Flavonoid dependent fertility

Abstract

The flavonoid dependent fertility provides a safe and convenient method for controlling fertilization between plants. Previously, quercetin, kaempferol, and other flavonoids restore fertility when applied to pollen or flowers by mutations of chalcone synthase in maize and petunia. Here, new gene targets and mutations thereof have been found in wheat, rice and maize. By mutating these new genes in each of the three genomes of wheat, we provide flavonoid dependent fertility in wheat. Also provided are rice FDF genes and improved FDF systems in maize.

Description

[0001] Related application information

[0002] This application claims priority under 35 U.S.C. §119 to U.S. application No. 63 / 369,975, filed on August 1, 2022 (the contents of which are incorporated herein by reference in their entirety). Technical Field

[0003] The present invention relates to methods of modulating plant fertility, and more particularly to generating flavonoid-dependent fertility in wheat and maize plants through mutations in novel genes, and restoring fertility in these plants by providing fertility-restoring flavonoids at the pollen locus of the plants.

[0004] Sequence Listing

[0005] Attached to this application is a sequence listing entitled 82692PCT.xml, created on July 24, 2023, which is approximately 200 kilobytes in size. The sequence listing is incorporated herein by reference in its entirety. The sequence listing is submitted herewith and complies with 37 C.FR §§1.831-1.835. Background Art

[0006] The control of male fertility in crop plants is a key factor required for maintaining seed purity and controlling material costs in hybrid seed production. In maize, the control of male fertility is often achieved by removing male reproductive tissue (emasculation). In cereals and maize, cytoplasmic male sterility (CMS) can also be used to control male fertility. Emergence is challenged by high costs in hybrid seed production, while CMS systems are challenged by the high cost of materials in CMS strain amplification, the increase in breeding complexity, and the instability of male fertility in hybrids. A class of alternative hybrid seed production technologies designed to overcome these challenges associated with emasculation and CMS is referred to as "conditional male sterility". In the process of cultivating conditional male sterile plants, growers can control whether pollen is fertile or sterile by selecting specific growth conditions or exogenous application of chemicals that affect pollen viability.

[0007] One form of conditional male sterility in maize and wheat is flavonoid-dependent fertility (FDF). Flavonoids are a class of secondary metabolites in plants that have a variety of biological roles, including tissue pigmentation, acting as signaling compounds, and mediating reactive oxygen species (ROS) homeostasis. Flavonoids are considered essential for maize and wheat reproduction because they regulate ROS homeostasis during pollen tube germination, which controls pollen tube rupture. These essential flavonoids accumulate in pollen grains before pollen matures, so when pollen falls off and lands on receptive stigmas, these compounds can be immediately utilized. Pollen tube rupture is usually a tightly regulated process that maintains the integrity of the pollen tube structure during growth through receptive stigmas. When the pollen tube reaches the synergids, the pollen tube begins to rupture, releasing sperm cells and allowing double fertilization required as part of plant sexual reproduction. For maize and wheat plants whose pollen grains lack these essential flavonoids, pollen tube growth fails prematurely. Pollen tubes either never successfully grow into the stigma or prematurely break before reaching the synergids. This failed pollen tube growth completely prevents fertilization, so plants of maize and wheat species whose pollen lacks flavonoids are male sterile.

[0008] The use of flavonols to restore fertility has been previously observed in maize (see, e.g., EH Coe et al., White pollen in maize, J. Heredity 72:318-320 (1981)). Early examples were the result of random mutations affecting two copies of chalcone synthase. A copy of chalcone synthase (white pollen 1, WHP1) on maize chromosome 2 is expressed in anthers at the late stage of pollen development. Plants homozygous for the whp1 mutant allele will produce and shed pollen that is more yellowish in color than typical pollen (commonly known as "white pollen"). If this white pollen is applied to standard wild-type maize filaments, normal pollen tube growth and fertilization can occur. The second chalcone synthase (colorless 2, C2) on maize chromosome 4 is expressed in a variety of tissue types, including filaments. If the mutations in whp1 and c2 are homozygous in the same plant, the plant will produce and shed pollen that is more pale yellow in color and will not grow normal pollen tubes when self-pollinated or fertilized by cross-pollination with another maize plant. It was later discovered that this white pollen is conditionally sterile and that full fertility can be restored by adding exogenous flavonoids during the interaction of pollen and filaments (Y. Mo et al., Biochemical complementation of chalcone synthase mutants defines a role for flavonols in functional pollen, Proc. Nat'l Acad. Sci. USA 89:7213-7217 (1992)). The application of such exogenous flavonoids can be accomplished by coating the mature white pollen grains with dry powdered flavonoids or by applying the flavonoids directly to the filaments before applying the white pollen.

[0009] The discovery of the c2 / whp1 conditional male sterility system has prompted patent applications seeking to commercialize the technology in maize through a variety of approaches, including the use of GM technology and inducible promoters (possibly referring to U.S. Patents 5,432,068 and 5,733,759). Although theoretically a promising alternative to emasculation or CMS, the technology has not yet been commercialized for maize seed production. The most critical disadvantage is the c2 mutation, which affects both male and female reproductive tissues in addition to pollen. The absence of this critical chalcone synthase inhibits downstream synthesis of flavonoids and anthocyanins in filaments, anthers, kernel aleurone, and other tissues. The loss of downstream metabolites potentially disrupts ROS signaling, inhibits responses to abiotic stresses in tissues that affect female receptivity and yield, and may alter the visual phenotype of the plant in ways that growers do not like. In addition, the c2 / whp1 system is not easily translated to crops other than maize. Summary of the invention

[0010] Here, we report the discovery of a novel FDF system based on anther-specific chalcone synthase gene mutations in maize and wheat. This novel system, termed flavonoid-dependent fertility ("FDF"), exhibits complete male sterility that can be restored to male fertility by application of exogenous flavonoids, but without the critical drawbacks of c2 / whp1. The FDF mutant allele does not affect the downstream synthesis of flavonoids and anthocyanins in tissues other than the anther tapetum cell layer, and therefore does not affect the color or stress response capacity of female reproductive tissues.

[0011] Within the scope of the present invention is a method of seed production, the method comprising: (a) obtaining a first plant that is a flavonoid-dependent fertile ("FDF") plant having a mutation in an FDF gene; (b) obtaining a second plant that is male fertile; (c) pollinating the FDF plant with pollen from the second plant; and (d) obtaining progeny seeds. The FDF gene can be TaFDF1A, TaFDF1B, TaFDF1D, or a combination thereof (SEQ ID NOs: 1-3), or a homolog thereof; or ZmFDF1, or ZmFDF2, or a combination thereof (SEQ ID NOs 4-5), or a homolog thereof, such as OsFDF in rice. In one aspect, the first plant and the second plant are monocots. In one aspect, the monocot is selected from the group consisting of wheat, maize, and rice.

[0012] Flavonoid-dependent fertility ("FDF") plants comprising a mutation in an FDF gene are also within the scope of the present invention. The mutation in the FDF gene can be a knockout mutation, and the knockout mutation in the FDF gene can be homozygous. The FDF gene can be TaFDF1A, TaFDF1B, TaFDF1D, or a combination thereof (SEQ ID NO: 1-3), or a homolog thereof; or ZmFDF1, or ZmFDF2, or a combination thereof (SEQ ID NO 4-5), or a homolog thereof, such as OsFDF in rice. In addition, the FDF plant further comprises a mutation in C2 / WHP or a homolog thereof. In one embodiment, the plant is a wheat plant. In an alternative embodiment, the plant is a maize plant.

[0013] Another embodiment of the present invention is a method for propagating FDF plants, the method comprising: (a) obtaining at least one plant, wherein the plant is a flavonoid-dependent fertile ("FDF") plant comprising a mutation in an FDF gene; (b) applying a composition comprising a flavonoid to the plant of step (a); (c) allowing self-pollination to occur; and (d) obtaining progeny seeds thereof.

[0014] The flavonoid can be quercetin; quercetin can be a liquid solution (optionally mixed in water, propylene glycol or other solvents or solutions). Quercetin can be mixed in a solvent or solution at a concentration of 1 mg / L to 100 mg / L, 2.5 mg / L to 50 mg / L or about 5 mg / L; alternatively, the liquid solution is saturated with quercetin. Alternatively, the composition comprising quercetin is a powder, which can be a mixture of quercetin and a carrier compound. The mixture of quercetin and a carrier compound can be a ratio between 1000:1 and 1:1000. The carrier compound can be crystalline silica, talc, a metal powder or a mica mineral. In a liquid formulation or a powder formulation, the flavonoid can be applied to the female organs of the flower of the parent plant. The flavonoid composition can be topically applied by smearing, spraying, sprinkling and watering.

[0015] Another embodiment of the present invention is a method of hybrid seed production, the method comprising: (a) obtaining at least one inbred female FDF plant, wherein the inbred female FDF plant comprises a mutation in the FDF gene; (b) obtaining pollen from at least one inbred male plant, wherein the inbred male plant produces fertile pollen; (c) pollinating the inbred female FDF plant with fertile pollen from the inbred male plant; and (d) obtaining hybrid progeny thereof. Another embodiment of the method is a plant produced by the previous method.

[0016] Another embodiment of the present invention is a method for restoring the fertility of FDF plants by applying a composition comprising flavonoids to FDF plants. FDF plants can be maize, wheat or rice. If wheat, the FDF wheat plant comprises a mutation in a gene selected from the group consisting of SEQ ID NO:1-3 and combinations thereof. If maize, the FDF maize plant comprises a mutation in a gene selected from the group consisting of SEQ ID NO:4-5 and combinations thereof, and optionally further comprises a mutation in C2 (SEQ ID NO:26) and / or WHP (SEQ ID NO:28). If the FDF plant is rice, the FDF rice plant comprises a mutation in SEQ ID NO:57. Flavonoids can include quercetin, which can be mixed in a liquid such as water, propylene glycol or other solvents or solutions. Alternatively, flavonoids can be mixed in powder form with carrier compounds such as crystalline silica, talc, metal powders and mica minerals. The composition can be applied to plant parts such as anthers, silks, stigmas, florets, spikes, leaf whorls, leaf crowns and / or roots by painting, spraying, sprinkling and root dredging.

[0017] Brief description of the sequences in the sequence listing

[0018] SEQ ID NO: 1 is TraesCS1A02G160300, a gene on chromosome 1A of common wheat (Triticum aestivum) (also known as TaFDF1A). This gene encodes a functional copy of chalcone synthase that is expressed in developing anthers. FDF in wheat requires loss of function of this gene.

[0019] SEQ ID NO: 2 is TraesCS1B02G176300, a gene on wheat chromosome 1B (also known as TaFDF1B). This gene encodes a functional copy of chalcone synthase that is expressed in developing anthers. FDF in wheat requires loss of function of this gene.

[0020] SEQ ID NO: 3 is TraesCS1D02G157500, a gene on chromosome 1D of common wheat (also known as TaFDF1D). This gene encodes a functional copy of chalcone synthase that is expressed in developing anthers. FDF in wheat requires loss of function of this gene.

[0021] SEQ ID NO:4 is Zm00001d032662, a gene on maize chromosome 1 (also known as ZmFDF1, ZmCHS_Chr1, chalcone synthase 2 (chls2) or GRMZM2G380650). The sequence was downloaded from maizeGDB.org and referenced the Zm-B73-REFERENCE-GRAMENE-4.0 genome version. The gene encodes a functional copy of chalcone synthase that is primarily expressed in meiotic tassels and developing anthers, with low or zero expression in other tissues. FDF in corn may require loss of function of this gene.

[0022] SEQ ID NO:5 is Zm00001d013991, a gene on maize chromosome 5 (also known as ZmFDF2, ZmCHS_Chr5, chalcone synthase 11 (chls11) or GRMZM2G477683). This sequence was downloaded from maizeGDB.org and referenced the Zm-B73-REFERENCE-GRAMENE-4.0 genome version. This gene encodes a functional copy of chalcone synthase that is primarily expressed in meiotic tassels and developing anthers, with low or zero expression in other tissues. FDF in corn may require the loss of function of this gene.

[0023] SEQ ID NO: 6 is the edited TraesCS1A02G160300. It contains a single base pair deletion relative to SEQ ID NO: 1.

[0024] SEQ ID NO: 7 is the edited TraesCS1B02G176300. It contains a two base pair deletion relative to SEQ ID NO: 2.

[0025] SEQ ID NO: 8 is the edited TraesCS1D02G157500. It contains a single base pair deletion relative to SEQ ID NO: 3.

[0026] SEQ ID NO:9 is a DNA sequence encoding a guide RNA sequence targeting SEQ ID NO:1.

[0027] SEQ ID NO: 10 is a DNA sequence encoding a guide RNA sequence targeting SEQ ID NO: 2. We found no evidence of genome editing with this guide.

[0028] SEQ ID NO:11 is a repeat of SEQ ID NO:10.

[0029] SEQ ID NO: 12 is construct 25206.

[0030] SEQ ID NO: 13 is TraesCS2A03G12234, the wheat chromosome A ortholog of maize c2 / whp1.

[0031] SEQ ID NO: 14 is TraesCS2B03G140060, the wheat chromosome B ortholog of maize c2 / whp1.

[0032] SEQ ID NO: 15 is TraesCS2D03G11827, the wheat chromosome D ortholog of maize c2 / whp1.

[0033] SEQ ID NO: 16 is construct 27726.

[0034] SEQ ID NO: 17 is construct 27738.

[0035] SEQ ID NO: 18 is construct 27769.

[0036] SEQ ID NOs: 19-24 are primers LZ311, LZ312, LZ313, LZ314, LZ315 and LZ316, respectively; see Table 5.

[0037] SEQ ID NO:25 is GRMZM2G380650; this is a repeat of SEQ ID NO:4.

[0038] SEQ ID NO: 26 is the reference sequence of C2 in Zea mays.

[0039] SEQ ID NO: 27 is the reference sequence of c2 in Zea mays.

[0040] SEQ ID NO: 28 is the reference sequence of Whp1 in Zea mays.

[0041] SEQ ID NO:29 is a DNA sequence encoding a guide RNA sequence named ZmFDF1\target37.

[0042] SEQ ID NO:30 is a DNA sequence encoding a guide RNA sequence named ZmFDF1\target74.

[0043] SEQ ID NO:31 is a DNA sequence encoding a guide RNA sequence named ZmFDF2\target72.

[0044] SEQ ID NO:32 is a DNA sequence encoding a guide RNA sequence named ZmFDF2\target74.

[0045] SEQ ID NO:33 is a DNA sequence encoding a guide RNA sequence named ZmFDF2\target2.

[0046] SEQ ID NO:34 is a DNA sequence encoding a guide RNA sequence named ZmFDF2\target16.

[0047] SEQ ID NOs: 35-52 are the primers and probes listed in Table 9.

[0048] SEQ ID NO:53 is the nucleotide sequence of Os05g0212900 presented in reverse orientation (3'-5').

[0049] SEQ ID NO:54 is the nucleotide sequence of Os07g0214900 presented in reverse orientation (3'-5').

[0050] SEQ ID NO:55 is the nucleotide sequence of Os07g0501100 presented in reverse orientation (3'-5').

[0051] SEQ ID NO:56 is the nucleotide sequence of Os11g0530600 presented in reverse orientation (3'-5').

[0052] SEQ ID NO:57 is the nucleotide sequence of OsFDF (also known as Os10g0484800), presented in the 5'-3' orientation.

[0053] definition

[0054] Unless otherwise defined below, all technical and scientific terms used herein are intended to have the same meaning as commonly understood by those of ordinary skill in the art. Reference to the technology employed herein is intended to refer to the technology commonly understood in the art, including variations of those technologies and / or alternatives to equivalent technologies that are clear to those of ordinary skill in the art. Although it is believed that the following terms will be well understood by those of ordinary skill in the art, the following definitions are set forth to facilitate the interpretation of the subject matter disclosed by the present invention.

[0055] The terms "a / an" and "the" as used in this application (including the claims) mean "one or more". For example, the phrase "a / a cell" refers to one or more cells, and in some embodiments may refer to tissues and / or organs. Similarly, the phrase "at least one / a kind", when used herein to refer to an entity, refers to, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100 or more of the entity, including but not limited to all integer values ​​between 1 and 100 and integers greater than 100.

[0056] As used herein, the term "about" means that a person skilled in the art would readily know that the typical error range for the corresponding value (eg, ±20%, ±10%, or ±5%) is within the intended meaning of the recited value.

[0057] The term "one or more alleles" refers to any one of one or more alternative forms of a gene, all of which are related to at least one trait or feature. In diploid cells, two alleles of a given gene occupy corresponding loci on homologous chromosome pairs. In some cases (e.g., for QTL), it is more accurate to replace "allele" with "haplotype" (i.e., alleles of chromosome segments), but in these cases, the term "allele" should be understood to include the term "haplotype". If two individuals (e.g., two plants) have the same allele at a specific locus, then if the allele is inherited from a common ancestor (i.e., the allele is a copy of the same parent's allele), the allele is referred to as "due to inheritance and the same". Alternatives are alleles "same by state" (i.e., the allele appears to be the same, but derived from two different copies of the allele). Generation information identification is useful for linkage studies; both generation identification and state information identification can be used for association studies, although pedigree information identification may be particularly useful.

[0058] As used herein, the term "and / or," when used in the context of listing entities, refers to the entities either alone or in combination. Thus, for example, the phrase "A, B, C, and / or D" includes A, B, C, and D individually, but also includes any and all combinations and subcombinations of A, B, C, and D (e.g., AB, AC, AD, BC, BD, CD, ABC, ABD, and BCD). In some embodiments, the one or more elements referred to by "and / or" may also be present alone in single or multiple occurrences in one or more combinations and / or one or more subcombinations.

[0059] "Anther-specific genes" are defined as genes that are highly expressed in developing anthers (including in developing microspores) and lowly or zero expressed in all other tissues tested (including female reproductive tissues).

[0060] The term "backcrossing" is understood within the scope of the present disclosure to refer to a process in which hybrid progeny are repeatedly crossed back to one of the parents.

[0061] As used herein, "carrier" means a compound that acts as an agent accompanying the collected pollen, preferably in powdered form. Suitable carrier compounds can be, but are not limited to, talc powder, silica powder, and the like.

[0062] As used herein, the term "comprising" or "comprise" is open ended. When used in conjunction with a subject nucleic acid (or amino acid sequence), it refers to a nucleic acid sequence (or amino acid sequence) that includes the subject sequence as a part or as its entire sequence.

[0063] As used herein, the term "superior line" or "inbred line" refers to any line resulting from breeding and selection for superior agronomic performance. A superior line has stable genetics, i.e., it is reasonably or nearly isogenic in its genome. In other words, a superior line is reasonably or nearly homozygous for all alleles in its genome.

[0064] The term "FDF gene" (and "FDF allele", used interchangeably throughout) refers to a gene that, when mutated or otherwise defective or suppressed, results in a plant being infertile due to a flavonoid deficiency; in addition, the deficiency can be compensated by the application of flavonols and / or flavonoids. For example, wheat FDF genes include TraesCS1A02G160300 (SEQ ID NO: 1; also known as TaFDF1A), TraesCS1B02G176300 (SEQ ID NO: 2; also known as TaFDF1B), and TraesCS1D02G157500 (SEQ ID NO: 3; also known as TaFDF1D). For example, maize FDF genes include Zm00001d032662 (SEQ ID NO:4; also known as ZmFDF1 or ZmCHS_Chr1) and Zm00001d013991 (SEQ ID NO:5; also known as ZmFDF2 or ZmCHS_Chr5).

[0065] The term "gene" refers to a unit of inheritance comprising a DNA sequence that occupies a specific location on a chromosome and contains the inheritance instructions for a specific characteristic or trait in an organism.

[0066] "Gene editing" generally refers to the use of site-directed nucleases (including but not limited to CRISPR / Cas, zinc fingers, meganucleases, etc.) to cut a nucleotide sequence at a desired location. This may result in insertion / deletion ("indel") mutations (i.e., "SDN1"), base editing (i.e., "SDN2"), or allele insertion or replacement (i.e., "SDN3"). SDN2 or SDN3 gene editing may include providing one or more recombination templates (e.g., in a vector) containing a gene sequence of interest that can be used for homology-directed repair (HDR) in plants (i.e., to be introduced into a plant genome).

[0067] The fracture in the plant genome can be introduced into the inside, upstream and / or downstream of the target sequence. In certain embodiments, double-stranded DNA breaks are produced inside or near the target sequence locus. In certain embodiments, fractures are produced upstream and downstream of the target sequence locus, which can cause it to be excised from the genome. In certain embodiments, one or more single-stranded DNA breaks (nicks) are produced inside, upstream and / or downstream of the target sequence (for example, using nickase Cas9 variants). Such fractures can be repaired by non-homologous end joining (NHEJ) process, which may result in the production of small insertions or deletions (indel) in the repair site. Such indels may result in frameshift mutations, thereby causing premature termination codons or other types of loss-of-function mutations in the targeted gene.

[0068] In some embodiments, gene editing may involve transient, inducible, or constitutive expression of a gene editing component or system in a target plant. Gene editing may also involve genomic integration or episomal presence of a gene editing component or system in a target plant.

[0069] In certain embodiments, nucleic acid modification or mutation is achieved by a (modified) zinc finger nuclease (ZFN) system. The ZFN system uses an artificial restriction endonuclease generated by fusing a zinc finger DNA binding domain with a DNA cleavage domain that can be engineered to target a desired DNA sequence. Exemplary methods for genome editing using ZFNs can be found, for example, in the following: U.S. Patent Nos. 6,534,261; 6,607,882; 6,746,838; 6,794,136; 6,824,978; 6,866,997; 6,933,113; and 6,979,539.

[0070] In certain embodiments, nucleic acid modification is achieved by (modified) meganucleases, which are endodeoxyribonucleases characterized by large recognition sites (double-stranded DNA sequences of 12 to 40 base pairs). Exemplary methods using meganucleases can be found in: U.S. Pat. Nos.: 8,163,514; 8,133,697; 8,021,867; 8,119,361; 8,119,381; 8,124,369; and 8,129,134.

[0071] In certain embodiments, nucleic acid modification is achieved by a (modified) CRISPR / Cas complex or system. In certain embodiments, the CRISPR / Cas system or complex is a Class 2 CRISPR / Cas system. In certain embodiments, the CRISPR / Cas system or complex is a Type II, Type V or Type VI CRISPR / Cas system or complex. Instead of generating customized proteins to target specific sequences, the CRISPR / Cas system can program a single Cas protein to recognize a specific nucleic acid target via an RNA guide sequence (gRNA). In other words, the short RNA guide sequence can be used to recruit the Cas enzyme protein to a specific nucleic acid target locus of interest (which may contain or consist of RNA and / or DNA).

[0072] Typically, CRISPR / Cas or CRISPR systems, as used herein in the above-mentioned documents, collectively refer to transcripts and other elements involved in the expression of CRISPR-related ("Cas") genes or directing their activity, including sequences encoding Cas genes and one or more of the following: tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr-pairing sequences (including "direct repeat sequences" and partial direct repeat sequences processed by tracrRNA in the context of endogenous CRISPR systems), guide sequences (also referred to as "spacers" in the context of endogenous CRISPR systems), or one or more "RNAs" as used herein (e.g., one or more RNAs for guiding Cas such as Cas9, such as CRISPR RNA and, where applicable, trans-activating (tracr) RNA or single guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from CRISPR loci. Typically, CRISPR systems are characterized by elements that promote the formation of CRISPR complexes at the site of the target sequence (also referred to as prototype spacers in the context of endogenous CRISPR systems). In the context of forming a CRISPR complex, a "target sequence" refers to a sequence to which a guide sequence is designed to have complementarity, wherein hybridization between the target sequence and the guide sequence promotes formation of a CRISPR complex. The target sequence may comprise any polynucleotide, such as a DNA or RNA polynucleotide.

[0073] In certain embodiments, gRNA is a chimeric guide RNA or a single guide RNA (sgRNA). In certain embodiments, gRNA comprises a guide sequence and a tracr pairing sequence (or a co-directional repeat sequence). In certain embodiments, gRNA comprises a guide sequence, a tracr pairing sequence (or a co-directional repeat sequence) and a tracr sequence. In certain embodiments, a CRISPR / Cas system or complex as described herein does not include and / or does not rely on the presence of a tracr sequence (e.g., if the Cas protein is Cas12a).

[0074] Cas proteins as mentioned herein, such as but not limited to Cas9, Cas12a (formerly known as Cpf1), Cas12b (formerly known as C2c1), Cas13a (formerly known as C2c2), C2c3, Cas13b proteins, can be derived from any suitable source, and therefore can include different orthologs from a variety of (prokaryotic) organisms, as well documented in the art. In certain embodiments, the Cas protein is (modified) Cas9, preferably (modified) Staphylococcus aureus Cas9 (SaCas9) or (modified) Streptococcus pyogenes Cas9 (SpCas9). In certain embodiments, the Cas protein is Cas12a, optionally from an aminoacidococcus species, such as an aminoacidococcus species BV3L6 Cpf1 (AsCas12a), or a Lachnospiraceae bacteria Cas12a, such as Lachnospiraceae bacteria MA2020 or Lachnospiraceae bacteria MD2006 (LBCas12a). See U.S. Patent No. 10,669,540. Alternatively, the Cas12a protein may be from Moraxella bovoculi AAX08_00205 [Mb2Cas12a] or Moraxella bovoculi AAX11_00205 [Mb3Cas12a]. See WO 2017 / 189308. In certain embodiments, the Cas protein is (modified) C2c2, preferably Leptotrichia wadei C2c2 (LwC2c2) or Listeria newyorkensis FSL M6-0635C2c2 (LbFSLC2c2). In certain embodiments, the (modified) Cas protein is C2c1. In certain embodiments, the (modified) Cas protein is C2c3. In certain embodiments, the (modified) Cas protein is Cas13b. Other Cas enzymes can be obtained by those skilled in the art.

[0075] The term "genotype" and its variants refer to the genetic components of an organism, including, for example, whether a diploid organism is heterozygous (i.e., has two different alleles for a given gene or QTL) or homozygous (i.e., has the same allele for a given gene or QTL) for one or more genes or loci (e.g., SNPs, haplotypes, gene mutations, indels). As used herein, the term "at least heterozygous" for a particular allele means that there is at least one copy of the allele. For example, a maize plant that is at least heterozygous for the HI allele of a gene has one or two copies of the HI allele (i.e., is heterozygous or homozygous).

[0076] The term "germplasm" refers to the totality of the genotypes of a population or other group of individuals (e.g., species or plant strains). The phrase "adapted germplasm" refers to plant material that has been proven to have genetic advantages; for example, for a given environment or geographic region, the phrases "unadapted germplasm," "original germplasm," and "exotic germplasm" refer to plant material of unknown or unproven genetic value; for example, for a given environment or geographic region; thus, the phrase "unadapted germplasm" refers in some embodiments to plant material that does not belong to an established breeding population and has no known relationship to a member of an established breeding population.

[0077] The term "haplotype" can refer to the set of alleles of an individual inherited from one parent. Thus, a diploid individual has two haplotypes. The term "haplotype" can be used in a more limited sense to refer to physically linked and / or unlinked genetic markers (e.g., sequence polymorphisms) associated with a phenotypic trait. The phrase "haplotype block" (sometimes also referred to simply as a haplotype in the literature) refers to a set of two or more genetic markers that are physically linked on a single chromosome (or a portion thereof). Typically, each block has several common haplotypes, and a subset of genetic markers (i.e., "haplotype tags") can be selected to uniquely identify each of these haplotypes.

[0078] The term "hybrid vigor" refers to hybrid vigor, i.e., the function of improving or increasing any biological quality (e.g., size, growth rate, fertility, yield, etc.) of its parents in hybrid offspring. For example, as described above, hybrid offspring from inbred plant strains of different hybrid vigor groups may show more hybrid vigor than their parental strains. The first generation offspring of such hybridization usually show the desired characteristics of both parents to a greater extent. If the first generation hybrids are mated together, such hybrid vigor may be reduced in subsequent generations.

[0079] The terms "hybrid group" and "hybrid pool" are used interchangeably and refer to a group of genotypes or inbred strains that exhibit similar heterotic responses when crossed with genotypes or inbred strains from other genetically different germplasm groups. The strains contained within a hybrid group have a closer genetic relationship relative to the strains of the more distant genetic relationship compared between hybrid groups. In general, hybrids from two inbred strains within the same hybrid group that are crossed together exhibit much less heterosis than hybrids from inbred strains from one hybrid group crossed with inbred strains from different hybrid groups. A specific hybrid group can include multiple strains with different genetics.

[0080] In the context of plant breeding, the terms "hybrid", "hybrid plant" and "hybrid progeny" refer to plants that are the offspring of genetically different parents produced by crossing plants of different lines or varieties or species, including but not limited to a cross between two inbred lines (e.g., genetically heterozygous or mostly heterozygous individuals). The phrase "single cross FI hybrid" refers to an FI hybrid produced by a cross between two inbred lines.

[0081] The phrase "inbred strain" refers to a population that is homozygous or nearly homozygous genetically. For example, an inbred strain can be obtained by several cycles of brother / sister breeding or self-fertilization. In certain embodiments, an inbred strain is pure bred for one or more phenotypic traits of interest. "Inbreeding", "inbred individuals" or "inbred progeny" are separate samples from an inbred strain. The term "inbreeding" refers to an individual or strain that is substantially homozygous. When used in a breeding program, an inbred strain may also be referred to as a "parental strain".

[0082] The terms "introgression," "introgressed," and "introgressing" refer to natural and artificial processes in which genomic regions are moved from one species, variety, or cultivar to another species, variety, or cultivar by crossing the other species, variety, or cultivar. The process can optionally be accomplished by backcrossing to the recurrent parent.

[0083] The term "loss of function mutation" is a change in the DNA sequence of a gene that causes the mutant gene product to lack the molecular function of the wild-type gene (i.e., "mutation"). There are four major genetic variations that can cause loss of function mutations: 1) mutations that cause premature stop codons to produce truncated protein sequences; 2) mutations that occur at canonical splicing sites that affect splicing (leading to the inclusion of introns or exclusion of exons in mRNA transcripts); 3) insertion or deletion variants that are located in three non-integer multiples of the gene coding region, which cause frameshifts by destroying the full-length transcript; and 4) mutations that cause the loss of the start codon (transcription start codon, such as ATG), which will prevent gene transcription if there is no selectable start codon near the mutation. In addition, mutations in gene promoters or untranslated regions (UTRs) can reduce or eliminate gene expression, leading to loss of function. The term "knockout mutation" is used interchangeably.

[0084] The terms "nucleic acid" and "polynucleotide" are used interchangeably and as used herein, refer to both the sense and antisense strands of RNA, cDNA, genomic DNA, mitochondrial DNA, and synthetic forms and mixed polymers of the above items. In specific embodiments, nucleotides refer to ribonucleotides, deoxynucleotides, or modified forms of any type of nucleotides and combinations thereof. The term also includes, but is not limited to, single-stranded and double-stranded forms of DNA and / or RNA. In addition, the polynucleotides disclosed herein (e.g., circular DNA templates), the nucleic acid concatemers disclosed herein may include any or both of naturally occurring nucleotides and modified nucleotides linked together by naturally occurring and / or non-naturally occurring nucleotide linkages. Nucleic acid molecules may be chemically or biochemically modified, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, tags, methylation, substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), side groups (e.g., polypeptides), intercalators (e.g., acridine, psoralens, etc.), chelators, alkylating agents, and modified linkages (e.g., α-anomeric nucleic acids, etc.). The above terms are also intended to include any topological conformations, including single-stranded, double-stranded, partially duplexed, triplexed, hairpin-shaped, circular, and padlock-shaped conformations. Unless otherwise indicated, reference to a nucleic acid sequence encompasses its complement. Therefore, reference to a nucleic acid molecule with a specific sequence is understood to encompass its complementary chain with its complementary sequence. When nucleotide sequences specifically hybridize in a solution, these nucleotide sequences are "complementary" (e.g., according to Watson-Crick base pairing principles). The term also includes codon-optimized nucleic acids encoding the same polypeptide sequence. It is also understood that the nucleic acid can be unpurified, purified, or attached to, for example, a synthetic material such as a bead or column matrix.

[0085] As used herein, the terms "nucleotide sequence", "polynucleotide", "nucleic acid sequence", "nucleic acid molecule" and "nucleic acid fragment" refer to polymers of single-stranded or double-stranded RNA or DNA, optionally containing synthetic, non-natural, and / or altered nucleotide bases. "Nucleotide" is a monomeric unit from which DNA or RNA polymers are constructed and consists of a purine or pyrimidine base, a pentose sugar and a phosphate group. Nucleotides (usually found in their 5'-monophosphate form) are represented by their single-letter names as follows: "A" represents adenylic acid or deoxyadenylic acid (for RNA or DNA, respectively), "C" represents cytidylic acid or deoxycytidylic acid, "G" represents guanylic acid or deoxyguanylic acid, "U" represents uridylic acid, "T" represents deoxythymidylic acid, "R" represents purine (A or G), "Y" represents pyrimidine (C or T), "K" represents G or T, "H" represents A or C or T, "I" represents inosine, and "N" represents any nucleotide.

[0086] The term "offspring" plant refers to any plant of the progeny of the nutrition or sexual reproduction of one or more parental plants or their descendants. For example, offspring plants can be obtained by cloning or selfing of parental plants or by the hybridization of two parental plants, and include selfing bodies and F1 or F2 or even more distant generations. F1 is the first generation offspring generated by parents (wherein at least one party is the donor used as a trait for the first time), and the offspring of the second generation (F2) or subsequent generations (F3, F4, etc.) are samples generated by the selfing of F1, F2, etc. Therefore, F1 can be a hybrid produced by hybridization between two true breeding parents, and F2 can be an offspring produced by self-pollination of the F1 hybrid.

[0087] The term "PCR (polymerase chain reaction)" is understood within the scope of the present invention to mean a method which generates relatively large amounts of specific regions of DNA, making possible different analyses based on those regions.

[0088] "Phenotype" is understood within the scope of this disclosure to refer to one or more distinguishable characteristics of a genetically controlled trait. The phrase "phenotypic trait" refers to the outward or other detectable characteristic in an individual resulting from the interaction of the individual's genome with the environment.

[0089] A "plant" is any plant at any stage of development, in particular a seed plant. In particular, in the context of the present disclosure, a plant refers to a maize plant.

[0090] A "plant cell" is a structural and physiological unit of a plant, comprising a protoplast and a cell wall. A plant cell may be in the form of an isolated single cell or a cultured cell, or as part of a higher organized unit such as, for example, a plant tissue, a plant organ, or a whole plant.

[0091] The term "population" means a genetically heterogeneous collection of plants sharing a common genetic derivation.

[0092] As used herein, the term "primer" refers to an oligonucleotide that is capable of annealing to a nucleic acid target (in some embodiments, specifically annealing to a nucleic acid target) when placed under conditions that induce synthesis of a primer extension product (e.g., in the presence of nucleotides and an agent for polymerization (e.g., a DNA polymerase) and at a suitable temperature and pH) to allow a DNA polymerase and / or reverse transcriptase to attach thereto, thereby serving as a starting point for DNA synthesis. In some embodiments, one or more primers are employed to amplify plant nucleic acids (e.g., using a polymerase chain reaction; PCR).

[0093] As used herein, the term "probe" refers to a nucleic acid (e.g., a single-stranded nucleic acid or a double-stranded or higher-order nucleic acid chain, or a subsequence thereof) that can form a hydrogen-bonded duplex with a complementary sequence in a target nucleic acid sequence. Typically, the probe has a sufficient length to form a stable and sequence-specific duplex molecule with its complementary sequence, and this can be used in some embodiments to detect a target sequence present in multiple nucleic acids.

[0094] The term "progeny" refers to one or more descendants of a particular cross. Typically, progeny are produced by breeding of two individuals, but some species (particularly some plants and hermaphroditic animals) can self-fertilize (i.e., the same plant serves as a donor of both male and female gametes). The one or more descendants can be, for example, F1, F2, or any subsequent generation.

[0095] The term "random mutagenesis" refers to an alternative mutation method. In certain embodiments, nucleic acid modification is achieved by random mutagenesis. Cells or organisms can be exposed to mutagens, such as UV radiation or mutagenic chemicals (such as ethyl methanesulfonate (EMS)), and then select mutants with desired characteristics. For example, mutants can be identified by TILLING (targeted induced local damage in genome). The method combines mutagenesis (such as mutagenesis using chemical mutagens such as ethyl methanesulfonate (EMS)) with sensitive DNA screening techniques for identifying single base mutations / point mutations in target genes. The TILLING method relies on the formation of DNA heteroduplexes, which are formed by amplifying multiple alleles by PCR, then heating and slowly cooling. "Bubble" is formed when two DNA chains do not match, and then cut by single-stranded nuclease. The products are then separated according to size, such as by HPLC. See McCallum et al., Targeted screening for induced mutations, Nat. Biotechnol. 18(4):455-57 (2000) and McCallum et al., Targeting induced local lesions IN genomes (TILLING) for plant functional genomics, Plant Physiol. 123(2):439-42 (2000).

[0096] As the phrases "sexual crossing" and "sexual reproduction" in the context of the presently disclosed subject matter refer to the fusion of gametes to produce offspring (e.g., by fertilization, such as in plants by pollination to produce seeds). In some embodiments, "sexual crossing" or "cross-fertilization" is the fertilization of one individual by another individual (e.g., cross-pollination in plants). In some embodiments, the term "selfing" refers to the production of seeds by self-fertilization or self-pollination; that is, the pollen and ovules are from the same plant.

[0097] The terms "solvent" and / or "solution" refer to a liquid in which the flavonoids can be mixed. Examples include water, alcohol, propylene glycol, DMSO, Tris buffer, and other liquids available to those skilled in the art. The flavonoids need not be dissolved in the liquid; a mixture or emulsion is sufficient.

[0098] "Transformable," "transformability," and the like refer to plants, plant lines, or plant cells (such as callus or protoplasts) that more readily accept foreign DNA and can stably integrate the foreign DNA into their genome.

[0099] The term "variety" or "cultivar" refers to a group of similar plants that can be distinguished from other varieties of the same species by structural or genetic characteristics and / or performance. DETAILED DESCRIPTION

[0100] An embodiment of the present invention is a method of seed production, comprising: (a) obtaining at least one first plant, wherein the first plant is a flavonoid-dependent sterility ("FDF") plant comprising a mutation in an FDF gene; (b) obtaining at least one second plant, wherein the second plant is male fertile; (c) allowing the at least one second plant to pollinate the at least one first plant; and (d) obtaining progeny seeds thereof. In one aspect, the first plant and the second plant are monocots. In another aspect, the monocot is selected from wheat, maize, and rice. In another aspect, the FDF gene is selected from the group consisting of SEQ ID NOs: 1-5 and 57.

[0101] Another embodiment of the present invention is a flavonoid-dependent fertility ("FDF") plant comprising a mutation in a FDF gene. In one aspect, the mutation in the FDF gene is a knockout mutation. In another aspect, the knockout mutation in the FDF gene of the plant is homozygous. In one aspect, the FDF plant is a wheat plant and the FDF gene is selected from TaFDF1A (SEQ ID NO: 1), TaFDF1B (SEQ ID NO: 2), TaFDF1D (SEQ ID NO: 3) and combinations thereof. In another aspect, the FDF plant is a maize plant and the FDF gene is ZmFDF1 (SEQ ID NO: 4) or ZmFDF2 (SEQ ID NO: 5) or both. In a further aspect, the FDF plant further comprises a mutation in C2 / WHP. In another aspect, the FDF plant is a rice plant and the FDF gene is OsFDF (SEQ ID NO: 57).

[0102] Another embodiment of the present invention is a method of propagating FDF plants, the method comprising: (a) obtaining at least one plant, wherein the plant is a flavonoid-dependent fertility ("FDF") plant comprising a mutation in an FDF gene; (b) applying a composition comprising a flavonoid to the plant of step (a); (c) allowing self-pollination to occur; and (d) obtaining progeny seeds thereof. In one aspect, the flavonoid is quercetin. In another aspect, the composition comprising quercetin is a liquid solution. In another aspect, the liquid solution comprises quercetin mixed in water, propylene glycol or other solvent or solution. In yet another aspect, the liquid solution comprises quercetin suspended in a solvent or solution at a concentration of 1 mg / L to 100 mg / L, 2.5 mg / L to 50 mg / L or about 5 mg / L. In yet another aspect, the liquid solution comprising quercetin is saturated. In an alternative aspect, the composition comprising quercetin is a powder. In one aspect, the powder comprises a mixture of quercetin and a carrier compound. In one aspect, the ratio of the mixture of quercetin and carrier compound is between 1000:1 and 1:1000. In another aspect, the carrier compound is selected from the group consisting of crystalline silica, talc, metal powders and mica minerals. In yet another aspect, the composition comprising flavonoids is applied to the female organs of flowers on at least one plant designated as a female plant. In yet another aspect, the composition is applied by a method selected from the group consisting of smearing, spraying, sprinkling and root irrigation. In one aspect, the FDF gene is a wheat gene selected from the group consisting of SEQ ID NO:1-3 and combinations thereof. In another, the FDF gene is a maize gene selected from the group consisting of SEQ ID NO:4-5 and combinations thereof. In yet another, the FGF gene is a rice gene comprising SEQ ID NO:57. One embodiment of the present invention is a plant produced by the above method.

[0103] Another embodiment of the present invention is a method of hybrid seed production, the method comprising: (a) obtaining at least one inbred female FDF plant, wherein the inbred female FDF plant comprises a mutation in an FDF gene; (b) obtaining pollen from at least one inbred male plant, wherein the inbred male plant produces fertile pollen; (c) pollinating the inbred female FDF plant with fertile pollen from the inbred male plant; and (d) obtaining hybrid progeny thereof. Another embodiment of the method is a plant produced by the previous method. In one aspect, the inbred female FDF plant is a maize plant, a wheat plant, or a rice plant. In another aspect, the FDF wheat plant comprises a mutation in a gene selected from the group consisting of SEQ ID NOs: 1-3 and combinations thereof. In yet another aspect, the FDF maize plant comprises a mutation in a gene selected from the group consisting of SEQ ID NOs: 4-5 and combinations thereof. In yet another aspect, the FDF rice plant comprises a mutation in SEQ ID NO: 57. An embodiment of the present invention is a plant produced by the above method.

[0104] Another embodiment of the present invention is a method for restoring the fertility of an FDF plant comprising a mutation in an FDF gene, the method comprising applying a composition comprising a flavonoid to the FDF plant. In one aspect, the FDF plant is a maize plant, a wheat plant or a rice plant. In another aspect, the FDF wheat plant comprises a mutation in a gene selected from the group consisting of SEQ ID NO: 1-3 and combinations thereof. In another aspect, the FDF maize plant comprises a mutation in a gene selected from the group consisting of SEQ ID NO: 4-5 and combinations thereof. In another aspect, the FDF maize plant further comprises a mutation in a chalcone synthase gene. In another aspect, the FDF rice plant comprises a mutation in SEQ ID NO: 57. In one aspect, the flavonoid comprises quercetin. In another aspect, quercetin is mixed in water, propylene glycol or other solvents or solutions. In alternative aspects, the composition comprising a flavonoid further comprises a carrier compound. In one aspect, the carrier compound is selected from the group consisting of crystalline silica, talc, metal powders and mica minerals. In one aspect, the composition comprising flavonoids is applied to anthers, silks, stigmas, florets, spikes, leaf whorls, leaf crowns or roots. In one aspect, the composition is applied by a method selected from the group consisting of painting, spraying, sprinkling and root irrigation.

[0105] Examples

[0106] 1. Identification of the FDF gene in maize.

[0107] The anther-specific chalcone synthase gene was originally discovered in maize during a study of the causal mutations of the colorless 2 (c2) and white pollen 1 (whp1) alleles disclosed in the maize mutant line 224H. In the 224H line, white pollen 1 is homozygous for the recessive whp1 mutant allele and segregates for the mutant colorless 2 allele c2. Male sterility phenotypes were observed in individual plants homozygous for c2, while plants heterozygous or homozygous for C2 were male fertile. In the 224H line, the zygosity for colorless 2 was visually tracked by the presence or absence of a purple aleurone layer. Wild-type C2 homozygous or heterozygous embryos will be able to synthesize anthocyanins in the grain aleurone layer, which will produce visually purple grains. Embryos homozygous for the mutant c2 cannot synthesize anthocyanins in the grain aleurone layer, thereby producing a colorless aleurone layer, exposing the colorless (white) endosperm below (in the 224H line, the endosperm is always white; endosperm color is controlled by an independent, unrelated gene Y1). Although these mutant alleles are known to the maize research community, we cannot identify the source of the causal mutation that released these mutant alleles. In order to identify the causal mutation of colorless 2, we planted 224H seed segregation populations of c2 (mutant) and C2 (wild type). Tissue samples were collected and the entire colorless 2 gene was amplified using a PCR primer set. PCR products were sequenced by Sanger sequencing. SNP haplotype variants different from C2 in the B73, NP2222 and W22 reference genomes (Table 1) were identified as being separated in a 1:2:1 ratio in the 224H plant population. It was found that the SNP haplotypes of multiple seedlings derived from white grains were homozygous. The whp1 allele was also amplified by PCR primers and the PCR products were sequenced by Sanger sequencing for comparison with the B73, AX5707, and W22 reference genomes. The putative causal mutation for the whp1 mutant allele was not identified.

[0108] Table 1: Haplotypes identified in the c2 allele. Male sterile individual 224H plants were found to be homozygous for this haplotype.

[0109]

[0110] NA = Not Applicable

[0111] Further investigation of the c2 SNP haplotype focused on the C1973T SNP that results in the R72C amino acid substitution. This amino acid substitution is not part of the catalytic triad of chalcone synthase. Without being bound by theory, the free amino acid substitution from the positively charged side chain on arginine to the thiol side chain on cysteine ​​may have a deleterious effect on protein function. The first evidence supporting the C1973T SNP as the cause was found in publications detailing that the arginine at amino acid position 72 in C2 is part of a conserved motif required for coenzyme A binding in chalcone synthase (see, e.g., J. L. Ferrer et al., Structure of chalcone synthase and the molecular basis of plant polyketide biosynthesis, Nature Structural Biol. 6(8):775-784 (1999) and M. B. Austin & J. P. Noel, The chalcone synthase superfamily of type III polyketide synthases, Nat. Prod. Rep. 20:79-110 (2003)). Additional evidence was found by comparing the variant c2 amino acid sequence containing the R72C substitution with the wild-type C2 sequence using the PROVEAN software (Y. Choi & A. P. Chan, PROVEAN web server: a tool to predict the functional effect of aminoacid substitutions and indels, Bioinformatics 31(16): 2745-2747 (2015). The software returned a score of -6.055 for the R72C amino acid substitution and predicted that the mutation was deleterious to protein function.Finally, the project team identified that similar amino acid substitutions were found in a mutant line of Matthiola incana (V. Hemleben et al., Characterization and structural features of a chalcone synthase mutation in a white-flowering line of Matthiola incana R. Br. (Brassicaceae), Plant Mol. Biol. 55: 455-465 (2004). Hemleben et al. characterized the R72S amino acid substitution, which eliminated chalcone synthase protein activity and resulted in a colorless flower phenotype in violet, further emphasizing that substitutions at the R72 position would result in loss of function and subsequent inability to synthesize flavonoids required for pigment synthesis.

[0112] Based on the identification of the causal mutation of the c2 mutant allele in maize, we began to look for orthologs of c2 in wheat that could be targeted using gene editing (e.g., but not limited to CRISPR-based editing). During this search in wheat, other chalcone synthase genes in maize were identified, including chalcone synthase 2 (chls2) GRMZM2G380650 (SEQ ID NO: 4). Including chls2 in the analysis allowed the project team to identify other chalcone synthase genes with anther-specific expression patterns (TraesCS1A02G160300 (SEQ ID NO: 1), TraesCS1B02G176300 (SEQ ID NO: 2), and TraesCS1D02G157500 (SEQ ID NO: 3)). These additional chalcone synthase genes were found to have anther-specific expression patterns and were included in candidate genome editing targets to generate FDS in wheat. According to the Kyoto Enzyme Genes and Genomes (KEGG) database (www.genome.jp / kegg / annotation / enzyme.html), there are six chalcone synthases in maize that catalyze the same enzymatic step in the flavonoid biosynthetic pathway (this step is designated as EC2.3.1.74). These six chalcone synthases include C2, WHP1, and CHLS2. To determine whether orthologs other than CHLS2 should be included as candidates for editing to create an FDF system in maize, expression patterns across cellular tissues were analyzed using RNA-seq data from a range of developmental stages from the following two datasets: RNA expression publicly hosted at MaizeGDB.org and an in-house RNA-seq atlas. Table 2 summarizes the qualitative results of the six maize genes that catalyze EC step 2.3.1.74; four genes (C2, WHP1, CHLS2, and CHLS11) are all highly expressed in anther tissue. BLAST search revealed that CHLS11 was found to be the closest hit to CHLS2, with 92% sequence identity at the nucleotide level, and even higher when only coding sequences were compared. CHLS11 was also included as a potential editing candidate for maize FDF due to its expression pattern, zymogram, and sequence similarity.

[0113] Table 2. Summary of maize gene identifiers and relative expression of CHS genes catalyzing enzymatic step 2.3.1.74 in flavonoid biosynthesis

[0114]

[0115] 1A. Identification of candidate genes for FDF in rice

[0116] After identifying Zmchls2 and Zmchls11 as targets of FDF, we sought to identify chalcone synthase genes in rice that could serve as targets for creating an FDF system. In rice (Oryza sativa), ZmC2 and ZmWHP have only one ortholog: OsCHS1 (Os11g0530600). Mutations in OsCHS1 have previously been reported to cause male sterility (Wang, Lanxiang, Pui Ying Lam, Andy CW Lui, Fu-Yuan Zhu, Mo-Xian Chen, Hongjia Liu, Jianhua Zhang, and Clive Lo. 2020. "Flavonoids Are Indispensable for Complete Male Fertility in Rice." Journal of Experimental Botany 71(16):4715-28. doi.org / 10.1093 / jxb / eraa204). However, no reference to one or more orthologs of ZmCHLS2 and ZmCHLS11 has been reported previously. Comparison of genomic DNA sequences, spliced ​​RNA sequences, and protein amino acid sequences between maize ZmCHLS2 and ZmCHLS11 and the rice genome using BLAST searches (NCBI, genome assembly IRGSP-1.0) revealed that the best match was the rice gene OsFDF, which has 85% sequence similarity to the maize ortholog (Table 3).

[0117] Table 3. Sequence similarity between maize chalcone synthase orthologs and OsFDF.

[0118]

[0119] The rice gene OsFDF was not assigned to any metabolic step in the KEGG database, so it is unclear whether it catalyzes KEGG enzymatic step 2.3.1.74, which is catalyzed by the maize genes ZmC2, ZmWHP1, ZmCHLS2, and ZmCHLS11 in flavonoid biosynthesis. However, Uniprot predicted it to be a “chalcone and stilbene synthase” based on the protein domain and structure (www.uniprot.org / uniprotkb / Q7X795 / entry); this is the same protein class to which the maize FDF gene was assigned. There were four other rice genes predicted to catalyze step 2.3.1.74 (Table 4), but when their expression patterns were compared with OsFDF in the in-house RNA-seq dataset, only one of these four genes showed a highly male reproductive tissue-enriched expression pattern (Os07g0411300) required for FDF. OsFDF was most strongly expressed in male tissues (Table 4). The combination of its sequence similarity, the presence of the chalcone stilbene synthase protein domain, and the requisite male-enriched gene expression pattern make OsFDF an ideal mutation candidate for establishing the FDF system in rice.

[0120] Table 4. Relative tissue expression patterns of rice chalcone synthase genes

[0121]

[0122] 2. Wheat transformation.

[0123] A binary vector was designed that generates loss-of-function alleles at the TraesCS1A02G160300, TraesCS1B02G176300, and TraesCS1D02G157500 genome editing targets. A wheat codon-optimized version of the Cas9 enzyme derived from Streptococcus pyogenes was selected to generate double-stranded cuts in the targeted genes. This version of Cas9 contains L1181V and L1196V amino acid substitutions and nuclear localization sequences at the N- and C-termini of the amino acid sequence. Cas9 transcription is driven by the constitutive sugarcane ubiquitin 4 promoter derived from Saccharum officinarum. This promoter is enhanced by three elements; the nopaline synthase enhancer derived from Agrobacterium tumefaciens, the figwort mosaic virus enhancer derived from figwort mosaic cauliflower virus, and the cauliflower mosaic virus 35S enhancer region derived from cauliflower mosaic cauliflower virus. A terminator sequence based on the maize metallothionein-like gene was included after Cas9 to terminate transcription.

[0124] Based on the consensus sequence derived from Chinese spring wheat, two independent dual guide RNAs targeting the first exon in TraesCS1A02G160300, TraesCS1B02G176300 and TraesCS1D02G157500 were designed. One guide RNA targets the fourth codon of each of the three chalcone synthase gene targets and is designated as TaGHSG target 1. The second guide RNA targets the fifty-first codon of each of the three chalcone synthase gene targets and is designated as TaGHSG target 2. Guide RNA transcription is driven by the constitutive sugarcane ubiquitin 4 promoter (without enhancer element) derived from sugarcane. For effective guide RNA processing, TaGHSG target 1 and TaGHSG target 2 are separated by hammerhead RNA sequences from the trans-activating crRNA sequence derived from Streptococcus pyogenes. After transcription, these units form a combined guide RNA and crRNA sequence (referred to herein as "double guide" system) in each of the two guide RNAs. The guide RNA and crRNA sequences of these combinations are designated as rCrRNATaCHSG-01 and rCrRNATaCHSG-02. Additional sequences of trans-activated crRNA are included in the design and named rTracrRNA. In order to further facilitate efficient guide RNA processing, the design includes multiple copies of self-cleaving hammerhead RNA derived from tobacco etch virus, designated as rHH, and hammerhead elements with matching sequences specific to each guide RNA and crRNA complex, designated as rHHTaCHSG-01 and rHHTaCHSG-02, respectively. Multiple copies of self-cleaving ribozymes derived from hepatitis delta virus are designated as rHDV. These elements are connected together in the following order to form a continuous sequence: rHH, rTracrRNA, HDV, rHH, rHH, rTracrRNA, rHV, rHH, rHHTaCHSG-01, rCrRNATaCHSG-01, rHDV, rHHTaCHSG-02, rCrRNATaCHSG-02, rHDV. Transcription of this guide RNA and processing element complex is terminated by the nopaline synthase terminator derived from Agrobacterium tumefaciens.

[0125] Phosphomannose isomerase (PMI; see U.S. Patent No. 5,767,378) was selected as a selectable marker for stable plant transformation. PMI transcription is driven by a ubiquitin promoter, and transcription is terminated by a ubiquitin terminator, both of which are derived from maize. Cas9 expression cassettes, guide RNA expression and processing cassettes, and PMI selectable marker cassettes are connected to a standard binary vector containing left and right border elements and backbone elements derived from Agrobacterium tumefaciens. The backbone elements include a copy of an aminoglycoside 3' adenylate transferase gene derived from Escherichia coli, which confers resistance to spectinomycin and streptomycin for the maintenance of vectors in Escherichia coli and Agrobacterium. The backbone also includes a bacterial expression cassette of two Agrobacterium tumefaciens genes driven by the VirG promoter, including VirG transcriptional activation virulence factors and RepA plasmid partitioning proteins. The backbone includes the oVS1 origin of replication element derived from Pseudomonas species (for plasmid replication in Agrobacterium tumefaciens) and the ColE1 origin of replication element (for plasmid replication in Escherichia coli). All of the above elements were assembled into a single binary vector in September and October 2019, verified by sequencing, and designated as Syngenta construct 25206.

[0126] Syngenta construct 25206 was stably transformed into Fielder wheat varieties using Agrobacterium-mediated plant transformation. Thirty-four T0 wheat plants were produced by transformation experiments. All T0 plants were self-pollinated to produce T1 seeds. T1 seed batches from low copy number T0 parents were planted and screened for editing activity at the target sites of TraesCS1A02G160300, TraesCS1B02G176300, and TraesCS1D02G157500. Screening was completed by using PCR to amplify the regions around the rCrRNATaCHSG-01 and rCrRNATaCHSG-02 cleavage sites of each gene and sequencing the resulting PCR products. The primers used for PCR amplification are shown in Table 5. The PCR conditions for all primers were 66°C annealing, 60 seconds extension time, and 40 amplification cycles. This PCR screen identified edits at the rCrRNATaCHSG-01 cleavage site in TraesCS1A02G160300 and TraesCS1B02G176300 in separate T1 plants. All genomic edits in this study were generated at the rCrRNATaCHSG-01 cleavage site, and no evidence of edits was found at the rCrRNATaCHSG-02 cleavage site throughout the study. Pollen was collected from a T1 plant containing an edit at the TraesCS1B02G176300 target and used to pollinate two T1 plants containing edits at the TraesCS1A02G160300 target. T2 seeds generated from crosses between sister T1 plants were planted and screened for edits at all targets using the same method. This screen confirmed that the edits at TraesCS1A02G160300 and TraesCS1B02G176300 had merged into one T2 plant, and that a single T2 sister plant had both an edit at the TraesCS1A02G160300 target and a new edit at the rCrRNATaCHSG-01 cleavage site within TraesCS1D02G157500. T2 plants with genome edits at both the TraesCS1A02G160300 and TraesCS1B02G176300 editing targets were self-pollinated for phenotypic analysis in the T3 generation. T3 progeny of this plant were selected for individuals homozygous for genome edits at TraesCS1A02G160300 and TraesCS1B02G176300, grown to maturity, and observed for male fertility. No evidence of male sterility or altered plant phenotype was observed in individuals homozygous for genome editing at TraesCS1A02G160300 and TraesCS1B02G176300. The functional wild-type allele of TraesCS1D02G157500 was subsequently demonstrated to be sufficient to maintain full male fertility.

[0127] Table 5. PCR primers and conditions.

[0128]

[0129] Pollen from T2 segregants with genome editing to TraesCS1B02G176300 is used to pollinate individuals with editing to TraesCS1A02G160300 and TraesCS1D02G157500 targets. The T3 progeny of this hybridization is planted, screened by PCR and sequencing, and individuals with genome editing at the rCrRNATaCHSG-01 cleavage site in all three targets are selected. The T3 progeny selected is allowed to self-pollinate naturally, to produce the T4 generation separated for individuals with homozygous editing at all three targets. Plant T4 generations and screen by PCR product sequencing. Due to the poor transmission rate of pollen in the case of editing all three targets, separation abnormalities (Table 6) are observed in T4 generations. A T4 plant is identified as having homozygous loss-of-function editing (Table 6) for all three target sites. Select five of the six possible target copies (six are the total copy numbers from three diploid A, B and D wheat genomes) with the allele of knocking out editing to carry out phenotypic analysis and be used as pollen donors.Other T4 plants include individuals with a functional copy of TraesCS1B02G176300 and individuals with a functional copy of TraesCS1D02G157500.The allele of knocking out editing at the TraesCS1A02G160300 target is fixed as homozygote by T4 generations in this strain.All editors from T4 generations individual through sequencing are as described in Table 6.

[0130] Table 6: Comparison of expected and observed segregation rates for three editing targets in T4 seedlings. All 192 seedlings screened were mixed self-pollination progenies of individual T3 plants that were homozygous for the TraesCS1A02G160300 edited allele, heterozygous for the TraesCS1B02G176300 and TraesCS1D02G157500 edited alleles. Sequencing confirmed that the edited alleles in all 192 T4 progenies screened were as shown in Table 6.

[0131]

[0132] NA = Not Applicable

[0133] Table 7: Edited alleles generated at the anther-specific chalcone synthase target in the common wheat variety Fielder. All deletions occurred in the fourth codon of exon 1 of the coding sequence and resulted in a frameshift affecting all downstream codons.

[0134]

[0135] 3. Perform phenotypic analysis on homozygous edited wheat plants.

[0136] The phenotype of a single T4 plant homozygous for the knockout edited allele at all six copies of the editing target during flowering is similar to the phenotype commonly seen in timopheevii CMS wheat. See, e.g., J. Song, A chimeric gene (orf256) is expressed as protein only in cytoplasmic male-sterile lines of wheat, Plant Mol. Biol. 26: 535-539 (1994). During flowering, the outer florets on each spike extend yellow anthers. It was observed that these anthers did not crack along the lodicules and did not shed pollen as observed in fertile wheat. A few days after the anther extension began, secondary dehiscence was observed, during which all individual florets opened to allow external pollen to contact the stigma. In contrast to Timofewe CMS wheat, all directly observed florets had the expected three fully formed anthers per floret, and the anthers themselves were similar in size to male fertile Fielder plants. No cases of anther dysplasia or deformities, common in Timofewe CMS wheat, were recorded. Six ears showing secondary dehiscence were pollinated using pollen from sister segregating plants with a complete copy of anther-specific chalcone synthase (TraesCS1B02G176300 or TraesCS1D02G157500). All individuals with a complete copy of anther-specific chalcone synthase exhibited a normal phenotype throughout their life cycle and set seeds normally at harvest; in addition, individuals containing a copy of a functional anther-specific chalcone synthase were observed to shed normal amounts of fertile yellow pollen during pollen collection. No differences were observed between individuals with a single intact copy of TraesCS1B02G176300 and individuals with a single intact copy of TraesCS1D02G157500.

[0137] Table 8: Seed set outcomes of self- and cross-pollination between sisters in a segregating population of common wheat for genome-edited loss-of-function alleles at TraesCS1A02G160300, TraesCS1B02G176300, and TraesCS1D02G157500.

[0138]

[0139] NA = Not Applicable; for cases where all ears from a single plant were threshed as a single mixed population, no count of harvested ears was recorded.

[0140] Uppercase letters = intact WT allele, lowercase letters = loss-of-function edited allele

[0141] 4. Wheat fertility restoration

[0142] It is speculated that applying exogenous flavonoids to developing wheat anthers will restore male fertility in complete knockout plants (aabbdd genotype). Male fertility restoration treatment can be applied to a single spike on a plant that is homozygous for the alleles edited through knockout at all three targets. The treatment method can be composed of the following: quercetin applied exogenously at one or more microspore development stages. Quercetin can be applied exogenously as a dry powder, dissolved in a solvent or solution, or suspended in a formulation. Liquid solutions and suspensions of quercetin dihydrate may include inert ingredients that enhance the permeability of quercetin into plant tissues. Liquid and dry powder treatments can be applied to the stigma in a single floret of a wheat spike, the entire wheat spike, the entire tiller of the spike origin, or the entire plant by soaking or watering.

[0143] 5. Corn transformation.

[0144] Based on the genome sequence of the NP2222v5 ​​strain, six guide RNA sequences (gRNAs) targeting the second exon in ZmFDF1 and ZmFDF2 for gene editing were designed. Syngenta designed and validated these gRNAs before vector design and assembly from September to November 2021. These gRNAs are designed by identifying all possible PAM recognition sites in the gene coding sequence (TTTN for LbCas12a), and then selecting guides that are closest to the target gene transcription start site, least likely to make any off-target edits (i.e., the least potential sequence matches elsewhere in the genome (especially in other chalcone synthase genes)), and likely to disrupt the conserved catalytic triad in exon 2, which is known to be essential for the function of chalcone synthases in several species (MBAustin & J.P. Noel, The chalconesynthase superfamily of type III polyketide synthases, Nat. Prod. Rep. [Natural Product Reports] 20: 79-110 (2003)). The list is then further prioritized by screening for optimal gRNA parameters, including excluding TTTT PAM sequences, excluding gRNAs that are not predicted to produce the desired hairpin structure, and gRNAs with high GC content (> 50%) or long T segments in the sequence. By using the polyethylene glycol transfection scheme, the ribonucleoprotein (RNP) complex (i.e., a synthetic gRNA (purchased from IDT) pre-compounded with purified LbCas12a protein) was delivered to maize leaf protoplasts, and the editing efficiency of 36 potential gRNAs was tested in a transient protoplast assay. After RNP delivery, protoplast DNA was extracted and the target editing region was amplified therefrom by polymerase chain reaction (PCR). Subsequently, the product of the PCR reaction was analyzed for editing evidence using T7E1-based enzyme assays and the next generation (Illumina sequencing) to confirm the presence and frequency of editing at the target site, and to rank gRNA according to its efficiency. According to these data, the six gRNAs (Table 9) that were finally entered into the vector for stable transformation were selected: two targeting ZmFDF1, two targeting ZmFDF2, and two simultaneously targeting ZmFDF1 and ZmFDF2.

[0145] Table 9: Guide RNAs designed to target ZmFDF1 and ZmFDF2.

[0146]

[0147] Table 10: Primers designed to amplify edits made using the guide RNAs described above.

[0148]

[0149] Syngenta designed a series of three binary vectors between January and March 2022 that can produce loss-of-function alleles at ZmFDF1, ZmFDF2, or ZmFDF1 and ZmFDF2 genome editing targets. In order to produce double-stranded cuts in targeted genes, a maize codon-optimized version of the Cas12a enzyme derived from Lachnospiraceae bacteria was selected. This version of Cas12a contains a C965S amino acid substitution, an intron derived from the Arabidopsis BAF60 gene (encoding a protein belonging to the chromodomain remodeling complex), and a nuclear localization sequence derived from simian virus 40 at the N-terminus and C-terminus of the amino acid sequence. Cas12a transcription is driven by the constitutive sugarcane ubiquitin 4 promoter derived from sugarcane (Saccharum officinarum). Transcription of Cas12a is terminated by the nopaline synthase terminator derived from Agrobacterium tumefaciens. In all vectors, guide RNA transcription was driven by the rice U3 promoter for pol III-dependent transcription of noncoding RNAs. For efficient guide RNA processing, all six guides were individually linked to a transactivating crRNA sequence derived from Streptococcus pyogenes to form a combined guide RNA and crRNA sequence.

[0150] For all 3 vectors, phosphomannose isomerase (PMI) was selected as the selectable marker for stable plant transformation. PMI transcription is driven by ubiquitin promoter, and transcription is terminated by ubiquitin terminator, both of which are derived from maize. Cas9 expression cassette, guide RNA expression and processing cassette and PMI selectable marker cassette are connected to standard binary vector, which contains left and right boundary elements and main chain elements derived from Agrobacterium tumefaciens. Main chain elements include a copy of aminoglycoside 3' adenylate transferase gene derived from Escherichia coli, which confers resistance to spectinomycin and streptomycin for the maintenance of vectors in Escherichia coli and Agrobacterium. Main chain also includes bacterial expression cassettes of two Agrobacterium tumefaciens genes driven by VirG promoter, including VirG transcriptional activation virulence factor and RepA plasmid distribution protein. Main chain includes oVS1 replication origin element (for plasmid replication in Agrobacterium tumefaciens) and ColE1 replication origin element (for plasmid replication in Escherichia coli) derived from Pseudomonas species. All the above elements were assembled into three binary vectors in April 2022, verified by sequencing, and designated as Syngenta constructs 27726, 27738, and 27769.

[0151] Constructs 27726, 27738 and 27769 will be transformed into maize inbred strain NP2222 by Agrobacterium-mediated transformation respectively.The transformant that is found to have the selectable marker of stable integration will be regenerated as seedling, and grow to maturity under greenhouse conditions.The existence of editing at the target will be confirmed by the following: first, the Taqman determination of the editor at the cleavage site intended to detect each gRNA, and the second determination intended to detect the wild type (uncut allele). In the T0 plant individuals where Taqman detects the existence of the cutting allele, the subsequent analysis is the next generation sequencing (Illumina), to characterize the definite insertion and deletion of the existence and select the T0 plant with the highest possibility of chalcone synthase loss of function.In this analysis, any insertion and deletion that causes the shift of the reading frame in the exon is interpreted as a loss-of-function allele.

[0152] 6. Perform phenotypic analysis on the edited maize plants.

[0153] T0 maize plants with different edits were observed for phenotypes associated with the sequenced loss-of-function alleles in ZmFDF1 and ZmFDF2. In general, plant male reproductive phenotypes were associated with the total number of loss-of-function alleles of ZmFDF1 and ZmFDF2, with the maximum possible total number of loss-of-function alleles being four. No abnormal phenotypes were observed in T0 plants with one or two loss-of-function alleles. It is noteworthy that complete loss of function at one of the editing targets due to frameshift alleles in both copies of ZmFDF1 or ZmFDF2 does not lead to deviations from the normal phenotype as long as there are no loss-of-function alleles in both copies of the other corresponding gene. This suggests that ZmFDF1 and ZmFDF2 are functional duplications. Some T0 plants exhibited abnormal anthers, which were reduced in size after being extended compared to most plants. These abnormally small anthers shed less pollen than most plants. It is observed that abnormal pollen falls off for about 10 microliters of pollen per tassel per day, while maize is typically 500-1000 microliters per tassel per day. However, this small amount of pollen is the standard yellow color commonly observed in maize. T0 plants with abnormal pollen and anthers are those with frameshift insertions and deletions, which cause three loss-of-function alleles in four possible gene editing targets. T0 plants with abnormal anthers that fall off a small amount of pollen are artificially self-pollinated and self-fertilized. T0 individuals with four loss-of-function alleles (frameshift insertions and deletions of two copies of ZmFDF1 and two copies of ZmFDF2) are not present in the T0 generation. Common phenotypes associated with c2 / whp1 maize (colorless aleurone layer, colorless tassel silk, white pollen, premature cessation of pollen tube growth) are not observed in this population.

[0154] Table 11: Observed anther and pollen phenotypes of FDF T0 maize plants associated with genotype. All loss-of-function alleles were indels that resulted in a reading frame shift in exon 2 of ZmFDF1 and / or ZmFDF2. There were no T0 individuals in the T0 generation that had four loss-of-function alleles (frameshift indels of two copies of ZmFDF1 and two copies of ZmFDF2).

[0155]

[0156]

[0157] ND = No Data

[0158] Planting T1 seeds obtained by self-pollination T0 plants, to further characterize the association between loss-of-function genotype and plant reproductive phenotype. Genotyping selected homozygous progeny for the loss-of-function allele of ZmFDF1 or ZmFDF2, ZmFDF1 or ZmFDF2 any one completely lost function and had a functional copy of another corresponding gene progeny and homozygous progeny for the loss-of-function allele of ZmFDF1 and ZmFDF2. As a part of pollen viability screening, T1 plants are self-pollinated to produce T2 seeds. T2 seeds are also planted in greenhouses, and the same genotype group is selected for phenotypic analysis. In the T1 and T2 plants screened, similar associations between edited allele genotypes and plant phenotypes are observed. Due to the complete loss of function at one of the editing targets caused by the frameshift allele in two copies of ZmFDF1 or ZmFDF2, as long as two copies of another corresponding gene do not have loss-of-function editing alleles, it will not cause deviations from normal phenotypes. The T1 and T2 plants with three kinds of four kinds of possible display loss-of-function frameshift alleles continue to show abnormal pollen and anther phenotype. In these individuals, anther is generally smaller than the standard anther of strain, and the amount of pollen that these smaller anthers fall off is still less. It is observed that the abnormal pollen shedding range is about 10 microliters per tassel per day to the pollen between 100 microliters per tassel per day, while maize is typically 500-1000 microliters per tassel per day by comparison. In the whole range of the amount of pollen falling off every day, pollen is the standard yellow color observed in maize. The pollen samples of all tested T1 and T2 plants are all self-fertilized.

[0159] It was observed that T1 and T2 plants with homozygous frameshift indels that resulted in complete loss of function of ZmFDF1 and ZmFDF2 displayed a range of phenotypes. Selected progeny displayed reduced anther size and pollen shedding phenotypes similar to sisters with one intact copy of ZmFDF1 or ZmFDF2. Pollen from these progeny was self-fertilizing. Other sister progeny showed loss of normal anther development, with anthers not fully mature, unable to extend from the florets, and not containing any viable pollen grains. These individuals were all functionally male sterile due to the complete absence of pollen. Further selection for specific indels in ZmFDF1 and ZmFDF2 will be needed to demonstrate whether the complete loss-of-function phenotype is characterized by a spectrum of anther and pollen phenotypes, or is a consistent antherless, pollenless phenotype. Selected indels that result in frameshifts in coding sequences can result in complete loss of function of the gene target, while other indels that result in frameshifts may not completely eliminate function based on the exact location in the target coding sequence. Further phenotypic analysis may reveal that anther and pollen phenotypic profiles are influenced by environmental factors and are independent of specific indels in ZmFDF1 and ZmFDF2.

[0160] It is speculated that applying exogenous flavonoids to the developing anthers of antherless, pollenless genotypes will restore male fertility. Male fertility restoration treatments can be applied to the developing tassel by delivering the treatment down the impeller, injecting the treatment into the cavities around the developing tassel through the impeller, or injecting the developing tassel directly into the developing tassel when the developing tassel grows out of the impeller. Male fertility restoration treatments can also be applied as foliar treatments of the impeller, the entire leaf canopy, or as irrigation. Treatment methods can consist of quercetin applied exogenously at one or more microspore development stages. Quercetin can be applied exogenously as a dry powder, dissolved in a solvent or solution, or suspended in a formulation. Liquid solutions and suspensions of quercetin dihydrate can contain inert ingredients that enhance the permeability of quercetin into plant tissues.

[0161] Table 12: Observed anther and pollen phenotypes of FDF T1 and T2 maize plants in relation to genotype. All loss-of-function alleles were indels that resulted in a reading frame shift in exon 2 of ZmFDF1 and / or ZmFDF2.

[0162]

[0163] 7. Construct information.

[0164] Table 13: Components found in construct 25206 (SEQ ID NO: 12).

[0165]

[0166]

[0167] Table 14: Components found in construct 27726 (SEQ ID NO: 16).

[0168]

[0169]

[0170] Table 15: Components found in construct 27738 (SEQ ID NO: 17).

[0171]

[0172]

[0173] Table 16: Components found in construct 27769 (SEQ ID NO: 18).

[0174]

[0175]

Claims

1. A method for seed production, the method comprising: a. obtaining at least one first plant, wherein the first plant is a flavonoid-dependent fertility plant comprising a mutation in a flavonoid-dependent fertility ("FDF") gene; b. obtaining at least one second plant, wherein said second plant is male fertile; c. allowing the at least one second plant to pollinate the at least one first plant; as well as d. Obtain its offspring seeds.

2. The method of claim 1, wherein the first plant and the second plant are monocots.

3. The method of claim 1, wherein the monocot is selected from the group consisting of wheat, maize and rice.

4. A flavonoid-dependent fertility ("FDF") plant comprising a mutation in a FDF gene.

5. The FDF plant of claim 4, wherein the mutation in the FDF gene is a knockout mutation.

6. The FDF plant of claim 5, wherein the plant is homozygous for a knockout mutation in the FDF gene.

7. The FDF plant of claim 3, wherein the FDF plant is a wheat plant.

8. The FDF plant of claim 6, wherein the FDF gene is selected from the group consisting of TaFDF1A (SEQ ID NO: 1), TaFDF1B (SEQ ID NO: 2), TaFDF1D (SEQ ID NO: 3), and combinations thereof.

9. The FDF plant of claim 3, wherein the FDF plant is a maize plant.

10. The FDF plant of claim 9, wherein the FDF gene is ZmFDF1 (SEQ ID NO: 4), ZmFDF2 (SEQ ID NO: 5), and combinations thereof.

11. The FDF plant of claim 10, wherein the FDF plant further comprises a mutation in C2 (SEQ ID NO: 26) and / or WHP (SEQ ID NO: 28).

12. The FDF plant of claim 3, wherein the FDF plant is a rice plant.

13. The FDF plant of claim 12, wherein the FDF gene is OsFDF (SEQ ID NO: 57).

14. A method for propagating FDF plants, the method comprising: a. obtaining at least one plant, wherein the plant is a flavonoid-dependent fertility ("FDF") plant comprising a mutation in the FDF gene; b. applying a composition comprising flavonoids to the plant of step a.; c. allow self-pollination to occur; and d. Obtain its offspring seeds.

15. The method of claim 14, wherein the flavonoid is quercetin.

16. The method of claim 15, wherein the composition comprising quercetin is a liquid solution.

17. The method of claim 16, wherein the liquid solution comprises quercetin mixed in water, propylene glycol or other solvent or solution.

18. The method of claim 17, wherein the liquid solution comprises quercetin suspended in a solvent or solution at a concentration of 1 mg / L to 100 mg / L, 2.5 mg / L to 50 mg / L, or about 5 mg / L.

19. The method of claim 17, wherein the liquid solution comprising quercetin is saturated.

20. The method of claim 15, wherein the composition comprising quercetin is a powder.

21. The method of claim 20, wherein the powder comprises a mixture of quercetin and a carrier compound.

22. The method of claim 21, wherein the ratio of the mixture of quercetin and carrier compound is between 1000:1 and 1:1000.

23. The method of claim 21, wherein the carrier compound is selected from the group consisting of crystalline silica, talc, metal powders, and mica minerals.

24. The method of claim 14, wherein the flavonoid-containing composition is applied to the female organs of a flower on at least one plant designated as a female plant.

25. The method of claim 24, wherein the composition is applied by a method selected from the group consisting of painting, spraying, misting and root irrigation.

26. The method of claim 14, wherein the FDF gene is a wheat gene selected from the group consisting of SEQ ID NO: 1-3 and combinations thereof.

27. The method of claim 14, wherein the FDF gene is a maize gene selected from the group consisting of SEQ ID NOs: 4-5 and combinations thereof.

28. The method of claim 14, wherein the FGF gene is a rice gene comprising SEQ ID NO:

57.

29. A plant produced by the method of claim 14.

30. A method for hybrid seed production, the method comprising: a. obtaining at least one inbred female FDF plant, wherein the inbred female FDF plant comprises a mutation in the FDF gene; b. obtaining pollen from at least one inbred male plant, wherein the inbred male plant produces fertile pollen; c. pollinating the inbred female FDF plants with fertile pollen from the inbred male plants; and d. Obtaining its hybrid offspring.

31. The method of claim 30, wherein the inbred female FDF plant is a maize plant, a wheat plant, or a rice plant.

32. The method of claim 31, wherein the FDF wheat plant comprises a mutation in a gene selected from the group consisting of SEQ ID NOs: 1-3 and combinations thereof.

33. The method of claim 31, wherein the FDF maize plant comprises a mutation in a gene selected from the group consisting of SEQ ID NOs: 4-5 and combinations thereof.

34. The method of claim 31, wherein the FDF rice plant comprises the mutation in SEQ ID NO:

57.

35. A plant produced by the method of claim 30.

36. A method of restoring fertility in a FDF plant comprising a mutation in an FDF gene, the method comprising applying to the FDF plant a composition comprising a flavonoid.

37. The method of claim 36, wherein the FDF plant is a maize plant, a wheat plant, or a rice plant.

38. The method of claim 37, wherein the FDF wheat plant comprises a mutation in a gene selected from the group consisting of SEQ ID NOs: 1-3 and combinations thereof.

39. The method of claim 37, wherein the FDF maize plant comprises a mutation in a gene selected from the group consisting of SEQ ID NOs: 4-5 and combinations thereof.

40. The method of claim 39, wherein the FDF maize plant further comprises a mutation in C2 (SEQ ID NO: 26) and / or WHP (SEQ ID NO: 28).

41. The method of claim 37, wherein the FDF rice plant comprises the mutation in SEQ ID NO:

57.

42. The method of claim 36, wherein the flavonoid comprises quercetin.

43. The method of claim 42, wherein the quercetin is mixed in water, propylene glycol or other solvent or solution.

44. The method of claim 36, wherein the composition comprising a flavonoid further comprises a carrier compound.

45. The method of claim 44, wherein the carrier compound is selected from the group consisting of crystalline silica, talc, metal powders, and mica minerals.

46. ​​The method of claim 36, wherein the composition comprising flavonoids is applied to anthers, filaments, stigmas, florets, spikes, leaf whorls, leaf crowns, or roots.

47. The method of claim 46, wherein the composition is applied by a method selected from the group consisting of painting, spraying, misting and root irrigation.

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