US Patent Applic. Pub. No.
  • 20190082718, incorporated herein by reference in its entirety seed with low fiber content (e.g., US Patent Applic. Pub. No. 20190082718, incorporated herein by reference in its entirety); (iii) seed with reduced glucosinolate content (e.g., US Patent Applic. Pub. No. 20190225977, incorporated herein by reference in its entirety); (iv) seed with reduced erucic acid content (e.g., US Patent Applic. Pub. No. 20200131523, incorporated herein by reference in its entirety); (v) an early transition trait; and/or (vi) a lodging trait.
  • low fiber content e.g., US Patent Applic. Pub. No. 20190082718, incorporated herein by reference in its entirety
  • seed with reduced glucosinolate content e.g., US Patent Applic. Pub. No. 20190225977, incorporated herein by reference in its entirety
  • seed with reduced erucic acid content e.g., US
  • Such linked and unlinked genetic markers which are characteristic of Brassica varieties and elite germplasm can include genotypic markers (e.g., genomic DNA sequence polymorphisms such as single nucleotide polymorphisms (SNPs), simple sequence repeats (SSR), DNA insertions, DNA deletions, and/or DNA inversions). Markers and associated methods that can be adapted for use in identifying, breeding, and/or introgressing iPSR mutations disclosed herein in various Brassica plants (e.g., Brassica napus, Brassica juncea, Brassica carinata, Brassica rapa (syn. B. campestris), Brassica oleracea, and Brassica nigra) include those disclosed in US Patent Applic. Pub.
  • genotypic markers e.g., genomic DNA sequence polymorphisms such as single nucleotide polymorphisms (SNPs), simple sequence repeats (SSR), DNA insertions, DNA deletions, and/or DNA inversions.
  • T. arvense germplasm doi.org/10.1186/sl2915-021-01079-0, can be used to introgress iPSR mutations into different T. arvense germplasm and/or to identify T. arvense plants provided herein which comprises two genomic DNA sequence polymorphisms which are not found together in any single naturally occurring Thlaspi arvense isolate.
  • Methods of obtaining a Brassica plants comprising the iPSR mutations comprising introducing a mutation in one or more nucleotides of at least one wild-type IND gene of a Brassica plant are also provided.
  • the iPSR mutations can be introduced into one or more of the wild-type IND genes by a variety of methods. Methods for introduction of the iPSR mutations include, but are not limited to, traditional mutagenesis (e.g., Ethyl Methane Sulfonate (EMS), fast neutrons (FN), or gamma rays), and TILLING.
  • EMS Ethyl Methane Sulfonate
  • FN fast neutrons
  • gamma rays gamma rays
  • the iPSR mutation results from introduction of a DSB at a target site in the IND gene (e.g., SEQ ID NO: 2, 4, 5, 6, 7, 8, 9, 10 or an allelic variant thereof in the region encoding the 18 amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of the IND gene as shown in Figure 3) to induce non-homologous end joining (NHEJ) at the site of the break followed by recovery of desired iPSR mutants.
  • a target site in the IND gene e.g., SEQ ID NO: 2, 4, 5, 6, 7, 8, 9, 10 or an allelic variant thereof in the region encoding the 18 amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of the IND gene as shown in Figure 3
  • NHEJ non-homologous end joining
  • the iPSR mutation results from introduction of a DSB at a target site in the IND gene (e.g., SEQ ID NO: 2, 4, 5, 6, 7, 8, 9, 10 or an allelic variant thereof in the region encoding the 18 amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of the IND gene as shown in Figure 3) followed by homology-directed repair (HDR), microhomology-mediated end joining (MMEJ), or NHEJ to introduce a desired donor DNA template polynucleotide at the DSB, followed by recovery of the iPSR mutation.
  • a target site in the IND gene e.g., SEQ ID NO: 2, 4, 5, 6, 7, 8, 9, 10 or an allelic variant thereof in the region encoding the 18 amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of the IND gene as shown in Figure 3
  • HDR homology-directed repair
  • MMEJ microhomology-
  • Methods for introduction of the DSB and iPSR mutations also include use of gene editing reagents which can comprise meganucleases, zinc finger nucleases, transcription activatorlike effector nucleases (TALENS), clustered regularly interspaced short palindromic repeat (CRISPR)-associated Cas nuclease (e.g., Cas9, Casl2a, Cmsl, S.
  • gene editing reagents which can comprise meganucleases, zinc finger nucleases, transcription activatorlike effector nucleases (TALENS), clustered regularly interspaced short palindromic repeat (CRISPR)-associated Cas nuclease (e.g., Cas9, Casl2a, Cmsl, S.
  • TALENS transcription activatorlike effector nucleases
  • CRISPR clustered regularly interspaced short palindromic repeat
  • aureus Cas9 variants a Cas9, a nCas9 nickase, a type V Cas nuclease, a Casl2a nuclease, a nCasl2a nickase, a Casl2d (CasY), a Casl2e (CasX), a Casl2b (C2cl), a Casl2c (C2c3), a Casl2i, a Casl2f, a Casl2j, a Casl4, or eSpCas9 nuclease) in combination with guide RNAs, and the like.
  • Methods where the aforementioned gene editing reagents, and in particular, CRISPR/Cas systems comprising a Cas nuclease and a guide RNA directed to nucleotide sequences encoding the final 18 C-terminal amino acids of the IND gene are contemplated.
  • Methods for modifying genomes by use of Cpfl or Csml nucleases are disclosed in US Patent Application Publication 20180148735, which is incorporated herein by reference in its entirety, can be adapted for introduction of the iPSR mutations disclosed herein.
  • PAM sequences include 5’-NGG (Streptococcus pyogenes), 5’-NNAGAA (Streptococcus thermophilus CRISPR1), 5’-NGGNG (Streptococcus thermophilus CRISPR3), 5’-NNGRRT or 5’-NNGRR (Staphylococcus aureus Cas9, SaCas9), and 5’-NNNGATT (Neisseria meningitidis).
  • T-rich PAM sites e.g., 5’-TTN or 5’-TTTV, where "V" is A, C, or G
  • V is A, C, or G
  • Casl2a can also recognize a 5’-CTA PAM motif.
  • Other examples of potential Cas 12a PAM sequences include TTN, CTN, TCN, CCN, TTTN, TCTN, TTCN, CTTN, ATTN, TCCN, TTGN, GTTN, CCCN, CCTN, TTAN, TCGN, CTCN, ACTN, GCTN, TCAN, GCCN, and CCGN (wherein N is defined as any nucleotide).
  • nucleotide substitutions at a target site in the IND gene include base-editing systems (e.g., CBE (cytosine base editors) or ABE (adenine base editors)).
  • Such CBE and ABE systems can be adapted for use in making iPSR mutations including nonsense mutations which introduce a premature “stop” codon in the in the region encoding the 18 amino acid residues located C-terminal to the conserved basic Helix- Loop Helix (bHLH) domain of the IND gene.
  • Examples of ABE and CBE systems which can be adapted for use in making iPSR mutations disclosed herein include those disclosed in Nat Biotechnol 39, 917 (2021). doi.org/10.1038/s41587-021-01015-l, and in US20170121693, US20210130805, and WO2020214842, which are each incorporated herein by reference in their entireties.
  • the genome editing reagents described herein can be introduced into a pennycress plant by any appropriate method.
  • nucleic acids encoding the genome editing reagents can be introduced into a plant cell using Agrobacterium- or Ensifer mediated transformation, particle bombardment, liposome delivery, nanoparticle delivery, electroporation, polyethylene glycol (PEG) transformation, or any other method suitable for introducing a nucleic acid into a plant cell.
  • the Site-Specific Nuclease (SSN) or other expressed gene editing reagents can be delivered as RNAs or as proteins to a plant cell and the RT, if one is used, can be delivered as DNA.
  • Brassica plants comprising the iPSR phenotypes can be identified by a variety of techniques that distinguish plants with pod shatter resistance from plants which are prone to pod shatter (e.g., plants comprising a wild-type IND gene).
  • pod shatter in iPSR plants and suitable controls e.g., plants lacking an iPSR allele or containing a wild-type IND gene
  • suitable controls e.g., plants lacking an iPSR allele or containing a wild-type IND gene
  • controlled environments e.g., growth chambers and/or greenhouses
  • Methods of measuring pod shatter include: (i) percent shattered pods determination by visual evaluation of plants (US20220298519, incorporated herein by reference in its entirety) where a reduction in the percent of shattered pods in comparison to a control indicates pod shatter resistance; and (ii) measurement of weight of seeds dropped per unit area under the plant preharvest and/or during harvest (US20190053458, incorporated herein by reference in its entirety).
  • Methods of measuring pod shatter resistance also include: (i) methods which measure the effect of mechanical force on pod shatter (e.g., measurement of the effect of mechanical agitation for specific speeds and times on pod shatter as disclosed US20220298519, incorporated herein by reference in its entirety) where a reduction in the percent of shattered pods in comparison to a control indicates pod shatter resistance; and (ii) measurement of the amount of force required to cause pod opening (e.g., use of a gram force meter to measure force needed to break open a pod US20190053458, incorporated herein by reference in its entirety) where an increase in the amount of force required is indicative of pod shatter resistance.
  • methods which measure the effect of mechanical force on pod shatter e.g., measurement of the effect of mechanical agitation for specific speeds and times on pod shatter as disclosed US20220298519, incorporated herein by reference in its entirety
  • measurement of the amount of force required to cause pod opening e.g., use of a gram force meter to
  • Brassica plants, Brassica plant parts, Brassica seed, and Brassica seed lots comprising the aforementioned or otherwise disclosed iPSR mutations are also provided herein.
  • seed lots comprising a population of Brassica plant seed comprising untreated or treated Brassica seed are provided.
  • populations of seed in the seed lots can comprise at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% Brassica plant seed comprising the iPSR mutation.
  • a seed lot can comprise at least 1, 2, 5, 10, 20, 50, 100, 500, or 1,000 kg of seed.
  • Use of any of the aforementioned treated or untreated Brassica plant seed lots to make animal feed (e.g., livestock or poultry feed), non-defatted Brassica sp. seed meal, or defatted Brassica sp. seed meal is also provided.
  • Use of any of the aforementioned Brassica sp. seed lots to provide whole, cracked or rolled seed to animals (e.g., poultry) in scratch grain is also provided.
  • Embodiments of the methods and Brassica plants provided herein include the following numbered embodiments.
  • [0071] 1. A method of obtaining Brassica plant with at least one mutant ind gene which can confer a pod shatter resistance trait comprising:(i) crossing a Brassica plant comprising at least one wild-type IND gene with a Brassica plant comprising at least one mutant ind gene encoding a mutant ind protein wherein one or more amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of said mutant ind protein are substituted and/or deleted; and; (ii) isolating Fl seed and/or Fl progeny plants comprising the mutant ind gene from the cross.
  • bHLH conserved basic Helix-Loop Helix
  • the encoded mutant ind protein comprises: (i) a deletion and/or substitution of 5 to 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the C-terminal amino acid residues corresponding to the final 5 to 18 C-terminal amino acid residues of the wild-type IND protein encoded by the wild-type IND gene; or (ii) a deletion and/or substitution of 6 to 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 18 C-terminal amino acid residues comprising the C-terminal consensus sequence of SEQ ID NO: 32 located at the C- terminus of the wild-type IND protein encoded by the wild-type IND gene.
  • a method of obtaining a Brassica plant with at least one mutant ind gene which can confer a pod shatter resistance trait comprising introducing a mutation in one or more nucleotides of at least one wild-type IND gene of a Brassica plant to obtain a Brassica plant comprising a mutant ind gene encoding a mutant ind protein wherein one or more amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of said mutant ind protein are substituted and/or deleted, optionally wherein the mutant ind protein comprises: (i) a deletion and/or substitution of 5 to 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the C-terminal amino acid residues corresponding to the final 5 to 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, or 18 C-terminal amino acid residues of the wild-type IND protein encoded by the wild-type IND gene; or (ii) a deletion and/or substitution of 6 to 18 of the 18 C-terminal amino acid residues comprising
  • said gene-editing reagents comprise: (i) an RNA directed endonuclease and a guide RNA directed to the IND gene; (ii) a transcription activator-like effector nuclease (TALEN) directed to the IND gene; (iii) a zinc-finger nuclease (ZFN) directed to the IND gene; or (iv) any one of (i), (ii), or (iii) and DNA donor template.
  • TALEN transcription activator-like effector nuclease
  • ZFN zinc-finger nuclease
  • the guide RNA comprises the RNA molecule of any one of SEQ ID NO: 21 to 27 or a fragment thereof which can hybridize to a target IND gene.
  • an adenine base-editor (ABE) and a guide RNA directed to the IND gene (i) an adenine base-editor (ABE) and a guide RNA directed to the IND gene; or (ii) a cytosine base-editor and a guide RNA directed to the IND gene.
  • ABE adenine base-editor
  • the Brassica plant is a Thlaspi arvense plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 2 or an allelic variant thereof or wherein the wild-type IND gene encodes a protein comprising the polypeptide of SEQ ID NO: 12 or an allelic variant thereof;
  • the Brassica plant is a Brassica napus plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 9 or an allelic variant thereof or wherein the wild-type IND gene encodes a protein comprising the polypeptide of SEQ ID NO: 19 or an allelic variant thereof, optionally further comprising introducing a mutation in the wild-type IND coding sequence of SEQ ID NO: 10 or an allelic variant thereof or in the wild-type IND coding sequence which encodes SEQ ID NO: 20 or an allelic variant thereof;
  • the Brassica plant is Brassica carin
  • the Brassica plant is a Thlaspi arvense plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 2 or an allelic variant thereof and wherein the mutation is located at nucleotide positions corresponding to nucleotide 496 to 546 of SEQ ID NO: 2, optionally wherein the mutation corresponds to a C531A or C531G mutation in SEQ ID NO: 2, corresponds to a deletion of nucleotides comprising nucleotides 529 to 546 of SEQ ID NO: 2, or corresponds to a frameshift or nonsense mutation in the Y176, Y177, or H178 codon of SEQ ID NO:2; (ii) the Brassica plant is a Brassica napus plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 9 or an allelic variant thereof; wherein the mutation is located at nucleotide positions corresponding
  • the Brassica plant is a Thlaspi arvense plant wherein the wild-type IND gene encodes a protein comprising the polypeptide of SEQ ID NO: 12 or an allelic variant thereof and wherein the mutant encodes a protein lacking at least 5 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 166 to 182 of SEQ ID NO: 12, wherein the mutant encodes a protein lacking at least 5 or 6 to 8, 9, or 10 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 173 to 182 of SEQ ID NO: 12, or wherein the mutant encodes a protein of SEQ ID NO: 43, 46, 78, or an allelic variant thereof lacking said amino acid residues having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 43, 46, or 78; (ii) the Brassica plant is a Brass
  • a Brassica plant comprising at least one mutant ind gene encoding a mutant ind protein wherein one or more amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of said mutant ind protein are substituted and/or deleted, wherein said Brassica plant is not a Thlaspi arvense plant and optionally wherein the mutant ind protein comprises: (i) a deletion and/or substitution of 5 to 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the C-terminal amino acid residues corresponding to the final 5 to 18 C-terminal amino acid residues of the corresponding wild-type IND protein; or (ii) a deletion and/or substitution of 6 to 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 18 C-terminal amino acid residues comprising the C-terminal consensus sequence of SEQ ID NO: 32 located at the C-terminus of the corresponding wild-type IND protein.
  • bHLH conserved basic Helix-Loop Helix
  • Brassica napus Brassica carinata, or Camelina sativa plant.
  • Brassica plant is a Brassica napus plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 9 or an allelic variant thereof or wherein the wild-type IND gene encodes the corresponding wild-type IND protein comprising the polypeptide of SEQ ID NO: 19 or an allelic variant thereof;
  • the Brassica plant is a Brassica carinata plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 4, SEQ ID NO: 5, or an allelic variant thereof or wherein the wild-type IND gene encodes the corresponding wildtype IND protein comprising the polypeptide of SEQ ID NO: 14, SEQ ID NO: 15, or an allelic variant thereof; or
  • the Brassica plant is a Camelina sativa plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 6, SEQ ID NO: 7, or an allelic variant thereof or wherein the wild-type IND gene encodes the corresponding wild-type IND gene encodes
  • Brassica plant is a Brassica napus plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 9 or an allelic variant thereof; wherein the mutation is located at nucleotide positions corresponding to nucleotide 484 to 534 of SEQ ID NO: 9 or optionally wherein the mutation corresponds to a C519A or C591G mutation in SEQ ID NO: 9 , corresponds to a deletion of nucleotides comprising nucleotides 517 to 534 of SEQ ID NO: 9, or corresponds to a frameshift or nonsense mutation in the Y172, Y173, or H174 codon of SEQ ID NO: 9; optionally further comprising introducing a mutation located at nucleotide positions corresponding to nucleotide 536 to 546 of SEQ ID NO: 10; (ii) the Brassica plant is a Brassica carinata plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 4, SEQ ID
  • Brassica plant is a Brassica napus plant wherein the wild-type IND gene encodes a protein comprising the polypeptide of SEQ ID NO: 19 or an allelic variant thereof and wherein the mutant encodes a protein lacking at least 5 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 161 to 178 of SEQ ID NO: 19; (ii) the Brassica plant is a Brassica carinata plant wherein the wild-type IND gene encodes a protein comprising the polypeptide of SEQ ID NO: 14, SEQ ID NO: 15, or an allelic variant thereof and wherein the mutant encodes a protein lacking at least 5 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 179 to 195 of SEQ ID NO: 14 and/or corresponding to amino acid residues 176 to 192 of SEQ ID NO: 15; or (iii) the Brassica plant is a Camelina sativa plant wherein the wild-type IND gene encode
  • a seed lot comprising a plurality of the Brassica seed of embodiment 29.
  • a method for harvesting seed from a Brassica crop comprising harvesting seed from a plurality of the Brassica plants of any one of embodiments 19 to 26.
  • a Thlaspi arvense plant comprising a mutant ind gene encoding a mutant ind protein wherein one or more amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of said mutant ind protein are substituted and/or deleted, wherein said Thlaspi arvense plant comprises two genomic DNA sequence polymorphisms which are not found together in any single naturally occurring Thlaspi arvense isolate and optionally wherein the mutant ind protein comprises: (i) a deletion and/or substitution of 5 to 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the C-terminal amino acid residues corresponding to the final 5 to 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, or 18 C-terminal amino acid residues of the corresponding wild-type IND protein; or (ii) a deletion and/or substitution of 6 to 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 18 C-terminal amino acid residues comprising the C-terminal consensus sequence of SEQ ID NO:
  • Thlaspi arvense plant of embodiment 32 or 33 wherein said Thlaspi arvense plant: (i) lacks one or more genetic polymorphisms characteristic of the wild-type Thlaspi arvense cultivar 2032, representative seed of the cultivar having been deposited under NCMA Accession Number 202210002; (ii) lacks one or more traits characteristic of the wild-type Thlaspi arvense cultivar 2032 , representative seed of the cultivar having been deposited under NCMA Accession Number 202210002; and/or (iii) wherein the Thlaspi arvense plant lacks a black seed trait, a high fiber seed trait, a reduced yield, and/or an increased lodging trait. [00105] 35.
  • Thlaspi arvense plant of any one of embodiments 32 to 36 wherein said plant is homozygous for the mutant ind gene or genes and exhibits reduced pod shatter in comparison to a control plant lacking the mutant ind gene.
  • Thlaspi arvense plant cell or plant part obtained from the Thlaspi arvense plant of any one of embodiments 32 to 38, wherein said cell, plant propagule, or plant part comprises the mutant IND gene.
  • Thlaspi arvense plant part of embodiment 39 wherein the part is a seed, pollen, ovule, root, pod, stem, or leaf.
  • [00112] 42 A seed lot comprising a plurality of the Thlaspi arvense seed of embodiment 41.
  • a method for harvesting seed from a Thlaspi arvense crop comprising harvesting seed from a plurality of the Thlaspi arvense plants of any one of embodiments 32 to 38.
  • Genomic DNA was extracted from the candidate T. arvense reduced shatter line designated 2032 (T. arvense cultivar 2032, deposited as deposited under NCMA Accession Number 202210002) using column-based plant DNA mini -prep protocols, and whole-genome resequencing was performed with 2x150 bp read technology on Illumina platform. Sequence reads were mapped to the MN106-Reference genome (Nunn et al. Plant Biotechnol J., 2022, doi: 10.1111/pbi.13775) using bwa alignment and SNPs were identified using GATK HaplotypeCaller.
  • SNPs were analyzed using genome annotations to assess the change in amino acid sites with the presence of nucleotide changes within the coding regions.
  • the nucleotide substitution at position C531 A (relative to the wild-type T. arvense INDI gene of SEQ ID NO: 2) resulting in premature stop codon at Y177* (relative to the wild-type T. arvense INDI protein of SEQ ID NO: 12) was identified in the INDEHISCENT1 gene of T. arvense cultivar 2032. This premature stop codon results in the expression of an indl protein which lacks the 6 C-terminal amino acids of the wild-type INDI protein.
  • the indl allele with the C531 A nucleotide substitution is referred to as indl-4, comprises SEQ ID NO: 73, and encodes the indl-4 protein of SEQ ID NO: 74.
  • T. arvense peptide sequences were aligned with the peptide sequences of other Brassica species ( Figure 4). The DNA and amino acid sequence alignments highlight the conserved regions of the IND genes.
  • Allele-specific markers were designed to genotype plants containing the C531 A mutation identified from whole-genome resequencing data and genotyping was performed on the DNA extracted using quick-extract protocol from LGC genomics.
  • Table 3 Summary of Phenotypes in Greenhouse observations [00120] To further confirm the trait and genotype interaction, the cultivars derived from T. arvense cultivar 2032 were planted in replicated trials at Mt. Pulaski, IL, USA (MPI) and Havana, IL, USA (HVI) in the fall of 2021. These cultivars were evaluated for shattering at harvest on a scale of 0 to 100 percent. Phenotypic measurements are collected just prior to harvest when plants are fully mature. Percent shatter is based on the estimation of pods lost to natural dehiscence.
  • Table 4 Field observations of 29 cultivars derived from T. arvense cultivar 2032 parent carrying reduced shatter mutation in indl gene. Shatter rating columns represent the percent of pods dehisced after harvest maturity in field conditions at Mt. Pulaski (MPI) and Havana, Illinois (HVI).
  • Table 5 shows that the 182002-B-B-31 line obtained from a cross between T. arvense cultivar 2032 and a second unrelated parent retains the reduced shattering trait of T. arvense cultivar 2032 but lacks the increased lodging and decreased yield traits of T. arvense cultivar 2032.
  • Protospacer coding sequences described below in Table 5 can be targeted in three Brassica plants (Ta - Thlaspi arvense, At - Arabidopsis thaliana; Cs - Camelina sativa. Ta_At_Cs_PS_2) can also be targeted for many of the Brassica plants such as Brassica napus, Camelina sativa, and Brassica carinata.
  • Suitable gRNAs can be designed based on the Cas9 nuclease and protospacer adjacent motif (PAM) - ‘NGG’ present in the target Brassica plant IND gene sequence (e.g., SEQ ID NO: 2, 4, 5, 6, 7, 8, and 9).
  • Protospacers can be designed to target the different region of IND gene including iPSR mutations in pennycress, Arabidopsis and Camelina using the webtool “CHOPCHOP” (on the https internet site chopchop.cbu.uib.no/; Labun et al. Nucleic Acids Research (2019) doi.org/10.1093/nar/gkz365) and “CRISPOR” (on the http internet site crispor.tefor.net/;
  • CRISPR/SpCas9 DNA constructs designed to target regions of the IND genes will be delivered using a disarmed Agrobacterium tumefaciens strain (GV3101) and a standard floral dip or other transformation method. Presence of the edits in the IND genes of T1 plants will confirmed through PCR or fluorescent marker screening. Seed from the progeny T2 generation will be screened for lines with IND gene edits that lack the transgene using PCR and sanger sequencing.
  • RPS5a and a Cassava mosaic virus promoter, respectively.
  • T1 seeds were screened under red light system to identify the DsRED2 expressing transformants. T1 plants were grown from the DsRED2 expressing transformants and T2 seeds were harvested for further genotyping.
  • a gram force tension gauge (SSEYL ATG-100-2 Tension Gauge) attached to a two-inch alligator clip was used to determine the force required to break apart seedpods at the septum.
  • One side of a pennycress pod was clipped, and the other side was pulled manually until the pod breaks apart. For each line, 5 pods each from five different plants were used for the measurements.
  • Camelina plants comprising the mutations in one, two, and/or all three of the Camelina INDI genes located on chromosomes 2, 8, and 13 will be crossed and/or selfed to obtain progeny plants homozygous for the indl mutations shown in in Figures Figure 25, Figure 26, and in Table 8.
  • Camelina plants comprising the following IND gene edits C10012D7_Cs_Chr2(+A) (encoding the SEQ ID NO: 68 protein), C10012D7_Cs_Chr8(+A) (encoding the SEQ ID NO: 69 protein), C10012D7_Cs_Chrl3(+A) (encoding the SEQ ID NO: 70 protein), C10012D10_Cs_Chr2(+T) (encoding the SEQ ID NO: 71 protein), C10012D10_Cs_Chr2(+A) (encoding the SEQ ID NO: 68 protein), C10012D10_Cs_Chr8(+T) (encoding the SEQ ID NO: 72 protein), and/or C10012D10_Cs_Chrl3(+A) (SEQ ID NO: 70) will be crossed and/or selfed to obtain progeny Camelina plants homozygous for at least one, at least two, or all three of the IND gene edit
  • At least some of the progeny Camelina plants homozygous for at least one, at least two, or all three of the IND gene edits on chromosome 2 (Chr2), chromosome 8 (Chr8) and chromosome 13 (Chrl3) will exhibit improved threshability in comparison to control Camelina plants homozygous for amorphic (null) alleles of at least one, at least two, or all three of the IND genes on chromosome 2 (Chr2), chromosome 8 (Chr8) and chromosome 13 (Chrl3).
  • Table 9 presents a description of the biological sequences provided herewith in the sequence listing.

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Abstract

Brassica plants comprising mutations in the endogenous INDEHISCENT (IND) gene which confer an improved pod shatter resistance trait are provided. Features of the Brassica plants having improved pod shatter resistance include improved agronomic features in comparison to Brassica plants having complete loss-of-function alleles of the IND gene.

Description

BRASSICA IND GENE MUTATIONS CONFERRING IMPROVED POD SHATTERRESISTANCE
INVENTORS: RATAN CHOPRA , MARK MESSMER, AND KRISHAN M. RAI
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. § 119 to provisional patent application U.S. Serial No. 63/382,369, filed November 4, 2022. The provisional patent application is incorporated herein by reference in its entirety, including without limitation, the specification, claims, and abstract, as well as any figures, tables, appendices, biological sequences, biological sequence listings, or drawings thereof.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [0002] Not applicable.
INCORPORATION OF SEQUENCE LISTING
[0003] A sequence listing contained in the file named “P14008WO00.xml” which is 87,115 bytes in size (measured in MS-Windows), which was created on November 2, 2023, and which comprises 74 polynucleotide and polypeptide sequences, is electronically filed herewith and is incorporated herein by reference in its entirety.
BACKGROUND
[0004] Achieving reduced pod dehiscence or reduced pod shatter in the Brassica species currently grown across the globe remains a challenge. Premature pod shatter results in yield loss. In Brassica plants such as oilseed rape, pod shatter-related yield losses can average 20% (Child et al., 1998, J Exp Bot 49: 829-838; and U.S. Patent 8,809,635).
[0005] Loss-of-function mutations in an INDEHISCENT (IND) gene which encodes an atypical basic helix-loop-helix transcription factor can provide a reduced pod-shatter phenotype in Arabidopsis (Liljegren et al., 2004, Cell 116, 843-853). Previously disclosed methods to achieve this trait include generation of point mutations in an INDEHISCENT (IND) gene through chemical mutagenesis, reducing expression of an IND gene using RNAi, or other geneediting based techniques for introducing mutations in an IND gene (US Patent Nos. 7,528,294 and 8809635; US Patent Applic. Pub. Nos. US20190053458 and US20220298519; Chopra et al. 2020; Nat Food 1, 84-91; Zhai et al. 2019, Theor Appl Genet 132, 2111-2123). While some of these approaches to reducing IND gene expression resulted in the reduced pod shatter trait, undesirable agronomic performance has been observed in plants having complete loss of function (z.e., amorphic) alleles of the IND gene(s). Such undesirable agronomic characteristics of certain pod-shatter resistant Brassica can include reduced threshability (z.e., mechanical separation of the seeds from the pods; U.S. Patent 8,809,635).
[0006] There is thus a need for improved alleles of the IND gene or genes of various Brassica sp. including Brassica napus. Brassica carinata, Camelina sativa, and Thlaspi arvense which exhibit reduced pod shatter without also causing undesirable agronomic characteristics.
SUMMARY
[0007] Methods of obtaining Brassica plants with at least one mutant IND gene copy which can confer a pod shatter resistance trait comprising: (i) crossing a Brassica plant comprising at least one wild-type IND gene with a Brassica plant comprising at least one mutant ind gene encoding a mutant ind protein wherein one or more amino acid residues located C- terminal to the conserved basic Helix-Loop Helix (bHLH) domain of said mutant ind protein are substituted and/or deleted; and (ii) isolating Fl seed and/or Fl progeny plants comprising the mutant ind gene from the cross are provided.
[0008] Methods of obtaining Brassica plants with at least one mutant ind gene which can confer a pod shatter resistance trait comprising introducing a mutation in one or more nucleotides of at least one IND gene of a Brassica plant to obtain a Brassica plant comprising a mutant ind gene encoding a mutant ind protein wherein one or more amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of said mutant ind protein are substituted and/or deleted are provided.
[0009] Brassica plants and plant parts comprising at least one mutant ind gene encoding a mutant ind protein wherein one or more amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of said mutant ind protein are substituted and/or deleted, wherein the Brassica plant or plant part is not a Thlaspi arvense plant are provided.
[0010] Thlaspi arvense plants and plant parts comprising a mutant ind gene encoding a mutant ind protein wherein one or more amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of said mutant ind protein are substituted and/or deleted, wherein the Thlaspi arvense plants comprise two genomic DNA sequence polymorphisms which are not found together in any single naturally occurring Thlaspi arvense isolate are provided. DESCRIPTION OF THE DRAWINGS
[0011] Figure 1 shows the results of field tests in Mt. Pulaski Illinois (“MPT’; left-most columns in each set of wild-type, indl-4 heterozygotes, and indl-4 homozygotes) and Havana, Illinois (“HVI”; right-most columns in each set of wild-type, indl-4 heterozygotes, and indl-4 homozygotes) that were evaluated in the spring of 2022.
[0012] Figure 2 shows the result of evaluating pod shatter resistance in a greenhouse with wild-type, indl-4 heterozygotes, and indl-4 homozygotes.
[0013] Figure 3 A-E shows the alignment of genomic or coding sequences of the indicated Brassica IND genes. The region highlighted in this alignment in boldface and underlining represents the bHLH domain and region with dashed underlining in the C-terminal protein coding region identifying a region which can be targeted with guide RNAs and Cas9 nucleases in gene editing methods.
[0014] Figure 4A-B shows the alignment of polypeptide sequences of the indicated Brassica IND proteins. The region highlighted in this alignment in boldface and underlining represents the bHLH domain and the C-terminal region where amino acid residues are in lower case and italicized with dashed underlining represents the amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain. A * symbol is located above the six C- terminal amino acids of the indicated Brassica IND proteins which comprise the consensus amino acid sequence YHNS(Q/D)(P/S/T) (SEQ ID NO: 32).
[0015] Figure 5 shows the T. arvense INDI DNA coding sequence (SEQ ID NO: 2) with the initiator methionine (Ml) codon through to the C -terminal threonine 182 (T182) codon and stop codon as well as the T. arvense INDI polypeptide (SEQ ID NO: 12).
[0016] Figure 6A shows the B. carinata INDla DNA coding sequence (SEQ ID NO: 4) with the initiator methionine (Ml) codon through to the C -terminal threonine 195 (T195) codon and stop codon as well as the IND polypeptide (SEQ ID NO: 14). Figure 6B shows the B. carinata IND-b DNA coding sequence (SEQ ID NO: 5) with the initiator methionine (Ml) codon through to the C-terminal threonine 192 (T192) codon and stop codon as well as the IND polypeptide (SEQ ID NO: 15).
[0017] Figure 7A shows the C. sativa IND-a DNA coding sequence (SEQ ID NO: 6) with the initiator methionine (Ml) codon through to the C -terminal serine 167 (SI 67) codon and stop codon as well as the encoded IND polypeptide (SEQ ID NO: 16). Figure 7B shows the C. sativa IND-b DNA coding sequence (SEQ ID NO: 7) with the initiator methionine (Ml) codon through to the C -terminal serine 167 (SI 67) codon and stop codon as well as the encoded IND polypeptide (SEQ ID NO: 17). Figure 7C shows the C. sativa IND-c DNA coding sequence (SEQ ID NO: 8) with the initiator methionine (Ml) codon through to the C -terminal serine 168 (SI 68) codon and stop codon as well as the encoded IND polypeptide (SEQ ID NO: 18).
[0018] Figure 8 shows the B. napus IND-a DNA coding sequence (SEQ ID NO: 9) with the initiator methionine (Ml) codon through to the C -terminal threoninel78 (T178) codon and stop codon as well as the encoded IND-a polypeptide (SEQ ID NO: 19).
[0019] Figure 9A and B show an alignment of DNA molecules for wild-type (TalNDI WT, SEQ ID NO: 2) and pARV55 (TaINDl_PSl)-Cas9 -generated alleles (B56924A1(+A) (SEQ ID NO: 33); B56914A5(+G) (SEQ ID NO: 34); B56930A5(+T) (SEQ ID NO: 35)).
[0020] Figure 10 shows an alignment of protein molecules for wild-type (TalNDI WT (SEQ ID NO: 12)) and pARV55 (TaINDl_PSl)-Cas9 -generated alleles (B56924A1(+A) (SEQ ID NO: 43); B56914A5(+G) (SEQ ID NO: 44); B56930A5(+T) (SEQ ID NO: 45)).
[0021] Figure 11 A and B show an alignment of DNA molecules for pennycress wild-type (TalNDI WT (SEQ ID NO: 2) and pARV56 (TaINDl_PS2)-Cas9 -generated alleles (B56929A1(+A) (SEQ ID NO: 36); B56922A5(+C) (SEQ ID NO: 37); B56941A1(+T) (SEQ ID NO: 38).
[0022] Figure 12 shows an alignment of protein molecules for penny cress wild-type (TalNDI WT (SEQ ID NO: 12) and pARV56 (TaINDl_PS2)-Cas9 -generated alleles (B56929A1(+A) (SEQ ID NO: 46); B56922A5(+C) (SEQ ID NO: 47); B56941A1(+T) (SEQ ID NO: 48)).
[0023] Figure 13 A and B show an alignment of DNA molecules for penny cress wild-type (TalNDI WT (SEQ ID NO: 2) and pARV58 (TaINDl_PS4)-Cas9 -generated alleles (B57581B1(+A) (SEQ ID NO: 39) and B57582A4(+T) (SEQ ID NO: 40).
[0024] Figure 14 shows an alignment of protein molecules for penny cress wild-type (TalNDI WT (SEQ ID NO: 12) and pARV58 (TaINDl_PS4)-Cas9 -generated alleles (B57581B1 (+A) (SEQ ID NO: 49) and B57582A4(+T) (SEQ ID NO: 50).
[0025] Figure 15A and B show an alignment of DNA molecules for penny cress wild-type (TalNDI WT (SEQ ID NO: 2) and pARV59 (TaINDl_PS5)-Cas9 -generated alleles B57663B2 (+A) (SEQ ID NO: 41) and B57449A1 (-19) (SEQ ID NO: 42).
[0026] Figure 16 shows an alignment of protein molecules for penny cress wild-type (TalNDI WT (SEQ ID NO: 12) and pARV59 (TaINDl_PS5)-Cas9 -generated B57663B2 (+A) (SEQ ID NO: 51) and B57449Al (-19) (SEQ ID NO: 52).
[0027] Figure 17A and B show penny cress reduced pod shattering reduction by the indl- 4 (SEQ ID NO: 73) or Cas9 edited INDI alleles depicted in Figures 9, 10, 11, and 12. Fig. 17A shows measurement of force required to break the pennycress pods comprising the indl-4 mutation (2032:WG; SEQ ID NO: 73) or Cas9 edited INDI alleles compared to the wild-type genotypes B3:WG and B28:WG. Fig. 17B shows a comparison of desirable cell separation layers observed in 2032-WG comprising the indl-4 mutation (2032:WG; SEQ ID NO: 73) and B56929A1 compared to the wild-type layers observed in the control genotype of B28:WG by toluidine blue staining of cross-sections.
[0028] Figure 18 A and B show an alignment of DNA molecules for Arabidopsis wild-type (AtINDl (SEQ ID NO: 1) and pARV56 (TaINDl_PS2)-Cas9 -generated alleles (At_indl_Pl(+A) (SEQ ID NO: 53); At_indl_P2(+T) (SEQ ID NO: 54); At_indl_P3(-34) (SEQ ID NO: 55)).
[0029] Figure 19 shows an alignment of protein molecules for Arabidopsis wild-type (AtINDl (SEQ ID NO: 11) and a pARV56 (TaINDl_PS2)-Cas9 -generated alleles (At_indl_Pl(+A) (SEQ ID NO: 58); At_indl_P2(+T) (SEQ ID NO: 59); (At_indl_P3(-34) (SEQ ID NO: 60)).
[0030] Figure 20 shows an alignment of DNA molecules for Arabidopsis wild-type (AtINDl (SEQ ID NO: 1) and a pARV72 (At_Cs_PS3)-Cas9 -generated allele (At_indl_P4(+A) (SEQ ID NO: 56).
[0031] Figure 21 shows an alignment of protein molecules for Arabidopsis wild-type (AtINDl (SEQ ID NO: 11) and a pARV72 (At_Cs_PS3)-Cas9 -generated allele (At_indl_P4(+A) (SEQ ID NO: 61).
[0032] Figure 22 shows an alignment of DNA molecules for Arabidopsis wild-type (AtINDl (SEQ ID NO: 1) and a pARV73 (At_Cs_PS4)-Cas9 -generated allele (At_indl_P5(+G) (SEQ ID NO: 57).
[0033] Figure 23 shows an alignment of protein molecules for Arabidopsis wild-type (AtINDl (SEQ ID NO: 11) and a pARV73 (At_Cs_PS4)-Cas9 -generated allele (At_indl_P5(+G) (SEQ ID NO: 62).
[0034] Figure 24A, B, and C show Arabidopsis reduced pod shattering optimization by a Cas9 edited INDI allele depicted in Figures 18 and 19. Fig. 24A shows pods of a control and indl mutant At_indl_Pl(+A) (SEQ ID NO: 53 and 58) subjected to force. Fig. 24B shows percentage of pods shattered for At_indl_Pl(+A) (SEQ ID NO: 53 and 58) Arabidopsis pods compared to the wild-type pods following subjection to force. Fig. 24C shows a comparison of desirable cell separation layers observed in the At_indl_Pl(+A) (SEQ ID NO: 53 and 58) allele compared to the wild-type Col-0 control.
[0035] Figure 25 A, B, and C show an alignment of DNA molecules for Camelina sativa wild-type (Cs_INDl_Chr2_WT (SEQ ID NO: 6); CsINDl_Chr8_WT (SEQ ID NO: 7); and CsINDl_Chrl3_WT (SEQ ID NO: 8)) and pARV56 (TaINDl_PS2) generated alleles (C10012D7_Cs_Chr2(+A) (SEQ ID NO: 63); C10012D7_Cs_Chr8(+A) (SEQ ID NO: 64); C10012D7_Cs_Chrl3(+A) (SEQ ID NO: 65); C10012D10_Cs_Chr2(+T) (SEQ ID NO: 66); C10012D10_Cs_Chr2(+A) (SEQ ID NO: 63): C10012D10_Cs_Chr8(+T) (SEQ ID NO: 67); and C10012D10_Cs_Chrl3(+A) (SEQ ID NO: 65)).
[0036] Figure 26 shows an alignment of protein molecules for Camelina sativa wild-type (Cs_INDl_Chr2_WT (SEQ ID NO: 16); CsINDl_Chr8_WT (SEQ ID NO: 17); and CsINDl_Chrl3_WT (SEQ ID NO: 18)) and pARV56 (TaINDl_PS2) generated alleles (C10012D7_Cs_Chr2(+A) (SEQ ID NO: 68); C10012D7_Cs_Chr8(+A) (SEQ ID NO: 69); C10012D7_Cs_Chrl3(+A) (SEQ ID NO: 70); C10012D10_Cs_Chr2(+T) (SEQ ID NO: 71); C10012D10_Cs_Chr2(+A) (SEQ ID NO: 68): C10012D10_Cs_Chr8(+T) (SEQ ID NO: 72); and C10012D10_Cs_Chrl3(+A) (SEQ ID NO: 70)).
DETAILED DESCRIPTION
[0037] The term "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
[0038] As used herein, the term “alleles” refers to alternate forms of a DNA sequence at a genetic locus, that is, a position on a chromosome of a gene or other chromosome marker.
[0039] As used herein, the phrase "Brassica plant" refers to a plant in the family Brassicaceae.
[0040] As used herein, the terms “correspond,” “corresponding,” and the like, when used in the context of an amino acid position, mutation, and/or substitution in any given IND gene, coding sequence, or protein with respect to the reference IND gene, coding sequence, or protein, all refer to the position, mutation, and/or substitution of the nucleotide or amino acid residue in the given IND sequence that has identity or similarity to the amino acid residue in the reference IND nucleotide or polypeptide sequence when the given IND nucleotide or polypeptide sequence is aligned to the reference IND nucleotide or polypeptide sequence using a pairwise sequence alignment algorithm (e.g, CLUSTAL O 1.2.4 with default parameters).
[0041] As used herein, the term “cultivar” refers to a plant that has been cultivated. A cultivar is generally developed using crossing, selfing, and/or selection and is maintained by any suitable method of propagation, through open pollination, selfing, or the like. Details of cultivar development can be found in “Principles of Cultivar Development” by Fehr, Macmillan Publishing Company (1993), which is herein incorporated by reference in its entirety. [0042] As used herein, the terms “elite” and “elite line” refer to any line that has resulted from breeding and selection for desirable agronomic performance (typically commercial production). Generally, individuals in a line have similar parentage and one or more similar traits. In some cases, an "elite line" or "elite variety” can be an agronomically superior line or variety that has resulted from many cycles of breeding and selection for superior agronomic performance. An "elite inbred line" is an elite line that is an inbred, and that has been shown to be useful for producing sufficiently high yielding and agronomically fit hybrid varieties (an "elite hybrid variety"). Similarly, "elite germplasm" is a germplasm resulting from breeding and selection for desirable agronomic performance (typically commercial production). Such germplasm may be agronomically superior germplasm, derived from and/or capable of giving rise to a plant with superior agronomic performance, such as an existing or newly developed elite line of a Brassica plant.
[0043] As used herein, a “frameshift mutation” refers to a mutation in a nucleic acid sequence that results in the wild-type reading frame being shifted to different reading frame such that translation of an mRNA having a frameshift mutation results in a departure from the wildtype reading frame.
[0044] As used herein, the phrase “nonsense mutation” refers to any mutation that results in the appearance of a stop codon where previously there was a codon specifying an amino acid. The presence of this premature stop codon results in the production of a truncated protein. Stop codons include TAA, TAG, and TGA codons.
[0045] As used herein, the term “amorphic allele” refers to an allele of a gene having no gene activity in comparison to the wild-type allele of the gene. Amorphic alleles are also known as null or “knock-out” alleles.
[0046] As used herein, the term “isomorphic allele” refers to an allele of a gene having wild-type gene activity.
[0047] As used herein, the term “hypomorphic allele” refers to an allele of a gene having reduced or partially reduced but not null gene activity in comparison to the wild-type allele of the gene. In embodiments, a hypomorphic allele can exhibit a subset of the phenotypes observed in an amorphic allele. An example of an hypomorphic ind gene allele includes an ind allele which exhibits reduced pod shatter but does not also exhibit undesirable agronomic traits characteristic of amorphic ind alleles.
[0048] As used herein, “gene activity” comprises any measure of gene function. Gene activity measures thus include measures of gene-mediated phenotypes e.g., pod shatter resistance and undesirable agronomic traits) and/or gene-encoded protein activity (e.g., transcriptional activation activity). In certain embodiments, transcriptional activation activity can be assayed by monitoring expression of genes that are regulated by the gene with transcriptional activation activity.
[0049] As used herein, the term “IND” refers to a wild-type INDEHISCENT gene or protein while the term “ind” refers to a mutant INDEHISCENT gene or protein.
[0050] As used herein, the terms “include,” “includes,” and “including” are to be construed as at least having the features to which they refer while not excluding any additional unspecified features.
[0051] As used herein, the term “plant” includes a whole plant and any descendant, cell, tissue, or part of a plant. The term “plant parts” include any part(s) of a plant, including, for example and without limitation: seed (including mature seed and immature seed); a plant cutting; a plant cell; a plant cell culture; or a plant organ (e.g., pollen, embryos, flowers, fruits, shoots, leaves, roots, stems, and explants). A plant tissue or plant organ may be a seed, protoplast, callus, or any other group of plant cells that is organized into a structural or functional unit. A plant cell or tissue culture may be capable of regenerating a plant having the physiological and morphological characteristics of the plant from which the cell or tissue was obtained, and of regenerating a plant having substantially the same genotype as the plant. Regenerable cells in a plant cell or tissue culture may be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, flowers, pods, or stalks. In contrast, some plant cells are not capable of being regenerated to produce plants and are referred to herein as “non-regenerable” plant cells.
[0052] As used herein, the term “variety” means a group of similar plants that by structural features, genetic features, and/or performance which can be distinguished from other varieties within the same species. In certain embodiments, the term variety refers to the botanical taxonomic designation whereby variety is ranked below species or subspecies, as well as the legal definition whereby the term “variety” refers to a commercial plant that is protected under the terms outlined in the International Convention for the Protection of New Varieties of Plants.
[0053] Where a term is provided in the singular, other embodiments described by the plural of that term are also provided.
[0054] To the extent to which any of the preceding definitions is inconsistent with definitions provided in any patent or non-patent reference incorporated herein by reference, any patent or non-patent reference cited herein, or in any patent or non-patent reference found elsewhere, it is understood that the preceding definition will be used herein.
[0055] Brassica plants with desirable ind alleles, referred to herein as improved pod shatter resistant (“iPSR”) Brassica, are provided. Also provided are iPSR Brassica plant cells, plant propagules, or plant parts which include seeds are also provided. Such desirable ind alleles, when in the homozygous state, can result in Brassica plants with useful pod shatter resistance traits while exhibiting improved agronomic performance in comparison to a control plant homozygous for an amorphic allele of the ind gene. Such improved agronomic performance can include traits such as better threshability. Also provided herein are methods of obtaining iPSR Brassica by breeding, gene editing, and/or mutagenesis. Also provided herein are methods of using the iPSR Brassica including methods of harvesting seed from Brassica crops comprising the iPSR Brassica.
[0056] An alignment of IND protein sequences of various Brassica set forth in Figure 4 illustrates extensive conservation of the bHLH domain. Desirable ind alleles provided herein which can confer an improved Pod Shatter Resistance (iPSR) phenotype include ind gene alleles comprising mutations encoding an ind protein where at least 5 or 6 of the amino acid residues located at the C-terminal end of a wild-type Brassica IND protein are deleted and/or substituted with different amino acid residues (e.g., including non-conservative amino acid substitutions). The wild-type IND C-terminal six amino acids comprise the consensus amino acid sequence YHNS(Q/D)(P/S/T) (SEQ ID NO: 32). Examples of the types of mutations (“iPSR mutations” or “iPSR alleles”) which can provide an iPSR Brassica plant include those provided in Table 1 and otherwise described herein. Combinations of such iPSR mutations which include substitutions and deletions of at least the wild-type IND C-terminal five or six amino acids are also contemplated. In certain embodiments, such mutations can be located or introduced at a target Brassica IND gene codon corresponding to the codon of the first N-terminal amino acid of the 18 amino acid C- terminal fragment located immediately C-terminal to the bHLH domain (e.g., the glycine codon in Figure 4) through all following C-terminal amino acid residues to the codon for the tyrosine or histidine residue of the SEQ ID NO: 19 consensus sequence. Brassica plants and the corresponding IND genes which can contain or be made to contain such iPSR mutations include: (i) Brassica napus plants wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 9 or an allelic variant thereof or encodes a protein comprising the polypeptide of SEQ ID NO: 19 or an allelic variant thereof; (ii) Brassica carinata plants wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 4, SEQ ID NO: 5, or an allelic variant thereof or encodes a protein comprising the polypeptide of SEQ ID NO: 14, SEQ ID NO: 15, or an allelic variant thereof; (iii) a Camelina sativa plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or an allelic variant thereof or encodes a protein comprising the polypeptide of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or an allelic variant thereof; and (iv) Thlaspi arvense plants wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO:2, or an allelic variant thereof or encodes a protein comprising the polypeptide of SEQ ID NO: 12 or an allelic variant thereof. In certain embodiments, such iPSR mutations encode an ind protein wherein at least 6 to 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 18 amino acid residues comprising the C-terminal consensus sequence of SEQ ID NO: 32 located at the C-terminus of a wild-type IND gene or an allelic variant thereof are deleted and/or are substituted with non-conservative amino acid residues. Brassica plants and the corresponding ind genes which can encode such iPSR mutations include: (i) Brassica napus plants wherein the mutated ind gene encodes a mutant ind protein wherein the final 5 or 6 to 7, 8, 10, or 18 C-terminal amino acid residues of the wild-type IND protein comprising the polypeptide of SEQ ID NO: 19 or an allelic variant thereof are deleted and/or substituted (e.g., with a non-conservative amino acid residue) in the mutant ind protein; (ii) Brassica carinata plants wherein the mutated ind gene(s) encode a mutant ind protein wherein the final 5 or 6 to 7, 8, 10, or 18 C-terminal amino acid residues of the wild-type IND protein comprising the polypeptide of SEQ ID NO: 14, SEQ ID NO: 15, and/or an allelic variant thereof are deleted and/or substituted (e.g., with a non-conservative amino acid residue) in the mutant ind protein; (iii) a Camelina sativa plant wherein the mutant ind gene(s) encode a mutant ind protein wherein the final 5 or 6 to 7, 8, 10, or 18 C-terminal amino acid residues of the wild-type IND protein comprising the polypeptide of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and/or an allelic variant thereof are deleted and/or substituted e.g., with a non-conservative amino acid residue) in the mutant ind protein; and (iv) Thlaspi arvense plants wherein the mutant ind gene encodes a mutant ind protein wherein the final 5 or 6 to 7, 8, 10, or 18 C-terminal amino acid residues of the wild-type IND protein comprising the polypeptide of SEQ ID NO: 12 or an allelic variant thereof are deleted and/or substituted (e.g., with a non-conservative amino acid residue) in the mutant ind protein.
[0057] Table 1. Non-Limiting Description of Brassica plant iPSR mutations in IND gene(s)
Figure imgf000012_0001
Figure imgf000013_0001
Figure imgf000014_0001
Figure imgf000015_0001
[0058] In certain embodiments, certain Brassica IND coding sequences that can be targeted for introduction of iPSR mutations can include the wild-type IND coding sequences set forth in SEQ ID NO: 1-9 as well as other wild-type IND coding sequences with substantial sequence identity thereto (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 1-9). Allelic variants of wild-type IND genes found in distinct Brassica isolates or varieties that exhibit wild-type pod-shatter phenotypes can be targeted for introduction of iPSR mutations. Introduction and fixation of the iPSR mutations in a homozygous state in such Brassica plants comprising wild-type IND coding sequences set forth in SEQ ID NO: 1-9, wildtype IND coding sequences with substantial sequence identity thereto, or allelic variants thereof that exhibit wild-type pod-shatter phenotypes can provide Brassica plants, seeds, seed lots, and plants exhibiting improved pod-shatter resistance in comparison to control Brassica plants comprising wild-type IND coding sequences set forth in SEQ ID NO: 1-9, wild-type IND coding sequences with substantial sequence identity thereto, or allelic variants thereof. In certain embodiments, such wild-type IND allelic variants can comprise polynucleotide sequences that have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity across the entire length of the polynucleotide sequences of the wild-type IND coding regions of SEQ ID NO: 2, 4, 5, 6, 7, 8, or 9. In certain embodiments, such allelic variants can also encode polypeptide sequences that have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity across the entire length of the polypeptide sequence of SEQ ID NO: 12, 14, 15, 16, 18, or 19.
[0059] To obtain Thlaspi arvense (i.e., T. arvense or field pennycress) plants comprising an iPSR trait, iPSR mutations in the INDI gene of T. arvense are fixed in a homozygous state. Such iPSR mutations include mutations in an ind gene encoding a mutant ind protein wherein one or more amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of said mutant ind protein are substituted and/or deleted (e.g,. any of the aforementioned mutations set forth in Table 1). In certain embodiments, iPSR mutations in the INDI gene of T. arvense include: (i) frameshift and/or nonsense mutations introduced in SEQ ID NO: 2 or an allelic variant thereof at a codon corresponding to G165 (GGA) through to H178 (CAC) of SEQ ID NO: 2 (Figure 5); (ii) frameshift and/or nonsense mutations introduced in SEQ ID NO: 2 or an allelic variant thereof at a codon corresponding to D170 to anyone one of Y176, Y177, and/or H178 (CAC) of SEQ ID NO: 2; (iii) frame shift and/or nonsense mutations introduced in SEQ ID NO: 2 or an allelic variant thereof at a codon corresponding to SI 72, Cl 73, LI 74, or Cl 75 to anyone one of Y176, Y177, and/or H178 (CAC) of SEQ ID NO: 2; (iv) frame shift and/or nonsense mutations introduced in SEQ ID NO: 2 or an allelic variant thereof at a codon corresponding to Y176, Y177, and/or H178 (CAC) of SEQ ID NO: 2; (v) missense mutations at codons Y177, H178, N179, S180, Q181, and/or T182 in SEQ ID NO: 2 or an allelic variant thereof; (vi) a nonsense mutation corresponding to a C531A or C531G mutation in SEQ ID NO: 2 or an allelic variant thereof; and/or a (vii) a deletion of nucleotides comprising any one of nucleotides 493, 495, 500, 505, 510, 515, 520, 525, 526, 527, 528, or 529 to 544, 545, or 546 of SEQ ID NO: 2 or an allelic variant thereof. In certain embodiments, such iPSR mutations encode an ind protein wherein 1, 2, 3, 4, 5, or 6 to 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 18 amino acid residues located at the C-terminus of SEQ ID NO: 12 or an allelic variant thereof (e.g., amino acids encompassed by the C-terminal consensus sequence of SEQ ID NO: 32) are deleted and/or substituted with non-conservative amino acid residues (e.g,. any of the aforementioned mutations set forth in Table 1). In certain embodiments, such iPSR mutations encode an ind protein wherein 6 to 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 18 amino acid residues comprising the C- terminal consensus sequence of SEQ ID NO: 32 located at the C-terminus of SEQ ID NO: 12 or an allelic variant thereof are deleted and/or substituted with non-conservative amino acid residues. Such iPSR mutations can be introduced into a T. arvense plant by gene editing and/or breedingbased techniques. In certain embodiments, iPSR mutations in the INDI gene of T. arvense include: (i) mutations encoding a T. arvense ind protein lacking 10 C-terminal amino acids of the wildtype INDI gene of T. arvense (e.g., the indl gene of SEQ ID NO: 33, the indl protein of SEQ ID NO: 43, and allelic variants thereof encoding or comprising indl proteins lacking the 10 C- terminal amino acids of the INDI protein wherein the allelic variants have at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 33 and/or SEQ ID NO: 43); (ii) mutations encoding a T. arvense ind protein lacking 8 C-terminal amino acids of the wild-type INDI gene of T. arvense (e.g., the indl gene of SEQ ID NO: 36, the indl protein of SEQ ID NO: 46, and allelic variants thereof encoding or comprising indl proteins lacking the 8 C-terminal amino acids of the INDI protein wherein the allelic variants have at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 36 and/or SEQ ID NO: 46); and (iii) mutations encoding a T. arvense ind protein lacking 6 C-terminal amino acids of the wild-type INDI gene of T. arvense (e.g., the indl-4 gene of SEQ ID NO: 73, the indl-4 protein of SEQ ID NO: 74, and allelic variants thereof encoding or comprising indl proteins lacking the 6 C-terminal amino acids of the INDI protein wherein the allelic variants have at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 73 and/or SEQ ID NO: 74). In certain embodiments, the T. arvense plant comprising the iPSR mutation is: (i) a non-naturally occurring T. arvense plant; (ii) an elite T. arvense plant; (iii) a T. arvense plant variety; (iv) a T. arvense plant comprising two genomic DNA sequence polymorphisms which are not found together in any single naturally occurring T. arvense isolate; (v) a T. arvense plant which lacks a black seed trait (i.e., has yellow or brown seed in comparison to the black seed of Thlaspi arvense cultivar 2032), lacks a high fiber seed trait (i.e., has low fiber content seed in comparison to wild-type or cultivar 2032 T. arvense) lacks a reduced yield trait (i.e., exhibits increased seed yield in comparison to Thlaspi arvense cultivar 2032), and/or lacks an increased lodging trait (i.e., exhibits decreased lodging in comparison to Thlaspi arvense cultivar 2032); (vi) a T. arvense plant which lacks one or more genetic polymorphisms characteristic of the wild-type Thlaspi arvense cultivar 2032, representative seed of the cultivar having been deposited under NCMA Accession Number 202210002; (vii) a T. arvense plant which lacks one or more traits characteristic of the wild-type Thlaspi arvense cultivar 2032, representative seed of the cultivar having been deposited under NCMA Accession Number 202210002; and/or (viii) a T. arvense plant which is not Thlaspi arvense cultivar 2032, representative seed of the cultivar having been deposited under NCMA Accession Number 202210002. Traits characteristic of Thlaspi arvense cultivar 2032 include black seed, high fiber seed trait, reduced yield, and/or increased lodging. In certain embodiments, the T. arvense plant comprising any of the aforementioned the iPSR mutations is a non-naturally occurring T. arvense plant or an elite T. arvense plant cultivar comprising a yellow/brown seed with low fiber seed trait (e.g., a yellow/brown seed coat color and low fiber content seed trait set forth in US Patent No. 10,709,151, incorporated herein by reference in its entirety) and/or comprising a low glucosinolate seed content trait (e.g., as set forth in US Patent No. 10,988,772, incorporated herein by reference in its entirety).
[0060] A deposit of at least 625 seeds of the Thlaspi arvense cultivar 2032 has been made with the Provasoli-Guillard National Center for Marine Algae and Microbiota (NCMA) at Bigelow Laboratory for Ocean Sciences at 60 Bigelow Drive, East Boothbay, Maine 04544. The Thlaspi arvense cultivar 2032 seeds have been given the Accession Number 202210002 by the NCMA as the International Depository Authority. The seeds deposited with the NCMA on October 6, 2022, for Thlaspi arvense cultivar 2032 were harvested by hand in March 2020 from the CoverCress greenhouse at 1249 N. Warson Rd., St. Louis, Missouri 63132 prior to the filing date of this application. Access to this deposit will be available during the pendency of the application to the Commissioner of Patents and Trademarks and persons determined by the Commissioner to be entitled thereto upon request. Upon allowance of any claims in the application, the Applicant will make available to the public, pursuant to 37 C.F.R. § 1.808, sample(s) of the deposit of at least 625 seeds of the Thlaspi arvense cultivar 2032 deposited with the NCMA. This deposit of seed of the Thlaspi arvense cultivar 2032 will be maintained in the NCMA depository, which is a public depository, for a period of 30 years, or 5 years after the most recent request, or for the enforceable life of the patent, whichever is longer, and will be replaced if it becomes nonviable during that period. Additionally, Applicant has satisfied all the requirements of37 C.F.R. §§ 1.801-1.809, including providing an indication of the viability of the sample upon deposit. Applicant has no authority to waive any restrictions imposed by law on the transfer of biological material or its transportation in commerce. Applicant(s) do not waive any infringement of their rights granted under this patent or rights applicable to Thlaspi arvense cultivar 2032 under the Plant Variety Protection Act (7 USC 2321 et seq.).
[0061] To obtain Brassica carinata (i.e., B. carinata) plants comprising an iPSR trait, iPSR mutations in the IND la and/or IND lb gene of B. carinata are fixed in a homozygous state. Wild-type B. carinata plants typically comprise: (i) a wild-type IND la gene comprising the polynucleotide sequence of SEQ ID NO: 4 or an allelic variants thereof which encode the IND la protein of SEQ ID NO: 14 or an allelic variants thereof; and (ii) a wild-type IND lb gene comprising the polynucleotide sequence of SEQ ID NO: 5 or an allelic variant thereof which encode the IND-b protein of SEQ ID NO: 15 or an allelic variant thereof. In certain embodiments, iPSR . carinata plants provided herein can comprise an iPSR mutation in the IND la and IND lb gene which are both fixed in the homozygous state. In certain embodiments, iPSR B. carinata plants provided herein can comprise an iPSR mutation in the IND la gene while the IND-b gene comprises an amorphic or hypomorphic allele, and where both of the IND la and ind-b alleles are fixed in the homozygous state. In certain embodiments, iPSR B. carinata plants provided herein can comprise an iPSR mutation in the IND-b gene while the IND la gene comprises an amorphic or hypomorphic allele, and where both of the IND la and ind-b alleles are fixed in the homozygous state. Amorphic alleles of the IND la and IND lb gene include mutations in the bHLH domain or other mutations in those genes such as nonsense or frameshift mutations in codons for amino acid residues located N-terminal to the bHLH domain. Hypomorphic alleles include those analogous to hypomorphic alleles of B. napus IND genes set forth in US Patent No. 8,809,635, which is incorporated herein by reference in its entirety. The wild-type IND la and IND lb protein of B. carinata comprises a full length carboxy -terminal sequence of 18 amino acid residues which includes the C-terminal 6 amino acid consensus sequence of SEQ ID NO: 32 (Figure 4B). Such iPSR mutations include mutations in an indla and/or indlb gene encoding a mutant indla and/or indlb protein wherein one or more amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of the mutant indla and/or indlb protein are substituted and/or deleted (e.g., any of the aforementioned mutations set forth in Table 1). In certain embodiments, iPSR mutations in the IND la or IND lb gene of B. carinata include: (i) frameshift and/or nonsense mutations introduced in SEQ ID NO: 4 or an allelic variant thereof at a codon corresponding to G178 (GGG) through to H191 (CAC) of SEQ ID NO: 4 (Figure 6A) or frameshift and/or nonsense mutations introduced in SEQ ID NO: 5 or an allelic variant thereof at a codon corresponding to G175 (GGA) through to H188 (CAC) of SEQ ID NO: 5 (Figure 6B); (ii) frameshift and/or nonsense mutations introduced in SEQ ID NO: 4 or an allelic variant thereof at a codon corresponding to D183 to anyone one of Y189, Y190, and/or H191 (CAC) of SEQ ID NO: 4 or frameshift and/or nonsense mutations in SEQ ID NO: 5 or an allelic variant thereof at a codon corresponding to DI 80 to anyone one of Y186, Y187, and/or Hl 88 (CAC) of SEQ ID NO: 5 ; (iii) frame shift and/or nonsense mutations introduced in SEQ ID NO: 4 or an allelic variant thereof at a codon corresponding to SI 85, Cl 86, LI 87, or Cl 88 to anyone one of Y189, Y190, and/or Hl 91 (CAC) of SEQ ID NO: 4 or frame shift and/or nonsense mutations introduced in SEQ ID NO: 5 or an allelic variant thereof at a codon corresponding to any S182, C183, L184, or C185 to any one of Y186, Y187, and/or H188 (CAC) of SEQ ID NO: 5; (iv) frame shift and/or nonsense mutations introduced in SEQ ID NO: 4 or an allelic variant thereof at a codon corresponding to Y189, Y190, and/or H191 (CAC) of SEQ ID NO: 4 or frame shift and/or nonsense mutations introduced in SEQ ID NO: 5 or an allelic variant thereof at a codon corresponding to Y186, Y187, and/or H188 (CAC) of SEQ ID NO: 5; (v) missense mutations at codons Y189, Y190, H191, N192, S193, D194, and/or T195 in SEQ ID NO: 4 or an allelic variant thereof or missense mutations at codons Y186, Y187, H188, N189, S190, D191, and/or T192 in SEQ ID NO: 5 or an allelic variant thereof; (vi) a nonsense mutation corresponding to a C570A or C570G mutation in SEQ ID NO: 4 or an allelic variant thereof or a nonsense mutation corresponding to a C561A or C561G mutation in SEQ ID NO: 5 or an allelic variant thereof; and/or a (vii) a deletion of nucleotides comprising any one of nucleotides 532, 535, 540, 545, 550, 555, 560, 565, 566, 567, 568, or 569 or 570 to 583, 584, or 585 of SEQ ID NO: 4 or an allelic variant thereof or a deletion of nucleotides comprising any one of nucleotides 523, 525, 530, 535, 540, 545, 550, 555, 558, 559, 560, or 561 to 574, 575, or 576 of SEQ ID NO: 5 or an allelic variant thereof. In certain embodiments, such iPSR mutations encode an ind protein wherein 1, 2, 3, 4, 5, or 6 to 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 18 amino acid residues introduced at the C-terminus of SEQ ID NO: 14 or 15 or an allelic variant thereof (e.g., amino acids encompassed by the C-terminal consensus sequence of SEQ ID NO: 32) are deleted and/or substituted with non-conservative amino acid residues (e.g,. any of the aforementioned mutations set forth in Table 1). In certain embodiments, such iPSR mutations encode an ind protein wherein 6 to 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 18 amino acid residues comprising the C-terminal consensus sequence of SEQ ID NO: 32 introduced at the C-terminus of SEQ ID NO: 14, 15, or an allelic variant thereof are deleted and/or substituted with non-conservative amino acid residues. Such iPSR mutations can be introduced into a B. carinata plant by gene editing and/or breeding-based techniques. In certain embodiments, the B. carinata plant comprising the iPSR mutation is: (i) a non-naturally occurring B. carinata plant; (ii) an elite B. carinata plant; and/or (iii) a 7>. carinata plant variety.
[0062] Camelina sativa (C. saliva is diploid (2n=40) comprising a hexapioid genome and comprises three sub-genomes which are referred to as Cs-Gl, Cs-G2 and Cs-G3 (Kagale et al., Nat Commun 5, 3706 (2014). doi.org/10.1038/ncomms4706). To obtain Camelina sativa plants comprising an iPSR trait, iPSR mutations in at least one of: (i) the IND-a gene of C. sativa comprising the coding sequence of SEQ ID NO: 6 or an allelic variant thereof; (ii) the IND-b gene SEQ ID NO: 7 or an allelic variant thereof; and/or (iii) the IND-c gene comprising the coding sequence of SEQ ID NO: 8 are fixed in a homozygous state. Wild-type C. sativa plants typically comprise: (i) a wild-type IND-a gene comprising the polynucleotide sequence of SEQ ID NO: 6 or an allelic variants thereof which encode the IND protein of SEQ ID NO: 16 or an allelic variants thereof; (ii) a wild-type IND-b gene comprising the polynucleotide sequence of SEQ ID NO: 7 or an allelic variant thereof, where SEQ ID NO: 7 encodes the IND protein of SEQ ID NO: 17; and (ii) a wild-type IND-c gene which encodes the IND-c protein of SEQ ID NO: 18 or an allelic variant thereof. The wild-type IND proteins of C. sativa (SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18) each comprise a full length carboxy -terminal sequence of 18 amino acid residues which includes the C-terminal 6 amino acid consensus sequence of SEQ ID NO: 32 (Figure 4B). In certain embodiments, iPSR C. sativa plants provided herein can comprise an iPSR mutation in the IND-a, IND-b, and IND-c gene which are all fixed in the homozygous state. In certain embodiments, iPSR C. sativa plants provided herein can comprise an iPSR mutation in at least one of the IND-a, IND-b, or IND-c genes while the remaining IND genes comprise an amorphic or hypomorphic allele, and where all of the ind-a, ind-b, and ind-c alleles are fixed in the homozygous state. In certain embodiments, the iPSR C. sativa plants provided herein can comprise an iPSR mutation in at least two of the IND-a, IND-b, and IND-c genes, wherein any of the remaining IND-a, IND-b gene, or IND-c genes comprises an amorphic or hypomorphic mutation and wherein all mutations are fixed in the homozygous state. In certain embodiments, iPSR C. sativa plants provided herein can comprise an iPSR mutation in the IND-a gene (e.g., in SEQ ID NO: 6 or an allelic variant thereof) which is fixed in the homozygous state, a mutation comprising an amorphic or hypomorphic allele of the IND-b gene (e.g., in SEQ ID NO: 7 or an allelic variant thereof) which is fixed in the homozygous state, and an amorphic or hypomorphic allele of the IND-c gene (e.g., in SEQ ID NO: 8 or an allelic variant thereof) which is fixed in a homozygous state. In certain embodiments, iPSR C. sativa plants provided herein can comprise an iPSR mutation in the IND-b gene (e.g., in SEQ ID NO: 7 or an allelic variant thereof) which is fixed in the homozygous state, a mutation comprising an amorphic or hypomorphic allele of the IND-a gene e.g., in SEQ ID NO: 6 or an allelic variant thereof) which is fixed in the homozygous state, and an amorphic or hypomorphic allele of the IND-c gene (e.g., in SEQ ID NO: 8 or an allelic variant thereof) which is fixed in a homozygous state. In certain embodiments, iPSR C. sativa plants provided herein can comprise an iPSR mutation in the IND-c gene e.g., in SEQ ID NO: 8 or an allelic variant thereof) which is fixed in the homozygous state, a mutation comprising an amorphic or hypomorphic allele of the IND-a gene (e.g., in SEQ ID NO: 6 or an allelic variant thereof) which is fixed in the homozygous state, and an amorphic or hypomorphic allele of the IND-b gene (e.g., in SEQ ID NO: 7 or an allelic variant thereof) which is fixed in a homozygous state. Amorphic alleles of the IND-a, IND-b, and IND-c gene include mutations in the bHLH domain or other mutations in those genes such as nonsense or frameshift mutations in codons for amino acid residues located N-terminal to the bHLH domain. Hypomorphic alleles include those analogous to hypomorphic alleles of B. napus IND genes set forth in US Patent No. 8,809,635, which is incorporated herein by reference in its entirety. Such iPSR mutations include mutations in a C. sativa ind-a, ind-b, and/or ind-c gene encoding a mutant ind protein wherein one or more amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of said mutant ind-a protein are substituted and/or deleted (e.g., any of the aforementioned mutations set forth in Table 1). In certain embodiments, iPSR mutations in the IND-a, IND-b, or IND-c gene of C. sativa include: (i) frameshift and/or nonsense mutations introduced in SEQ ID NO: 6, 7, 8, or an allelic variant thereof at a codon corresponding to G150 (GGA) through to H163 (CAC) of SEQ ID NO: 6 or 7 or at a codon corresponding to G151 (GGA) through to Hl 64 (CAC) of SEQ ID NO: 8 (Figure 7A,B,C); (ii) frameshift and/or nonsense mutations introduced in SEQ ID NO: 6, 7, or an allelic variant thereof at a codon corresponding to Al 54 (GCT)to anyone one of Y161 (TAT), Y162 (TAC), and/or H163 (CAC) of SEQ ID NO: 6 or 7 or introduced in SEQ ID NO: 8 or an allelic variant thereof at a codon corresponding to Al 55 (GCT) to anyone one of Y162 (TAT), Y163 (TAC), and/or Hl 64 (CAC) of SEQ ID NO: 8; (iii) frame shift and/or nonsense mutations introduced in SEQ ID NO: 6, 7, or an allelic variant thereof at a codon corresponding to D155, P156, S157, Y158, L159, or C160 to anyone one of Y161, Y162, and/or H163 (CAC) of SEQ ID NO: 6 or 7 or introduced in SEQ ID NO: 8 or an allelic variant thereof at a codon corresponding to D156, P157, S158, Y159, L160, or C161 to anyone one of Y162, Y163, and/or H164 (CAC) of SEQ ID NO:8; (iv) frame shift and/or nonsense mutations introduced in SEQ ID NO: 6, 7 or an allelic variant thereof at a codon corresponding to Y161, Y162, and/or H163 (CAC) of SEQ ID NO: 6 or 7 or introduced in SEQ ID NO: 8 or an allelic variant thereof at a codon corresponding to Y162, Y163, and/or H164 (CAC) of SEQ ID NO: 8; (v) missense mutations at codons corresponding to Y162, H163, N164, S165, Q166, and/or S167 in SEQ ID NO: 6, 7, or an allelic variant thereof or at codons corresponding to Y163, Hl 64, N165, SI 66, QI 67, and/or SI 68 in SEQ ID NO: 8; and/or (vi) a nonsense mutation corresponding to a C486A or C486G mutation in SEQ ID NO: 6, 7, or an allelic variant thereof or a nonsense mutation corresponding to a C489A or C489G mutation in SEQ ID NO: 8 or an allelic variant thereof; and/or a (vii) a deletion of nucleotides comprising nucleotides 448, 450, 455, 460, 465, 470, 475, 480, 481, 482, or 483 to 499, 500, or 501 of SEQ ID NO: 6 or 7 or a deletion of nucleotides comprising nucleotides 448, 450, 455, 460, 465, 470, 475, 480, 484, 485, 486, 487, 488, or 489 to 502, 503, or 504 of SEQ ID NO: 8. In certain embodiments, such iPSR mutations encode an ind protein wherein 1, 2, 3, 4, 5, or 6 to 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 18 amino acid residues located at the C- terminus of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or an allelic variant thereof (e.g., amino acids encompassed by the C-terminal consensus sequence of SEQ ID NO: 32) are deleted and/or substituted with non-conservative amino acid residues (e.g,. any of the aforementioned mutations set forth in Table 1). In certain embodiments, such iPSR mutations encode an ind protein wherein 6 to 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 18 amino acid residues comprising the C-terminal consensus sequence of SEQ ID NO: 32 located at the C-terminus of SEQ ID NO: 16, 17, 18, or an allelic variant thereof are deleted and/or substituted with nonconservative amino acid residues. In certain embodiments, such iPSR mutations encode a C. sativa indla protein lacking 8 C-terminal amino acids of the wild-type INDla gene of C. sativa e.g., the indl-a gene of SEQ ID NO: 63, the indl-a protein of SEQ ID NO: 68, and allelic variants thereof encoding or comprising an indla protein lacking the 8 C-terminal amino acids of the INDl-a protein wherein the allelic variants have at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 63 and/or SEQ ID NO: 68). In certain embodiments, such iPSR mutations encode a C. sativa indl-b protein lacking 8 C-terminal amino acids of the wild-type INDlb gene of C. sativa (e.g., the indl-b gene of SEQ ID NO: 64, the indlb protein of SEQ ID NO: 69, and allelic variants thereof encoding or comprising an indl-b protein lacking the 8 C-terminal amino acids of the INDl-b protein wherein the allelic variants have at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 64 and/or SEQ ID NO: 69). In certain embodiments, such iPSR mutations encode a C. -sativa indl-c protein lacking 8 C-terminal amino acids of the wild-type INDl-c gene of C. sativa (e.g., the indie gene of SEQ ID NO: 65, the indlb protein of SEQ ID NO: 70, and allelic variants thereof encoding or comprising an indie protein lacking the 8 C- terminal amino acids of the INDl-c protein wherein the allelic variants have at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 65 and/or SEQ ID NO: 70). Such iPSR mutations can be introduced into a C. sativa plant by gene editing and/or breeding-based techniques. In certain embodiments, the C. sativa plant comprising the iPSR mutation is: (i) a non- naturally occurring C. sativa plant; (ii) an elite C. sativa plant; (iii) a C. sativa plant variety; and/or (iv) a C. sativa plant comprising two genomic DNA sequence polymorphisms which are not found together in any single naturally occurring C. sativa isolate.
[0063] To obtain Brassica napus plants comprising an iPSR trait, iPSR mutations in the IND-a gene of B. napus comprising the coding sequence of SEQ ID NO: 9 or an allelic variant thereof are fixed in a homozygous state. Wild-type B. napus plants typically comprise: (i) a wildtype IND-a gene comprising the polynucleotide sequence of SEQ ID NO: 9 or an allelic variants thereof which encode the IND-a protein of SEQ ID NO: 19 or an allelic variants thereof; and (ii) a wild-type IND-b gene comprising the polynucleotide sequence of SEQ ID NO: 10 or an allelic variant thereof. The wild-type IND-a protein of B. napus comprises a full length carboxy -terminal sequence of 18 amino acid residues which includes the C-terminal 6 amino acid consensus sequence of SEQ ID NO: 32 (Figure 4B). In contrast, the wild-type IND-b protein of B. napus comprises a truncated carboxy-terminal sequence of six amino acid residues which lacks the C- terminal 6 amino acid consensus sequence of SEQ ID NO: 32 (Figure 4B). In certain embodiments, iPSR . napus plants provided herein can comprise an iPSR mutation in the IND- a gene which is fixed in the homozygous state and a wild-type IND-b gene which is fixed in a homozygous state. In certain embodiments, iPSR B. napus plants provided herein can comprise an iPSR mutation in the IND-a gene which is fixed in the homozygous state and a mutation comprising an amorphic or hypomorphic allele of the IND-b gene which is fixed in a homozygous state. Amorphic alleles of the IND-b gene include mutations in the bHLH domain or other mutations in those genes such as nonsense or frameshift mutations in codons for amino acid residues located N-terminal to the bHLH domain and include mutations in the IND-b gene set forth in US Patent No. 8,809,635, which is incorporated herein by reference in its entirety. Hypomorphic alleles of the IND-b gene include mutations in the bHLH domain or other mutations in the IND-b gene set forth in US Patent No. 8,809,635, which is incorporated herein by reference in its entirety. Such iPSR mutations include mutations in an ind-a gene encoding a mutant ind-a protein wherein one or more amino acid residues located C-terminal to the conserved basic Helix- Loop Helix (bHLH) domain of said mutant ind-a protein are substituted and/or deleted (e.g., any of the aforementioned mutations set forth in Table 1). In certain embodiments, iPSR mutations in the INDI -a gene of B. napus include: (i) frameshift and/or nonsense mutations introduced in SEQ ID NO: 9 or an allelic variant thereof at a codon corresponding to G161 (GGG) through to H174 (CAC) of SEQ ID NO: 9 (Figure 8); (ii) frameshift and/or nonsense mutations introduced in SEQ ID NO: 9 or an allelic variant thereof at a codon corresponding to S165 to anyone one of Y172 (TAT), Y173 (TAC), and/or H174 (CAC) of SEQ ID NO: 9; (iii) frame shift and/or nonsense mutations introduced in SEQ ID NO: 9 or an allelic variant thereof at a codon corresponding to D166, P167, S168, R169. LI 70, or C171 to anyone one of Y172, Y173, and/or H174 (CAC) of SEQ ID NO: 9; (iv) frame shift and/or nonsense mutations introduced in SEQ ID NO: 9 or an allelic variant thereof at a codon corresponding to Y172, Y173, and/or H174 (CAC) of SEQ ID NO: 9; (v) missense mutations at codons Y173, H174, N175, S176, N177, and/or T178 in SEQ ID NO: 9 or an allelic variant thereof; (vi) a nonsense mutation corresponding to a C519A or C519G mutation in SEQ ID NO: 9 or an allelic variant thereof; and/or a (vii) a deletion of nucleotides comprising nucleotides 481, 485, 490, 495, 500, 505, 510, 513, 514, 515, 516, or 517 to 532, 533, or 534 of SEQ ID NO: 9 or an allelic variant thereof. In certain embodiments, such iPSR mutations are characteristic in that they encode an ind protein wherein 1, 2, 3, 4, 5, or 6 to 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 18 amino acid residues introduced at the C- terminus of SEQ ID NO: 19 or an allelic variant thereof (e.g., amino acids encompassed by the C- terminal consensus sequence of SEQ ID NO: 32) are deleted and/or substituted with nonconservative amino acid residues (e.g,. any of the aforementioned mutations set forth in Table 1). In certain embodiments, such iPSR mutations encode an ind protein wherein 6 to 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 18 amino acid residues comprising the C-terminal consensus sequence of SEQ ID NO: 32 introduced at the C-terminus of SEQ ID NO: 19 or an allelic variant thereof are deleted and/or substituted with non-conservative amino acid residues. Such iPSR mutations can be introduced into a B. napus plant by gene editing and/or breeding-based techniques. In certain embodiments, the B. napus plant comprising the iPSR mutation is: (i) a non- naturally occurring B. napus plant; (ii) an elite B. napus plant; (iii) a B. napus plant variety; (iv) a B. napus plant comprising two genomic DNA sequence polymorphisms which are not found together in any single naturally occurring B. napus isolate; and/or (iv) a B. napus plant which lacks a black seed trait (i.e., has yellow or brown seed) or lacks a high fiber seed trait. Such aforementioned B. napus plants include Brassica napus L. subsp. Napus, Brassica napus L. sub sp. napus forma annua, Brassica napus subsp. rapifera Metzg, and Brassica napus L. N ax . pabular ia plants. Such aforementioned B. napus plants are also alternatively referred to as Brassica campe str is and Brassica oleracea plants.
[0064] Methods of obtaining Brassica plants with at least one mutant ind gene which can confer a pod shatter resistance trait by plant breeding methods are also provided herein. In certain embodiments, the methods can comprise crossing a Brassica plant comprising at least one wildtype IND gene with a Brassica plant comprising at least one mutant ind gene containing an iPSR mutation; and (ii) isolating Fl seed and/or Fl progeny plants comprising the mutant ind gene from the cross. Such Fl seed and/or Fl progeny can be identified and isolated by non-destructively assaying for the presence of the ind mutation in a nucleic acid detection assay (e.g., a nucleic acid hybridization-, amplification-, and/or sequencing-based assay for the presence of the iPSR mutation in the IND gene(s)). Non-destructive assays can be accomplished by sampling a portion of the Brassica plant or plant part while leaving plant parts necessary for seed production and/or seed viability intact. In certain embodiments, the methods further comprise crossing the isolated Fl progeny plant comprising the mutant ind gene to a recurrent parent Brassica plant comprising a wild-type IND gene and isolating F2 seed and/or F2 progeny comprising the mutant ind gene and one or more genetic markers of the recurrent parent plant. Examples of methods for obtaining new Brassica varieties or elite germplasm containing the iPSR mutations can be achieved by introgressing the iPSR mutation from a first Brassica line into the genetic background of a Brassica varieties or elite germplasm lacking the iPSR trait by using the Brassica varieties or elite germplasm as a recurrent parent in a series of backcrosses, where progeny which contain the iPSR mutation are selected (e.g., by a non-destructive nucleic acid assay) and carried forward into additional crosses to the recurrent parent. When sufficient conversion of the germplasm containing the iPSR mutation is obtained in progeny backcrossed to the recurrent parent (e.g., wherein at least 95%, 98%, or 99% of the linked and unlinked genetic markers to those of the variety or elite germplasm has occurred), the progeny plant containing the iPSR mutation can be selfed to fix the iPSR mutation in the homozygous state and obtain a new Brassica plant variety or elite germplasm exhibiting the iPSR trait. Such linked and unlinked genetic markers which are characteristic of Brassica varieties and elite germplasm can include phenotypic markers: (i) yellow seed (e.g., US Patent Applic. Pub. No. 20190082718, incorporated herein by reference in its entirety); (ii) seed with low fiber content (e.g., US Patent Applic. Pub. No. 20190082718, incorporated herein by reference in its entirety); (iii) seed with reduced glucosinolate content (e.g., US Patent Applic. Pub. No. 20190225977, incorporated herein by reference in its entirety); (iv) seed with reduced erucic acid content (e.g., US Patent Applic. Pub. No. 20200131523, incorporated herein by reference in its entirety); (v) an early transition trait; and/or (vi) a lodging trait. Such linked and unlinked genetic markers which are characteristic of Brassica varieties and elite germplasm can include genotypic markers (e.g., genomic DNA sequence polymorphisms such as single nucleotide polymorphisms (SNPs), simple sequence repeats (SSR), DNA insertions, DNA deletions, and/or DNA inversions). Markers and associated methods that can be adapted for use in identifying, breeding, and/or introgressing iPSR mutations disclosed herein in various Brassica plants (e.g., Brassica napus, Brassica juncea, Brassica carinata, Brassica rapa (syn. B. campestris), Brassica oleracea, and Brassica nigra) include those disclosed in US Patent Applic. Pub. 20220298519, which is incorporated herein by reference in its entirety. Similar methods can be adapted for use in other Brassica plants including T. arvense where genomic sequence is available (Dorn et al., DNA Research, 2015, 22(2): 121-131, doi: 10.1093/dnares/dsu045; Nunn et al. Plant Biotechnol J., 2022, (5):944-963. doi: 10.1111/pbi.1377). In certain embodiments, SNPs identified in T. arvense, including the SNPs provided in Geng et al. BMC Biol 19, 143 (2021). doi.org/10.1186/sl2915-021-01079-0, can be used to introgress iPSR mutations into different T. arvense germplasm and/or to identify T. arvense plants provided herein which comprises two genomic DNA sequence polymorphisms which are not found together in any single naturally occurring Thlaspi arvense isolate.
[0065] Methods of obtaining a Brassica plants comprising the iPSR mutations comprising introducing a mutation in one or more nucleotides of at least one wild-type IND gene of a Brassica plant are also provided. The iPSR mutations can be introduced into one or more of the wild-type IND genes by a variety of methods. Methods for introduction of the iPSR mutations include, but are not limited to, traditional mutagenesis (e.g., Ethyl Methane Sulfonate (EMS), fast neutrons (FN), or gamma rays), and TILLING. In certain embodiments, the iPSR mutation results from introduction of a DSB at a target site in the IND gene (e.g., SEQ ID NO: 2, 4, 5, 6, 7, 8, 9, 10 or an allelic variant thereof in the region encoding the 18 amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of the IND gene as shown in Figure 3) to induce non-homologous end joining (NHEJ) at the site of the break followed by recovery of desired iPSR mutants. In certain embodiments, the iPSR mutation results from introduction of a DSB at a target site in the IND gene (e.g., SEQ ID NO: 2, 4, 5, 6, 7, 8, 9, 10 or an allelic variant thereof in the region encoding the 18 amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of the IND gene as shown in Figure 3) followed by homology-directed repair (HDR), microhomology-mediated end joining (MMEJ), or NHEJ to introduce a desired donor DNA template polynucleotide at the DSB, followed by recovery of the iPSR mutation. Methods for introduction of the DSB and iPSR mutations also include use of gene editing reagents which can comprise meganucleases, zinc finger nucleases, transcription activatorlike effector nucleases (TALENS), clustered regularly interspaced short palindromic repeat (CRISPR)-associated Cas nuclease (e.g., Cas9, Casl2a, Cmsl, S. aureus Cas9 variants, a Cas9, a nCas9 nickase, a type V Cas nuclease, a Casl2a nuclease, a nCasl2a nickase, a Casl2d (CasY), a Casl2e (CasX), a Casl2b (C2cl), a Casl2c (C2c3), a Casl2i, a Casl2f, a Casl2j, a Casl4, or eSpCas9 nuclease) in combination with guide RNAs, and the like. Methods where the aforementioned gene editing reagents, and in particular, CRISPR/Cas systems comprising a Cas nuclease and a guide RNA directed to nucleotide sequences encoding the final 18 C-terminal amino acids of the IND gene are contemplated. Methods for modifying genomes by use of Cpfl or Csml nucleases are disclosed in US Patent Application Publication 20180148735, which is incorporated herein by reference in its entirety, can be adapted for introduction of the iPSR mutations disclosed herein. Methods for modifying genomes by use of CRISPR-Cas systems are disclosed in US Patent Application Publications 20150344912, 20160138008, 20180179547, 20200172886, and 20220282244, which are incorporated herein by reference in its entirety, can also be adapted for introduction of the iPSR mutations disclosed herein. The particular Cas endonuclease selected can be used to identify the location of suitable PAM sites and design of crRNAs or sgRNAs. G-rich PAM sites, e.g., 5’-NGG are used for guide RNAs (e.g., crRNAs or sgRNAs) used with Cas9 proteins. Examples of PAM sequences include 5’-NGG (Streptococcus pyogenes), 5’-NNAGAA (Streptococcus thermophilus CRISPR1), 5’-NGGNG (Streptococcus thermophilus CRISPR3), 5’-NNGRRT or 5’-NNGRR (Staphylococcus aureus Cas9, SaCas9), and 5’-NNNGATT (Neisseria meningitidis). T-rich PAM sites (e.g., 5’-TTN or 5’-TTTV, where "V" is A, C, or G) are typically targeted for design of crRNAs or sgRNAs used with Casl2a proteins. In some instances, Casl2a can also recognize a 5’-CTA PAM motif. Other examples of potential Cas 12a PAM sequences include TTN, CTN, TCN, CCN, TTTN, TCTN, TTCN, CTTN, ATTN, TCCN, TTGN, GTTN, CCCN, CCTN, TTAN, TCGN, CTCN, ACTN, GCTN, TCAN, GCCN, and CCGN (wherein N is defined as any nucleotide). Cpfl endonuclease and corresponding guide RNAs and PAM sites are disclosed in US Patent Application Publication 2016/0208243 Al, which is incorporated herein by reference for its disclosure of DNA encoding Cpfl endonucleases and guide RNAs and PAM sites. Other related gene-editing reagents contemplated for introduction of nucleotide substitutions at a target site in the IND gene (e.g., SEQ ID NO: 2, 4, 5, 6, 7, 8, 9, 10 or an allelic variant thereof in the region encoding the 18 amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of the IND gene as shown in Figure 3) include base-editing systems (e.g., CBE (cytosine base editors) or ABE (adenine base editors)). Such CBE and ABE systems can be adapted for use in making iPSR mutations including nonsense mutations which introduce a premature “stop” codon in the in the region encoding the 18 amino acid residues located C-terminal to the conserved basic Helix- Loop Helix (bHLH) domain of the IND gene. Examples of ABE and CBE systems which can be adapted for use in making iPSR mutations disclosed herein include those disclosed in Nat Biotechnol 39, 917 (2021). doi.org/10.1038/s41587-021-01015-l, and in US20170121693, US20210130805, and WO2020214842, which are each incorporated herein by reference in their entireties.
[0066] The genome editing reagents described herein can be introduced into a pennycress plant by any appropriate method. In certain embodiments, nucleic acids encoding the genome editing reagents can be introduced into a plant cell using Agrobacterium- or Ensifer mediated transformation, particle bombardment, liposome delivery, nanoparticle delivery, electroporation, polyethylene glycol (PEG) transformation, or any other method suitable for introducing a nucleic acid into a plant cell. In certain embodiments, the Site-Specific Nuclease (SSN) or other expressed gene editing reagents can be delivered as RNAs or as proteins to a plant cell and the RT, if one is used, can be delivered as DNA. [0067] Brassica plants comprising the iPSR phenotypes can be identified by a variety of techniques that distinguish plants with pod shatter resistance from plants which are prone to pod shatter (e.g., plants comprising a wild-type IND gene). In some embodiments, pod shatter in iPSR plants and suitable controls (e.g., plants lacking an iPSR allele or containing a wild-type IND gene) can be measured in controlled environments (e.g., growth chambers and/or greenhouses) or in field trials. Methods of measuring pod shatter include: (i) percent shattered pods determination by visual evaluation of plants (US20220298519, incorporated herein by reference in its entirety) where a reduction in the percent of shattered pods in comparison to a control indicates pod shatter resistance; and (ii) measurement of weight of seeds dropped per unit area under the plant preharvest and/or during harvest (US20190053458, incorporated herein by reference in its entirety). Methods of measuring pod shatter resistance also include: (i) methods which measure the effect of mechanical force on pod shatter (e.g., measurement of the effect of mechanical agitation for specific speeds and times on pod shatter as disclosed US20220298519, incorporated herein by reference in its entirety) where a reduction in the percent of shattered pods in comparison to a control indicates pod shatter resistance; and (ii) measurement of the amount of force required to cause pod opening (e.g., use of a gram force meter to measure force needed to break open a pod US20190053458, incorporated herein by reference in its entirety) where an increase in the amount of force required is indicative of pod shatter resistance.
[0068] Brassica plants, Brassica plant parts, Brassica seed, and Brassica seed lots comprising the aforementioned or otherwise disclosed iPSR mutations are also provided herein. In certain embodiments, seed lots comprising a population of Brassica plant seed comprising untreated or treated Brassica seed are provided. Such populations of seed in the seed lots can comprise at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% Brassica plant seed comprising the iPSR mutation. A seed lot can comprise at least 1, 2, 5, 10, 20, 50, 100, 500, or 1,000 kg of seed. Use of any of the aforementioned treated or untreated Brassica plant seed lots to make animal feed (e.g., livestock or poultry feed), non-defatted Brassica sp. seed meal, or defatted Brassica sp. seed meal is also provided. Use of any of the aforementioned Brassica sp. seed lots to provide whole, cracked or rolled seed to animals (e.g., poultry) in scratch grain is also provided.
[0069] It is to be understood that while certain embodiments have been described in conjunction with the detailed description thereof and examples, the foregoing and following description is intended to illustrate and not limit the scope of the disclosure. Other aspects, advantages, and modifications are within the scope of the following embodiments and claims.
[0070] Embodiments of the methods and Brassica plants provided herein include the following numbered embodiments. [0071] 1. A method of obtaining Brassica plant with at least one mutant ind gene which can confer a pod shatter resistance trait comprising:(i) crossing a Brassica plant comprising at least one wild-type IND gene with a Brassica plant comprising at least one mutant ind gene encoding a mutant ind protein wherein one or more amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of said mutant ind protein are substituted and/or deleted; and; (ii) isolating Fl seed and/or Fl progeny plants comprising the mutant ind gene from the cross.
[0072] 2. The method of embodiment 1, wherein the encoded mutant ind protein comprises: (i) a deletion and/or substitution of 5 to 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the C-terminal amino acid residues corresponding to the final 5 to 18 C-terminal amino acid residues of the wild-type IND protein encoded by the wild-type IND gene; or (ii) a deletion and/or substitution of 6 to 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 18 C-terminal amino acid residues comprising the C-terminal consensus sequence of SEQ ID NO: 32 located at the C- terminus of the wild-type IND protein encoded by the wild-type IND gene.
[0073] 3. The method of embodiment 1 or 2, wherein the Fl seed and/or Fl progeny are isolated by non-destructively assaying for the presence of the mutation in a nucleic acid detection assay.
[0074] 4. The method of any of embodiments 1 to 3, further comprising the step of crossing the isolated Fl progeny plant comprising the mutant ind gene to a recurrent parent Brassica plant comprising a wild-type IND gene and isolating F2 seed and/or F2 progeny comprising the mutant ind gene and one or more genetic markers of the recurrent parent plant.
[0075] 5. The method of any of embodiments 1 to 4, further comprising the step of selfing the Fl progeny plants or progeny thereof and selecting an F2 progeny plant which is homozygous for the at least one mutant ind gene.
[0076] 6. A method of obtaining a Brassica plant with at least one mutant ind gene which can confer a pod shatter resistance trait comprising introducing a mutation in one or more nucleotides of at least one wild-type IND gene of a Brassica plant to obtain a Brassica plant comprising a mutant ind gene encoding a mutant ind protein wherein one or more amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of said mutant ind protein are substituted and/or deleted, optionally wherein the mutant ind protein comprises: (i) a deletion and/or substitution of 5 to 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the C-terminal amino acid residues corresponding to the final 5 to 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, or 18 C-terminal amino acid residues of the wild-type IND protein encoded by the wild-type IND gene; or (ii) a deletion and/or substitution of 6 to 18 of the 18 C-terminal amino acid residues comprising the C-terminal consensus sequence of SEQ ID NO: 32 located at the C-terminus of the wild-type IND protein encoded by the wild-type IND gene.
[0077] 7. The method of embodiment 6, wherein said mutation is introduced with one or more gene-editing reagents.
[0078] 8. The method of embodiment 7, wherein said gene-editing reagents comprise: (i) an RNA directed endonuclease and a guide RNA directed to the IND gene; (ii) a transcription activator-like effector nuclease (TALEN) directed to the IND gene; (iii) a zinc-finger nuclease (ZFN) directed to the IND gene; or (iv) any one of (i), (ii), or (iii) and DNA donor template.
[0079] 9. The method of embodiment 8, wherein the guide RNA comprises the RNA molecule of any one of SEQ ID NO: 21 to 27 or a fragment thereof which can hybridize to a target IND gene.
[0080] 10. The method of embodiment 7, wherein the gene-editing reagents comprise:
(i) an adenine base-editor (ABE) and a guide RNA directed to the IND gene; or (ii) a cytosine base-editor and a guide RNA directed to the IND gene.
[0081] 11. The method of embodiment 6, wherein the mutation is introduced by random mutagenesis and wherein the method further comprising screening progeny subjected to the mutagenesis by a DNA analysis technique to identify a plant comprising the mutation.
[0082] 12. The method of any one of embodiments 6 to 11, wherein the mutation is simultaneously introduced in more than one IND gene of the plant.
[0083] 13. The method of any one of embodiments 6 to 11, wherein the mutation is introduced in a first IND gene of the plant, a plant comprising the mutation in the first gene is isolated, and the mutation is introduced in a one or more additional IND genes of the plant comprising the mutation in the first IND gene.
[0084] 14. The method of any one of embodiments 1 to 13, wherein the Brassica plant is a Thlaspi arvense. Brassica napus, Brassica carinata, or Camelina sativa plant .
[0085] 15. The method of any one of embodiments 1 to 14, wherein: (i) the Brassica plant is a Thlaspi arvense plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 2 or an allelic variant thereof or wherein the wild-type IND gene encodes a protein comprising the polypeptide of SEQ ID NO: 12 or an allelic variant thereof; (ii) the Brassica plant is a Brassica napus plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 9 or an allelic variant thereof or wherein the wild-type IND gene encodes a protein comprising the polypeptide of SEQ ID NO: 19 or an allelic variant thereof, optionally further comprising introducing a mutation in the wild-type IND coding sequence of SEQ ID NO: 10 or an allelic variant thereof or in the wild-type IND coding sequence which encodes SEQ ID NO: 20 or an allelic variant thereof; (iii) the Brassica plant is Brassica carinata plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 4, SEQ ID NO: 5, or an allelic variant thereof or wherein the wild-type IND gene encodes a protein comprising the polypeptide of SEQ ID NO: 14, SEQ ID NO: 15, or an allelic variant thereof; or (iv) the Brassica plant is a Camelina sativa plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or an allelic variant thereof or wherein the wild-type IND gene encodes a protein comprising the polypeptide of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or an allelic variant thereof
[0086] 16. The method of any one of embodiments 1 to 14 wherein: (i) the Brassica plant is a Thlaspi arvense plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 2 or an allelic variant thereof and wherein the mutation is located at nucleotide positions corresponding to nucleotide 496 to 546 of SEQ ID NO: 2, optionally wherein the mutation corresponds to a C531A or C531G mutation in SEQ ID NO: 2, corresponds to a deletion of nucleotides comprising nucleotides 529 to 546 of SEQ ID NO: 2, or corresponds to a frameshift or nonsense mutation in the Y176, Y177, or H178 codon of SEQ ID NO:2; (ii) the Brassica plant is a Brassica napus plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 9 or an allelic variant thereof; wherein the mutation is located at nucleotide positions corresponding to nucleotide 484 to 534 of SEQ ID NO: 9 or optionally wherein the mutation corresponds to a C519A or C591G mutation in SEQ ID NO: 9 , corresponds to a deletion of nucleotides comprising nucleotides 517 to 534 of SEQ ID NO: 9, or corresponds to a frameshift or nonsense mutation in the Y172, Y173, or H174 codon of SEQ ID NO: 9; optionally further comprising introducing a mutation located at nucleotide positions corresponding to nucleotide 536 to 546 of SEQ ID NO: 10; (iii) the Brassica plant is a Brassica carinata plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 4, SEQ ID NO: 5, or an allelic variant thereof and wherein the mutation is located at nucleotide positions corresponding to nucleotide 535 to 585 of SEQ ID NO: 4, optionally wherein the mutation corresponds to a C570A or C570G mutation in SEQ ID NO: 4, corresponds to a deletion of nucleotides corresponding to nucleotides 515 to 534 of SEQ ID NO: 4, or corresponds to a frameshift or nonsense mutation in the Y189, Y190, or Hl 91 codon of SEQ ID NO: 4; and/or at nucleotide positions corresponding to nucleotide 526 to 576 of SEQ ID NO: 5, optionally wherein the mutation corresponds to a C561 A or C561G mutation in SEQ ID NO: 5, corresponds to a deletion of nucleotides comprising nucleotides 559 to 576 of SEQ ID NO: 5, or corresponds to a frameshift or nonsense mutation in the Y186, Y187, or Hl 88 codons of SEQ ID NO: 5; or(iv) the Brassica plant is a Camelina sativa plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or an allelic variant thereof and wherein the mutation is located at nucleotide positions corresponding to nucleotide 451 to 501 of SEQ ID NO: 6, optionally wherein the mutation corresponds to a C486A or C486G mutation in SEQ ID NO: 6, corresponds to a deletion of nucleotides comprising nucleotides 559 to 576 of SEQ ID NO: 6, or corresponds to a frameshift or nonsense mutation in the Y161, Y162, or H163 codons of SEQ ID NO: 6; and/or at nucleotide positions corresponding to nucleotide 451 to 501 of SEQ ID NO: 7 or optionally wherein the mutation corresponds to a C486A or C486G mutation in SEQ ID NO: 7, corresponds to a deletion of nucleotides comprising nucleotides 559 to 576 of SEQ ID NO: 7, or corresponds to a frameshift or nonsense mutation in the Y161, Y162, or H163 codons of SEQ ID NO: 7; and/or at nucleotide positions corresponding to nucleotide 454 to 504 of SEQ ID NO: 8, optionally wherein the mutation corresponds to a C489A or C489G mutation in SEQ ID NO: 8, corresponds to a deletion of nucleotides comprising nucleotides 487 to 504 of SEQ ID NO: 8, or corresponds to a frameshift or nonsense mutation in the Y162, Y163, or H164 codons of SEQ ID NO: 8.
[0087] 17. The method of any one of embodiments 1 to 14 wherein: (i) the Brassica plant is a Thlaspi arvense plant wherein the wild-type IND gene encodes a protein comprising the polypeptide of SEQ ID NO: 12 or an allelic variant thereof and wherein the mutant encodes a protein lacking at least 5 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 166 to 182 of SEQ ID NO: 12, wherein the mutant encodes a protein lacking at least 5 or 6 to 8, 9, or 10 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 173 to 182 of SEQ ID NO: 12, or wherein the mutant encodes a protein of SEQ ID NO: 43, 46, 78, or an allelic variant thereof lacking said amino acid residues having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 43, 46, or 78; (ii) the Brassica plant is a Brassica napus plant wherein the wildtype IND gene encodes a protein comprising the polypeptide of SEQ ID NO: 19 or an allelic variant thereof and wherein the mutant encodes a protein lacking at least 5 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 161 to 178 of SEQ ID NO: 19; (iii) the Brassica plant is a Brassica carinata plant wherein the wild-type IND gene encodes a protein comprising the polypeptide of SEQ ID NO: 14, SEQ ID NO: 15, or an allelic variant thereof and wherein the mutant encodes a protein lacking at least 5 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 179 to 195 of SEQ ID NO: 14 and/or corresponding to amino acid residues 176 to 192 of SEQ ID NO: 15; or (iv) the Brassica plant is a Camelina sativa plant wherein the wild-type IND gene encodes a protein comprising the polypeptide of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or an allelic variant thereof and wherein the mutant encodes a protein lacking at least 5 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 150 to 167 of SEQ ID NO: 16 or SEQ ID NO: 17 and/or lacking at least 5 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 151 to 168 of SEQ ID NO: 18, wherein the mutant encodes a protein lacking at least 5 or 6 to 8, 9, or 10 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 158 to 167 of SEQ ID NO: 16 or SEQ ID NO: 17 and/or lacking at least 5 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 159 to 168 of SEQ ID NO: 18, or wherein the mutant encodes a protein of SEQ ID NO: 68, 69, 70, or an allelic variant thereof lacking said amino acid residues and having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 68, 69, or 70.
[0088] 18. A Brassica plant made by the method of any one of embodiments 1 to 17, optionally wherein said Brassica plant is a Thlaspi arvense plant wherein said Thlaspi arvense plant comprises two genomic DNA sequence polymorphisms which are not found together in any single naturally occurring Thlaspi arvense isolate or is a Thlaspi arvense isolate lacking: (i) one or more genetic polymorphisms characteristic of the wild-type Thlaspi arvense cultivar 2032, representative seed of the cultivar having been deposited under NCMA Accession Number 202210002; (ii) one or more traits characteristic of the wild-type Thlaspi arvense cultivar 2032, representative seed of the cultivar having been deposited under NCMA Accession Number 202210002, and/or (iii) wherein the Thlaspi arvense plant lacks a black seed trait, a high fiber seed trait, reduced yield trait, and/or an increased lodging trait.
[0089] 19. A Brassica plant comprising at least one mutant ind gene encoding a mutant ind protein wherein one or more amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of said mutant ind protein are substituted and/or deleted, wherein said Brassica plant is not a Thlaspi arvense plant and optionally wherein the mutant ind protein comprises: (i) a deletion and/or substitution of 5 to 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the C-terminal amino acid residues corresponding to the final 5 to 18 C-terminal amino acid residues of the corresponding wild-type IND protein; or (ii) a deletion and/or substitution of 6 to 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 18 C-terminal amino acid residues comprising the C-terminal consensus sequence of SEQ ID NO: 32 located at the C-terminus of the corresponding wild-type IND protein.
[0090] 20. The Brassica plant of embodiment 19, wherein the Brassica plant is
Brassica napus. Brassica carinata, or Camelina sativa plant.
[0091] 21. The Brassica plant of embodiment 19 or 20, wherein said plant is homozygous for the mutant ind gene or genes and exhibits reduced pod shatter in comparison to a control plant lacking the mutant ind gene or genes. [0092] 22. The Brassica plant of any one of embodiments 19 to 21, wherein said plant exhibits improved agronomic performance in comparison to a control plant homozygous for an amorphic allele of the ind gene.
[0093] 23. The Brassica plant of any one of embodiments 19 to 22, wherein Brassica plant comprises elite Brassica plant germplasm.
[0094] 24. The Brassica plant of any one of embodiments 19 to 23, wherein: (i) the
Brassica plant is a Brassica napus plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 9 or an allelic variant thereof or wherein the wild-type IND gene encodes the corresponding wild-type IND protein comprising the polypeptide of SEQ ID NO: 19 or an allelic variant thereof; (ii) the Brassica plant is a Brassica carinata plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 4, SEQ ID NO: 5, or an allelic variant thereof or wherein the wild-type IND gene encodes the corresponding wildtype IND protein comprising the polypeptide of SEQ ID NO: 14, SEQ ID NO: 15, or an allelic variant thereof; or (iii) the Brassica plant is a Camelina sativa plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 6, SEQ ID NO: 7, or an allelic variant thereof or wherein the wild-type IND gene encodes the corresponding wild-type IND protein comprising the polypeptide of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or an allelic variant thereof.
[0095] 25. The Brassica plant of any one of embodiments 19 to 23, wherein: (i) the
Brassica plant is a Brassica napus plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 9 or an allelic variant thereof; wherein the mutation is located at nucleotide positions corresponding to nucleotide 484 to 534 of SEQ ID NO: 9 or optionally wherein the mutation corresponds to a C519A or C591G mutation in SEQ ID NO: 9 , corresponds to a deletion of nucleotides comprising nucleotides 517 to 534 of SEQ ID NO: 9, or corresponds to a frameshift or nonsense mutation in the Y172, Y173, or H174 codon of SEQ ID NO: 9; optionally further comprising introducing a mutation located at nucleotide positions corresponding to nucleotide 536 to 546 of SEQ ID NO: 10; (ii) the Brassica plant is a Brassica carinata plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 4, SEQ ID NO: 5, or an allelic variant thereof and wherein the mutation is located at nucleotide positions corresponding to nucleotide 535 to 585 of SEQ ID NO: 4, optionally wherein the mutation corresponds to a C570A or C570G mutation in SEQ ID NO: 4, corresponds to a deletion of nucleotides corresponding to nucleotides 515 to 534 of SEQ ID NO: 4, or corresponds to a frameshift or nonsense mutation in the Y189, Y190, or H191 codon of SEQ ID NO: 4; and/or at nucleotide positions corresponding to nucleotide 526 to 576 of SEQ ID NO: 5, optionally wherein the mutation corresponds to a C561 A or C561G mutation in SEQ ID NO: 5, corresponds to a deletion of nucleotides comprising nucleotides 559 to 576 of SEQ ID NO: 5, or corresponds to a frameshift or nonsense mutation in the Y186, Y187, or Hl 88 codons of SEQ ID NO: 5; or (iii) the Brassica plant is a Camelina sativa plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or an allelic variant thereof and wherein the mutation is located at nucleotide positions corresponding to nucleotide 451 to 501 of SEQ ID NO: 6, optionally wherein the mutation corresponds to a C486A or C486G mutation in SEQ ID NO: 6, corresponds to a deletion of nucleotides comprising nucleotides 559 to 576 of SEQ ID NO: 6, or corresponds to a frameshift or nonsense mutation in the Y161, Y162, or H163 codons of SEQ ID NO: 6; and/or at nucleotide positions corresponding to nucleotide 451 to 501 of SEQ ID NO: 7 or optionally wherein the mutation corresponds to a C486A or C486G mutation in SEQ ID NO: 7, corresponds to a deletion of nucleotides comprising nucleotides 559 to 576 of SEQ ID NO: 7, or corresponds to a frameshift or nonsense mutation in the Y161, Y162, or H163 codons of SEQ ID NO: 7; and/or at nucleotide positions corresponding to nucleotide 454 to 504 of SEQ ID NO: 8, optionally wherein the mutation corresponds to a C489A or C489G mutation in SEQ ID NO: 8, corresponds to a deletion of nucleotides comprising nucleotides 487 to 504 of SEQ ID NO: 8, or corresponds to a frameshift or nonsense mutation in the Y162, Y163, or H164 codons of SEQ ID NO: 8.
[0096] 26. The Brassica plant of any one of embodiments 19 to 23, wherein: (i) the
Brassica plant is a Brassica napus plant wherein the wild-type IND gene encodes a protein comprising the polypeptide of SEQ ID NO: 19 or an allelic variant thereof and wherein the mutant encodes a protein lacking at least 5 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 161 to 178 of SEQ ID NO: 19; (ii) the Brassica plant is a Brassica carinata plant wherein the wild-type IND gene encodes a protein comprising the polypeptide of SEQ ID NO: 14, SEQ ID NO: 15, or an allelic variant thereof and wherein the mutant encodes a protein lacking at least 5 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 179 to 195 of SEQ ID NO: 14 and/or corresponding to amino acid residues 176 to 192 of SEQ ID NO: 15; or (iii) the Brassica plant is a Camelina sativa plant wherein the wild-type IND gene encodes a protein comprising the polypeptide of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or an allelic variant thereof and wherein the mutant encodes a protein lacking at least 5 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 150 to 167 of SEQ ID NO: 16, 17 and/or lacking at least 5 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 151 to 168 of SEQ ID NO: 18, wherein the mutant encodes a protein lacking at least 5 or 6 to 8, 9, or 10 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 158 to 167 of SEQ ID NO: 16 or SEQ ID NO: 17 and/or lacking at least 5 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 159 to 168 of SEQ ID NO: 18, or wherein the mutant encodes a protein of SEQ ID NO: 68, 69, 70, or an allelic variant thereof lacking said amino acid residues and having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ
ID NO: 68, 69, or 70.
[0097] 27. A Brassica plant cell or plant part obtained from the Brassica plant of any one of embodiments 19 to 26, wherein said cell, plant propagule, or plant part comprises the mutant ind gene.
[0098] 28. The Brassica plant cell of embodiment 27, wherein the plant cell is plant cell callus.
[0099] 29. The Brassica plant part of embodiment 27, wherein the part is a seed, pollen, ovule, root, pod, stem, or leaf.
[00100] 30. A seed lot comprising a plurality of the Brassica seed of embodiment 29.
[00101] 31. A method for harvesting seed from a Brassica crop comprising harvesting seed from a plurality of the Brassica plants of any one of embodiments 19 to 26.
[00102] 32. A Thlaspi arvense plant comprising a mutant ind gene encoding a mutant ind protein wherein one or more amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of said mutant ind protein are substituted and/or deleted, wherein said Thlaspi arvense plant comprises two genomic DNA sequence polymorphisms which are not found together in any single naturally occurring Thlaspi arvense isolate and optionally wherein the mutant ind protein comprises: (i) a deletion and/or substitution of 5 to 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the C-terminal amino acid residues corresponding to the final 5 to 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, or 18 C-terminal amino acid residues of the corresponding wild-type IND protein; or (ii) a deletion and/or substitution of 6 to 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of the 18 C-terminal amino acid residues comprising the C-terminal consensus sequence of SEQ ID NO: 32 located at the C-terminus of the corresponding wild-type IND protein.
[00103] 33. The Thlaspi arvense plant of embodiment 32, wherein the Thlaspi arvense plant comprises elite Thlaspi arvense plant germplasm.
[00104] 34. The Thlaspi arvense plant of embodiment 32 or 33, wherein said Thlaspi arvense plant: (i) lacks one or more genetic polymorphisms characteristic of the wild-type Thlaspi arvense cultivar 2032, representative seed of the cultivar having been deposited under NCMA Accession Number 202210002; (ii) lacks one or more traits characteristic of the wild-type Thlaspi arvense cultivar 2032 , representative seed of the cultivar having been deposited under NCMA Accession Number 202210002; and/or (iii) wherein the Thlaspi arvense plant lacks a black seed trait, a high fiber seed trait, a reduced yield, and/or an increased lodging trait. [00105] 35. The Thlaspi arvense plant of any one of embodiments 32 to 24, wherein the mutation corresponds to a C531 A or C531G mutation in SEQ ID NO: 2, corresponds to a deletion of nucleotides comprising nucleotides 529 to 546 of SEQ ID NO: 2, or corresponds to a frameshift or nonsense mutation in the Y176, Y177, or H178 codon of SEQ ID NO:2.
[00106] 36. The Thlaspi arvense plant of any one of embodiments 32 to 35, wherein the mutant encodes a protein lacking at least the six C-terminal amino acid residues of the polypeptide corresponding to amino acid residues 177 to 182 of SEQ ID NO: 12 or wherein the mutant encodes a protein lacking at least 5 or 6 to 8, 9, or 10 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 173 to 182 of SEQ ID NO: 12, or wherein the mutant encodes a protein of SEQ ID NO: 43, 46, 78, or an allelic variant thereof lacking said amino acid residues having at least 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 43, 46, or 78.
[00107] 37. The Thlaspi arvense plant of any one of embodiments 32 to 36, wherein said plant is homozygous for the mutant ind gene or genes and exhibits reduced pod shatter in comparison to a control plant lacking the mutant ind gene.
[00108] 38. The Thlaspi arvense plant of embodiment 37, wherein said plant exhibits improved agronomic performance in comparison to a control plant homozygous for an amorphic allele of the IND gene.
[00109] 39. A Thlaspi arvense plant cell or plant part obtained from the Thlaspi arvense plant of any one of embodiments 32 to 38, wherein said cell, plant propagule, or plant part comprises the mutant IND gene.
[00110] 40. The Thlaspi arvense plant cell of embodiment 39, wherein the plant cell is plant cell callus.
[00111] 41. The Thlaspi arvense plant part of embodiment 39, wherein the part is a seed, pollen, ovule, root, pod, stem, or leaf.
[00112] 42. A seed lot comprising a plurality of the Thlaspi arvense seed of embodiment 41.
[00113] 43. A method for harvesting seed from a Thlaspi arvense crop comprising harvesting seed from a plurality of the Thlaspi arvense plants of any one of embodiments 32 to 38.
[00114] The disclosure will be further described in the following examples, which do not limit the scope of the disclosure described in the claims and embodiments. EXAMPLES
Example 1. Identification of Underlying Gene Mutations in a T. arvense Reduced Shatter Line
[00115] Genomic DNA was extracted from the candidate T. arvense reduced shatter line designated 2032 (T. arvense cultivar 2032, deposited as deposited under NCMA Accession Number 202210002) using column-based plant DNA mini -prep protocols, and whole-genome resequencing was performed with 2x150 bp read technology on Illumina platform. Sequence reads were mapped to the MN106-Reference genome (Nunn et al. Plant Biotechnol J., 2022, doi: 10.1111/pbi.13775) using bwa alignment and SNPs were identified using GATK HaplotypeCaller. SNPs were analyzed using genome annotations to assess the change in amino acid sites with the presence of nucleotide changes within the coding regions. The nucleotide substitution at position C531 A (relative to the wild-type T. arvense INDI gene of SEQ ID NO: 2) resulting in premature stop codon at Y177* (relative to the wild-type T. arvense INDI protein of SEQ ID NO: 12) was identified in the INDEHISCENT1 gene of T. arvense cultivar 2032. This premature stop codon results in the expression of an indl protein which lacks the 6 C-terminal amino acids of the wild-type INDI protein. The indl allele with the C531 A nucleotide substitution is referred to as indl-4, comprises SEQ ID NO: 73, and encodes the indl-4 protein of SEQ ID NO: 74. To determine the conservative nature of the mutation sites across species, T. arvense peptide sequences were aligned with the peptide sequences of other Brassica species (Figure 4). The DNA and amino acid sequence alignments highlight the conserved regions of the IND genes. [00116] Allele-specific markers were designed to genotype plants containing the C531 A mutation identified from whole-genome resequencing data and genotyping was performed on the DNA extracted using quick-extract protocol from LGC genomics. PCR reactions were carried out ThermoFisher QuantStudio 6.0 using the recommended protocols. Co-segregation analysis was performed in the greenhouse grown 54 F2 plants derived from cross between a line carrying the reduced shatter marker and a line carrying the normal shatter trait. Greenhouse observations were made for presence and absence of the reduced shatter trait. It was found that the plants carrying the C531 A indl mutation in the homozygous state (“homozyg.”) had this reduced shatter trait in the greenhouse conditions.
[00117] Table 2. Summary of Greenhouse Experiments
Figure imgf000038_0001
Figure imgf000039_0001
[00118] Based on observations in the greenhouse it was found that plants with wild-type INDI allele had no reduced shatter phenotype, whereas 64.7% of the plants classified as having the C531A indl mutation (indl-4; SEQ ID NO: 73) in the homozygous state exhibited a reduced shatter phenotype. Penetrance of the trait could have been influenced by the greenhouse conditions. Also, the method used to assess shattering for this experiment was a qualitative approach. Shattering resistance was not, for example, quantified using a force meter.
[00119] Table 3. Summary of Phenotypes in Greenhouse observations
Figure imgf000039_0002
[00120] To further confirm the trait and genotype interaction, the cultivars derived from T. arvense cultivar 2032 were planted in replicated trials at Mt. Pulaski, IL, USA (MPI) and Havana, IL, USA (HVI) in the fall of 2021. These cultivars were evaluated for shattering at harvest on a scale of 0 to 100 percent. Phenotypic measurements are collected just prior to harvest when plants are fully mature. Percent shatter is based on the estimation of pods lost to natural dehiscence. DNA was extracted using column based mini-prep methods and genotyping was performed on the seedlings of these 29 cultivars using the allele-specific marker designed to target the indl gene mutation discussed above. Phenotypic data and genotypic data associated with these lines is described below and in Figures 1 and 2. Co-segregation analysis with the trait was significantly associated with the reduced shatter measurement observed in the field conditions.
[00121] Table 4. Field observations of 29 cultivars derived from T. arvense cultivar 2032 parent carrying reduced shatter mutation in indl gene. Shatter rating columns represent the percent of pods dehisced after harvest maturity in field conditions at Mt. Pulaski (MPI) and Havana, Illinois (HVI).
Figure imgf000040_0001
Figure imgf000041_0001
[00122] Table 5. Comparison of T. arvense cultivar 2032 with the 182002-B-B-31 line obtained from T. arvense cultivar 2032
Figure imgf000041_0002
[00123] Table 5 shows that the 182002-B-B-31 line obtained from a cross between T. arvense cultivar 2032 and a second unrelated parent retains the reduced shattering trait of T. arvense cultivar 2032 but lacks the increased lodging and decreased yield traits of T. arvense cultivar 2032.
Example 2. Identification of Protospacer regions for guide RNAs directed to Brassica IND genes
[00124] Protospacer coding sequences described below in Table 5 can be targeted in three Brassica plants (Ta - Thlaspi arvense, At - Arabidopsis thaliana; Cs - Camelina sativa. Ta_At_Cs_PS_2) can also be targeted for many of the Brassica plants such as Brassica napus, Camelina sativa, and Brassica carinata. Suitable gRNAs can be designed based on the Cas9 nuclease and protospacer adjacent motif (PAM) - ‘NGG’ present in the target Brassica plant IND gene sequence (e.g., SEQ ID NO: 2, 4, 5, 6, 7, 8, and 9).
[00125] Protospacers can be designed to target the different region of IND gene including iPSR mutations in pennycress, Arabidopsis and Camelina using the webtool “CHOPCHOP” (on the https internet site chopchop.cbu.uib.no/; Labun et al. Nucleic Acids Research (2019) doi.org/10.1093/nar/gkz365) and “CRISPOR” (on the http internet site crispor.tefor.net/;
Concordet and Haeussler. Nucleic Acids Research (2018) doi.org/10.1093/nar/gky354). These protospacers can be assembled for use with a Cas9 system. To generate mutations in these Brassicas, CRISPR/SpCas9 DNA constructs designed to target regions of the IND genes will be delivered using a disarmed Agrobacterium tumefaciens strain (GV3101) and a standard floral dip or other transformation method. Presence of the edits in the IND genes of T1 plants will confirmed through PCR or fluorescent marker screening. Seed from the progeny T2 generation will be screened for lines with IND gene edits that lack the transgene using PCR and sanger sequencing.
[00126] Table 6. Guide RNA protospacer sequence examples
Figure imgf000042_0001
Example 3. Generation of Pennycress, Arabidopsis, and Camelina plants with Mutant ind Genes by Gene Editing
[00127] Protospacer designing and construct preparation
[00128] Validated INDI gene sequences from Penny cress, Arabidopsis, and Camelina were used to design protospacers unique and common to the INDI loci in all three plant species. Editing efficiency and frameshift efficiency along with other parameters were compared using two webbased protospacer design software’s CHOPCHOP and Cas-Designer (Park et al. Bioinformatics 31 (24): 4014-4016 (2015), doi.org/10.1093/bioinformatics/btv537. Off-targeting potential was evaluated using BlastN similarity search program against the respective genomes. [00129] The designed protospacers were cloned in a binary Agrobacterium plant transformation vector between Aarl restriction sites using Golden Gate assembly. The expression of the guide RNA containing the protospacer was driven by Arabidopsis U6 promoter whereas expression of Cas9 nuclease and the scorable marker DsRED2 were driven by an Arabidopsis
RPS5a and a Cassava mosaic virus promoter, respectively.
[00130] Table 7
Figure imgf000043_0001
[00131] Agrobacterium transformation and floral dip
[00132] Sanger sequencing verified constructs (pARV55, pARV56, pARV58, pARV59, pARV72 and pARV73) were electroporated into the Agrobacterium tumefaciens GV3101 strain and selected on Rifampicin, Gentamycin and construct specific Kanamycin antibiotics. Agrobacterium culture (OD 1.0-1.2) from positively screened single agrobacterium colony was used to transform the Pennycress, Arabidopsis and Camelina plants using a floral dip method. Pennycress and Camelina flowers were dipped in a suspension of the Agrobacterium for 5-8 min under a 27-28 mmHg vacuum in order to maximize the number of transformation events whereas Arabidopsis flowers were dipped in a suspension of the Agrobacterium without vacuum. Once matured, the T1 seeds were screened under red light system to identify the DsRED2 expressing transformants. T1 plants were grown from the DsRED2 expressing transformants and T2 seeds were harvested for further genotyping.
[00133] Screening for edited plants
[00134] Plants from independent T2 lines of Penny cress, Arabidopsis, and Camelina were genotyped for Cas9 nuclease-mediated editing. In brief, PCR product was amplified using target site flanking primer pairs and Sanger sequencing. The resulting sequences were aligned against the wildtype INDI sequence to identify the plants with insertions, deletions, and/or substitutions (InDeis). [00135] Reduced pod shatter phenotyping
[00136] A gram force tension gauge (SSEYL ATG-100-2 Tension Gauge) attached to a two-inch alligator clip was used to determine the force required to break apart seedpods at the septum. One side of a pennycress pod was clipped, and the other side was pulled manually until the pod breaks apart. For each line, 5 pods each from five different plants were used for the measurements.
[00137] In Arabidopsis, 5 siliques from each plant were manually detached and kept in 1.5 ml microcentrifuge tube. Tubes with pods from Col-0 and edited T2 plants were simultaneously vortexed for 10 seconds and scored based on number and extant of pods shattered. The experiment was replicated by 5 independent people to reduce handling bias.
[00138] Partially mature pods were observed under the microscope to understand the separation layer formation in the edited plants. In brief, free-hand sections were stained with 0.05% toluidine blue O and washed thrice with MilliQ™ water before transferred to a microscope slide for observation under a compound microscope using the 20x objective.
[00139] Screening for edited plants in pennycress
[00140] Several protospacers were designed to target 5’ to 3’ region of INDI gene in pennycress, Arabidopsis, and Camelina and subsequently cloned to generate constructs pARV55, pARV56, pARV58, pARV59, pARV72 and pARV73. Pennycress was transformed with 4 constructs (pARV55, pARV56, pARV58, pARV59) whereas Arabidopsis and Camelina were transformed with 3 constructs (pARV56, pARV72 and pARV73). The TDNA-free T2 plants from each positive line were further screened for the presence or absence of editing in the INDI gene(s). [00141] In penny cress, PCR followed by Sanger sequencing of the INDI locus from pARV55 derived T2 plants resulted into identification of 3 different insertion events after the 517th nucleotide (+A, +G and +T in gene edited lines B56924A1 (SEQ ID NO: 43), B56914A5 (SEQ ID NO: 34), and B56930A5 (SEQ ID NO: 35), respectively) whereas pARV56 derived T2 plants resulted in +A, +C and +T insertion events after the 524th nucleotide of the INDI gene in gene edited lines B56929A1, B56922A5 and B56941 Al, respectively (Figure 1 IB). Interestingly, insertion in two of the edited plants namely B56924A1 (+A insertion after 517th nucleotide) and B56929A1 (+A insertion after 524th nucleotide) resulted into early stop codon immediately after the conserved domain and resultant protein was like the Ta_indl-4 allele (Figure 12). When we additionally created edits in the non-iPSR regions of the INDI gene which targeted the 5’ coding region of the gene and the bHLH domain, we were unable to break the seed pods using the methods described here and threshability of the seed pods was significantly reduced in comparison to the edits in the iPSR regions of the INDI gene.
[00142] Screening for edited plants in Arabidopsis [00143] In Arabidopsis, screening of pARV56- derived T2 plants resulted in identification of 2 insertion events after the 572nd nucleotide (+A in At indl Pl and +T in At_indl_P2) and one deletion event after 560th nucleotide (-34 in At indl Pl) (Figure 18B). The +A insertion in At indl Pl and -34 bases deletion in At_indl_P3 resulted in early stop codon after conserved domain whereas +T insertion in At_indl_P2 resulted in frameshift with one amino acid extended protein (Figure 19). Similar to penny cress, we created additional edits in the non-iPSR regions of the Arabidopsis INDI gene (Figure 23).
[00144] Screening for edited plants in Camelina
[00145] In Camelina, screening of pARV56- derived T2 plants resulted in identification of several T2 plants with each of 3 homeologs having different combination of edits. In T2 plant C10012D7, gene on Chr8 and Chrl3 were found to have +A insertion after 476th and 479th nucleotide, respectively whereas edit on Chr2 homologue was detected to be biallelic in nature (+A after 476th nucleotide and other edits). Similarly, In T2 plant C10012D10, the IND gene on Chr8 and Chrl3 were found to have +T insertion (after 476th nucleotide) and +A insertion (after 479th nucleotide), respectively whereas the edit on Chr2 IND homologue was again detected to be biallelic in nature (+A and +T after 476th nucleotide and other edits).
[00146] Reduced pod shatter phenotyping in pennycress
[00147] Multiple pods from each edited T2 plants were phenotype for difference in force needed to break open the pod using a gram force tension gauge attached to a two-inch alligator clip. Upon applying the force, the seedpods of all the edited alleles required more force to break open than the wild type seed pods (Fig. 17 A. However, the force required to break open the seedpods of both the alleles with +A insertion (B56924A1 and B56929A1) was approximately 3 times more wild type (Fig. 17A). Other alleles in this region showed a stronger reduction in shatter. Alleles with edits in the iPSR regions showed significantly reduced pod breakage and strongly supports the causation of the observed reduced pod shatter phenotype by these mutations. For 5’ edits or the bHLH domain edits in the non-iPSR regions of the INDI gene which created nonsense or frameshift mutations, we were unable to collect any measurements because the seed pods harvested from these edited plants were too difficult to separate using the above methods to separate pods of the iPSR mutants or even with manual threshing.
[00148] Reduced pod shatter phenotyping in Arabidopsis
[00149] The size and shape of the Arabidopsis siliques makes it difficult to use the gram force tension gauge, so the vortexing method described above was used. The manual shattering observations performed by 5 different individuals clearly showed the lesser number of open siliques in the At indl Pl allele with +A insertion compared to wild type siliques (Figure 10C). Upon applying the external force, only 20% of At_indl_Pl siliques were shattered whereas 60% siliques break open in the wild type (Figure IOC) which clearly indicates towards the reduce pod shatter phenotype acquired upon the +A insertion in edited alleles. We expect similar results when edited in this iPSR region of the Arabidopsis.
[00150] Toluidine blue O staining assay
[00151] The phenotype observed in the pennycress pods and Arabidopsis siliques were clearly supported by the 0.05% toluidine blue O staining assay. In pennycress, 2032-WG with the indl-4 allele (SEQ ID NO: 73 and 74) retains the separation layer but is less well-defined than the B28:WG wildtype control (Fig. 17B) while B56929A1 with +A allele (SEQ ID NO: 36 and 46) retains the separation layer but is darker than the wildtype (Figure 17B). More blue color (dark in greyscale Fig. 17B) in the dehiscence zone indicates the presence of more separation layer. Similarly, in the Arabidopsis, staining of At indl Pl allele retained darker blue stain than wild type Col-0 siliques (Figure 24C). The toluidine blue O staining results clearly supported the reduced pod shatter phenotype observed in both pennycress and Arabidopsis seed pods.
[00152] Characterization of Camelina with indl gene edits
[00153] Cas9-mediated INDI gene editing results achieved with the pARV56 vector driving expression of the guide RNA comprising the PS2 protospacer are shown in Figures 25A, B, C, Figure 26, and in Table 8.
[00154] Table 8. Camelina T2 gene edited plants generated with the pARV56 construct
Figure imgf000046_0001
[00155] Camelina plants comprising the mutations in one, two, and/or all three of the Camelina INDI genes located on chromosomes 2, 8, and 13 will be crossed and/or selfed to obtain progeny plants homozygous for the indl mutations shown in in Figures Figure 25, Figure 26, and in Table 8. More specifically, Camelina plants comprising the following IND gene edits C10012D7_Cs_Chr2(+A) (encoding the SEQ ID NO: 68 protein), C10012D7_Cs_Chr8(+A) (encoding the SEQ ID NO: 69 protein), C10012D7_Cs_Chrl3(+A) (encoding the SEQ ID NO: 70 protein), C10012D10_Cs_Chr2(+T) (encoding the SEQ ID NO: 71 protein), C10012D10_Cs_Chr2(+A) (encoding the SEQ ID NO: 68 protein), C10012D10_Cs_Chr8(+T) (encoding the SEQ ID NO: 72 protein), and/or C10012D10_Cs_Chrl3(+A) (SEQ ID NO: 70) will be crossed and/or selfed to obtain progeny Camelina plants homozygous for at least one, at least two, or all three of the IND gene edits on chromosome 2 (Chr2), chromosome 8 (Chr8) and chromosome 13 (Chrl3). It is anticipated that at least some of the progeny Camelina plants homozygous for at least one, at least two, or all three of the IND gene edits on chromosome 2 (Chr2), chromosome 8 (Chr8) and chromosome 13 (Chrl3) will exhibit improved pod shatter resistance in comparison to wild-type Camelina plants lacking IND mutants. It is also anticipated that at least some of the progeny Camelina plants homozygous for at least one, at least two, or all three of the IND gene edits on chromosome 2 (Chr2), chromosome 8 (Chr8) and chromosome 13 (Chrl3) will exhibit improved threshability in comparison to control Camelina plants homozygous for amorphic (null) alleles of at least one, at least two, or all three of the IND genes on chromosome 2 (Chr2), chromosome 8 (Chr8) and chromosome 13 (Chrl3).
[00156] Table 9 presents a description of the biological sequences provided herewith in the sequence listing.
[00157] Table 9. Biological Sequences
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Figure imgf000051_0001

Claims

[00158] WHAT IS CLAIMED IS :
1. A method of obtaining Brassica plant with at least one mutant ind gene which can confer a pod shatter resistance trait comprising:
(i) crossing a Brassica plant comprising at least one wild-type IND gene with a Brassica plant comprising at least one mutant ind gene encoding a mutant ind protein wherein one or more amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of said mutant ind protein are substituted and/or deleted; and;
(ii) isolating Fl seed and/or Fl progeny plants comprising the mutant ind gene from the cross.
2. The method of claim 1, wherein the encoded mutant ind protein comprises: (i) a deletion and/or substitution of 5 to 7, 8, 10, or 18 of the C-terminal amino acid residues corresponding to the final 5 to 18 C-terminal amino acid residues of the wild-type IND protein encoded by the wild-type IND gene; or (ii) a deletion and/or substitution of 6 to 7, 8, 10, or 18 of the 18 C- terminal amino acid residues comprising the C-terminal consensus sequence of SEQ ID NO: 32 located at the C-terminus of of the wild-type IND protein encoded by the wild-type IND gene.
3. The method of claim 1, wherein the Fl seed and/or Fl progeny are isolated by non- destructively assaying for the presence of the mutation in a nucleic acid detection assay.
4. The method of claim 1, further comprising the step of crossing the isolated Fl progeny plant comprising the mutant ind gene to a recurrent parent Brassica plant comprising a wild-type IND gene and isolating F2 seed and/or F2 progeny comprising the mutant ind gene and one or more genetic markers of the recurrent parent plant.
5. The method of claim 1, further comprising the step of selfing the Fl progeny plants or progeny thereof and selecting an F2 progeny plant which is homozygous for the at least one mutant ind gene.
6. A method of obtaining a Brassica plant with at least one mutant ind gene which can confer a pod shatter resistance trait comprising introducing a mutation in one or more nucleotides of at least one wild-type IND gene of a Brassica plant to obtain a Brassica plant comprising a mutant ind gene encoding a mutant ind protein wherein one or more amino acid residues located C- terminal to the conserved basic Helix-Loop Helix (bHLH) domain of said mutant ind protein are substituted and/or deleted, optionally wherein the mutant ind protein comprises: (i) a deletion and/or substitution of 5 to 7, 8, 10, or 18 of the C-terminal amino acid residues corresponding to the final 5 to 7, 8, 10, or 18 C-terminal amino acid residues of the wild-type IND protein encoded by the wild-type IND gene; or (ii) a deletion and/or substitution of 6 to 7, 8, 10, or 18 of the 18 C- terminal amino acid residues comprising the C-terminal consensus sequence of SEQ ID NO: 32 located at the C-terminus of the wild-type IND protein encoded by the wild-type IND gene.
7. The method of claim 6, wherein said mutation is introduced with one or more gene-editing reagents.
8. The method of claim 7, wherein said gene-editing reagents comprise: (i) an RNA directed endonuclease and a guide RNA directed to the IND gene; (ii) a transcription activator-like effector nuclease (TALEN) directed to the IND gene; (iii) a zinc-finger nuclease (ZFN) directed to the IND gene; or (iv) any one of (i), (ii), or (iii) and DNA donor template.
9. The method of claim 8, wherein the guide RNA comprises the RNA molecule of any one of SEQ ID NO: 21 to 27 or a fragment thereof which can hybridize to a target IND gene.
10. The method of claim 7, wherein the gene-editing reagents comprise: (i) an adenine baseeditor (ABE) and a guide RNA directed to the IND gene; or (ii) a cytosine base-editor and a guide RNA directed to the IND gene.
11. The method of claim 6, wherein the mutation is introduced by random mutagenesis and wherein the method further comprising screening progeny subjected to the mutagenesis by a DNA analysis technique to identify a plant comprising the mutation.
12. The method of claim 6, wherein the mutation is simultaneously introduced in more than one IND gene of the plant.
13. The method of claim 6 wherein the mutation is introduced in a first IND gene of the plant, a plant comprising the mutation in the first gene is isolated, and the mutation is introduced in a one or more additional IND genes of the plant comprising the mutation in the first IND gene.
14. The method of any one of claims 1 to 13, wherein the Brassica plant is a Thlaspi arvense. Brassica napus, Brassica carinata, or Camelina sativa plant .
15. The method of any one of claims 1 to 13, wherein:
(i) the Brassica plant is a Thlaspi arvense plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 2 or an allelic variant thereof or wherein the wild-type IND gene encodes a protein comprising the polypeptide of SEQ ID NO: 12 or an allelic variant thereof;
(ii) the Brassica plant is a Brassica napus plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 9 or an allelic variant thereof or wherein the wild-type IND gene encodes a protein comprising the polypeptide of SEQ ID NO: 19 or an allelic variant thereof, optionally further comprising introducing a mutation in the wild-type IND coding sequence of SEQ ID NO: 10 or an allelic variant thereof or in the wild-type IND coding sequence which encodes SEQ ID NO: 20 or an allelic variant thereof;
(iii) the Brassica plant is a. Brassica carinata plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 4, SEQ ID NO: 5, or an allelic variant thereof or wherein the wild-type IND gene encodes a protein comprising the polypeptide of SEQ ID NO: 14, SEQ ID NO: 15, or an allelic variant thereof; or
(iv) the Brassica plant is a Camelina sativa plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 6, SEQ ID NO: 7, or an allelic variant thereof or wherein the wild-type IND gene encodes a protein comprising the polypeptide of SEQ ID NO:
16, SEQ ID NO: 17, SEQ ID NO: 18, or an allelic variant thereof.
16. The method of any one of claims 1 to 13, wherein:
(i) the Brassica plant is a Thlaspi arvense plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 2 or an allelic variant thereof and wherein the mutation is located at nucleotide positions corresponding to nucleotide 496 to 546 of SEQ ID NO: 2, optionally wherein the mutation corresponds to a C531A or C531G mutation in SEQ ID NO: 2, corresponds to a deletion of nucleotides comprising nucleotides 529 to 546 of SEQ ID NO: 2, or corresponds to a frameshift or nonsense mutation in the Y176, Y177, or Hl 78 codon of SEQ ID N0:2;
(ii) the Brassica plant is a Brassica napus plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 9 or an allelic variant thereof; wherein the mutation is located at nucleotide positions corresponding to nucleotide 484 to 534 of SEQ ID NO: 9 or optionally wherein the mutation corresponds to a C519A or C591G mutation in SEQ ID NO: 9 , corresponds to a deletion of nucleotides comprising nucleotides 517 to 534 of SEQ ID NO: 9, or corresponds to a frameshift or nonsense mutation in the Y172, Y173, or H174 codon of SEQ ID NO: 9; optionally further comprising introducing a mutation located at nucleotide positions corresponding to nucleotide 536 to 546 of SEQ ID NO: 10;
(iii) the Brassica plant is a Brassica carinata plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 4, SEQ ID NO: 5, or an allelic variant thereof and wherein the mutation is located at nucleotide positions corresponding to nucleotide 535 to 585 of SEQ ID NO: 4, optionally wherein the mutation corresponds to a C570A or C570G mutation in SEQ ID NO: 4, corresponds to a deletion of nucleotides corresponding to nucleotides 515 to 534 of SEQ ID NO: 4, or corresponds to a frameshift or nonsense mutation in the Y189, Y190, orH191 codon of SEQ ID NO: 4; and/or at nucleotide positions corresponding to nucleotide 526 to 576 of SEQ ID NO: 5, optionally wherein the mutation corresponds to a C561 A or C561G mutation in SEQ ID NO: 5, corresponds to a deletion of nucleotides comprising nucleotides 559 to 576 of SEQ ID NO: 5, or corresponds to a frameshift or nonsense mutation in the Y186, Y187, or H188 codons of SEQ ID NO: 5; or
(iv) the Brassica plant is a Camelina sativa plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or an allelic variant thereof and wherein the mutation is located at nucleotide positions corresponding to nucleotide 451 to 501 of SEQ ID NO: 6, optionally wherein the mutation corresponds to a C486A or C486G mutation in SEQ ID NO: 6, corresponds to a deletion of nucleotides comprising nucleotides 559 to 576 of SEQ ID NO: 6, or corresponds to a frameshift or nonsense mutation in the Y161, Y162, or H163 codons of SEQ ID NO: 6; and/or at nucleotide positions corresponding to nucleotide 451 to 501 of SEQ ID NO: 7 or optionally wherein the mutation corresponds to a C486A or C486G mutation in SEQ ID NO:7, corresponds to a deletion of nucleotides comprising nucleotides 559 to 576 of SEQ ID NO: 7, or corresponds to a frameshift or nonsense mutation in the Y161, Y162, or H163 codons of SEQ ID NO: 7; and/or at nucleotide positions corresponding to nucleotide 454 to 504 of SEQ ID NO: 8, optionally wherein the mutation corresponds to a C489A or C489G mutation in SEQ ID NO: 8, corresponds to a deletion of nucleotides comprising nucleotides 487 to 504 of SEQ ID NO: 8, or corresponds to a frameshift or nonsense mutation in the Y162, Y163, or Hl 64 codons of SEQ ID NO: 8.
17. The method of any one of claims 1 to 13, wherein:
(i) the Brassica plant is a Thlaspi arvense plant wherein the wild-type IND gene encodes a protein comprising the polypeptide of SEQ ID NO: 12 or an allelic variant thereof and wherein the mutant encodes a protein lacking at least 5 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 166 to 182 of SEQ ID NO: 12; (ii) the Brassica plant is a Brassica napus plant wherein the wild-type IND gene encodes a protein comprising the polypeptide of SEQ ID NO: 19 or an allelic variant thereof and wherein the mutant encodes a protein lacking at least 5 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 161 to 178 of SEQ ID NO: 19;
(iii) the Brassica plant is a Brassica carinata plant wherein the wild-type IND gene encodes a protein comprising the polypeptide of SEQ ID NO: 14, SEQ ID NO: 15, or an allelic variant thereof and wherein the mutant encodes a protein lacking at least 5 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 179 to 195 of SEQ ID NO: 14 and/or corresponding to amino acid residues 176 to 192 of SEQ ID NO: 15; or
(iv) the Brassica plant is a Camelina sativa plant wherein the wild-type IND gene encodes a protein comprising the polypeptide of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or an allelic variant thereof and wherein the mutant encodes a protein lacking at least 5 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 150 to 167 of SEQ ID NO: 16 or 17 and/or lacking at least 5 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 151 to 168 of SEQ ID NO: 18.
18. A Brassica plant made by the method of any one of claims 1 to 13, optionally wherein said Brassica plant is a Thlaspi arvense plant comprising two genomic DNA sequence polymorphisms which are not found together in any single naturally occurring Thlaspi arvense isolate or is a Thlaspi arvense plant lacking a black seed trait, a high fiber seed trait, a reduced yield trait, and/or an increased lodging trait.
19. A Brassica plant comprising at least one mutant ind gene encoding a mutant ind protein wherein one or more amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of said mutant ind protein are substituted and/or deleted, wherein said Brassica plant is not a Thlaspi arvense plant and optionally wherein the mutant ind protein comprises: (i) a deletion and/or substitution of 5 to 7, 8, 10, or 18 of the C-terminal amino acid residues corresponding to the final 5 to 7, 8, 10, or 18 C-terminal amino acid residues of the corresponding wild-type IND protein; or (ii) a deletion and/or substitution of 6 to 7, 8, 10, or 18 of the 18 C-terminal amino acid residues comprising the C-terminal consensus sequence of SEQ ID NO: 32 located at the C-terminus of the corresponding wild-type IND protein.
20. The Brassica plant of claim 19, wherein the Brassica plant is Brassica napus, Brassica carinata, or Camelina sativa plant.
21. The Brassica plant of claim 19, wherein said plant is homozygous for the mutant ind gene or genes and exhibits reduced pod shatter in comparison to a control plant lacking the mutant ind gene or genes.
22. The Brassica plant of claim 19, wherein said plant exhibits improved agronomic performance in comparison to a control plant homozygous for an amorphic allele of the ind gene.
23. The Brassica plant of claim 19, wherein Brassica plant comprises elite Brassica plant germplasm.
24. The Brassica plant of claim 19, wherein:
(i) the Brassica plant is a Brassica napus plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 9 or an allelic variant thereof or wherein the wildtype IND gene encodes the corresponding wild-type IND protein comprising the polypeptide of SEQ ID NO: 19 or an allelic variant thereof;
(ii) the Brassica plant is a Brassica carinata plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 4, SEQ ID NO: 5, or an allelic variant thereof or wherein the wild-type IND gene encodes the corresponding wild-type IND protein comprising the polypeptide of SEQ ID NO: 14, SEQ ID NO: 15, or an allelic variant thereof; or
(iii) the Brassica plant is a Camelina sativa plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 6, SEQ ID NO: 7, or an allelic variant thereof or wherein the wild-type IND gene encodes the corresponding wild-type IND protein comprising the polypeptide of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or an allelic variant thereof.
25. The Brassica plant of claim 19, wherein:
(i) the Brassica plant is a Brassica napus plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 9 or an allelic variant thereof; wherein the mutation is located at nucleotide positions corresponding to nucleotide 484 to 534 of SEQ ID NO: 9 or optionally wherein the mutation corresponds to a C519A or C591G mutation in SEQ ID NO: 9 , corresponds to a deletion of nucleotides comprising nucleotides 517 to 534 of SEQ ID NO: 9, or corresponds to a frameshift or nonsense mutation in the Y172, Y173, or H174 codon of SEQ ID NO: 19; optionally further comprising introducing a mutation located at nucleotide positions corresponding to nucleotide 536 to 546 of SEQ ID NO: 10; (ii) the Brassica plant is a Brassica carinata plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 4, SEQ ID NO: 5, or an allelic variant thereof and wherein the mutation is located at nucleotide positions corresponding to nucleotide 535 to 585 of SEQ ID NO: 4, optionally wherein the mutation corresponds to a C570A or C570G mutation in SEQ ID NO: 4, corresponds to a deletion of nucleotides corresponding to nucleotides 515 to 534 of SEQ ID NO: 4, or corresponds to a frameshift or nonsense mutation in the Y189, Y190, orH191 codon of SEQ ID NO: 4; and/or at nucleotide positions corresponding to nucleotide 526 to 576 of SEQ ID NO: 5, optionally wherein the mutation corresponds to a C561 A or C561G mutation in SEQ ID NO: 5, corresponds to a deletion of nucleotides comprising nucleotides 559 to 576 of SEQ ID NO: 5, or corresponds to a frameshift or nonsense mutation in the Y186, Y187, or Hl 88 codons of SEQ ID NO: 5; or
(iii) the Brassica plant is a Camelina sativa plant wherein the wild-type IND coding sequence comprises the DNA molecule of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or an allelic variant thereof and wherein the mutation is located at nucleotide positions corresponding to nucleotide 451 to 501 of SEQ ID NO: 6, optionally wherein the mutation corresponds to a C486A or C486G mutation in SEQ ID NO: 6, corresponds to a deletion of nucleotides comprising nucleotides 559 to 576 of SEQ ID NO: 6, or corresponds to a frameshift or nonsense mutation in the Y161, Y162, or H163 codons of SEQ ID NO: 6; and/or at nucleotide positions corresponding to nucleotide 451 to 501 of SEQ ID NO: 7 or optionally wherein the mutation corresponds to a C486A or C486G mutation in SEQ ID NO: 7, corresponds to a deletion of nucleotides comprising nucleotides 559 to 576 of SEQ ID NO: 7, or corresponds to a frameshift or nonsense mutation in the Y161, Y162, or H163 codons of SEQ ID NO: 7; and/or at nucleotide positions corresponding to nucleotide 454 to 504 of SEQ ID NO: 8, optionally wherein the mutation corresponds to a C489A or C489G mutation in SEQ ID NO: 8, corresponds to a deletion of nucleotides comprising nucleotides 487 to 504 of SEQ ID NO: 8, or corresponds to a frameshift or nonsense mutation in the Y162, Y163, or H164 codons of SEQ ID NO: 8.
26. The Brassica plant of claim 19, wherein:
(i) the Brassica plant is a Brassica napus plant wherein the wild-type IND gene encodes a protein comprising the polypeptide of SEQ ID NO: 19 or an allelic variant thereof and wherein the mutant encodes a protein lacking at least 5 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 161 to 178 of SEQ ID NO: 19;
(ii) the Brassica plant is a Brassica carinata plant wherein the wild-type IND gene encodes a protein comprising the polypeptide of SEQ ID NO: 14, SEQ ID NO: 15, or an allelic variant thereof and wherein the mutant encodes a protein lacking at least 5 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 179 to 195 of SEQ ID NO: 14 and/or corresponding to amino acid residues 176 to 192 of SEQ ID NO: 15; or
(iii) the Brassica plant is a Camelina sativa plant wherein the wild-type IND gene encodes a protein comprising the polypeptide of SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, or an allelic variant thereof and wherein the mutant encodes a protein lacking at least 5 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 150 to 167 of SEQ ID NO: 16, 17 and/or lacking at least 5 of the C-terminal amino acid residues of the polypeptide sequence corresponding to amino acid residues 151 to 168 of SEQ ID NO: 18.
27. A Brassica plant cell or plant part obtained from the Brassica plant of any one of claims 19 to 26, wherein said cell, plant propagule, or plant part comprises the mutant IND gene.
28. The Brassica plant cell of claim 27, wherein the plant cell is plant cell callus.
29. The Brassica plant part of claim 27, wherein the part is a seed, pollen, ovule, root, pod, stem, or leaf.
30. A seed lot comprising a plurality of the Brassica seed of claim 29.
31. A method for harvesting seed from a Brassica crop comprising harvesting seed from a plurality of the Brassica plants of claim 19.
32. A Thlaspi arvense plant comprising a mutant ind gene encoding a mutant ind protein wherein one or more amino acid residues located C-terminal to the conserved basic Helix-Loop Helix (bHLH) domain of said mutant ind protein are substituted and/or deleted, wherein said Thlaspi arvense plant comprises two genomic DNA sequence polymorphisms which are not found together in any single naturally occurring Thlaspi arvense isolate and optionally wherein the mutant ind protein comprises: (i) a deletion and/or substitution of 5 to 7, 8, 10, or 18 of the C- terminal amino acid residues corresponding to the final 5 to 7, 8, 10, or 18 C-terminal amino acid residues of the corresponding wild-type IND protein; or (ii) a deletion and/or substitution of 6 to 7, 8, 10, or 18 of the 18 C-terminal amino acid residues comprising the C-terminal consensus sequence of SEQ ID NO: 32 located at the C-terminus of the corresponding wild-type IND protein.
33. The Thlaspi arvense plant of claim 32, wherein the Thlaspi arvense plant comprises elite Thlaspi arvense plant germplasm.
34. The Thlaspi arvense plant of claim 32, wherein said Thlaspi arvense plant lacks a black seed trait, a high fiber seed trait, a reduced yield, and/or an increased lodging trait.
35. The Thlaspi arvense plant of claim 32, wherein the mutation corresponds to a C531A or C531G mutation in SEQ ID NO: 2, corresponds to a deletion of nucleotides comprising nucleotides 529 to 546 of SEQ ID NO: 2, or corresponds to a frameshift or nonsense mutation in the Y176, Y177, or H178 codon of SEQ ID NO:2.
36. The Thlaspi arvense plant of claim 32, wherein the mutant encodes a protein: (i) lacking at least the six C-terminal amino acid residues of the polypeptide corresponding to amino acid residues 177 to 182 of SEQ ID NO: 12; or (ii) .
37. The Thlaspi arvense plant of any one of claims 32 to 36, wherein said plant is homozygous for the mutant ind gene or genes and exhibits reduced pod shatter in comparison to a control plant lacking the mutant ind gene.
38. The Thlaspi arvense plant of claim 37, wherein said plant exhibits improved agronomic performance in comparison to a control plant homozygous for an amorphic allele of the IND gene.
39. A Thlaspi arvense plant cell or plant part obtained from the Thlaspi arvense plant of any one of claims 32 to 36, wherein said cell, plant propagule, or plant part comprises the mutant ind gene.
40. The Thlaspi arvense plant cell of claim 39, wherein the plant cell is plant cell callus.
41. The Thlaspi arvense plant part of claim 39, wherein the part is a seed, pollen, ovule, root, pod, stem, or leaf.
42. A seed lot comprising a plurality of the Thlaspi arvense seed of claim 41.
43. A method for harvesting seed from a Thlaspi arvense crop comprising harvesting seed from a plurality of the Thlaspi arvense plants of any one of claims 32 to 36.
PCT/US2023/078712 2022-11-04 2023-11-03 Brassica ind gene mutations conferring improved pod shatter-resistance WO2024098012A2 (en)

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