Restoration of male fertility in wheat

Patent metadata

Field	Value
Publication number	US-2017369902-A1
Application number	US-201515536336-A
Country	US
Kind code	A1
Filing date	Dec 15, 2015
Priority date	Dec 16, 2014
Publication date	Dec 28, 2017
Grant date	—

Abstract

Manipulation of male fertility in a polyploid species requires attention to the interaction of male-fertility alleles of multiple genomes. In hexaploid wheat, single-genome heterozygotes for Ms26 provide differential levels of male fertility across genomes. Hexaploid wheat homozygous for mutations in the Ms26 gene on the A, B, and D genomes is male-sterile. Male fertility may be restored by sufficient levels of expression of Ms26 using native Ms26 or a transgene, which may be native to wheat or to another species, or a combination of native and transgenic alleles. CRISPR/Cas9 technology may be used to generate mutations in Ms26 in wheat or rice.

First claim

We claim:

1 . A method of controlling male fertility in a polyploid species, comprising modulating expression of a male fertility gene differentially across genomes.

2 . The method of claim 1 , wherein the species is wheat.

3 . The method of claim 2 , wherein the gene is Ms26.

4 . The method of claim 3 , wherein two genomes are homozygous for the recessive allele of Ms26 and the third genome is heterozygous for the dominant allele of Ms26.

5 . The method of claim 4 , wherein expression is modulated by transforming the plant with a transgenic construct comprising an Ms26 polynucleotide encoding an Ms26 polypeptide.

6 . The method of claim 3 , wherein two genomes are homozygous for the recessive allele of Ms26 and the third genome is homozygous for the dominant allele of Ms26.

7 . The method of claim 6 , wherein expression is modulated by transforming the plant with a transgenic construct comprising an Ms26 polynucleotide encoding a functional Ms26 polypeptide.

8 . The method of claim 3 , wherein all three genomes are homozygous for the recessive allele of Ms26.

9 . The method of claim 8 , wherein expression is modulated by transforming the plant with a transgenic construct comprising an Ms26 polynucleotide encoding a functional Ms26 polypeptide.

10 . A male-sterile wheat plant comprising double or triple homozygous mutations in a gene encoding a gene product necessary for male fertility.

11 . The plant of claim 10 , further comprising a transgenic construct comprising a polynucleotide encoding a polypeptide which restores male fertility to the plant.

12 . The plant of claim 10 , wherein the gene is Ms26.

13 . The plant of claim 11 , wherein the transgenic construct comprises an Ms26 polynucleotide.

14 . The plant of claim 13 , wherein the Ms26 polynucleotide is native to a species other than wheat.

15 . The plant of claim 11 , wherein the transgenic construct further comprises
(a) A promoter operably linked to the polynucleotide encoding a polypeptide which restores male fertility to the plant, wherein said promoter drives expression in the plant; (b) A pollen-specific promoter operably linked to a polynucleotide encoding a gene product which interferes with starch accumulation; and (c) A seed-specific promoter operably linked to a polynucleotide encoding a marker protein.

16 . The plant of claim 4 , wherein expression of the dominant allele of Ms26 is enhanced by one or more of the methods selected from the group consisting of: modification of the promoter; operable linkage to a different promoter; incorporation of transcriptional enhancer elements in the construct; modification of the structural gene to improve splicing of the primary transcript; removal of mRNA destabilizing elements, optimization of translation initiation or elongation; and addition or removal of sequences to increase the half-life of the primary encoded RNA or the spliced transcript.

17 . The plant of claim 11 , wherein expression of the polynucleotide is enhanced by one or more of the methods selected from the group consisting of: modification of the promoter;
operable linkage to a different promoter; incorporation of transcriptional enhancer elements in the construct; modification of the structural gene to improve splicing of the primary transcript; removal of mRNA destabilizing elements, optimization of translation initiation or elongation; and addition or removal of sequences to increase the half-life of the primary encoded RNA or the spliced transcript.

18 . A method for modifying expression of Ms26 in a wheat plant by modifying a target site in a wheat Ms26 gene, the method comprising providing a guide crRNA molecule to a plant cell having a Cas endonuclease, wherein said guide RNA and Cas endonuclease are capable of forming a complex that enables the Cas endonuclease to introduce a double strand break at said target site in the Ms26 gene.

19 . The method of claim 18 , wherein said guide crRNA molecule has the sequence of SEQ ID NO: 12.

Assignees

• Pioneer Hi Bred Int

Inventors

• Cigan Andrew Mark
• Singh Manjit

Classifications

• C07K14/415

  from plants · CPC title

• A01H1/02

  Methods or apparatus for hybridisation; Artificial pollination {; Fertility} · CPC title

• A01H5/10

  Seeds · CPC title

• C12N15/8289 Primary

  Male sterility · CPC title

• C12N15/8213

  Targeted insertion of genes into the plant genome by homologous recombination · CPC title