Methods and compositions for reducing pod shatter in canola
This invention relates to compositions and methods for modifying SHATTERPROOF MADS-BOX(SHP) genes in canola plants, optionally to reduce pod shattering. The invention further relates to canola plants having reduced pod shatter produced using the methods and compositions of the invention.
1 . A method for producing a canola plant or plant part thereof comprising at least one cell having a mutated endogenous SHATTERPROOF MADS-BOX(SHP) gene, the method comprising:
contacting a target site in an endogenous SHP gene in the canola plant or plant part thereof with a guide nucleic acid and a nuclease comprising a cleavage domain and a nucleic acid binding domain, wherein the guide nucleic acid binds to the target site and the target site is in a region of the endogenous SHP gene having at least 95% sequence identity to any one of SEQ ID NOs:103, 112, 243, or 261, wherein the nucleic acid binding domain binds to the target site in the endogenous SHP gene, and wherein the endogenous SHP gene
(a) comprises a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs:69, 70, 100, 101, 148, 149, 177, 178, 206, 207, 240 or 241;
(b) comprises a region having at least 95% sequence identity to a nucleotide sequence of any one of SEQ ID NOs:72-96, -103-144, 151-173, 180-202, 209-236, 243-288 or -324-338;
(c) encodes a SHP polypeptide having at least 95% sequence identity to any one of SEQ ID NOs:71, 102, 150, 179, 208, or 242; and/or
(d) encodes a region of a SHP polypeptide having at least 95% sequence identity to any one of SEQ ID NOs:97-99, 145-147, 174-176, 203-205, 237-239 or 289-291, thereby producing the canola plant or plant part thereof comprising the at least one cell having a mutation in the endogenous SHP gene to provide the mutated endogenous SHP gene.
2 . The method of claim 1 , wherein the mutation is an out-of-frame insertion or an out-of-frame deletion that results in a premature stop codon.
3 . The method of claim 1 , wherein the mutated endogenous SHP gene comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs:298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 319, 321, 322, or 323.
4 . A guide nucleic acid that binds to a target site in a Brassica napus SHATTERPROOF MADS-BOX (SHP) gene, wherein the target site is in a region of the Brassica napus SHP gene having at least 95% sequence identity to any one of SEQ ID NOs: 103, 112, 243, or 261.
5 . The guide nucleic acid of claim 4 , wherein the guide nucleic acid comprises a spacer comprising a nucleotide sequence of any one of SEQ ID NOs:292-297.
6 . The method of claim 1 , wherein the mutation results in a mutated SHP polypeptide that comprises an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs:299, 301, 303, 305, 307, 309, 311, 313, 315, or 317.
7 . A Brassica napus plant or plant part thereof comprising a mutation in an endogenous SHATTERPROOF MADS-BOX (SHP) gene that encodes a SHP polypeptide, wherein the mutation is introduced using the guide nucleic acid of claim 4 .