Methods and compositions relating to maintainer lines for male-sterility

A male-fertile maintainer plant for a male-sterile polyploid plant comprising:

• a first genome comprising:
• on a first chromosome of a pair of homologous chromosomes, at least one functional allele of a MF gene at the MF gene locus and at least one allele of a seed color gene;
• on a second chromosome of the pair of homologous chromosomes, a loss-of-function allele of the MF gene at the MF gene locus and at least one ectopic functional allele of a PV gene;
• and loss-of-function alleles of the PV gene at the native PV gene loci;
• and
• at least one further genome, each of the further genomes comprising loss-of-function alleles of the MF gene at the native MF gene loci and loss-of-function alleles of the PV gene at the native PV gene loci.

A method of preparing a male-fertile maintainer plant for a male-sterile polyploid plant, the method comprising engineering a plant to comprise:

• in a first genome:
• on a first chromosome of a pair of homologous chromosomes, at a single target locus, at least one functional ectopic allele of a MF gene and at least one functional ectopic allele of a seed color gene;
• on a second chromosome of the pair of homologous chromosomes, at the target locus corresponding to the target locus of the first chromosome of the pair of homologous chromosomes, at least one functional ectopic allele of a PV gene;
• and
• loss-of-function alleles of the endogenous MF genes at the native MF gene loci and loss-of-function alleles of the endogenous PV genes at the native PV gene loci.

A method of preparing a male-fertile maintainer plant for a male-sterile polyploid plant, the method comprising, simultaneously or sequentially:

• inserting, on a first chromosome of a pair of homologous chromosomes in a first genome, at a single target locus, a construct comprising at least one functional ectopic allele of a MF gene and at least one functional ectopic allele of a seed color gene, optionally wherein the inserting comprises nuclease cleavage of the target locus and recombination or end-joining of the construct;
• inserting, on a second chromosome of the pair of homologous chromosomes in the first genome, at the target locus corresponding to the target locus of the first chromosome of the pair of homologous chromosomes, a construct comprising at least one functional ectopic allele of a PV gene, optionally wherein the inserting comprises nuclease cleavage of the target locus and/or recombination or end-joining of the construct;
• and
• mutating the the endogenous MF genes at the native MF gene loci and the endogenous PV genes at the native PV gene loci to create loss-of-function alleles, optionally wherein the loss-of-function alleles are caused by contacting the genome with a site-specific guided nuclease and one or more guide RNA sequences or multi-guide constructs.

A method of preparing a male-fertile maintainer plant for a male-sterile polyploid plant, the method comprising:

• i. inserting, on a first chromosome of a pair of homologous chromosomes in a first genome, at a single target locus, a cassette comprising in 5′ to 3′ or 3′ to 5′ order:
• a first recognition site for a first recombinase;
• at least one functional ectopic nuclease null allele of a MF gene and at least one functional ectopic allele of a seed color gene in either relative order;
• a first recognition site for a second recombinase;
• a selection gene;
• a second recognition site for the first recombinase;
• at least one functional ectopic nuclease-null allele of a PV gene;
• a second recognition site for the second recombinase;
• thereby providing a full-cassette insertion plant;
• ii. contacting a first progeny of the full-cassette insertion plant, or a cell thereof, with the first recombinase,
• thereby excising:
• one recognition site for the first recombinase, the at least one functional ectopic nuclease null allele of a MF gene and at least one functional ectopic allele of a seed color gene, the first recognition site for the second recombinase, and the selection gene from the genome of the first progeny and
• thereby providing an excised first progeny comprising:
• one recognition site for the first recombinase, the at least one functional ectopic nuclease-null allele of a PV gene, and the second recognition site for the second recombinase portions of the construct;
• iii. contacting a second progeny of the full-cassette insertion plant, or a cell thereof, with the second recombinase,
• thereby excising:
• one recognition site for the second recombinase, the selection gene, the second recognition site for the first recombinase and at least one functional ectopic nuclease-null allele of a PV gene, and
• thereby providing an excised second progeny comprising:
• one recognition site for the second recombinase, the first recognition site for the first recombinase, and the at least one functional ectopic nuclease null allele of a MF gene and at least one functional ectopic allele of a seed color gene portions of the construct;
• iv. crossing the excised first progeny provided in step ii) and the excised second progeny provided in step iii), thereby providing a third progeny comprising, in a first genome,
• on a first chromosome of a pair of homologous chromosomes, at a single target locus, the at least one functional ectopic nuclease-null allele of a MF gene and the at least one functional ectopic allele of a seed color gene;
• on a second chromosome of the pair of homologous chromosomes, at the target locus corresponding to the target locus of the first chromosome of the pair of homologous chromosomes, the at least one functional ectopic nuclease-null allele of a PV gene;
• and
• v. mutating the endogenous MF genes at the native MF gene loci and the endogenous PV genes at the native PV gene loci to create loss-of-function alleles, optionally wherein the loss-of-function alleles are caused by contacting the genome with a site-specific guided nuclease and one or more guide RNA sequences or multi-guide constructs, thereby providing the male-fertile maintainer plant.

A method of preparing a male-fertile maintainer plant for a male-sterile polyploid plant, the method comprising:

• i. contacting a cell comprising a PV locus in a first chromosome and a second chromosome of a pair of homologous chromosomes in a first genome, with:
• 1. a site-specific guided nuclease;
• 2. one or more guide RNA sequences or multi-guide constructs specific to one or more sequences at the PV locus;
• and
• 3. an targeting insertion cassette comprising in 5′ to 3′ or 3′ to 5′ order:
• a first recognition site for a first recombinase and a second recognition site for the first recombinase;
• thereby providing a targeting insertion plant;
• ii. contacting the targeting insertion plant, or first progeny of the targeting insertion plant, or a cell thereof with the first recombinase and a cassette comprising in 5′ to 3′ or 3′ to 5′ order:
• 1) a first recombination site for the first recombinase;
• 2) at least one functional ectopic nuclease null allele of a MF gene and at least one functional ectopic allele of a seed color gene in either relative order;
• and
• 3) a second recombination site for the first recombinase;
• thereby providing a cassette insertion plant;
• iii. selecting a cassette insertion plant comprising a cassette insertion at one allele of the PV locus, or crossing a cassette insertion plant comprising a cassette insertion at both alleles of the PV locus with a plant with a functional PV allele at the PV locus,
• thereby providing a cassette insertion plant with a cassette insertion at one PV allele in the first genome and a functional PV allele at the second PV allele in the first genome,
• iv. contacting the cassette insertion plant selected in iii), or a first progeny or cell thereof, with:
• 1. a site-specific guided nuclease (e.g., CRISPR);
• 2. one or more guide RNA sequences or multi-guide constructs flanking the insertion sites, thereby excising the inserted recombination sites;
• 3. one or more guide RNA sequences or multi-guide constructs specific to the functional endogenous MF genes and/or flanking the functional endogenous MF genes, thereby mutating the functional endogenous MF genes at the functional native MF gene loci to create loss-of-function alleles;
• thereby providing the male-fertile maintainer plant.