Methods for regulating self-incompatibility for generating hybrids

Abstract

Disclosed are methods of producing hybrid plants. The methods may include introducing a first self-incompatible plant and a second self-incompatible plant and crossing the first self¬ incompatible plant with the second self-incompatible plant to produce a self-compatible hybrid plant. The methods may further include introducing a first plant including a first set of genes and a first cassette and introducing a second plant including a second set of genes and a second cassette, such that the first set of genes is regulated by the second cassette, and the second set of genes is regulated by the first cassette. Also disclosed are methods of propagating plants. Methods of propagating plants may include introducing a self- incompatible plant including a gene encoding a self-incompatibility trait and may further include introducing a maintainer plant that is near isogenic with the self-incompatible plant.

Description

METHODS FOR REGULATING SELF-INCOMPATIBILITY FOR GENERATINGHYBRIDSREFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of United States provisional application No. 63 / 584,946, filed September 25, 2023, herein incorporated by reference in its entirety.INCORPORATION OF SEQUENCE LISTING

[0002] The sequence listing that is contained in the file named “MONS578WO_ST26.xml,” which is 33.1 kilobytes as measured in Microsoft Windows operating system and was created on September 24, 2024, is filed electronically herewith and incorporated herein by reference.FIELD OF THE INVENTION

[0003] The present disclosure relates to the field of agricultural biotechnology, and more specifically to crop breeding.BACKGROUND OF THE INVENTION

[0004] Breeders are continually developing new methods and systems for generating hybrid plants. These methods commonly require utilization of large areas of agricultural land and associated resources for maintaining the agricultural land. As breeders look to accelerate crop development using less resources, it is critical to develop methods and systems that facilitate more efficient hybrid plant generation.SUMMARY OF THE INVENTION

[0005] The present disclosure provides methods of producing crop plants and crop plants that are useful for making hybrid plants. Crop plants include any plant that is grown for commercial agricultural purposes and include soybean, corn, cotton, canola, sugar cane, rye, barley, oats, sorghum, tomato, sunflower, rice, quinoa, and wheat. The crop plants can be self-incompatible, meaning they cannot self-pollinate. The methods involve using self-incompatible crop plants that upon crossing with other self-incompatible crop plants produce hybrids and, in some embodiments, collecting the seeds from the cross. In some embodiments, at least some of the hybrids produced from the cross are self-compatible. In other embodiments, all of the hybrids produced from the cross are self-compatible. The self-incompatible plants can additionally comprise a counteracting factorthat contributes to the suppression of the self-incompatibility construct. In some embodiments, the first self-incompatible plant also comprises a counteracting factor that can act to suppress the selfincompatibility gene in a second plant. In further embodiments, the counteracting factor is a suppression construct. In some embodiments, the second plant is self-incompatible and further comprises a counteracting factor that specifically suppresses a self-incompatibility gene in the first plant. When a cross is made between these two plants the self-incompatibility in the first generation is suppressed that the resulting plant are self-compatible. In yet other embodiments a first selfincompatible plant includes a self-incompatibility gene located at a first locus and a second selfincompatible plant includes a self-incompatibility gene at a locus and the loci are distinct such that they are not genetically linked. Upon crossing the plants some of the plants in the first generation will not include a self-incompatibility gene. In some embodiments, the first self-incompatible plant, the second self-incompatible plant, or both the first self-incompatible plant and the second selfincompatible plant additionally include a counteracting factor. Some of the self-incompatible plants described herein are inbred, meaning that they are substantially homozygous. Inbreds can also be made by doubling haploid plants using methods known in the art.

[0006] The present disclosure also provides methods of propagating a crop plant that is homozygous for a self-incompatibility trait. These methods include growing a crop plant that is homozygous for a self-incompatibility trait and that further comprises a counteracting factor that is capable of suppressing the self-incompatibility trait and allowing the crop plant to self-pollinate to produce a first generation. The crop plant can be heterozygous or homozygous for the counteracting factor. In some embodiments, the counteracting factor is part of a maintainer locus that functions to allow for self-propagation and selection. The maintainer locus also includes a pollen block preventing homozygosity of the counteracting factor and a seed phenotype marker. The first generation of plants, therefore, include plants with the maintainer locus and plants without the maintainer locus but all plants will carry the self-incompatibility trait.

[0007] The crop plants disclosed herein that are homozygous for a self-incompatibility trait can may further comprise a counteracting factor that is inducible. The plants can be grown and associated with an inducer that in turn causes the self-incompatibility to be suppressed and the selfincompatible plants will self. One of ordinary skill in the art will appreciate that there many methods that can be used to make plants that include recombinant expression cassettes or constructs which include combinations of various nucleic acid sequences that give rise to the phenotype ofself-incompatibility. A particular method includes transforming a crop plant with a recombinant nucleic acid sequence, or construct, that comprises self-incompatibility coding sequences. The construct can encode both of two corresponding (two 5 allele proteins that act together to prevent self-pollination) .S' locus coding sequences, and optionally a counteracting factor. In some instances when only a single insertion occurs during the transformation, a selfing step can be included to produce a homozygote. Transformation can be accomplished using any method know in the art, for instance Agrobacterium mediated, nanoparticle exposure, electroporation, chemical cellpermeation, or bombardment mediated transformation steps can be used. The incorporation of the recombinant nucleic acid sequence into the genome of the crop plant can be mediated by any mechanism known in the art, for example spontaneous insertions may be recovered or a transposase system can be used. Site specific integration methods using nucleases, recombinases, or targeted transposition can also be used and are further described herein. The transgenic crop plant produced by the methods can also include recombinant counteracting factors.

[0008] The disclosure also includes compositions such as self-incompatible crop plants and selfcompatible hybrid progeny thereof. In one example, crop plants comprising a genomically integrated first cassette expressing a first 5 allele protein and a second cassette expressing a corresponding second .S’ allele protein are provided, wherein upon expression of the first .S’ allele protein and the second .S' allele protein the crop plant displays a self-incompatibility trait, and wherein the plant further comprises a counteracting factor that is capable of regulating the selfincompatibility trait. In some embodiments, the counteracting factor is inducible. In some embodiments, the counteracting factor is inactive and the crop plant is self-incompatible. In some embodiments the counteracting factor is active and the crop plant is self-compatible. In some embodiments the counteracting factor that allows for suppression of the self-incompatibility trait can be activated or triggered by applying a chemical to the plant. One of ordinary skill in the art will appreciate that established molecular biology techniques allow for the use of various proteins, ligands, nucleic acid sequences, and combinations thereof to regulate the activity of a selfincompatibility trait. Some gene regulation systems comprise multiple components and may be referred to as multicomponent systems. In some embodiments, the gene regulation system is activated by alcohol, copper sulfate, tetracycline derivatives, methoxyfenozide, salt, benzenesulfonamide herbicide safeners, or combinations thereof. In other embodiments, the multicomponent system is selected from the group consisting of AlcR, ACE1, TetR, GAL4 DNAbinding domain fused to a modified PYR1 , ACE1 fused to a yeast transcription activation domain, HAB1 protein fused to a transcription activation domain, P-AtAT5G52310:A, and any combination thereof. Methods to control the counteracting factor may also include gene regulation systems triggered by environmental inducers such as blue light, temperature, drought stress, and combinations thereof. In yet other embodiments the counteracting factor can be based on a genetic element that suppresses the self-incompatibility trait. The genetic element can be a nucleic acid sequence that is expressed from a promoter, which can be inducible. The nucleic acid sequence can be selected from antisense, co-suppression, viral-suppression, hairpin suppression, stem-loop suppression, RNAi-based approaches, double- stranded RNA, small RNA-based approaches, artificial transcription factor repressors, and deletion or inversion of a DNA sequence by a sitespecific recombinase. Proteins from such systems can be engineered into constructs and transformed into crop plants and used to produce hybrids. Exemplary systems include those associated with Papaveraceae, Brassicaceae, Poaceae, Asteraceae, Convolvulaceae, Betulaceae, Caryophyllaceae, Sterculiaceae, and Polemoniaceae. In particular embodiments, the Papaver proteins PrpS and PrsS can be used. The experimental designs taught herein can be used to characterize the combinations of proteins that give rise to the desired level of self-incompatibility. In yet other embodiments crop plants that comprise at least one of a Prs gene and a Prp gene and are self-incompatible are produced.

[0009] The present disclosure also provides a method of producing self-compatible hybrid crop plants. In some embodiments, the method comprises crossing two self-incompatible plants, a first plant and a second plant, wherein the first self-incompatible plant comprises a first cassette that regulates the self-incompatibility trait of the second plant and wherein the second self-incompatible plant comprises a second cassette that regulates the self-incapability trait of the first plant. Upon crossing the resulting hybrid is self-compatible because the self-incompatibility traits are down regulated or suppressed. Counteracting factors can also be designed to express sequences that give rise to targeted protein degradation of the self-incompatibility proteins or suppress the transcription of the self-incompatibility proteins. Also described herein are pairs of breeding crop plants that include the self-incompatibility loci described and the counteracting factors specific for plants that are in the breeding pair. These breeding pairs, upon crossing, are able to produce self-compatible hybrids due to the reciprocal action of the counteracting factor, suppressing the self-incompatiblegene from the opposite parental plant.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 shows a schematic drawing to produce a self-incompatible plant by combining self-incompatibility components from two parents into a single progeny. A cross is made between a parent that has a female tissue self-incompatibility gene (S allele A) and another parent that has the corresponding male tissue self-incompatibility gene (corresponding S allele for A). The resulting progeny plants from homozygous plant crosses will include self-incompatible plants.

[0011] FIG. 2 shows a graphical representation of a self-incompatible plant that also contains a counteracting factor which is specific for suppressing the self-incompatibility allele found in a second plant. The second plant is also self-incompatible and contains a counteracting factor which is specific for suppressing the self-incompatibility trait found in the first plant. Upon crossing the plants, the progeny will be self-compatible.

[0012] FIG. 3 shows a graphical representation of an inducible expression suppressor system. In this example the ethanol inducible system that uses AlcR and AlcA is shown. In this example, the AlcR protein is expressed from an AlcR cassette and is present in cells expressing a selfincompatibility component, S1A. When ethanol is present, the AlcR protein interacts with ethanol and then can bind a DNA sequence present in the AlcA promoter (represented by a green box) resulting in expression of a counteracting factor. In this example, the counteracting factor is a miRNA (miRNAA) that targets SIAmRNA and thereby suppresses expression of the SIAprotein. Also shown in this illustration is an expression cassette for a miRNA (miRNAB) that targets a complementary SI trait, SIB. The miRNABcassettes functions to suppress the SIBtrait in a hybrid that contains both the construct that is shown and a second construct that encodes the SIBtrait and a counteracting factor that suppresses the SIAtrait.

[0013] FIG. 4 shows a graphical representation of use of the self-incompatibility system in combination with inducible suppression to increase seed production of the self-incompatible line. In this example, a first variety of soybean is shown (left hand side, light blue box) that is homozygous for a transgenic locus that comprises 1) expression cassettes that express a pair of corresponding SI proteins from the Papaver SI self-incompatibility locus (SI, blue box without border that says SI), an inducible counteracting factor that suppresses the SI1trait (purple box), and a counteracting factor that suppresses the SI3trait (green box with black border that says antiS3). A second variety of soybean is shown (light green box in upper left) that is homozygous for atransgenic locus that comprises 1) expression cassettes that express a pair of corresponding SI proteins from the Papaver S3 self-incompatibility locus (green box without border that says S3), an inducible counteracting factor that suppresses the SI1trait (purple box), and a counteracting factor that suppresses the SI1trait (light blue box with black border that says antiS 1). The spray bottle illustrates that the two varieties can be propagated and increased by growing the variety in the presence of the inducer chemical that suppresses the SI trait to allow the plants to self-pollinate and produce seeds. The light blue circle with an “X” in it indicates that crosses between the two varieties can be performed to produce hybrid seeds. In the hybrid plants, the counteracting factor, antiS3, suppresses the SI3 trait and the counteracting factor, antiS 1, suppresses the Sil trait. Because both the Sil and SI3 traits are suppressed, the hybrids are self-fertile.

[0014] FIG. 5 shows a graphical representation of the pollen viability rates of Ro single copy SI or S3 soybean transgenic plants compared to non-transgenic control plants of the same genetic background. Pollen viability for the non-transgenic plants averaged about 80% in this assay. Viable pollen was produced from plants containing the various constructs.

[0015] FIG. 6 shows images from an in vivo pollination assay to evaluate pollen tube growth.

[0016] FIG. 7 shows a graphical representation of the proportion of SI or S3 homozygotes in Ri progeny from 9 constructs. For each construct, approximately 40 Ri seeds from 5 or 6 individual plants containing independent, single-copy transgene insertions events were genotyped to determine the copy number of the transgene. Each dot in this graph represents the individual events. The green line represents the number of homozygous seeds expected if the transgene had no effect on male transmission. For events from 4 of the constructs (constructs 1, 2, 3, and 4) very few or no homozygous seeds were recovered as expected if the transgene prevented successful fertilization by pollen containing the transgene.

[0017] FIG. 8 shows a graphical representation of the genotype proportion ratios observed for 5 or 6 different soybean transgenic events per construct. The same genotype data that was used to create FIG. 7 was used to create FIG. 8. In a case where the transgene has no effect on male transmission, the expected ratios are 1 (homozygous for transgene) ; 2 (hemizygous for the transgene) : 1 (no transgene) whereas if fertilization of eggs was prevented in pollen that contain the same transgene, the expected ratios are 1 (hemizygous) : 1 (no transgene). Events from 4 of the constructs had segregation ratios of 1:1. Other events from those constructs had nearly 1:1 ratios with very few offspring that have 2 copies of the transgene (homozygous).

[0018] FIG. 9 shows a images of seed pods from control and homozygous plants and progeny plants obtained from crossing the homozygous plants. Images from a control plant that is the same soybean variety as the transgenic plants are shown in the first box labeled “Control.” Developing pods of various sizes are shown. In the box labeled “SI or S3 homo (self-incompatible),” images of flowers after plants are allowed to self-pollinate are shown. Tiny pods that are not developing can be seen. This result shows that plants that are homozygous for either the SI or S3 transgenic event are self-incompatible. In the box labeled “Crosses between S 1 and S3 homo plants,” a flower is shown that was manually pollinated with pollen from a reciprocal transgenic event (i.e., S3 pollen onto a SI flower in box 1 and SI pollen on to a S3 flower in box 2). The pod is clearly developing indicating that a successful fertilization has occurred.BRIEF DESCRIPTION OF THE SEQUENCES

[0019] SEQ ID NO: 1 is a 417-nucleotide sequence representing the coding region of the Papaver rhoeas PrsS3 gene.

[0020] SEQ ID NO:2 is a 576-nucleotide sequence representing the coding region of the Papaver rhoeas PrpS3 gene.

[0021] SEQ ID NO:3 is a 420-nucleotide sequence representing the coding region of the Papaver rhoeas PrsSl gene.

[0022] SEQ ID NO:4 is a 555-nucleotide sequence representing the coding region of the Papaver rhoeas PrpSl gene.

[0023] SEQ ID NO:5 is the promoter sequence from the tobacco NTP303 gene.

[0024] SEQ ID NO:6 is the promoter sequence from the Arabidopsis SLR1 gene.

[0025] SEQ ID NO:7 is the promoter sequence from the soybean CHIA1 gene.

[0026] SEQ ID NO:8 is the promoter sequence from the tomato LAT25 gene.

[0027] SEQ ID NO:9 is the promoter sequence from the soybean Rsp-1 gene.

[0028] SEQ ID NO:11 is the promoter sequence from the soybean Gm.08G269800 gene.

[0029] SEQ ID NO: 12 is the promoter sequence from the cotton Ghstp gene.

[0030] SEQ ID NO: 13 is the promoter sequence from the cotton peroxidase gene.

[0031] SEQ ID NO: 14 is the promoter sequence from the maize GRMZM2G010868 gene.

[0032] SEQ ID NO: 15 is the promoter sequence from the maize GRMZM2G487322 gene.

[0033] SEQ ID NO: 16 is a 180-nucleotide antisense sequence targeting the PrsS3 gene.

[0034] SEQ ID NO: 17 is a 180-nucleotide antisense sequence targeting the PrsSl gene.DETAILED DESCRIPTION

[0035] The goal of crop breeding is to produce agricultural plants with desirable traits, such as increased grain yield, improved nutrition quality, and enhanced adaptation to environmental changes. One of the ways breeders have produced such plants is by crossing inbred parent varieties derived from diverse gene pools to generate hybrid plants. The superior performance of a hybrid plant compared to either of its parent is a phenomenon known as heterosis or “hybrid vigor.” Although the genetic basis of heterosis is not fully understood, the effect is well document and is widespread among sexual species including crop plants. Some explanations for heterosis suggest that the relatively higher genetic diversity of the hybrid compared to the two inbred parents results in a combination of beneficial traits from each parent that enables the hybrids to outperform either of their inbred parents.

[0036] In order to obtain the benefits of heterosis for crops, hybrid seeds may be produced by controlled cross-pollination, which requires controlled application of pollen from one line to the flowers of a second line to produce the desired Fi seed while also preventing unwanted self- pollination through use of a pollination control system. Manual emasculation, namely the physical removal of either the male floral structure from hermaphrodite flowers or removal of whole male flowers from monoecious plants, is a common method of pollination control in hybrid seed production. This process is time consuming and labor-intensive for most crop species but is especially difficult in soybean (Glycine max). This is largely due to the structure of the flower itself, which is small in size, fragile, and contains both the pistil and the stamens situated in a manner that facilitates the anthers to shed mature pollen directly onto the stigma of the same flower. Further, because pollen is shed shortly before or immediately after the flower opens, self-pollination typically occurs before the flowers arc fully open. As a result, natural outcrossing occurs at rates below 1%. Because manual emasculation and controlled pollination is labor, time, and cost intensive in soybean, producing hybrid seed so that growers can benefit from heterosis in this crop has continued to be a challenge.

[0037] Self-incompatibility and male sterility are two mechanisms that encourage crosspollination. Self-incompatibility refers to the inability of a plant to set seed when self-pollinated, even though it produces viable pollen. It differs from male sterility in that self-incompatible plants produce fertile pollen while male sterile plants do not produce pollen or produce non-viable pollen.Self-incompatibility may be caused by genetic interactions between pistil and pollen that allow stigmas to distinguish between pollen derived from “self’ versus pollen from other plants and to prevent self-pollination of the plant. Self-incompatibility systems promote cross-pollination and genetic diversity.

[0038] Self-incompatibility has several advantages over physical emasculation and male- sterility systems. In addition to the effort and time required to perform emasculation, plants may be damaged by the physical removal of plant parts and thereby reduce the amount of hybrid seed that an emasculated plant may produce. For both emasculation and male- sterility systems of hybrid seed production, space is needed to grow the pollen producing plants and seed is not harvested from the male plants. In contrast, in a self-incompatibly based production system, hybrid seed may be harvested from every plant in a hybrid production field. For crops where insect pollinators are used to move pollen among lines, another benefit of self-incompatibility compared to male-sterility is that all flowers produce pollen and are attractive to insects that seek pollen.

[0039] In a natural system, self-incompatibility is usually controlled by a single locus, the 5 locus, which has many alleles. When male pollen and female pistil share the same .S' locus allele, the pollen is identified as incompatible and is inhibited. Self-incompatibility is present in many different plant species and there are many different genetic mechanisms that cause selfincompatibility. Two categories of self-incompatibility have been identified and are distinguished from one another based on the phenotype of the pollen: sporophytic self-incompatibility and gametophytic self-incompatibility .

[0040] In sporophytic self-incompatibility, the pollen phenotype is determined by the genetic composition of the pollen parent plant and not by the genetic composition of the pollen itself. Thus, although pollen is produced that has either one or the other of the two S locus alleles present in the sporophyte, all of the pollen has the same allelic phenotype and will be incompatible on flowers of plants that have the corresponding 5 locus allele. This form of self-incompatibility was identified in Brassicaceae, Asleraceae. Convolvulaceae, Betulaceae, Caryophyllaceae, Sterculiaceae, and Polemoniaceae.

[0041] In gametophytic self-incompatibility, the genotype of the pollen grain itself determines the phenotype of the pollen. Incompatibility occurs only when the S-haplotype or allele of the haploid pollen is the same as either of the two -haplotypcs expressed in the diploid stigma. Importantly, pollen is rejected on any stigma with a matching S-haplotype. One example of agametophytic self-incompatibility system that is well characterized is the one found in Papaver rhoeas. In the Papaver system, the .S'-locus comprises two tightly linked genes that encode the PrsS and PrpS proteins, which are the female and male .S'-dctcrm inants, respectively. PrsS is a small stigma-expressed secreted protein whereas PrpS is a pollen-expressed transmembrane protein. The self-incompatibility response is characterized as a series of intracellular signaling events triggered by an allele- specific PrpS-PrsS protein interaction that results in rapid inhibition of pollen tube growth and, ultimately, programmed cell death.

[0042] In the plants and methods described herein, .S' locus proteins expressed from a genetic construct are used for conferring and / or regulating self-incompatibility in crop plants. The constructs include three components: a cassette that expresses in pollen a pollen- specific S protein allele; a cassette that expresses in the stigma / pistil the corresponding pistil- specific .S' protein allele; and a counteracting factor which negates the effect or phenotype of a trait. An example of the first component is a cassette that comprises a coding sequence encoding a PrpS protein from a first Papaver S locus driven by a pollen-specific promoter. An example of the second component is a cassette that comprises a coding sequence encoding the corresponding PrsS protein from the same .S' locus allele of Papaver driven by a stigma-specific promoter. The third component is designed to downregulate the expression of the PrsS coding region. In an aspect, the third component is a genetic element that suppresses a PrsS allele that is different from the allele present in the construct. Genetic sequences or components that give rise to suppression of self-incompatibility can be collectively referred to as counteracting factors. Unlike previous methods, which required crosses and more generations to bring both components together, the new approach described in the present disclosure allows for the simultaneous expression of several genes, leading to the rapid generation of self-incompatibility and suppression of self-incompatibility. Furthermore, while the Papaver self-incompatibility system was demonstrated to function in Arabidopsis thaliana, the present disclosure provides self-incompatible soybean plants, whereby the self-incompatibility is conferred by the Papaver self-incompatibility system. This was surprising as there are potentially differences between soybean and Arabidopsis in some programmed cell death processes (Nawade el al., Front Plant Sci. 14:1216082, 2023).I. Self-Incompatibility Control and Counteracting Factors

[0043] As described herein there are multiple methods for regulating expression of a gene in plants using constructs that comprise a suppression element. The suppression function of thesuppression element is not limited to a specific mechanism and is inclusive of, suppressors that are nucleic acid based, amino acid based and combinations thereof. For example, suppressors can be molecules that are antisense, co-suppression, viral-suppression, hairpin suppression, stem-loop suppression, RNAi-based approaches, small RNA-based approaches, artificial transcription factor repressors, and deletion or inversion of a DNA sequence by a site-specific recombinase. Plants are designed which comprise part of a pair or set of constructs comprising suppression elements which, when combined, result in downregulation of a gene by targeting the gene itself or a promoter associated with such gene. The gene may be a transgene or a native gene. The promoter may be heterologous to the downregulated gene. Combinations may occur by crossing such plants. In certain embodiments, the parental phenotype is not present in the progeny. In certain embodiments, the phenotype is related to self-incompatibility. For example, selfincompatible plants may be crossed to produce progeny that comprise suppressed selfincompatibility genes; these progeny can be useful in hybrid seed production.

[0044] As used herein, the terms “suppress,” “suppression,” “inhibit,” “inhibition,” “inhibiting,” “knock-out,” “knock-down,” and “down regulation” refer to a lowering, reduction, or elimination of the expression level of an mRNA and / or protein encoded by a target gene in a plant, plant cell, or plant tissue at one or more stage(s) of plant development, as compared to the expression level of such target mRNA and / or protein in a wild-type or control plant, cell, or tissue at the same stage(s) of plant development. "Silencing," as used herein with respect to the target gene, refers generally to the suppression of levels of mRNA or protein / enzyme expressed by the target gene and / or the level of the enzyme activity or protein functionality. The terms "suppression," "suppressing," and "silencing," used interchangeably herein, include lowering, reducing, declining, decreasing, inhibiting, eliminating, or preventing. " As used herein, “gene suppression” or “gene silencing” is intended to refer to any of the well-known methods for reducing the levels of protein produced as a result of gene transcription to mRNA and subsequent translation of the mRNA. Gene suppression is also intended to mean the reduction of protein expression from a gene or a coding sequence including posttranscriptional gene suppression and transcriptional suppression. Posttranscriptional gene suppression is mediated by the homology between all, or a part of, an mRNA transcribed from a gene or coding sequence targeted for suppression and the corresponding RNA used for suppression and refers to the substantial and measurable reduction of the amount of available mRNA available in the cell for binding by ribosomes.

[0045] Transcriptional suppression is mediated by the presence in the cell of a gene suppression agent, exhibiting substantial sequence identity to a promoter DNA sequence or the complement thereof to effect what is referred to as promoter trans suppression. Gene suppression may be effective against a native plant gene associated with a trait, e.g., to provide plants with reduced levels of a protein encoded by the native gene or with enhanced or reduced levels of an affected metabolite. Gene suppression can also be effective against a transgene that was introduced into a plant and is associated with a trait. Post-transcriptional gene suppression by antisense or sense- oriented RNA to regulate gene expression in plant cells is disclosed in U.S. Pat. Nos. 5,107,065, 5,759,829, 5,283,184, and 5,231,020.

[0046] As used herein, the term “antisense” refers to DNA or RNA sequences that are complementary to a specific DNA or RNA sequence. The term “antisense strand” refers to a nucleic acid strand that is complementary to the “sense” strand. The “sense strand” of a gene or locus is the strand of DNA or RNA that has the same sequence as an RNA molecule transcribed from the gene or locus (with the exception of uracil in RNA and thymine in DNA).

[0047] "Antisense RNA" refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target isolated nucleic acid fragment. The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence. "Antisense inhibition" refers to the production of antisense RNA transcripts capable of suppressing the expression of the target gene or gene product. “Sense" RNA refers to RNA transcript that includes the mRNA and can be translated into protein within a cell or in vitro.

[0048] ' ’Co-suppression" refers to the production of sense RNA transcripts capable of suppressing the expression of the target gene or gene product. Co-suppression constructs in plants have been previously designed by focusing on overexpression of a nucleic acid sequence having homology to a native mRNA, in the sense orientation, which results in the reduction of all RNA having homology to the overexpressed sequence (see, Vaucheret et al., Plant J. 16:651-659, 1998 and Gura, Nature 404:804-808, 2000).

[0049] Any method known in the art for suppression of a target gene may be used to suppress a gene according to embodiments of the present disclosure, including expression of antisense RNAs, double stranded RNAs (dsRNAs) or inverted repeat RNA sequences, or via co-suppression or RNA interference (RNAi) through expression of small interfering RNAs (siRNAs), short hairpin RNAs(shRNAs), trans-acting siRNAs (ta-siRNAs), or micro RNAs (miRNAs). Furthermore, sense and / or antisense RNA molecules may be used that target the coding and / or non-coding genomic sequences or regions within or near the target gene to cause silencing of the gene. Accordingly, any of these methods may be used for the targeted suppression of the gene in a tissue- specific or tissue-preferred manner. See, e.g., U.S. Patent Application Publication Nos. 2009 / 0070898, 2011 / 0296555, and 2011 / 0035839.

[0050] Suppression of a target gene may also occur by targeted promoter inactivation. Promoter inverted repeats are also suitable to suppress the expression of endogenous or transgenic genes. Transcripts from a promoter inverted repeat are known to silence another locus containing the same promoter, accompanied by promoter methylation. Such targeted promoter inactivation is possible by identifying the promoter region of interest linked to the target gene and constructing promoter inverted repeat constructs.

[0051] One of ordinary skill in the art will appreciate that transcription factors (TFs) regulate gene expression and that TFs can be designed to have desired activities. TF-repressors expressed from a transgene can target and suppress a gene of interest.

[0052] Custom transcription repressor proteins may be designed to bind the promoter or 5'UTR sequence of a target gene of interest. When the custom repressor is present, it will bind the gene of interest and block expression. Custom TF-repressors can be created using a DNA binding proteins such as zinc-fingers, TALEs, or CRISPR. Binding to the promo ter / 5'UTR may sterically hinder binding of other transcription factors or impede progress of the transcription complex. Transcription repression domains can be fused to the DNA binding domain to cause repression or enhance repression.

[0053] Expression of the custom TF-repressor by a tissue specific promoter, a cell-type specific promoter, inducible promoter or other promoter with a desired expression pattern. Additionally, expression and activity of the custom TF-repressor can be accomplished by various gene regulation mechanisms to produce a desired expression profile. When the custom TF-repressor is present and active, it will suppress the target gene. Thus, use of custom TF-repressors can allow those skilled in the art to effect suppression of a target gene of interest

[0054] One or more custom TF-repressor genes can be present in one or both parents of a hybrid and a target of each custom TF-repressor can be present in the other parent. In this case, the repression effect will manifest in the hybrid.

[0055] Any TF repressor that functions to down regulate the self-incompatibility can be used. There arc numerous publications describing the design and use of custom TF-rcprcssors in plants. Detailed protocols teach how to select the target binding site for desired suppression and how to engineer the corresponding DNA binding protein. By following the teachings that are publicly available, one of ordinary skill can successfully create a custom TF-repressor for effectively any target gene.

[0056] TF repressors can also be used to target promoters that are designed to incorporate target DNA binding sites for known DNA binding proteins. By doing so, existing DNA binding proteins may be converted into transcription factors. This approach is an older method that was more likely to be used before the invention of several excellent custom DNA binding protein systems. As with ZFs, TALEs or CRISPR, other DNA binding domains may be used as transcription factors in plants. Some DNA binding proteins already have transcription suppression activity whereas others require the fusion of a suppression domain in order to be used to repress gene expression. Examples of DNA binding proteins that have been used as plant transcription factors include Gal4, TetR and LacI, AlcR and ACE1.

[0057] One of ordinary skill in the art will appreciate that inducible suppression of the selfincompatibility can be included in the crop plant. Inducible suppression allows for the creation of seeds from a plant that, in the absence of the inducer, would be self-incompatible. In the presence of the inducer, that plant can self and therefore, inbred lines can be created, and seeds can be increased for the subsequent production of commercial hybrid production.

[0058] Inducible suppressors include for example, DNA binding proteins that are affected by the presence of a ligand. For example, AlcR binds the AlcA sequence only in the presence of ethanol, ACE1 binds its target only in the presence of copper sulfate and TetR binds the TetO sequence only in the absence of the antibiotic doxycycline / tetracycline or derivatives. In this way, gene activity (or suppression) can be made dependent on the presence of a compound. Exemplary systems that can be used in the embodiments described herein include those described in Kocaoglan et al., J Exp Bol. 74(13):3864-3876,2023; Becker el al., Biophys J. 2020 Nov 17;119(10):2045-2054, 2020; Deng et al., Methods Mol Biol. 1937:47-58, 2019.

[0059] One of ordinary skill in the art will also appreciate that site-specific recombinases (SSRs) and their corresponding target sequences can be used to activate and inactivate genes. As described herein the counteracting factor included in a crop plant can be designed so that it selectively targetsa recognition sequence in a plant in a cross. The site-specific recombinase can then target the deletion, insertion, inversion or otherwise inactivate of the suppression allele in the other parental plant. These systems, particularly the CRE / Lox system have been shown to work in many plant species. One of ordinary skill in the art that is familiar with the Cre / Lox systems, the analogous FLP / FRT, or other systems appreciates how to apply these systems to inactivate specific genes.

[0060] SSR target sites may be placed on either side of a portion of DNA such that the target sites are oriented in the same direction. This configuration allows the portion of DNA to be deleted by the action of the SSR. When the SSR is Cre and the target sites are Lox sites, the sequence is said to be flanked by lox sites or "floxed." The FLP recombinase and corresponding FRT target sites may also be used in the same way as may the many other known SSR systems that have been demonstrated in various eukaryotes including plants. The recombinase can also be expressed in a different plant and then introduced through crossing to selectively activate through deletion (e.g., when a construct is designed to have an inactivation domain inserted within two Lox sequences) or inactivate through deletion, insertion, or inversion as further described herein. In the event that the self-incompatibility trait is excised the resulting plant will be able to self-fertilize.

[0061] To affect the inactivation of a floxed gene or a gene flanked by other SSR sites, the recombinase is activated. Upon recombination by the SSR, the gene is deleted. A gene may also comprise a floxed sequence or a sequence flanked by other SSR sites such that when the corresponding SSR acts, the said sequence is deleted thereby inactivating the gene.

[0062] The use of SSRs and corresponding sites in this manner allows gene inactivation to be caused by activation of the corresponding recombinase. Activation may be accomplished by expression of the recombinase but other methods of activation such as protein dimerization, conditional intron splicing, cellular localization, protein stabilization and others have been demonstrated.

[0063] SSRs can be utilized for gene inactivation by making a genetic cross where the SSR is provided to the hybrid progeny from one parent and the gene to be inactivated is provided by the other parent. If the SSR is expressed and active in the zygote, then the target gene is inactivated (i.e., via a deletion or inversion) early in development and will be inactive in all cells of the organism. If inactivation is preferred at a particular developmental stage, the SSR is expressed at that stage. Expression of the SSR can be accomplished with an inducible promoter so that when induction conditions are present, the SSR is expressed and the target gene is inactivated. Methodsof making SSR systems are known in the art (Tian et al., J Biol Chem. 2021 Ian-Iun;296: 100509. doi: 10.1016 / j.jbc.2021.100509. Epub 2021 Mar 4. PMID: 33676891; PMCID: PMC8050033: Lyznik etal., Transgenic Plant Journal 1(1): 1-9, 2007. Ye etal., Plant Cell Rep. 2023 Jan;42(l):45- 55. doi: 10.1007 / s00299-022-02935-l. Epub 2022 Oct 31. PMID: 36316413: Li et al., Plant Physiol. 151(3): 1087-95, 2009).II. Genome Editing in Plants

[0064] The present disclosure provides, in certain embodiments, plants, plant parts, plant cells, and seeds produced through genome modification using site-specific integration or genome editing. Genome editing can be used to make one or more edit(s) or mutation(s) at a desired target site in the genome of a plant, such as to change expression and / or activity of one or more genes, or to integrate an insertion sequence or transgene at a desired location in a plant genome. Any site or locus within the genome of a plant may potentially be chosen for making a genomic edit (or gene edit) or site-directed integration of a transgene, construct, or transcribable DNA sequence.

[0065] As used herein, a “target site” or “a target sequence” for genome editing refers to the location of a polynucleotide sequence within a plant genome that is bound and cleaved by a sitespecific nuclease to introduce a strand break into the nucleic acid backbone of the polynucleotide sequence and / or its complementary DNA strand within the plant genome. A target site may comprise, for example, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 1 , at least 28, at least 29, or at least 30 consecutive nucleotides. A “target site” for an RNA-guided nuclease may comprise the sequence of either complementary strand of a double-stranded nucleic acid (DNA) molecule or chromosome at the target site. A sitespecific nuclease may bind to a target site, such as via a non-coding guide RNA (e.g., without being limiting, a CRISPR RNA (crRNA) or a single-guide RNA (sgRNA) as described further herein). A non-coding guide RNA provided herein may be complementary to a target site (e.g., complementary to either strand of a double-stranded nucleic acid molecule or chromosome at the target site). It will be appreciated that perfect identity or complementarity may not be required for a non-coding guide RNA to bind or hybridize to a target site. It will be appreciated that perfect identity or complementarity may not be required for a non-coding guide RNA to bind or hybridize to a target site. For example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 mismatches (or more) between a target site and a non-coding RNA may be tolerated.A “target site” also refers to the location of a polynucleotide sequence within a plant genome that is bound and cleaved by any other site-specific nuclease that may not be guided by a non-coding RNA molecule, such as a zinc finger nuclease (ZFN), a transcription activator- like effector nuclease (TALEN), a meganuclease, etc., to introduce a DSB or single- stranded nick into the polynucleotide sequence and / or its complementary DNA strand. As used herein, a “target region” or a “targeted region” refers to a polynucleotide sequence or region that is flanked by two or more target sites. Without being limiting, in some embodiments a target region may be subjected to a mutation, deletion, insertion or inversion. As used herein, “flanked” when used to describe a target region of a polynucleotide sequence or molecule, refers to two or more target sites of the polynucleotide sequence or molecule surrounding the target region, with one target site on each side of the target region. According to some embodiments, a site-specific nuclease may be co-delivered with a donor template molecule to serve as a template for making a desired edit, mutation or insertion into the genome at the desired target site through repair of the double strand break (DSB) or nick created by the site-specific nuclease. According to some embodiments, a site-specific nuclease may be codelivered with a DNA molecule comprising a selectable or screenable marker gene.

[0066] A site-specific nuclease provided herein may be selected from the group consisting of a zinc-finger nuclease (ZFN), a TALE-endonuclease (TALEN), a meganuclease, an RNA-guided endonuclease, a recombinase, a transposase, or any combination thereof. See, e.g., Khandagale et al. (Plant Biotechnol Rep 10:327-343, 2016); and Gaj et al. (Trends Biotechnol. 31(7):397-405, 2013). Zinc finger nucleases (ZFN) are synthetic proteins consisting of an engineered zinc finger DNA-binding domain fused to a cleavage domain (or a cleavage half-domain), which may be derived from a restriction endonuclease (e.g., Fokl). The DNA binding domain may be canonical (C2H2) or non-canonical (e.g., C3H or C4). The DNA-binding domain can comprise one or more zinc fingers (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more zinc fingers) depending on the target site but may typically be composed of 3-4 (or more) zine-fingers. Multiple zinc fingers in a DNA-binding domain may be separated by linker sequence(s). ZFNs can be designed to cleave almost any stretch of double-stranded DNA by modification of the zinc finger DNA-binding domain. ZFNs form dimers from monomers composed of a non-specific DNA cleavage domain (e.g., derived from the Fokl nuclease) fused to a DNA-binding domain comprising a zinc finger array engineered to bind a target site DNA sequence. The amino acids at positions -1, +2, +3, and +6 relative to the start of the zinc finger a-helix, which contribute to site-specific binding to the target site, can be changedand customized to fit specific target sequences. The other amino acids may form a consensus backbone to generate ZFNs with different sequence specificities.

[0067] Methods and rules for designing ZFNs for targeting and binding to specific target sequences are known in the art. See, e.g., U.S. Patent App. Pub. Nos. 2005 / 0064474, 2009 / 0117617, and 2012 / 0142062. The FokI nuclease domain may require dimerization to cleave DNA and therefore two ZFNs with their C-terminal regions are needed to bind opposite DNA strands of the cleavage site (separated by 5-7 bp). The ZFN monomer can cut the target site if the two-ZF-binding sites are palindromic. A ZFN, as used herein, is broad and includes a monomeric ZFN that can cleave double stranded DNA without assistance from another ZFN. The term ZFN may also be used to refer to one or both members of a pair of ZFNs that are engineered to work together to cleave DNA at the same site. Because the DNA-binding specificities of zinc finger domains can be re-engineered using one of various methods, customized ZFNs can theoretically be constructed to target nearly any target sequence (e.g., at or near a gene in a plant genome). Publicly available methods for engineering zinc finger domains include Context-dependent Assembly (CoDA), Oligomerized Pool Engineering (OPEN), and Modular Assembly.

[0068] Transcription activator-like effectors (TALEs) can be engineered to bind practically any DNA sequence, such as at or near the genomic locus of a gene in a plant. TALE has a central DNA- binding domain composed of 13-28 repeat monomers of 33-34 amino acids. The amino acids of each monomer are highly conserved, except for hypervariable amino acid residues at positions 12 and 13. The two variable amino acids are called repeat-variable diresidues (RVDs). The amino acid pairs NI, NG, HD, and NN of RVDs preferentially recognize adenine, thymine, cytosine, and guanine / adenine, respectively, and modulation of RVDs can recognize consecutive DNA bases. This simple relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA binding domains by selecting a combination of repeat segments containing the appropriate RVDs.

[0069] TALENs are artificial restriction enzymes generated by fusing the TALE DNA binding domain to a nuclease domain. In some aspects, the nuclease is selected from a group consisting of PvuII, MutH, TevI, FokI, Alwl, Mlyl, Sbfl, Sclal, Sisi, CleDORF, Clo051, and Pept071. When each member of a TALEN pair binds to the DNA sites flanking a target site, the FokI monomers dimerize and cause a double- stranded DNA break at the target site. The term TALEN, as used herein, is broad and includes a monomeric TALEN that can cleave double stranded DNA without assistancefrom another TALEN. The term TALEN also refers to one or both members of a pair of TALENs that work together to cleave DNA at the same site.

[0070] Besides the wild-type FokI cleavage domain, variants of the FokI cleavage domain with mutations have been designed to improve cleavage specificity and cleavage activity. The FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALEN DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites are parameters for achieving high levels of activity. PvuII, MutH, and TevI cleavage domains are useful alternatives to FokI and FokI variants for use with TALEs. PvuII functions as a highly specific cleavage domain when coupled to a TALE (see Yank et al., PLoS One 8:e82539, 2013). MutH is capable of introducing strand-specific nicks in DNA (see Gabsalilow et al., Nucleic Acids Research. 41:e83, 2013). TevI introduces doublestranded breaks in DNA at targeted sites (see Beurdeley et al., Nature Communications 4:1762, 2013).

[0071] The relationship between amino acid sequence and DNA recognition of the TALE binding domain allows for designable proteins. Software programs such as DNAWorks can be used to design TALE constructs. Other methods of designing TALE constructs are known to those of skill in the art. See Doyle et al. (Nucleic Acids Research 4O:W117-122, 2012); Cermak et al. (Nucleic Acids Research 39:e82, 2011); and tale-nt.cac.cornell.edu / about. In another aspect, a TALEN provided herein is capable of generating a targeted DSB.

[0072] A site-specific nuclease may be a meganuclease. Meganucleases, which are commonly identified in microbes, such as the LAGLIDADG family of homing endonucleases, are unique enzymes with high activity and long recognition sequences (> 14 bp) resulting in site-specific digestion of target DNA. Engineered versions of naturally occurring meganucleases typically have extended DNA recognition sequences (for example, 14 to 40 bp). The engineering of meganucleases can be more challenging than ZFNs and TALENs because the DNA recognition and cleavage functions of meganucleases are intertwined in a single domain. Specialized methods of mutagenesis and high-throughput screening have been used to create novel meganuclease variants that recognize unique sequences and possess improved nuclease activity.

[0073] A site-specific nuclease may be an RNA-guided nuclease. According to some embodiments, an RNA-guided endonuclease may be selected from the group consisting of Casl,CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csx l2), CaslO, Csyl, Csy2, Csy3, Cscl, Csc2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, Cpfl, CasX, CasY, and homologs or modified versions of any thereof, as well as Argonaute (non-limiting examples of Argonaute proteins include Thermus thermophilus Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), Natronobacterium gregoryi Argonaute (NgAgo), and homologs or modified versions of any thereof). According to some embodiments, an RNA-guided endonuclease is a Cas9 or Cpfl enzyme. The RNA-guided nuclease may be delivered as a protein with or without a guide RNA, or the guide RNA may be complexed with the RNA-guided nuclease enzyme and delivered as a ribonucleoprotein (RNP).

[0074] For RNA-guided endonucleases, a guide RNA molecule may be further provided to direct the endonuclease to a target site in the genome of the plant via base-pairing or hybridization to cause a DSB or nick at or near the target site. The guide RNA may be transformed or introduced into a plant cell or tissue as a gRNA molecule, or as a recombinant DNA molecule, construct or vector comprising a transcribable DNA sequence encoding the guide RNA operably linked to a promoter. As understood in the art, a guide RNA may comprise, for example, a CRISPR RNA (crRNA), a single-chain guide RNA (sgRNA), or any other RNA molecule that may guide or direct an endonuclease to a specific target site in the genome. A prototypical CRISPR-associated protein, Cas9 from .S', pyogenes, naturally binds two RNAs, a CRISPR RNA (crRNA) guide and a transacting CRISPR RNA (tracrRNA), to assemble a CRISPR ribonucleoprotein (crRNP). A “singlechain guide RNA” (or “sgRNA”) is an RNA molecule comprising a crRNA covalently linked a tracrRNA by a linker sequence, which may be expressed as a single RNA transcript or molecule. The guide RNA comprises a guide or targeting sequence (also referred to herein as a “spacer sequence”) that is identical or complementary to a target site within the plant genome, such as at or near a gene. The guide RNA is typically a non-coding RNA molecule that does not encode a protein. The guide sequence of the guide RNA may be at least 10 nucleotides in length, such as 12-40 nucleotides, 12-30 nucleotides, 12-20 nucleotides, 12-35 nucleotides, 12-30 nucleotides, 15- 30 nucleotides, 17-30 nucleotides, or 17-25 nucleotides in length, or about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides in length. The guide sequence may be at least 95%, at least 96%, at least 97%, at least 99% or 100% identical or complementary to at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19,at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, or more consecutive nucleotides of a DNA sequence at the genomic target site.

[0075] As used herein, with respect to a given sequence, a “complement,” a “complementary sequence,” and a “reverse complement” are used interchangeably. All three terms refer to the inversely complementary sequence of a nucleotide sequence, i.e., to a sequence complementary to a given sequence in reverse order of the nucleotides.

[0076] The introduction of a DSB or nick may also be used to introduce targeted mutations in the genome of a plant. According to this approach, mutations, such as deletions, insertions, inversions, and / or substitutions may be introduced at a target site via imperfect repair of the DSB or nick to produce a knock-out or knock-down of a gene. Such mutations may be generated by imperfect repair of the targeted locus even without the use of a donor template molecule. A “knock-out” of a gene may be achieved by inducing a DSB or nick at or near the endogenous locus of the gene that results in non-expression of the protein or expression of a non-functional protein, whereas a “knockdown” of a gene may be achieved in a similar manner by inducing a DSB or nick at or near the endogenous locus of the gene that is repaired imperfectly at a site that does not affect the coding sequence of the gene in a manner that would eliminate the function of the encoded protein. For example, the site of the DSB or nick within the endogenous locus may be in the upstream or 5’ region of the gene (e.g., a promoter and / or enhancer sequence) to affect or reduce its level of expression.

[0077] Similarly, such targeted knock-out or knock-down mutations of a gene may be generated with a donor template molecule to direct a particular or desired mutation at or near the target site via repair of the DSB or nick. The donor template molecule may comprise a homologous sequence with or without an insertion sequence and comprising one or more mutations, such as one or more deletions, insertions, inversions and / or substitutions, relative to the targeted genomic sequence at or near the site of the DSB or nick. For example, targeted knock-out or knock-down mutations of a gene may be achieved by substituting, inserting, deleting or inverting at least a portion of the gene, such as by introducing a frame shift or premature stop codon into the coding sequence of the gene or disrupting a promoter sequence or the sequence of another non-coding regulatory element of the gene. A deletion of a portion of a gene may also be introduced by generating DSBs or nicks at two target sites and causing a deletion of the intervening target region flanked by the target sites.III. Recombinant DNA Constructs

[0078] Recombinant DNA constructs and vectors are provided comprising one or more polynucleotide sequences encoding self-incompatibility proteins, wherein the coding sequences are operably linked to a tissue-specific promoter. A polynucleotide sequence of a recombinant DNA construct and vector that encodes a self-incompatibility protein may be operably linked to a pollenspecific or pistil-specific promoter. For down regulation of a self-incompatibility phenotype conferred by the self-incompatibility proteins described herein, recombinant DNA constructs and vectors are further provided which, when transformed or stably integrated into the genome of the plant, results in suppression of a target gene in the plant. The target gene may be endogenous or transgenic to the plant.

[0079] As used herein, a “gene” refers to a nucleic acid sequence forming a genetic and functional unit and coding for one or more sequence-related RNA and / or polypeptide molecules. A gene generally contains a coding region operably linked to appropriate regulatory sequences that regulate the expression of a gene product (e.g., a polypeptide or a functional RNA). A gene can have various sequence elements, including, but not limited to, a promoter, an untranslated region(s) (UTR), exons, introns, and other upstream or downstream regulatory sequences. Genes can range in complexity from a single promoter, single open reading frame and a terminator, to highly complex structures including polycistronic elements and the like. A gene may be native to the host organism meaning it has not been altered or moved within the host genome, or a gene can be non-native meaning that a gene can be native to a host organism but yet using recombinant techniques added at a non-native location or duplicated at the same location as the native gene. Non-native genes also include artificial genes that can be artificially added to the genome of the host through, for example, transformation methods or traditional breeding methods in instances where one of the parental plants contains the non-native gene.

[0080] As used herein, “locus” is a chromosomal location or region where a polymorphic nucleic acid, trait determinant, gene, or marker is located. A “locus” can be shared by two homologous chromosomes to refer to their corresponding locus or region. As used herein, “allele” refers to an alternative nucleic acid sequence of a gene or at a particular locus (e.g., a nucleic acid sequence of a gene or locus that is different than other alleles for the same gene or locus). Such an allele can be considered (i) wild-type or (ii) mutant if one or more mutations or edits are present in the nucleic acid sequence of the mutant allele relative to the wild-type allele. A mutant allele for a gene mayhave a reduced or eliminated activity or expression level for the gene relative to the wild-type allele. For diploid organisms such as soybean, a first allele can occur on one chromosome, and a second allele can occur at the same locus on a second homologous chromosome.

[0081] In general, the term "variant" refers to molecules with some differences, generated synthetically or naturally, in their nucleotide or amino acid sequences as compared to a reference (native) polynucleotides or polypeptides, respectively. These differences include substitutions, insertions, deletions or any desired combinations of such changes in a native polynucleotide or amino acid sequence.

[0082] As used herein, the term “expression” refers to the biosynthesis of a gene product, and typically the transcription and / or translation of a nucleotide sequence, such as an endogenous gene, a heterologous gene, a transgene or an RNA and / or protein coding sequence, in a cell, tissue, organ, or organism, such as a plant, plant part or plant cell, tissue or organ.

[0083] The term “recombinant” in reference to a polynucleotide (DNA or RNA) molecule, protein, construct, vector, etc., refers to a polynucleotide or protein molecule or sequence that is man-made and not normally found in nature, and / or is present in a context in which it is not normally found in nature, including a polynucleotide (DNA or RNA) molecule, protein, construct, etc., comprising a combination of two or more polynucleotide or protein sequences that would not naturally occur together in the same manner without human intervention, such as a polynucleotide molecule, protein, construct, etc., comprising at least two polynucleotide or protein sequences that are operably linked but heterologous with respect to each other. For example, the term “recombinant” can refer to any combination of two or more DNA or protein sequences in the same molecule (e.g., a plasmid, construct, vector, chromosome, protein, etc.) where such a combination is man-made and not normally found in nature. As used in this definition, the phrase “not normally found in nature” means not found in nature without human introduction. A recombinant polynucleotide or protein molecule, construct, etc., can comprise polynucleotide or protein sequence(s) that is / are (i) separated from other polynucleotide or protein sequence(s) that exist in proximity to each other in nature, and / or (ii) adjacent to (or contiguous with) other polynucleotide or protein sequence(s) that are not naturally in proximity with each other. Such a recombinant polynucleotide molecule, protein, construct, etc., can also refer to a polynucleotide or protein molecule or sequence that has been genetically engineered and / or constructed outside of a cell. For example, a recombinant DNA molecule can comprise any engineered or man-made plasmid, vector,etc., and can include a linear or circular DNA molecule. Such plasmids, vectors, etc., can contain various maintenance elements including a prokaryotic origin of replication and selectable marker, as well as one or more transgenes or expression cassettes perhaps in addition to a plant selectable marker gene, etc. The term “operably linked” refers to a functional linkage between a promoter or other regulatory element and an associated transcribable DNA sequence or coding sequence of a gene (or transgene), such that the promoter, etc., operates or functions to initiate, assist, affect, cause, and / or promote the transcription and expression of the associated transcribable DNA sequence or coding sequence, at least in certain cell(s), tissue(s), developmental stage(s), and / or condition(s).

[0084] Reference in this application to an “isolated DNA molecule” or an “isolated polynucleotide,” or an equivalent term or phrase, is intended to mean that the DNA molecule or polynucleotide is one that is present alone or in combination with other compositions, but not within its natural environment. For example, nucleic acid elements such as a coding sequence, intron sequence, untranslated leader sequence, promoter sequence, transcriptional termination sequence, and the like, that are naturally found within the DNA of the genome of an organism are not considered to be “isolated” so long as the element is within the genome of the organism and at the location within the genome in which it is naturally found. However, each of these elements, and subparts of these elements, would be “isolated” within the scope of this disclosure so long as the element is not within the genome of the organism and at the location within the genome in which it is naturally found. Similarly, a nucleotide sequence encoding a protein or any naturally occurring variant of that protein would be an isolated nucleotide sequence so long as the nucleotide sequence was not within the DNA of the organism in which the sequence encoding the protein is naturally found. A synthetic nucleotide sequence encoding the amino acid sequence of the naturally occurring protein would be considered to be isolated for the purposes of this disclosure. For the purposes of this disclosure, any transgenic nucleotide sequence, i.e.. the nucleotide sequence of the DNA inserted into the genome of the cells of a plant or bacterium, or present in an extrachromosomal vector, would be considered to be an isolated nucleotide sequence whether it is present within the plasmid or similar structure used to transform the cells, within the genome of the plant or bacterium, or present in detectable amounts in tissues, progeny, biological samples or commodity products derived from the plant or bacterium.

[0085] As commonly understood in the art, the term “promoter” can generally refer to a DNA sequence that contains an RNA polymerase binding site, transcription start site, and / or TATA box and assists or promotes the transcription and expression of an associated transcribable polynucleotide sequence and / or gene (or transgene). A promoter can be synthetically produced, varied, or derived from a known or naturally occurring promoter sequence or other promoter sequence. A promoter can also include a chimeric promoter comprising a combination of two or more heterologous sequences. A promoter of the present disclosure can thus include variants or fragments of promoter sequences that are similar in composition, but not identical to, other promoter sequence(s) known or provided herein. A promoter provided herein, or variant or fragment thereof, may comprise a “minimal promoter” which provides a basal level of transcription and is comprised of a TATA box or equivalent DNA sequence for recognition and binding of the RNA polymerase II complex for initiation of transcription. A promoter can be classified according to a variety of criteria relating to the pattern of expression of an associated coding or transcribable sequence or gene (including a transgene) operably linked to the promoter, such as constitutive, developmental, tissue- specific, inducible, etc. Promoters that drive expression in all or most tissues of the plant are referred to as “constitutive” promoters. Promoters that drive expression during certain periods or stages of development are referred to as “developmental” promoters. Promoters that drive enhanced expression in certain tissues of the plant relative to other plant tissues are referred to as “tissue- enhanced” or “tissue-preferred” promoters. Thus, a “tissue-preferred” promoter causes relatively higher or preferential expression in a specific tissue(s) of the plant, but with lower levels of expression in other tissue(s) of the plant. Promoters that express within a specific tissue(s) of the plant, with little or no expression in other plant tissues, are referred to as “tissue-specific” promoters. An “inducible” promoter is a promoter that initiates transcription in response to an environmental stimulus such as cold, drought or light, or other stimuli, such as wounding or chemical application. A promoter can also be classified in terms of its origin, such as being heterologous, homologous, chimeric, synthetic, etc.

[0086] As used herein, a “plant-expressible promoter” refers to a promoter that can initiate, assist, affect, cause, and / or promote the transcription and expression of its associated transcribable DNA sequence, coding sequence or gene in a plant cell or tissue.

[0087] The term “heterologous” in reference to a promoter or other regulatory sequence in relation to an associated polynucleotide sequence (e.g., a transcribable DNA sequence or codingsequence or gene) is a promoter or regulatory sequence that is not operably linked to such associated polynucleotide sequence in nature without human introduction - c.g., the promoter or regulatory sequence has a different origin relative to the associated polynucleotide sequence and / or the promoter or regulatory sequence is not naturally occurring in a plant species to be transformed with the promoter or regulatory sequence.

[0088] As used herein, in the context of a protein-coding gene, an “exon” refers to a segment of a DNA or RNA molecule containing information coding for a protein or polypeptide sequence.

[0089] As used herein, an “intron” of a gene refers to a segment of a DNA or RNA molecule, which does not contain information coding for a protein or polypeptide, and which is first transcribed into an RNA sequence but then spliced out from a mature RNA molecule.

[0090] As used herein, an “untranslated region (UTR)” of a gene refers to a segment of an RNA molecule or sequence (e.g., a mRNA molecule) expressed from a gene (or transgene) but excluding the exon and intron sequences of the RNA molecule. An “untranslated region (UTR)” also refers to a DNA segment or sequence encoding such a UTR segment of an RNA molecule. An untranslated region can be a 5'-UTR or a 3'-UTR depending on whether it is located at the 5' or 3' end of a DNA or RNA molecule or sequence relative to a coding region of the DNA or RNA molecule or sequence (i.e., upstream (5') or downstream (3') of the exon and intron sequences, respectively).

[0091] As used herein, “upstream” refers to a nucleic acid sequence that is positioned before the 5' end of a linked nucleic acid sequence. As used herein, “downstream” refers to a nucleic acid sequence is positioned after the 3' end of a linked nucleic acid sequence. As used herein, “5'” refers to the start of a coding DNA sequence or the beginning of an RNA molecule. As used herein, “3'” refers to the end of a coding DNA sequence or the end of an RNA molecule. It will be appreciated that an “inversion” refers to reversing the orientation of a given polynucleotide sequence.

[0092] As used herein, a “transcription termination sequence” refers to a nucleic acid sequence containing a signal that triggers the release of a newly synthesized transcript RNA molecule from an RNA polymerase complex and marks the end of transcription of a gene or locus.

[0093] As used herein, a “vector” is a polynucleotide or other molecule that transfers nucleic acids between cells. Vectors are often derived from plasmids, bacteriophages, or viruses and optionally comprise pails which mediate vector maintenance and enable its intended use. A “cloning vector” or “shuttle vector” or “sub cloning vector” contains operably linked parts thatfacilitate subcloning steps (e.g., a multiple cloning site containing multiple restriction endonuclease sites). The term “expression vector” as used herein refers to a vector comprising operably linked polynucleotide sequences that facilitate expression of a coding sequence in a particular host organism (e.g., a bacterial expression vector or a plant expression vector).

[0094] As used herein, “expression cassette” means a distinct component of vector DNA consisting of coding and non-coding sequences, including 5’ and 3’ regulatory sequences that control expression in a transformed / transfected cell. As used herein, “coding sequence” refers to the portion of DNA sequence bounded by a start and a stop codon that encodes the amino acids of a protein. As used herein, the term “non-coding sequence” refers to the portions of a DNA sequence that are transcribed to produce a messenger RNA, but that do not encode the amino acids of a protein, such as 5’ untranslated regions, introns and 3’ untranslated regions. Non-coding sequence also refers to RNA molecules such as micro-RNAs, interfering RNA or RNA hairpins, that when expressed down-regulate expression of an endogenous gene or another transgene. As used herein, the term “regulatory sequence” means a segment of a nucleic acid molecule which is capable of increasing or decreasing the expression of a gene. Regulatory sequences include promoters, terminators, enhancer elements, silencing elements, 5’ UTR and 3’ UTR (untranslated region). As used herein, the term “transfer cassette” means a T-DNA comprising an expression cassette or expression cassettes flanked by the right border and the left border. As used herein, the term “T- DNA” means a portion of a Ti plasmid that is inserted into the genome of a host plant cell.

[0095] As used herein, a "homolog" or "homologues" means a protein in a group of proteins that perform the same biological function and that provide a common trait in plants of this disclosure. Homologs are expressed by homologous genes. With reference to homologous genes, homologs include orthologs, for example, genes expressed in different species that evolved from common ancestral genes by speciation and encode proteins retain the same function, but do not include paralogs, z.e., genes that arc related by duplication but have evolved to encode proteins with different functions. Homologous genes include naturally occurring alleles and artificially-created variants.

[0096] Homologs are inferred from sequence similarity, by comparison of protein sequences, for example, manually or by use of a computer-based tool. For optimal alignment of sequences to calculate their percent identity, various pair-wise or multiple sequence alignment algorithms and programs are known in the art, such as ClustalW or Basic Local Alignment Search Tool® (BLAST),etc., that can be used to compare the sequence identity or similarity between two or more nucleotide or protein sequences. BLAST can also be used, for example to search query protein sequences of a base organism against a database of protein sequences of various organisms, to find similar sequences. The generated summary Expectation value (E-value) can be used to measure the level of sequence similarity. Because a protein hit with the lowest E-value for a particular organism may not necessarily be an ortholog or be the only ortholog, a reciprocal query is used to filter hit sequences with significant E-values for ortholog identification. The reciprocal query entails search of the significant hits against a database of protein sequences of the base organism. A hit can be identified as an ortholog, when the reciprocal query's best hit is the query protein itself or a paralog of the query protein. With the reciprocal query process orthologs are further differentiated from paralogs among all the homologs, which allows for the inference of functional equivalence of genes.

[0097] The terms “percent identity,” “% identity” or “percent identical” or “percent sequence identity” as used herein in reference to two or more nucleotide or protein sequences is calculated by (i) comparing two optimally aligned sequences (nucleotide or protein) over a window of comparison, (ii) determining the number of positions at which the identical nucleic acid base (for nucleotide sequences) or amino acid residue (for proteins) occurs in both sequences to yield the number of matched positions, (iii) dividing the number of matched positions by the total number of positions in the window of comparison, and then (iv) multiplying this quotient by 100% to yield the percent identity. If the “percent identity” or “percent sequence identity” is being calculated in relation to a reference sequence without a particular comparison window being specified, then the percent identity is determined by dividing the number of matched positions over the region of alignment by the total length of the reference sequence. Accordingly, for purposes of the present application, when two sequences (query and subject) are optimally aligned (with allowance for gaps in their alignment), the “percent identity” for the query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions in the query sequence over its length (or a comparison window), which is then multiplied by 100%. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity can be adjustedupwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions arc said to have “sequence similarity” or “similarity.” Sequences having a percent identity to a base sequence may exhibit the activity of the base sequence. Degeneracy of the genetic code provides the possibility to substitute at least one base of the protein encoding sequence of a gene with a different base without causing the amino acid sequence of the polypeptide produced from the gene to be changed.

[0098] As used herein, “alignment” refers to a number of nucleotide bases or amino acid residue sequences aligned by lengthwise comparison so that components in common (i. e. , nucleotide bases or amino acid residues at corresponding positions) may be visually and readily identified. The fraction or percentage of components in common is related to the homology or identity between the sequences. An alignment may be used to identify conserved domains and relatedness within these domains. An alignment may suitably be determined by means of computer programs known in the art. Specialist databases also exist for the identification of domains, for example, SMART (Schultz et al., Proc. Natl. Acad. Sci. USA 95:5857-5864, 1998; Letunic et al., Nucleic Acids Res. 30: 242- 244, 2002), InterPro (Mulder etal., Nucleic Acids Res. 31:315-318, 2002), PROSITE (Bairoch and Bucher, Nucleic Acids Res., 22:3583-3589, 1994; Hofmann et al., Nucleic Acids Res. 27:215-219, 1999), or Pfam (Bateman et al., Nucleic Acids Res. 30(l):276-280, 2002). A set of tools for in silico analysis of protein sequences is available on the ExPASY proteomics server (hosted by the Swiss Institute of Bioinformatics (Gasteiger et al., Nucleic Acids Res. 31:3784-3788, 2003).

[0099] The terms “percent complementarity” or “percent complementary,” as used herein in reference to two nucleotide sequences, is similar to the concept of percent identity but refers to the percentage of nucleotides of a query sequence that optimally base-pair or hybridize to nucleotides of a subject sequence when the query and subject sequences are linearly arranged and optimally base paired without secondary folding structures, such as loops, stems or hairpins. Such a percent complementarity may be between two DNA strands, two RNA strands, or a DNA strand and an RNA strand. The “percent complementarity” is calculated by (i) optimally base-pairing or hybridizing the two nucleotide sequences in a linear and fully extended arrangement (i.e., without folding or secondary structures) over a window of comparison, (ii) determining the number of positions that base-pair between the two sequences over the window of comparison to yield the number of complementary positions, (iii) dividing the number of complementary positions by the total number of positions in the window of comparison, and (iv) multiplying this quotient by 100%to yield the percent complementarity of the two sequences. Optimal base pairing of two sequences may be determined based on the known pairings of nucleotide bases, such as G-C, A-T, and A-U, through hydrogen bonding. If the “percent complementarity” is being calculated in relation to a reference sequence without specifying a particular comparison window, then the percent identity is determined by dividing the number of complementary positions between the two linear sequences by the total length of the reference sequence. Thus, for purposes of the present disclosure, when two sequences (query and subject) are optimally base-paired (with allowance for mismatches or non-base-paired nucleotides but without folding or secondary structures), the “percent complementarity” for the query sequence is equal to the number of base-paired positions between the two sequences divided by the total number of positions in the query sequence over its length (or by the number of positions in the query sequence over a comparison window), which is then multiplied by 100%.

[0100] As used herein, a “fragment” of a polynucleotide refers to a sequence comprising at least about 50, at least about 75, at least about 95, at least about 100, at least about 125, at least about 150, at least about 175, at least about 200, at least about 225, at least about 250, at least about 275, at least about 300, at least about 500, at least about 600, at least about 700, at least about 750, at least about 800, at least about 900, or at least about 1000 contiguous nucleotides, or longer, of a DNA molecule or protein as disclosed herein. Methods for producing such fragments from a starting promoter molecule are well known in the art. Fragments of a DNA molecule or protein may exhibit the activity of the DNA molecule or protein from which they are derived.

[0101] A plant selectable marker transgene in a transformation vector or construct of the present disclosure may be used to assist in the selection of transformed cells or tissue due to the presence of a selection agent, such as an antibiotic or herbicide, wherein the plant selectable marker transgene provides tolerance or resistance to the selection agent. Thus, the selection agent may bias or favor the survival, development, growth, proliferation, etc., of transformed cells expressing the plant selectable marker gene, such as to increase the proportion of transformed cells or tissues in the Ro plant. Commonly used plant selectable marker genes include, for example, those conferring tolerance or resistance to antibiotics, such as kanamycin and paromomycin (nptll), hygromycin B (aph IV), streptomycin or spectinomycin (aadA) and gentamycin (aac3 and aacC4), or those conferring tolerance or resistance to herbicides such as glufosinate (bar or pat), dicamba (DM0) and glyphosate (proA or EPSPS). Plant screenable marker genes may also be used, which providean ability to visually screen for transformants, such as luciferase or green fluorescent protein (GFP), or a gene expressing a beta glucuronidase or uidA gene (GUS) for which various chromogenic substrates are known. Plant transformation may also be carried out in the absence of selection during one or more steps or stages of culturing, developing or regenerating transformed explants, tissues, plants and / or plant parts.IV. Transformation Methods

[0102] Methods and compositions are provided for transforming a plant cell, tissue or explant with a recombinant DNA molecule or construct encoding one or more molecules required for selfincompatibility. Suitable methods for transformation of host plant cells include virtually any method by which DNA or RNA can be introduced into a cell (for example, where a recombinant DNA construct is stably integrated into a plant chromosome or where a recombinant DNA construct or an RNA is transiently provided to a plant cell) and are well known in the art. Two effective methods for cell transformation are bacterially-mediated transformation, such as Agrobacterium- mediated or / ? / uAz / ?mzzz- mediated transformation, and microprojectile or particle bombardment- mediated transformation. Microprojectile bombardment methods are illustrated, for example, in U.S. Patent Nos. 5,550,318; 5,538,880; 6,160,208; and 6,399,861. Agrobacterium-mediated transformation methods are described, for example in U.S. Patent No. 5,591,616. Other methods for plant transformation, such as microinjection, electroporation, vacuum infiltration, pressure, sonication, silicon carbide fiber agitation, PEG-mediated transformation, etc., are also known in the art.

[0103] Transformation of plant material is practiced in tissue culture on nutrient media, for example a mixture of nutrients that allow cells to grow in vitro. Recipient cell targets include, but are not limited to, meristem cells, shoot tips, hypocotyls, calli, immature or mature embryos, and gametic cells such as microspores and pollen. Callus can be initiated from tissue sources including, but not limited to, immature or mature embryos, hypocotyls, seedling apical meristems, microspores and the like. Cells containing a transgenic nucleus are grown into transgenic plants, also referred to as Ro plants. As used herein, “Ro plant” refers to an initial regenerated transformant. As used herein, “Ri seed” refers to seed produced from selfing Ro plants. As used herein, “Ri plant” refers to a plant grown from Ri seed. As used herein, “R2 seed” refers to seed produced from selfing Ri plants. As used herein, “R2 plant” refers to a plant grown from R2 seed.

[0104] Any suitable method or technique for transformation of a plant cell known in the art may be used according to present methods. In transformation, DNA is typically introduced into only a small percentage of target plant cells in any one transformation experiment. Marker genes are used to provide an efficient system for identification of those cells that are stably transformed by receiving and integrating a recombinant DNA molecule into their genomes.

[0105] As used herein, the terms “regeneration” and “regenerating” refer to a process of growing or developing a plant from one or more plant cells through one or more culturing steps. Transformed or edited cells, tissues or explants containing a DNA sequence insertion or edit may be grown, developed or regenerated into transgenic plants in culture, plugs, or soil according to methods known in the art. Certain embodiments of the disclosure therefore relate to methods and constructs for regenerating a plant from a cell with modified genomic DNA resulting from genome editing. The regenerated plant can then be used to propagate additional plants.

[0106] According to an aspect of the present disclosure, regenerated plants or a progeny plant, plant part or seed thereof can be screened or selected based on a marker, trait, or phenotype produced by the edit or mutation, or by the site-directed integration of an insertion sequence, transgene, etc., in the developed or regenerated plant, or a progeny plant, plant part or seed thereof. If a given mutation, edit, trait, or phenotype is recessive, one or more generations or crosses (e.g., selfing) from the initial Ro plant may be necessary to produce a plant homozygous for the edit or mutation so the trait or phenotype can be observed. Progeny plants, such as plants grown from Ri seed or in subsequent generations, can be tested for zygosity using any known zygosity assay, such as by using a single nucleotide polymorphism (SNP) assay, DNA sequencing, thermal amplification, or polymerase chain reaction (PCR), and / or Southern blotting that allows for the distinction between heterozygote, homozygote and wild-type plants.

[0107] Methods and techniques are provided for screening for, and / or identifying, cells or plants, etc., for the presence of transgenes and selecting cells or plants comprising transgenes, which may be based on one or more phenotypes or traits, or on the presence or absence of a molecular marker or polynucleotide or protein sequence in the cells or plants. As used herein, a “molecular technique” refers to any method known in the fields of molecular biology, biochemistry, genetics, plant biology, or biophysics that involves the use, manipulation, or analysis of a nucleic acid, a protein, or a lipid. Without being limiting, molecular techniques useful for detecting the presence of a modified sequence in a genome include phenotypic screening; molecular marker technologies suchas SNP analysis by TaqMan® or Illumina / Infinium technology; Southern blot; PCR (including amplicon sequencing which consists of the generation of one or more unique PCR products across the genomic region of interest for further sequencing analysis, such as using Next-Gen Sequencing techniques known in the art. Sequence data from each sample is then mapped to a reference sequence to identify consensus differences); enzyme-linked immunosorbent assay (ELISA); and sequencing (e.g., Sanger, Illumina®, 454, Pac-Bio, Ion Torrent™). In one aspect, a method of detection provided herein comprises phenotypic screening. In another aspect, a method of detection provided herein comprises SNP analysis. In a further aspect, a method of detection provided herein comprises a Southern blot. In a further aspect, a method of detection provided herein comprises PCR. In a further aspect, a method of detection provided herein comprises amplicon sequencing. In an aspect, a method of detection provided herein comprises ELISA. In a further aspect, a method of detection provided herein comprises determining the sequence of a nucleic acid or a protein. Without being limiting, nucleic acids can be detected using hybridization. Hybridization between nucleic acids is discussed in detail in Sambrook el al. (1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0108] Nucleic acids can be isolated using techniques routine in the art. For example, nucleic acids can be isolated using any method including, without limitation, recombinant nucleic acid technology, and / or PCR. General PCR techniques are described, for example in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleic acid techniques include, for example, restriction enzyme digestion and ligation, which can be used to isolate a nucleic acid. Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides.

[0109] Detection (e.g., of an amplification product, of a hybridization complex, of a polypeptide) can be accomplished using detectable labels that may be attached or associated with a hybridization probe or antibody. The term “label” is intended to encompass the use of direct labels as well as indirect labels. Detectable labels include enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. The screening and selection of modified (e.g., edited) plants or plant cells can be through any methodologies known to those skilled in the art of molecular biology. Examples of screening and selection methodologies include, but are not limited to, Southern analysis, PCR amplification for detection of a polynucleotide (including amplicon sequencing), Northern blots, RNase protection, primer-extension, RT-PCR amplification for detecting RNA transcripts, Sanger sequencing, Next Generation sequencing technologies (c.g., Illumina®, PacBio®, Ion Torrent™, etc.) enzymatic assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides, and protein gel electrophoresis, Western blots, immunoprecipitation, and enzyme-linked immunoassays to detect polypeptides. Other techniques such as in situ hybridization, enzyme staining, and immunostaining also can be used to detect the presence or expression of polypeptides and / or polynucleotides. Methods for performing all of the referenced techniques are known in the art.

[0110] As used herein, the term “polypeptide” refers to a chain of at least two covalently linked amino acids. Polypeptides can be encoded by polynucleotides provided herein. An example of a polypeptide is a protein. Proteins provided herein can be encoded by nucleic acid molecules provided herein. Polypeptides can be purified from natural sources (e.g., a biological sample) by known methods such as DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. A polypeptide also can be purified, for example, by expressing a nucleic acid in an expression vector. In addition, a purified polypeptide can be obtained by chemical synthesis. The extent of purity of a polypeptide can be measured using any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0111] Polypeptides can be detected using antibodies. Techniques for detecting polypeptides using antibodies include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. An antibody provided herein can be a polyclonal antibody or a monoclonal antibody. An antibody having specific binding affinity for a polypeptide provided herein can be generated using methods well known in the art. An antibody provided herein can be attached to a solid support such as a microtiter plate using methods known in the art.V. Self-Incompatible Crop Plants

[0112] The present disclosure provides, in certain embodiments, crop plants produced to be selfincompatible. In an aspect, said self-incompatible crop plants comprise a recombinant DNA construct(s) that include three components: a cassette that expresses in pollen a pollen-specific protein from a 5 locus allele; a cassette that expresses in the stigma the corresponding stigma protein of the same .S' locus allele; and a counteracting factor which negates the self-incompatible phenotype conferred by 5 locus alleles distinct from those present in the construct. In some embodiments, the pollen-specific protein is the Papaver PrpS protein, and the stigma- specific protein is the Papaver PrsS protein. In certain embodiments, the pollen- specific protein is a Papaver PrpS protein selectedfrom PrpSl , PrpS3, or PrpS8 and the stigma-specific protein is a Papaver PrsS protein selected from PrsSl, PrsS3, or PrsS8. In some embodiments, the pollen-specific protein is the Brassica SP11 / SCR protein, and the stigma-specific protein is the Brassica SRK protein. Variants of these proteins that confer self-incompatibility are also included and the examples described herein can be used to identify active variants.

[0113] In a further aspect, the present disclosure provides a first self-incompatible soybean plant that comprises a recombinant DNA construct that includes a first expression cassette that comprises a coding sequence encoding a PrpS 1 protein from a first Papaver S locus driven by a pollen-specific promoter, a second expression cassette that comprises a coding sequence encoding the corresponding PrsS 1 protein from the same .S' locus allele of Papaver driven by a stigma-specific promoter, and a third expression cassette that comprises an antisense RNA sequence capable of suppressing expression of a PrsS3 gene and a second self-incompatible soybean plant that comprises a recombinant DNA construct that includes a first expression cassette that comprises a coding sequence encoding a PrpS3 protein from a first Papaver S locus driven by a pollen-specific promoter, a second expression cassette that comprises a coding sequence encoding the corresponding PrsS3 protein from the same .S' locus allele of Papaver driven by a stigma-specific promoter, and a third expression cassette that comprises a sequence capable of suppressing expression of a PrsSl gene.

[0114] Another aspect of the invention includes conditional self-incompatibility system for maintaining a self-incompatible plant line. In some embodiments, the compatibility system is inducible by application of a chemical or by other factors, such as photoperiod or light. In one embodiment, the chemical inducer is ethanol. In some embodiments, a self-incompatible plant comprises a recombinant DNA construct that includes a first cassette expressing pollen- specific .S' allele protein, a second cassette expressing a corresponding pistil-specific 5 allele protein, a third cassette expressing a gene encoding a transcription factor, a fourth cassette comprising a selfincompatibility suppressing gene that encodes a sequence targeting one or more .S' locus alleles distinct from those present in the first and second cassettes), and a fifth cassette comprising an inducible self-incompatibility suppressing gene that encodes a sequence targeting one or both of the 5 locus alleles present in the first and second cassette, wherein the gene will be expressed upon exogenous application of an inducer (sometimes also referred to as an elicitor) molecule.

[0115] In an embodiment, a gene encoding a transcription factor is expressed to produce said transcription factor. In the presence of an clicitor molecule, the transcription factor interacts with the elicitor molecule. The promotor-bound transcription factor may then bind a regulatory region of an conditional self-incompatibility suppressing gene. The combination of inducer, transcription factor, and regulatory region activates the conditional self-incompatibility suppressing gene. When activated, the inducible self-incompatibility suppressing gene suppresses expression of the selfincompatibility gene. Suppression of the self-incompatibility gene may confer self-compatibility onto a self-incompatible plant line.

[0116] In another embodiment, the gene encoding a transcription factor may be the AlcR gene. When expressed, the AlcR gene produces the AlcR transcription factor which may bind the promoter region of the self-incompatibility suppressing gene. In other embodiments, an inducible gene expression system other than AlcR / AlcA system may be utilized. When using other chemical inducible expression systems, the system should be applied in a manner that activation of the selfincompatibility suppressing gene is affected by application of the elicitor molecule. In other embodiments of the inducible compatibility system, the elicitor may be an alcohol other than ethanol, an aldehyde, rapamycin or other rapalog, ecdysone, tetracycline, dexamethasone, estradiol, mandipropamid, herbicide safeners, or any other compound or molecule which is corresponds to the respective inducible expression system such that upon addition of the elicitor, the suppressor is expressed, self-incompatibility genes are suppressed and a plant that is self-incompatible in the absence of the elicitor is thus rendered self-compatible.

[0117] In some embodiments, an elicitor may be delivered to the conditional self-incompatibility system by, for example, spraying a solution comprising the elicitor onto a self-incompatible plant comprising the conditional self-incompatibility system. In other embodiments, elicitor may be delivered by applying a solution containing elicitor to the roots of the plant containing the conditional self-incompatibility system or contacting the plant with a vapor containing elicitor or contacting the plant by any other means which enables a conditional self-incompatibility system to function as described.

[0118] In some embodiments, the recombinant DNA constructs of this invention includes DNA encoding a transcription regulatory element. Transcription regulatory elements include elements that regulate the expression level of one or more of the cassettes comprised in the recombinant DNA constructs of this invention (relative to their expression in the absence of such regulatory elements).Examples of suitable transcription regulatory elements include riboswitches (cis- or trans-acting), transcript stabilizing sequences, and miRNA recognition sites.

[0119] Another aspect of the invention includes a self-incompatible plant propagation system, wherein a self-incompatible plant comprises an herbicide tolerance trait that enables said selfincompatible plant to produce seeds after pollination and application of the herbicide. In some embodiments, the self-incompatible plant comprises a recombinant DNA construct that includes a first cassette expressing pollen- specific .S' allele protein, a second cassette expressing a corresponding pistil-specific .S’ allele protein, and a third cassette expressing a recombinant protein that confers herbicide tolerance to the plant. In some embodiments, the herbicide may be glyphosate or any other herbicide or agrochemical which enables the self-incompatible plant propagation system to function as described.

[0120] The maintainer plant is self-compatible, susceptible to herbicide, and is otherwise isogenic or near isogenic with the self-incompatible plant. The self-incompatible plant and maintainer plant may be grown near each other, for example in adjacent rows in a growing field. In the example embodiment, pollen produced by the maintainer plant pollinates the self-incompatible plant. Pollination may occur, for example, through the action of pollinating agents or insects such as bees. After pollination of self-incompatible plant with pollen from the maintainer plant, an herbicide is applied to self-incompatible plant and maintainer plant. Herbicide may be applied through, for example, a liquid spray, a powder spray, an irrigation drip, or any other means which enables selfincompatible plant propagation system to function as described.

[0121] In the example embodiment, the herbicide tolerance trait possessed by self-incompatible plant enables self-incompatible plant to produce seeds after pollination and exposure to the herbicide. Seeds harvested from self-incompatible plant comprise a gene encoding a selfincompatibility trait and a gene encoding a tolerance to the herbicide. In the example embodiment, seeds may be planted to propagate the self-incompatible plant line.

[0122] In some embodiments, the self-incompatible plant comprises a recombinant DNA construct that includes a first cassette expressing pollen- specific 5 allele protein, a second cassette expressing a corresponding pistil-specific 5 allele protein, and a third cassette expressing a gene associated with a phenotypic marker, such as seed coat color. In some embodiments, expression of the phenotypic marker gene may result in pigment accumulation in the cotyledons, colored protein accumulation (e.g. RFP or a chromoprotein), altered levels of oil, protein or starch, or any otherphenotype that enables self-incompatible plant propagation system to function as described. Such markers can be used to quickly identify progeny in a breeding program that include one or more of the desired alleles.

[0123] In an embodiment, the self-incompatible plant propagation system further includes a maintainer plant, which lacks the seed phenotype marker, is self-compatible, and is otherwise isogenic or near isogenic with the self-incompatible plant. The self-incompatible plant and maintainer plant may be grown near each other, for example in adjacent rows in a growing field. In another embodiment, pollen produced by the maintainer plant pollinates the self-incompatible plant. Pollination may occur, for example, through the action of pollinating agents or insects such as bees. In yet another embodiment, after pollination of the self-incompatible plant with pollen from the maintainer plant, seeds are produced by the self-incompatible plant and the maintainer plant. Seeds comprising a self-incompatibility gene are identifiable based on a phenotypic difference, such as seed color resulting from a seed phenotype marker gene. In other embodiments, seeds comprising or lacking a self-incompatibility gene are identifiable through other phenotypic difference including oil content, seed color, seed size, or any other means which enables the self-incompatible plant propagation system to function as described. In an embodiment, seeds may be planted to propagate a self-incompatible plant.

[0124] In some embodiments, the maintainer line can be a haploid inducing line that is either isogenic or not isogenic with the self-incompatible plant, such that upon crossing with the selfincompatible plant, seeds are produced, but yet the self-incompatible line is recovered. The selfincompatibility system can also be established to trigger upon crossing with a recombinase donor line. This is important in a breeding program where elite lines need to be created by successive crosses with parental plants having diverse genomes. Modem accelerated breeding programs operate using machine learning and modeling to predict outcomes and identify genomes that should be crossed. At certain points in the cycles of breeding genomes can be extracted from the breeding program for further testing and / or for commercial line production. The constructs described herein with the respective S alleles, promoters, terminators and counteracting sequences can be engineered to include sites that can be recognized by a recombinase and upon crossing with a recombinase donor plant the resulting progeny will be edited. Depending upon the desired scheme the edit can either activate the self-incompatibility functionality or de-activate it. In some instances, the site specific recombinase can be included in a haploid inducing line so that upon crossing the haploidgenerated is self-incompatible, or is no longer self-incompatible. See, Zhang et al., Theor Appl Genet. 107(7): 1157-68, 2003.

[0125] In some embodiments, the self-incompatible plants described herein may further comprise a maintainer locus. As used herein, a “maintainer locus” refers to a genetic locus that comprises a gene or genes that counteracts the self-incompatibility trait. In plants that have a selfincompatibility trait but that contain the maintainer locus, some pollen will be able to self-fertilize. The maintainer locus may be at a genetically distinct location from some or all of the genes that produce the self-incompatibility trait. In some cases, the maintainer locus may have an additional gene or genes that confer other traits. For example, the maintainer locus may contain a gene that produces a distinct seed phenotype that enables seeds containing the maintainer locus to be distinguished and selected away from seeds that lack the maintainer locus.

[0126] Self-incompatible plants further comprising a maintainer locus can be used to enable self- pollination in such plants or to produce hybrid seed. In some embodiments, a maintainer locus may comprise a pollen block to prevent transmission of the maintainer locus through pollen. As used herein, a “pollen block” refers to a genetic element that prevents fertilization by a pollen that comprises that element. A pollen block may be a transgene that expresses in pollen a cytotoxic protein that kills the pollen to prevent fertilization or expresses an enzyme that impairs the pollen function. Examples of proteins that have been used as pollen blocks include, but are not limited to, Zea mays alpha-amylase, Bacillus amyloliquefaciens barnase. EcoRI endonuclease, and various CRISPR components that cut sequences in the pollen genome.

[0127] Fertilization may also be prevented through suppression by the pollen block element of a pollen gene that is required for pollen function or for successful fertilization. In such embodiments, plants that comprise the maintainer locus may be self-pollinated to produce seeds that contain a single copy of the maintainer locus or lack the maintainer locus but seeds that are homozygous for the maintainer locus are not recovered. Furthermore, pollen from a plant containing a maintainer locus and a pollen block may pollinate other plants to produce seeds that lack the maintainer locus. The pollen block mechanism may function to only partially impair pollen function so that a pollen comprising the pollen block is not competitive with pollen that lack the pollen block. In this case, fertilization may be possible for pollen that contain the pollen block if insufficient pollen that lack the pollen block are present to outcompete the blocked pollen.

[0128] Pollen block elements may function in a context dependent manner, whereby the pollen block will prevent pollen fertilization in some genetic backgrounds but not in other backgrounds. For example, a transgene that suppresses a cytoplasmic male-sterility (CMS) restoration factor will prevent pollen from a plant with the corresponding gametophytic type CMS from functioning in plants that have the CMS cytoplasm.

[0129] In some embodiments, self-incompatible plant propagation system may be scaled to include up to 1, up to 10, up to 100, up to 1,000, or up to 10,000, up to 100,000, up to 1,000,000, up to 10,000,000, up to 100,000,000 up to 1,000,000,000, or up to 10,000,000,000 or more selfincompatible and maintainer plants in a plant population. In some embodiments, self-incompatible plant propagation system may be scaled to include any number of self-incompatible and maintainer plants that enable the self-incompatible plant propagation system to function as described.

[0130] In some embodiments, self-incompatible and maintainer plants may be present within self-incompatible plant propagation system in a ratio of up to 1:1,000, up to 1:100, up to 1:10, up to 1: 1, up to 10: 1, up to 100: 1, or up to 1,000: 1 or more in a plant population. In some embodiments, self-incompatible and maintainer plants may be present within the self-incompatible plant propagation system in any ratio that enables the self-incompatible plant propagation system to function as described.

[0131] In some embodiments, a self-incompatible plant propagation system and selfincompatible and maintainer plants may be housed within a greenhouse, a field, or in any environment which enables the self-incompatible plant propagation systems described herein to function as described.

[0132] In another aspect of the disclosure, a method for suppression of self-incompatible genes is provided to enable self-incompatibility in hybrid plants. In plants, the introduction of transgenes into the genome may result in the transgenes being silenced or even silencing of endogenous genes if they share sufficient homology with the inserted transgene(s). This is known as homologydependent gene silencing or co-suppression and typically occurs when multiple copies of particular sequences are present in the genome. Gene silencing may occur at the transcriptional or post- transcriptional level. Post-transcriptional silencing and amplification have been observed in transgene with redundant terminators. For example, antisense fragments that are ~200bp in length are designed from coding region of self-incompatible genes and placed at 3’ end of the selfincompatibility gene. The antisense fragment will be transcribed along with self-incompatibilitygene in a single transcript. When two parent plants with self-incompatihility genes are crossed, transcript of homologous antisense fragment may bind to the transcript from a self-incompatibility gene and form aberrant dsRNA, which may be processed into small RNA. Small RNA may trigger degradation of self-incompatibility gene transcript and restore fertility in hybrid seed. In addition, the same terminator sequence can used in both self-incompatibility vectors to enhance cosuppression of self-incompatibility genes. Once post-transcriptional silencing is triggered, homologous terminator transcript will rapidly amplify the small RNA and enhance suppression of the targeted self-incompatibility gene.VI. Definitions

[0133] Unless defined otherwise herein, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. Examples of resources describing many of the terms related to molecular biology used herein can be found in Alberts et al., Molecular Biology of The Cell, 5th Edition, Garland Science Publishing, Inc.: New York, 2007; Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer- Verlag: New York, 1991; King et al, A Dictionary of Genetics, 6th ed., Oxford University Press: New York, 2002; and Lewin, Genes IX, Oxford University Press: New York, 2007. The nomenclature for DNA bases as set forth at 37 C.F.R. § 1.822 is used.

[0134] The term “and / or” when used in a list of two or more items, means that any one of the listed items can be employed by itself or in combination with any one or more of the listed items. For example, the expression “A and / or B” is intended to mean either or both of A and B - i.e., A alone, B alone, or A and B in combination. The expression “A, B and / or C” is intended to mean A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination.

[0135] As used herein, terms in the singular and the singular forms “a,” “an,” and “the,” for example, include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “plant,” “the plant,” or “a plant” also includes a plurality of plants; also, depending on the context, use of the term “plant” can also include genetically similar or identical progeny of that plant; use of the term “a nucleic acid” optionally includes, as a practical matter, many copies of that nucleic acid molecule.

[0136] The term “about” as used herein, is intended to qualify the numerical values that it modifies, denoting such a value as variable within a margin of error. When no particular margin oferror, such as a standard deviation to a mean value, is recited, the term “about” should be understood to mean that range which would encompass the recited value and the range which would be included by rounding up or down to that figure, taking into account significant figures.

[0137] As used herein, “plant” refers to a whole plant, any part thereof, or a cell or tissue culture derived from a plant, comprising any of whole plants, plant components or organs (e.g., leaves, stems, roots, etc.), plant tissues, seeds, plant cells, and / or progeny of the same. A plant cell is a biological cell of a plant, taken from a plant or derived through culture from a cell taken from a plant. As used herein, a “plant part” may refer to any organ or intact tissue of a plant, such as a meristem, shoot organ / structure (e.g., leaf, stem or node), root, flower or floral organ / structure (e.g., bract, sepal, petal, stamen, carpel, anther and ovule), seed (e.g., embryo, endosperm, and seed coat), fruit (e.g., the mature ovary), propagule, or other plant tissues (e.g., vascular tissue, dermal tissue, ground tissue, and the like), or any portion thereof. Plant parts of the present invention may be viable, nonviable, regenerable, and / or non-regenerable. A “propagule” may include any plant part that is capable of growing into an entire plant.

[0138] As used herein, “germplasm” refers to living sources of genetic material. The germplasm can be part of an organism or cell or can be separate from the organism or cell. In general, germplasm provides genetic material with a specific molecular makeup that provides a physical foundation for some or all of the hereditary qualities of an organism or cell culture. As used herein, germplasm includes cells, seed or tissues from which new plants may be grown, or plant pails, such as leaves, stems, pollen, or cells that can be cultured into a whole plant.

[0139] As used herein, a “transgenic plant” refers to a plant whose genome has been altered by the integration or insertion of a recombinant DNA molecule, construct, or sequence.

[0140] As used herein a “component” refers to any molecule that contributes to selfincompatibility in a plant.

[0141] As used herein, “gene regulation” is a process by which an output of a gene is changed in response to a state of a cell or organism. Gene regulation may include changes in the amount of RNA produced, the levels of RNA that accumulate in a cell or organism, the rate of translation of a coding sequence into proteins, the amount and locations intron splicing, the location of a protein within a cell, the amount of a protein that accumulates in a cell or organism, activity of the protein from the gene, post-translational modification of proteins from the gene and the folding of proteins from the gene.

[0142] As used herein, the term “down regulation” refers to reduced activity or expression of a gene, protein, or phenotype (at least 10%, 20%, 30%, 50%, 60%, 70%, 80%, 90% or 100% reduction in activity or expression) as compared to its activity or expression in a plant of the same species and the same developmental stage not expressing the exogenous polynucleotide.

[0143] As used herein, “allele” refers to an alternative nucleic acid sequence at a particular locus; the length of an allele can be as small as 1 nucleotide base but is typically larger. For example, a first allele can occur on one chromosome, while a second allele occurs on a second homologous chromosome, e.g., as occurs for different chromosomes of a heterozygous individual, or between different homozygous or heterozygous individuals in a population.

[0144] As used herein, the term “population” refers to a grouping of one or more plants. In certain embodiments, a population of plants comprises at least about 10, 50 100, 250, 500, 1,000, 5,000, 10,000, 50,000, 100,000, 1,000,000, 10,000,000, 100,000,000 or more plants.

[0145] As used herein, “crossed” or “cross” means to produce progeny via fertilization (e.g., cells, seeds, or plants) and includes crosses between plants (sexual) and self-fertilization (selling).

[0146] As used herein, “genotype” is the genetic constitution of an individual (or group of individuals) at one or more genetic loci, as contrasted with the observable trait (the phenotype). Genotype is defined by the allele(s) of one or more known loci that the individual has inherited from its parents. The term genotype can be used to refer to an individual's genetic constitution at a single locus, at multiple loci, or, more generally, the term genotype can be used to refer to an individual's genetic make-up for all the genes in its genome. A “haplotype” is the genotype of an individual at a plurality of genetic loci. Typically, the genetic loci described by a haplotype are physically and genetically linked, i.e., on the same chromosome interval.

[0147] The terms “phenotype,” or “phenotypic trait,” or “trait” refers to one or more trait of an organism. The phenotype can be observable to the naked eye, or by any other means of evaluation known in the art, e.g., microscopy, biochemical analysis, genomic analysis, an assay for a particular disease tolerance, etc. In some cases, a phenotype is directly controlled by a single gene or genetic locus, i.e., a “single gene trait.” In other cases, a phenotype is the result of several genes.

[0148] The term “self-incompatible,” as used herein, refers to a genetic mechanism that prevents self-fertilization and thus forces outcrossing and allogamy. A “self-incompatibility response” as used herein is defined inhibition of self pollen triggered by an allele-specific male S determinant and female S determinant interaction. Phenotypes associated with a self-incompatibility responseas defined herein include disruption of pollen hydration and germination and / or cessation in pollen tube growth.

[0149] The term “self-compatible,” as is used herein, refers to a mechanism that has overcome the prevention of self-fertilization.

[0150] Other objects, features, and advantages of the present disclosure are apparent from detailed description provided herein. It should be understood, however, that the detailed description and any specific examples provided, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.EXAMPLESExample 1. Generation of Self-incompatibility in Arabidopsis thaliana.

[0151] Self-incompatibility is generally controlled by a single 5 locus region, composed of at least two tightly linked loci encoding the pollen (male) and pistil (female) 5-determinants that exhibit tight tissue- specific and developmental- stage- specific expression. The self-incompatibility system in Papaver rhoeas is well characterized and utilizes two genetically linked S-determinants: a pollen-expressed transmembrane protein, PrpS, and a stigma-expressed secreted protein, PrsS. Interaction of PrpS and PrsS results in rejection of incompatible pollen, triggering a signaling network causing rapid growth arrest and ensuing programmed cell death in incompatible pollen. The stigma and pollen -specific S genes exist in different allelic forms and thus enables control of pollination specificity. For example, a plant carrying the SI allele cannot pollinate itself or another plant comprising the SI allele but can pollinate plants of the same species carrying different S locus alleles.

[0152] A novel construct design was implemented to co-express either the SI and S3 alleles of the PrpS and PrsS genes from Papaver in a tandem vector stack. This enabled rapid generation of transgenic plants and testing of self-incompatibility in these plants. Constructs were designed to include an intergenic spacer region (ISR) between the PrpS and PrsS sequences to facilitate the expression of both genes. The expression of PrpSl (SEQ ID NO:4) or PrpS3 (SEQ ID NO:2) in the constructs is driven by the promoter of the pollen- specific NTP303 gene from tobacco (SEQ ID NO:5) while expression of PrsSl (SEQ ID NO:3) or PrsS3 (SEQ ID NO:1) in these constructs isdriven by the promoter of the stigma-specific SLR] gene from Arabidopsis (SEQ TD NO:6). Details of the components of the constructs arc shown in Table 1 below.Table 1. Allele-specific expression constructs for Arabidopsis.

[0153] To determine whether the constructs were functional in plants, A. thaliana ecotype Columbia (Col-0) lines was transformed with Construct 1 and Construct 2 using the floral dip method. To transgenic seeds were then screened on MS plates containing spectinomycin (75 pg / ml) as the selection agent, resulting in the acquisition of 9 independent lines obtained from Construct 1 and 7 independent lines obtained from Construct 2. Plates with seeds were placed at 4°C for two days before germination. Surviving seedlings were transplanted to soil pots in the greenhouse (20- 22°C, under 16 / 8 h photoperiod condition) two weeks later. Leaf samples were collected to evaluate the integration of T-DNA and copy number in the A. thaliana genome by a TaqMan assay. In addition, transgenic plants were crossed to Col-0 plants to examine crossability and function of pollen and stigma. The results showed that the Col-0 are cross compatible with the transgenic plants as expected.Example 2. Generation of Self- Incompatibility in Soybean.

[0154] Experiments were conducted to determine if the Papaver self-incompatibility system can be introduced into soybean. Constructs were generated as described in the Example above. Soybean pollen specific promoters and stigma specific promoters were mined and selected to generate additional constructs. Constructs were designed to include an intergenic spacer region (ISR) between the PrpS and PrsS sequences to facilitate the expression of both genes. The expression of PrpSl (SEQ ID NO:4) or PrpS3 (SEQ ID NO:2) in the constructs is driven by a pollen- specific promoter sequence from the tobacco NTP303 gene (SEQ ID NO:5), tomato LAT25 gene (SEQ ID NO:8), and cotton peroxidase gene (SEQ ID NO: 13) or from the soybean Rsp-1 (SEQ ID NO:9), MADS-box transcription factor 17 (SEQ ID NO: 10), and Gm.08G269800 (SEQ ID NO: 11) genes from while expression of PrsSl (SEQ ID NO:3) or PrsS3 (SEQ ID NO: 1) in these constructs is driven by a stigma- specific promoter sequence, such as from the Arabidopsis SLR1gene (SEQ ID N0:6), cotton Ghstp gene (SEQ ID NO: 12), or the soybean CHIA ] gene (SEQ ID NO:7). The Ghstp, Gm.Chial, Ghpop, and Gm.08G269800 promoter sequences were taken from the upstream region of the corresponding genes. The Ghstp promoter sequence was further modified to enhance stigma- specific expression by using the STIGMA AND STYLE STYLIST 1-3 (SSS1- SSS3) genes and three NGATHA3 (NGA3) transcription factor binding motifs that were previously shown to be involved in interactions with the SAS genes (Li et al., Current Biology 30(23):4780 - 4788, 2020). Details of the components of the constructs are shown in Table 2 below.Table 2. Allele-specific expression constructs for soybean.

[0155] Soybean A3555 plants were transformed with each of the 10 constructs using A. tumefaciens and standard methods known in the art. The copy number of the transgene was determined via a Taqman assay using leaf samples and events with a single copy of the T-DNA insertion were selected for further evaluation and characterization. 15 independent events were confirmed and transplanted to pots for assays. Pollen viability was examined through FlowCAM assay. Most transgenic plants demonstrated pollen viability comparable to that of control wild-type plants (FIG. 5). The transgenic plants do not appear to show any changes to agronomic performance compared to the nontransgenic controls. Ri seeds from each event were harvested for furtheranalysis. Ri seeds were planted to determine genetic segregation ratio and level of selfincompatibility.

[0156] A key feature of self-incompatibility in Papaver is the inhibition of pollen tube growth. An aniline blue staining method was developed to visualize pollen tube growth and track pollen tube extension in soybean flowers using microscopy. Flowers at different developmental stages were collected from control soybean plants to identify the most suitable stage for visualizing pollen tube growth and to refine the aniline blue staining protocol. After careful evaluation, flowers at the Al stage were identified as the optimal stage for this purpose. Subsequently, flowers at the Al stage from the transgenic soybean events were collected to visualize pollen tube growth. The results showed that flowers from transgenic plants demonstrated inhibition of pollen tube growth compared to controls, which indicates the presence of the self-incompatible response in transgenic plants (FIG. 6).

[0157] In order to confirm the effectiveness of the self-incompatibility trait in transgenic plants, an Ri genotyping experiment was performed. When no self-incompatibility occurs, the expected Ri plants ratio is 1:2:1 (S / S: S / -: - / -), however if self-incompatibility is present, the expected ratio is 0:1:1 (S / S: S / - : - / -) because pollen containing the transgenes will be inhibited. Ri seeds were harvested from the Ro plants and genotyped for the presence and copy number of the transgenes. For constructs 1 and 2, only a small proportion of the Ri progeny were homozygous and the Ri genotype ratios were close to 0:1:1 (FIG. 7 and FIG. 8).

[0158] Among the seeds of self-pollinated plants containing constructs 1 and 2, a small percentage of SI or S3 homozygous plants that were produced. For plants that are homozygous for the self-incompatibility transgene (2-copy), the pollen suppression effect may be even stronger than in Ri plants that are hemizygous (1-copy). Therefore, these plants were selected and grown to the reproductive stage and the flowers examined. For both Sl / Sl and S3 / S3 homozygous plants, plants can’t produce seeds through selfing, and sterility results from self-incompatibility (FIG. 9). In contrast to selfing, cross-pollination between Sl / Sl and S3 / S3 plants produced seeds inside seed pods and demonstrated that the plants are cross-compatible.

[0159] Since self-pollination is suppressed in homozygous Sl / Sl and homozygous S3 / S3 plants but reciprocal crosses produce Fi progeny, these two types of plants could be grown interspersed in a hybrid production field and all the resulting seeds harvested.Example 3. Generation of Self-Incompatibility in Cotton.

[0160] Experiments arc conducted to determine if the Papaver self-incompatibility system could be successfully introduced and used in cotton. Following a similar process as described above in Examples 1 and 2, constructs were designed to include an intergenic spacer region (ISR) between the PrpS and PrsS sequences to facilitate the expression of both genes. The expression of PrpSl (SEQ ID NO:4) or PrpS3 (SEQ ID NO:2) in the constructs is driven by a pollen- specific promoter sequence from tobacco NTP303 gene (SEQ ID NO:5) or cotton Ghstp gene (SEQ ID NO: 12), while expression of PrsSl (SEQ ID NO:3) or PrsS3 (SEQ ID NO:1) in these constructs is driven by a stigma- specific promoter sequence from the Arabidopsis SLR1 gene (SEQ ID NO:6) or the or cotton Ghstp gene (SEQ ID NO: 12). Details of the components of the constructs are shown in Table 3 below.Table 3. Allele-specific expression constructs for cotton.

[0161] Experiments are conducted to determine if the Papaver self-incompatibility system could be successfully introduced and used in maize. Leaf samples are used to identify transgenic plants with a single copy of the transgenic insert. Ro plants that contain only a single copy were selected for further evaluation and characterization.Example 4. Generation of Self- Incompatibility in Maize.

[0162] Experiments arc conducted to determine if the Papaver self-incompatibility system can be introduced into maize. Following a similar process as described above in Examples 1 and 2, constructs were designed to include an intergenic spacer region (ISR) between the PrpS and PrsSsequences to facilitate the expression of both genes. The expression of PrpSl (SEQ ID NO:4) or PrpS3 (SEQ ID NO:2) in the constructs is driven by a maize pollen- specific promoter from the GRMZM2G487322 gene (SEQ ID NO: 15) while expression of PrsSl (SEQ ID NO:3) or PrsS3 (SEQ ID NO:1) in these constructs is driven by a maize stigma- specific promoter sequence from the GRMZM2G010868 gene (SEQ ID NO: 14). Details of the components of the constructs are shown in Table 4 below.Table 4. Allele-specific expression constructs for maize.

[0163] Maize plants were transformed with these vectors using A. tumefaciens and standard methods known in the art. Leaf samples can be used to identify transgenic plants with a single copy of the transgenic insert. Ro plants that contain only a single copy can be selected for further evaluation and characterization.Example 5. Production of Self-Compatible Hybrid Crop Plants from Self-Incompatible Parents.

[0164] To facilitate the control of self-incompatibility in hybrids and to enable hybrid seed production, constructs 1 and 2, that include counteracting elements were transformed into soybean plants as described above. Specifically, an antisense sequence targeting PrsS3 (SEQ ID NO: 16) and an antisense sequence targeting PrsSl (SEQ ID NO: 17) were designed to induce homology sequence-based gene silencing when plants expressing SI and S3 are crossed. An 180bp antisense sequence was identified that was reverse complementary to coding sequence of each gene and had acceptable sequence complexity, aiming to trigger the silencing of prsSl or prsS3 gene expression upon crossing SISI and S3S3 lines. The seeds resulting from the cross will be grown and allowed to self-pollinate. Recovery of seeds will demonstrate that the plants are self-compatible.Example 6. Inducible Suppression Constructs for Use in Breeding Programs

[0165] The phenomenon of heterosis in Fi hybrid plants arises from crossing two distinct inbred plant lines. In some instances, this is accomplished through the generation of haploid plants and subsequent doubling to produce double haploids. These inbred / double haploid lines, if chosencorrectly, will give rise to hybrid seed that have agronomically valuable traits, such as increased crop yield and vigor. Specifically, inducible suppression of one of more genes that confer the selfincompatibility trait enables self-pollination for the purposes of inbred line maintenance and seed production in a controlled manner. If self-incompatibility is not suppressed, self-pollination is prevented in such inbred lines and inbred line seed production is hindered. (FIG. 4).

[0166] Several chemically induced expression systems that are used for controlled gene expression are known in the art and may be used with the self-incompatibility system of the present invention. Commonly used chemical inducers that may be used with the present invention include, but are not limited to, ethanol, copper, tetracycline, fungicides, insecticides, and salt. Chemical inducers may be applied to soil, plant surfaces, or plant growth media. A chemical inducer utilized with the present invention should not be a plant metabolite and should have no toxic effect on plant viability. Inducers can also be environmental, for example, blue light inducible promoters, photoperiod inducible genetic elements, and the like.

[0167] The self-incompatibility system described herein may be modified to include an ethanol- inducible suppression system, such as the AlcR-pAlcA system. For example, plants are transformed with a construct comprising a first cassette that expresses the transcription factor AlcR in tissues that also express Papaver self-incompatibility genes, such as the PrpSl and PrsSl genes, and a second cassette that comprises one or more artificial miRNAs that target at least one of the Papaver self-incompatibility genes, such as the PrsSl gene, whose expression is controlled by the promoter from the AlcA gene to produce self-incompatible plants. When suppression of the self incompatibility trait in such plants is desired, ethanol is applied to the plants to induce the suppression system. In the AlcR-pAlcA system as used herein, the presence of ethanol causes the AlcR transcription factor to bind to target sequences on the AlcA promoter and activate expression of the artificial miRNA sequences designed to target, for example, the PrsSl gene. Suppression of the PrsSl gene will result in a critical component of the self-incompatibility system to be absent and further will result in the plant being self-compatible.

[0168] Alternatively, the self-incompatibility system described herein may be modified to include a copper-inducible suppression system, such as the ACE1 system. For example, plants are transformed with a construct comprising a first cassette that expresses a fusion ACE1-TALE transcription factor in tissues that also express Papaver self-incompatibility genes, such as the PrpSl and PrsSl genes, and a second cassette that comprises one or more artificial miRNAs thattarget at least one of the Papaver self-incompatibility genes, such as the PrsSl gene, whose expression is controlled by a responsive promoter, such as a promoter that comprises a minimal 35S (-70) promoter and one or more Metal Responsive Elements (MREs) to produce selfincompatible plants. When suppression of the self incompatibility trait in such plants is desired, copper sulfate is applied to the plants to induce the suppression system. The application of copper sulfate may be applied over a specific period of time, such as a period of development associated with flower maturation. In the copper system as used herein, the presence of copper sulfate causes the ACE1-TALE transcription factor to bind to MREs adjoining the minimal promoter and results in expression of the artificial miRNA sequences designed to target, for example, the PrsSl gene. Suppression of the PrsSl gene will result in a critical component of the self-incompatibility system to be absent and further will result in the plant being self-compatible.Example 7. Use of Self-incompatibility to Produce Hybrid Broccoli[001691 In Brassica oleracea, hybrid plants can be produced using a cytoplasmic male- sterility (CMS) system or a self-incompatibility system to enable controlled pollinations. However, both systems have disadvantages. In order to use CMS in a new broccoli hybrid variety, the cytoplasm of the female inbred line is exchanged for a cytoplasm type that carries male-sterility conferring mitochondria. To use self-incompatibility using methods known in the art, a complex scheme involving multiple self-incompatibility alleles is required.

[0170] The self-incompatibility system of the present disclosure can be utilized to produce hybrid broccoli. A transgenic maintainer locus is introduced into a broccoli inbred line. The maintainer locus comprises at least three components: 1) an expression cassette that produces antisense RNA targeting the ARC1 gene; 2) a DsRed expression cassette that produces a detectable color in seeds that carry the maintainer locus; and 3) the bamase gene expressed with a pollen- specific promoter. Antisense RNA production has previously been shown to suppress the ARC1 gene to cause selfcompatibility in otherwise self-incompatible lines in Brassica species (Stone etal., 1999) The other two components of the maintainer locus, DsRed and bamase expression cassettes, have also previously been demonstrated (Kong et al., 2023 and Zhan et al., 1996). The novel combination of these three components to create a maintainer locus enables broccoli inbred varieties with the selfincompatibility trait to be self-pollinated.

[0171] To create broccoli hybrid seeds, a first inbred line that is homozygous for a functional allele of a B. oleraceae self-incompatibility locus and that is hemizygous for the maintainer locusis self-pollinated and seeds are produced. The DsRed containing seeds are hemizygous for the maintainer locus whereas the seeds lacking the DsRed will not carry the maintainer locus. Plants from the first line that lack the maintainer locus are grown in a field that also contain plants from a second inbred line. Pollinations between the second line and the first line occur such that pollen from plants of the second line fertilize flowers on plants of the first line. Pollen from plants of the first line does not self-fertilize. Seeds produced from plants of the first line are hybrids and lack the transgenic maintainer line.

Claims

WHAT IS CLAIMED IS:

1. A method of producing a self-compatible hybrid crop plant, the method comprising: crossing a first self-incompatible plant with a second self-incompatible plant to produce at least one hybrid plant, and wherein at least one of the at least one hybrid plants is selfcompatible.

2. The method according to claim 1, wherein the first self-incompatible plant further comprises a counteracting factor.

3. The method according to claim 2, wherein the second self-incompatible plant further comprises a counteracting factor.

4. The method according to claim 1, wherein the first self-incompatible plant comprises a self-incompatibility locus at a first position and wherein the second self-incompatible plant comprises a second self-incompatibility locus at a second position and wherein the loci are such that they are not genetically linked.

5. The method according to claim 4, further comprising collecting seeds, wherein at least a portion of the seeds lack both of said self-incompatibility loci.

6. The method according to claim 1, further comprising growing the at least one hybrid plant and producing seed.

7. The method according to claim 1, wherein at least one of the first self-incompatible plant or the second self-incompatible plant is an inbred line.

8. A method of propagating a crop plant that is homozygous for a self-incompatibility trait comprising: growing a crop plant that is homozygous for a self-incompatibility trait and that further comprises a counteracting factor that is capable of suppressing the self-incompatibility trait, and allowing the crop plant to sclf-pollinatc to produce a first generation.

9. The method of claim 8, wherein the crop plant is not homozygous for the counteracting factor.

10. The method of claim 9, wherein the counteracting factor is part of a maintainer locus that further comprises a pollen block preventing homozygosity of the counteracting factor and a seed phenotype marker.

11. The method of claim 10, further comprising selecting plants from the first generation that lack the maintainer locus and are self-incompatible.

12. The method of claim 10, wherein multiple plants of the first generation comprise the maintainer locus and other plants of the variety lack the maintainer locus.

13. The method according to claim 8, wherein the counteracting factor is inducible, and wherein the crop plant is exposed to the inducer and wherein the first-generation produces seed.

14. A method of making a self-incompatible crop plant, comprising: transforming a crop plant cell with a recombinant nucleic acid sequence comprising an expression construct comprising self- incompatibility coding sequences; regenerating a transformed crop plant, wherein the transformed crop plant comprises at least one copy of the expression construct; and optionally selfing the regenerated transformed crop plant to generate a homozygous selfincompatible crop plant.

15. The method according to claim 14, wherein the transformation is Agrobacterium mediated, nanoparticle exposure, electroporation, chemical cell-permeation, or bombardment mediated.

16. The method according to claim 14, wherein the transformation comprises introducing a ribonuclease and a transposase.

17. The method according to claim 14, wherein the crop plant cell additionally comprises a recombinant counteracting factor.

18. A crop plant comprising in its genome a first cassette expressing a first 5 allele protein and a second cassette expressing a corresponding second .S' allele protein, wherein expression of the first .S' allele protein and the second .S' allele protein confers to said plant a self-incompatibility trait, and wherein the plant further comprises a counteracting factor that is capable of regulating the self-incompatibility trait.

19. The crop plant of any one of claims 2, 8, 17, or 18, wherein the counteracting factor isinactive and the crop plant is self-incompatible.

20. The crop plant of any one of claims 2, 8, 17, or 18, wherein the counteracting factor is active and the plant is not self-incompatible.

21. The crop plant of any one of claims 2, 8, 17, or 18, wherein the counteracting factor is inducible.

22. The crop plant according to any one of claims 2, 7, 16, 17, or 18, wherein the counteracting factor is regulated by a multicomponent system.

23. The crop plant of claim 21, wherein the counteracting factor is induced by an inducer selected from blue light, temperature, ethanol, salt, mandipropamid, copper sulfate, tetracycline and derivatives thereof, and any combinations thereof.

24. The plant of claim 22, wherein the multicomponent system is selected from AlcR, ACE1, TetR, GAL4 DNA binding domain fused to a modified PYR1, ACE1 fused to a yeast transcription activation domain, HAB1 protein fused to a transcription activation domain, P-AtAT5G52310:A, and combinations thereof.

25. The crop plant according to 18, wherein the .S' allele protein is selected from a selfincompatibility system derived from Papaveraceae, Brassicaceae, Poaceae, Asteraceae, Convolvulaceae, Betulaceae, Caryophyllaceae, Sterculiaceae, and Polemoniaceae26. The crop plant of claim 18, wherein the first .S' allele protein is a PrpS protein and the second 5 allele protein is a PrsS protein.

27. The crop plant of claim 18, wherein the first .S' allele protein is derived from Papaver rhoeas.

28. The crop plant of claim 2, wherein said .S' allele proteins are SI alleles or S3 alleles.

29. The crop plant of any one of claims 2, 8, 17, or 18, wherein the counteracting factor is a genetic element that suppresses the self-incompatibility response in a plant.

30. The crop plant according to claim 29, wherein the genetic clement is selected from the group consisting of: antisense, co-suppression, viral-suppression, hairpin suppression, stem-loop suppression, RNAi-based approaches, double-stranded RNA, small RNA-based approaches, artificial transcription factor repressors, and deletion or inversion of a DNA sequence by a site-specific recombinase.

31. The method of claim 1, wherein the self-incompatibility trait in each of the first selfincompatible plant and the second self-incompatible plant is caused by self- incompatibility genes from a species other than the said plants that has been added to the plants.

32. The method of claim 1, wherein at least one of the first self-incompatible plant and the second self-incompatible plant comprises at least one of a Prs gene and a Prp gene.

33. The method of claim 1, wherein the first self-incompatible plant comprises a first cassette that regulates the self-incompatibility trait of the second plant and the second self-incompatible plant comprises a second cassette that regulates the self-incapability trait of the first plant.

34. The method of claim 33, wherein at least one of the first cassette and the second cassette regulates at least one of a Prs gene and a Prp gene.

35. The crop plant of any one of claims 2, 8, 17, or 18, wherein the counteracting factor causes targeted protein degradation.

36. The crop plant of any one of claims 2, 8, 17, or 18, wherein the counteracting factor suppresses transcription.

37. The crop plant of any one of claims 1, 8, 14, and 18, wherein the crop plant is selected from the group consisting of soybean, com, cotton, canola, sugar cane, barley, oats, sorghum, tomato, sunflower, rice, quinoa, rye, and wheat.

38. A breeding pair of crop plants, comprising a first self-incompatible plant and a second selfincompatible plant, wherein the first self-incompatible plant expresses a pollen- specific 5 allele protein, a corresponding pistil- specific 5 allele protein, and a genetic element that suppresses expression of a pollen- specific or pistil-specific .S' allele protein not expressed in said plant but expressed in said second self-incompatible plant, wherein the second self- incompatible plant expresses a pollen- specific .S' allele protein, a corresponding pistil- specific 5 allele protein, and a genetic element that suppresses expression of a pollen- specific or pistil- specific .S' allele protein not expressed in said plant but expressed in said first self-incompatible plant; wherein crossing the plants results in progeny plants that are self-compatible.

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