Parthenogenesis factors and methods of using same
Parthenogenesis is a natural form of asexual reproduction wherein growth and development of embryos occur without fertilization by sperm. Peptides are used as parthenogenesis factors, specifically comprising polypeptides or polynucleotides encoding gene products for generating doubled haploids or haploid plants from female gametes.
1 . A method of producing a genome-edited, doubled haploid maize plantlet or plant, the method comprising:
(a) providing to a maternal gametophyte:
(i) a polynucleotide sequence encoding a parthenogenesis factor, wherein the polynucleotide sequence is operably linked to a regulatory element active in an egg cell, and wherein the parthenogenesis factor is an Ovule Development Protein 2 (ODP2) polypeptide having at least 95% sequence identity to SEQ ID NO: 20 and comprising a first motif having an amino acid sequence of SEQ ID NO: 36 and a second motif having an amino acid sequence of SEQ ID NO: 38; and
(ii) a site-directed nuclease;
(b) modifying a DNA target site in the maternal gametophyte via the site-directed nuclease, resulting in a modified DNA target site;
(c) regenerating a T 0 plant containing the polynucleotide sequence encoding the parthenogenesis factor and the modified DNA target site;
(d) pollinating the T 0 plant with pollen;
(e) obtaining a genome-edited, doubled haploid embryo from a parthenogenic maternal gametophyte of the T 0 plant; and
(f) regenerating a genome-edited, doubled haploid maize plantlet or plant from the genome-edited, doubled haploid embryo.
2 . The method of claim 1 , further comprising providing the parthenogenic maternal gametophyte with a genetic chromosome doubling agent, wherein obtaining the genome-edited, doubled haploid embryo from the parthenogenic maternal gametophyte comprises
diploidization of maternal chromosomes of the parthenogenic maternal gametophyte via the genetic chromosome doubling agent resulting in a diploidized parthenogenic maternal gametophyte and obtaining the genome-edited, doubled haploid embryo from the diploidized parthenogenic maternal gametophyte.
3 . The method of claim 1 , wherein obtaining the genome-edited, doubled haploid embryo from the parthenogenic maternal gametophyte comprises:
obtaining a haploid embryo from the parthenogenic maternal gametophyte and contacting the haploid embryo with a chromosome doubling agent for a period sufficient to generate the genome-edited, doubled haploid embryo.
4 . The method of claim 3 , wherein the chromosome doubling agent is colchicine, a colchicine derivative, a carbamate, a benzamide, a benzoic acid, a dinitroaniline, a chloralin, a phosphoroamidate, or a pyridine.
5 . The method of claim 1 , wherein the pollen is from a haploid inducer.
6 . The method of claim 1 , wherein the pollen comprises a marker gene, wherein the marker gene is a selectable marker, a reporter gene, or a visible endogenous morphological marker.
7 . The method of claim 6 , wherein the selectable marker is GUS, PMI, or PAT, the reporter gene is GFP, RFP, or CFP, and the visible endogenous morphological marker is B1, R1-nj, R1-scm, or anthocyanin pigments.
8 . The method of claim 6 , wherein the genome-edited, doubled haploid embryo lacks the marker gene.
9 . The method of claim 6 , wherein obtaining the genome-edited, doubled haploid embryo from the parthenogenic maternal gametophyte and regenerating the genome-edited, doubled haploid maize plantlet or plant from the genome-edited, doubled haploid embryo comprises: obtaining mature seed from the T 0 plant, the mature seed having a diploidized maternal embryo lacking the marker gene and germinating the mature seed to obtain the genome-edited, doubled haploid maize plantlet or plant.
10 . The method of claim 2 , wherein the genetic chromosome doubling agent comprises a polynucleotide sequence encoding a polypeptide having at least 95% sequence identity to SEQ ID No: 111.
11 . The method of claim 1 , wherein the regulatory element comprises an egg cell promoter.
12 . The method of claim 1 , wherein the regulatory element comprises expression modulating element selected from SEQ ID Nos: 39-103 and/or an enhancer selected from SEQ ID Nos: 104-109.
13 . The method of claim 1 , wherein the site-directed nuclease is a meganuclease, a zinc-finger nuclease, a transcription-activator like effector nuclease, a Cas9 nuclease, a Cas alpha nuclease, a Cpf1 nuclease, a dCas9-FokI, a dCpf1-FokI, a chimeric Cas9-cytidine deaminase, a chimeric Cas9 adenine deaminase, a chimeric FEN1-Fok1, a Mega-TALs, a Cas9 nickase, a chimeric dCas9 non-FokI nuclease, or a dCpf1-non-FokI nuclease.
14 . A method of producing a doubled haploid maize plantlet or plant, the method comprising:
(a) providing to a maternal gametophyte a polynucleotide sequence encoding a parthenogenesis factor, wherein the polynucleotide sequence encoding a parthenogenesis factor is operably linked to a regulatory element active in an egg cell, and wherein the parthenogenesis factor is an Ovule Development Protein 2 (ODP2) polypeptide having at least 95% sequence identity to SEQ ID NO: 20 and comprising a first motif having an amino acid sequence of SEQ ID NO: 36 and a second motif having an amino acid sequence of SEQ ID NO: 38;
(b) regenerating a T 0 plant containing the polynucleotide sequence encoding the parthenogenesis factor;
(c) pollinating the T 0 plant with pollen;
(d) obtaining a doubled haploid embryo from a parthenogenic maternal gametophyte of the T 0 plant; and
(e) regenerating a doubled haploid maize plantlet or plant from the doubled haploid embryo.
15 . The method of claim 14 , wherein the pollen is from a haploid inducer.
16 . The method of claim 15 , wherein the pollen comprises a marker gene, wherein the marker gene is a selectable marker, a reporter gene, or a visible endogenous morphological marker.
17 . The method of claim 16 , wherein the selectable marker is GUS, PMI, or PAT, the reporter gene is GFP, RFP, or CFP, and the visible endogenous morphological marker is B1, R1-nj, R1-scm, or anthocyanin pigments.
18 . The method of claim 16 , wherein the doubled haploid embryo obtained from the parthenogenic maternal gametophyte lacks the marker gene.
19 . The method of claim 16 , wherein obtaining the doubled haploid embryo from the parthenogenic maternal gametophyte and regenerating the doubled haploid maize plantlet or plant from the doubled haploid embryo comprises: obtaining mature seed from the T 0 plant, the mature seed having a diploidized maternal embryo lacking the marker gene and germinating the mature seed to obtain the doubled haploid maize plantlet or plant.
20 . The method of claim 14 , wherein the genetic chromosome doubling agent comprises a polynucleotide sequence encoding a polypeptide having at least 95% sequence identity to SEQ ID No: 111.
21 . The method of claim 14 , wherein the regulatory element is an egg cell promoter.
22 . The method of claim 1 , wherein the site-directed nuclease is a Cas9 nuclease or a Cas alpha nuclease.
23 . The method of claim 22 , further comprising providing to the maternal gametophyte a guide polynucleotide.
24 . The method of claim 14 , further comprising providing the parthenogenic maternal gametophyte with a genetic chromosome doubling agent, wherein obtaining the doubled haploid embryo from the parthenogenic maternal gametophyte comprises diploidization of maternal chromosomes of the parthenogenic maternal gametophyte via the genetic chromosome doubling agent resulting in a diploidized parthenogenic maternal gametophyte and obtaining the doubled haploid embryo from the diploidized parthenogenic maternal gametophyte.
25 . The method of claim 24 , wherein the genetic chromosome doubling agent comprises a polynucleotide sequence encoding a polypeptide having at least 95% sequence identity to SEQ ID No: 111.
26 . The method of claim 14 , wherein obtaining the doubled haploid embryo from the parthenogenic maternal gametophyte comprises obtaining a haploid embryo from the parthenogenic maternal gametophyte and contacting the haploid embryo with a chromosome doubling agent for a period sufficient to generate the doubled haploid embryo.
27 . The method of claim 26 , wherein the chromosome doubling agent is colchicine, a colchicine derivative, a carbamate, a benzamide, a benzoic acid, a dinitroaniline, a chloralin, a phosphoroamidate, or a pyridine.
28 . The method of claim 1 , wherein the parthenogenesis factor is a fusion protein comprising the ODP2 polypeptide having at least 95% sequence identity to SEQ ID NO: 20 and a CBF1a domain having an amino acid sequence of SEQ ID NO: 449.
29 . The method of claim 1 , wherein the pollen is from a non-haploid inducer.
30 . The method of claim 11 , wherein the egg cell promoter is any one of SEQ ID Nos: 34 or 121-135.
31 . The method of claim 11 , wherein the egg cell promoter comprises an expression modulating element selected from SEQ ID Nos: 39-103 and/or an enhancer selected from SEQ ID Nos: 104-109.
32 . The method of claim 14 , wherein the pollen is from a non-haploid inducer.
33 . The method of claim 21 , wherein the egg cell promoter is any one of SEQ ID Nos: 34 or 121-135.
34 . The method of claim 21 , wherein the egg cell promoter comprises an expression modulating element selected from SEQ ID Nos: 39-103 and/or an enhancer selected from SEQ ID Nos: 104-109.