Methods and compositions for preventing hermaphroditism in cannabis plants and for making male sterile cannabis plants

Abstract

This document describes methods and compositions for preventing hermaphroditism in Cannabis plants, as well as methods and compositions for making male sterile Cannabis plants.

Description

[0001] METHODS AND COMPOSITIONS FOR PREVENTING HERMAPHRODITISM IN CANNABIS PLANTS AND FOR MAKING MALE STERILE CANNABIS PLANTS

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Application No. 63 / 634,015 filed on April 15, 2024.

[0004] TECHNICAL FIELD

[0005] This disclosure generally relates to methods and compositions for preventing hermaphroditism in Cannabis plants and for making male sterile Cannabis plants.

[0006] BACKGROUND

[0007] Cannabis is largely dioecious, producing male plants as the heterogametic sex (XY) and female plants as the homogametic sex (XX). Cannabis also can occur naturally in monoecious forms in which male and female reproductive parts exist on the same plant (e.g., male flowers form on a XX female flowering plant; female flowers form on a XY male flowering plant), known as hermaphroditism. In addition to occurring naturally, hermaphroditism in Cannabis can be purposefully induced (e.g., artificially) or can occur when Cannabis plants are stressed. For example, breeders can artificially induce hermaphroditism in XX female plants through the exogenous application of different chemicals for planned male flower emergence to create specific crosses, where pollen from a female plant is then used to self-pollinate to create feminized seed lines. Feminized seed lines are 100% homogametic (XX) female plants, which maximize flower production and, thus, profits, by filling cultivation space with only females. However, hermaphroditism can ensue (e.g., from environmental stresses) in any Cannabis plant, whether homogametic female (XX) or not, as an unanticipated and unintended occurrence.

[0008] Female hermaphroditism is the emergence of pistillate flowers on a Cannabis plant during the flowering period followed by anther formation, which produces viable pollen and results in the undesirable fertilizing of itself and surrounding plants. Unintended pollination from these hermaphrodites can turn valuable sinsemilla flowers (i.e., unpollinated female flowers) into pollinated flowers that produce unwanted seeds, destroying plant breeding schemes as well as harming female flower production during cultivation. Additionally, pollen fertilization causes significant reductions in cannabinoid and terpene production, driving down medicinal and economic value of that flower significantly.

[0009] Currently, the only commercial assays for biological sex in Cannabis only test for homogametic females (XX) and heterogametic males (XY). No commercial tests exist on the market to test for hermaphrodites, monoecious plants, flower fertility, or plants susceptible or resistant to hermaphroditism. Additionally, to date, there are no commercial-scale solutions to eliminate the impact of male flower reproduction, such as hermaphroditism, during Cannabis cultivation and breeding. Unfortunately, spontaneous hermaphrodites commonly occur in commercial settings, and unanticipated occurrence of hermaphroditism can result in commercial devastation during both Cannabis breeding and cultivation in the form of decreased quality and quantity of yield.

[0010] Lastly, male sterility in plants is a common tool used by breeders to maximize hybrid vigor, or heterosis, in hybrid crops. Hybrid vigor occurs when the offspring of a cross of two inbred lines outperform the parent lines, creating higher expression of a phenotype (e.g., yield). Male sterile lines are used to ensure outcrossing because their sterility prevents plants from natural self-pollination. Establishing stable male sterile plants for Fl hybrid production can be difficult, requiring significant efforts and investments. Producing hybrid seeds from selfpollinating plants requires extensive emasculation (i.e., the removal of functional pollen producing plant parts) to prevent self-pollination, which historically has been done manually, by machine or chemically. Consequently, the control of both male-sterility during hybrid production and restored-fertility in Fl hybrids are major bottlenecks in commercial hybrid seed production, and the creation of male sterile Cannabis plants is needed to create Cannabis plants having improved agricultural yield, health and vigor. Therefore, the development of male sterile plant lines and a sterility-restoration system would greatly improve Cannabis breeding potential, use and output. SUMMARY

[0011] This disclosure describes several genes related to male flower production in Cannabis. As described herein, expression of one or more of these genes can be altered (e.g., silenced) to control male fertility and the impact of hermaphroditism, or to produce male sterile plants.

[0012] Historically, the genus Cannabis included Cannabis sativa L., Cannabis indica Lam. and Cannabis ruderalis Janisch, which were considered highly related and often interbred in commercial varieties. It is now believed, however, that these lineages are all the same species, Cannabis sativa (Lapierre et al., 2023, Genome, 66(8):202-l l; McPartland, 2018, Cannabis Cannabinoid Res., 3 ( 1 ) :203 -12). Thus, as used herein, Cannabis refers to the entire genus.

[0013] In one aspect, compositions comprising an RNA nucleic acid molecule are provided, wherein the RNA nucleic acid molecule is at least 10 nucleotides in length and has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least one of the genes selected from: AMS, bHLH91, TM6, STP6, DYT1, MYB35, MYB35, aw3 MYB80, wherein the RNA nucleic acid molecule effectively silences expression of the at least one gene, wherein the AMS gene comprises any of SEQ ID NOs: 1 or 81-88; wherein the bHLH91 gene comprises any of SEQ ID NOs: 2 or 89-92; wherein the TM6 gene comprises any of SEQ ID NOs: 3 or 93-99; wherein the STP6 gene comprises any of SEQ ID NOs: 4 or 100-108; wherein the DY T1 gene comprises any of SEQ ID NOs: 5 or 109-115; wherein the MYB35-15 gene comprises any of SEQ ID NOs: 6 or 116-119; wherein the MYB35-16 gene comprises any of SEQ ID NOs: 7 or 120-124; wherein the MYB80 gene comprises any of SEQ ID NOs: 8 or 125-127.

[0014] In one embodiment, the composition comprises RNA nucleic acid molecules at least 10 nucleotides in length and having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least two of the genes selected from AMS, bHLH91, TM6, STP6, DYT1, MYB35, MYB35, and MYB80, wherein the RNA nucleic acid molecules effectively silence expression of the at least two genes.

[0015] In one embodiment, the composition is an aqueous solution. In one embodiment, the composition is formulated as a spray. In one embodiment, the composition is dried / lyophilized.

[0016] In one embodiment, the RNA nucleic acid molecules are double-stranded (ds) RNA nucleic acid molecules. In one embodiment, the dsRNA nucleic acid molecules are selected from any of SEQ ID NOs:33-43, wherein each of the dsRNA nucleic acid molecules effectively silence expression of the AMS gene. In one embodiment, the dsRNA nucleic acid molecules are selected from any of SEQ ID NOs:44-54, wherein each of the dsRNA nucleic acid molecules effectively silence expression of the bHLH91 gene. In one embodiment, the dsRNA nucleic acid molecules are selected from any of SEQ ID NOs: 55-65, wherein each of the dsRNA nucleic acid molecules effectively silence expression of the at TM6 gene. In one embodiment, the dsRNA nucleic acid molecules are selected from any of SEQ ID NOs: 66-80, wherein each of the dsRNA nucleic acid molecules effectively silence expression of the STP6 gene.

[0017] In one embodiment, the composition further comprises a delivery vehicle. In one embodiment, the delivery vehicle is a nanoparticle.

[0018] In another aspect, articles of manufacture are provided. Such articles of manufacture typically include RNA nucleic acid molecules, wherein the RNA nucleic acid molecules are at least 10 nucleotides in length and have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least one of the genes selected from AMS, bHLH91, TM6, STP6, DYT1, MYB35, MYB35, and MYB80, wherein the RNA nucleic acid molecules effectively silence expression of the at least one gene, wherein the AW.S’ gene comprises any of SEQ ID NOs: 1 or 81-88; wherein the bHLH91 gene comprises any of SEQ ID NOs: 2 or 89-92; wherein the TM6 gene comprises any of SEQ ID NOs: 3 or 93-99; wherein the STP6 gene comprises any of SEQ ID NOs: 4 or 100-108; wherein the DYT1 gene comprises any of SEQ ID NOs: 5 or 109-115; wherein the MYB35-15 gene comprises any of SEQ ID NOs: 6 or 116-119; wherein the MYB35- 16 gene comprises any of SEQ ID NOs: 7 or 120-124; wherein the MYB80 gene comprises any of SEQ ID NOs: 8 or 125-127; and a delivery vehicle.

[0019] In one embodiment, the delivery vehicle is a nanoparticle. In one embodiment, the nanoparticle is selected from chitosan salts (e.g., CQAS), carbon dots, liposomes, clay nanosheets, and gold nanoparticles. In one embodiment, the delivery vehicle is conjugated to the RNA nucleic acid molecules. In one embodiment, the delivery vehicle is not conjugated to the RNA nucleic acid molecules.

[0020] In one embodiment, the articles of manufacture further include a linker.

[0021] In one embodiment, the article of manufacture is formulated as a spray.

[0022] In still another aspect, methods of preventing hermaphroditism (e.g., in females) or the viability in male flowers (e.g., in male hermaphrodites or biological males) in Cannabis plants is provided. Such methods typically include contacting Cannabis plants with any of the compositions described herein. In one embodiment, the contacting is spraying. In one embodiment, the contacting is performed for about 10 seconds to about 60 seconds (per plant) at least one time a day. In one embodiment, the contacting is performed for about 10 seconds to about 60 seconds (per plant) a plurality of times each day.

[0023] In yet another aspect, methods of producing male sterile Cannabis plants (e.g., hermaphroditic female or a biological male) are provided. Such methods typically include introducing a nucleic acid molecule into Cannabis cells to produce genetically-engineered Cannabis cells, wherein the nucleic acid molecule comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more genes selected from AMS, bHLH91, TM6, STP6, DYT1, MYB35, MYB35, and MYB80 under direction of a promoter sequence, wherein the AMS gene comprises any of SEQ ID NOs: 1 or 81-88; wherein the bHLH91 gene comprises any of SEQ ID NOs: 2 or 89-92; wherein the TM6 gene comprises any of SEQ ID NOs: 3 or 93-99; wherein the STP6 gene comprises any of SEQ ID NOs: 4 or 100-108; wherein the DYT1 gene comprises any of SEQ ID NOs: 5 or 109-115; wherein the MYB35-15 gene comprises any of SEQ ID NOs: 6 or 116-119; wherein the MYB35- 16 gene comprises any of SEQ ID NOs: 7 or 120-124; wherein the MYB80 gene comprises any of SEQ ID NOs: 8 or 125-127; and regenerating a genetically engineered Cannabis plant from the genetically engineered Cannabis cells, wherein the genetically engineered Cannabis plant exhibits reduced or no expression from the one or more genes or reduced, altered, or lack of expression or function of the expressed protein.

[0024] In one embodiment, expression of the nucleic acid molecule triggers RNAi, thereby silencing expression of the at least one gene.

[0025] In one embodiment, pollen produced by the genetically engineered Cannabis plants is non-viable.

[0026] In one embodiment, the methods further include selecting for genetically engineered Cannabis plants.

[0027] In one aspect, methods of producing male sterile Cannabis plants (e.g., hermaphroditic female or a biological male) is provided. Such methods typically include introducing a nucleic acid molecule into Cannabis cells under conditions in which the genome of the Cannabis cells is edited to produce edited Cannabis cells, wherein the nucleic acid molecule has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more genes selected from AMS, bHLH91, TM6, STP6, DYTl, MYB35, MYB35, and MYB80, wherein the AMS gene comprises any of SEQ ID NOs: 1 or 81-88; wherein the bHLH91 gene comprises any of SEQ ID NOs: 2 or 89-92; wherein the TM6 gene comprises any of SEQ ID NOs: 3 or 93-99; wherein the STP6 gene comprises any of SEQ ID NOs: 4 or 100-108; wherein the DYTl gene comprises any of SEQ ID NOs: 5 or 109-115; wherein the MYB35-15 gene comprises any of SEQ ID NOs: 6 or 116-119; wherein the MYB35-16 gene comprises any of SEQ ID NOs: 7 or 120-124; wherein the MYB80 gene comprises any of SEQ ID NOs: 8 or 125-127; and regenerating an edited Cannabis plant from the edited Cannabis cells, wherein the edited Cannabis plant exhibits reduced or no expression from the one or more genes or reduced, altered, or lack of expression or function of the expressed protein.

[0028] In one embodiment, the nucleic acid molecule comprises one or more constructs comprising gene editing components for editing at least one of the genes selected from AMS, bHLH91, 1M6, STP6, DYTl, MYB35, MYB35, and MYB80, wherein the gene editing reduces or knocks-out expression from the one or more genes or the expressed protein exhibits reduced, altered, or lack of function.

[0029] In one embodiment, the gene editing components are selected from CRISPR / Cas components, TALEN components, or the like.

[0030] In one embodiment, the pollen produced by the edited Cannabis plants is non-viable.

[0031] In still another aspect, methods of making a Cannabis plant (e.g., hermaphroditic female or a biological male) male sterile are provided. Such methods typically include contacting Cannabis seeds with a mutagen to create mutant Cannabis seeds; producing mutant Cannabis plants from the mutant Cannabis seeds; and screening the mutant Cannabis plants for those having a mutation in one or more genes selected from AMS, bHLH91, TM6, STP6, DYTl, MYB35, MYB35, and MYB80, wherein the mutant Cannabis plants exhibit reduced or no expression from the one or more genes or reduced, altered, or lack of expression or function of the expressed protein.

[0032] In still another aspect, methods of evaluating a Cannabis plant for the propensity to trigger male flower formation (e.g., hermaphroditism) are provided. Such methods typically include determining the presence or absence of a variation in sequence within one or more genes selected from AMS, bHLH91, 1M6, STP6, DYTl, MYB35, MYB35, and MYB80, wherein the presence or absence of sequence variation within one or more of the genes is indicative of the propensity to trigger male flower formation (e.g., hermaphroditism) in the Cannabis plant.

[0033] In another aspect, methods of obtaining a male sterile or hermaphroditic-resistant Cannabis plant are provided. Such methods typically include a) crossing a first Cannabis plant and a second Cannabis plant, wherein the first Cannabis plant exhibits male sterility or hermaphroditic-resistance; b) hybridizing genomic DNAfrom cells obtained from the cross or from cells of later filial generations of the cross with one or more markers within one or more genes selected from AMS, bHLH91, TM6, STP6, DYT1, MYB35, MYB35, and MYB80 and; c) selecting a plant in which the genomic DNA hybridizes with one or more of the markers within one or more of the genes selected from AMS, bHLH91, TM6, STP6, DYT1, MYB35, MYB35, and MYB80 to obtain a male sterile or hermaphroditic-resistant plant.

[0034] In one aspect, compositions are provided that include an RNA nucleic acid molecule, where the RNA nucleic acid molecule has at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to at least one of the genes selected from AMS (SEQ ID NO: 1), bHLH91 (SEQ ID NO:2), TM6 (SEQ ID NO:3), STP6 (SEQ ID NO:4), DYT1 (SEQ ID NO:5), MYB35 (SEQ ID NO:6), MYB35 (SEQ ID NO:7), and MYB80 (SEQ ID NO: 8), where the RNA nucleic acid molecule effectively silences expression of the at least one gene.

[0035] In some embodiments, the composition is an aqueous solution. In some embodiments, the composition is formulated as a spray. In some embodiments, the composition is dried / lyophilized.

[0036] In some embodiments, the RNA is a double-stranded (ds) RNA.

[0037] In some embodiments, the composition comprises RNA nucleic acid molecules having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to at least two of the genes selected from AMS (SEQ ID NO: 1), bHLH91 (SEQ ID NO:2), TM6 (SEQ ID NO:3), STP6 (SEQ ID NO:4), DYT1 (SEQ ID NO:5), MYB35 (SEQ ID NO:6), MYB35 (SEQ ID NO:7), and MYB80 (SEQ ID NO:8), wherein the RNA nucleic acid molecules effectively silence expression of the at least two genes.

[0038] In some embodiments, the RNA nucleic acid molecules having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the AMS gene are selected from any of SEQ ID NOs:33-43, wherein each of the RNA nucleic acid molecules effectively silence expression of the AMS gene.

[0039] In some embodiments, the RNA nucleic acid molecules having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the bHLH91 gene are selected from any of SEQ ID NOs:44-54, wherein each of the RNA nucleic acid molecules effectively silence expression of the bHLH91 gene.

[0040] In some embodiments, the RNA nucleic acid molecules having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the TM6 gene are selected from any of SEQ ID NOs:55-65, wherein each of the RNA nucleic acid molecules effectively silence expression of the at TM6 gene.

[0041] In some embodiments, the RNA nucleic acid molecules having at least 80% sequence identity (e g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to the STP6 gene are selected from any of SEQ ID NOs:66-80, wherein each of the RNA nucleic acid molecules effectively silence expression of the STP6 gene.

[0042] In some embodiments, the composition further comprises a delivery vehicle. In some embodiments, the delivery vehicle is a nanoparticle.

[0043] In another aspect, articles of manufacture are provided that include RNA nucleic acid molecules, wherein the RNA nucleic acid molecules have at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to at least one of the genes selected from AMS (SEQ ID NO: 1), bHLH91 (SEQ ID NO:2), TM6 (SEQ ID NO:3), STP6 (SEQ ID NO:4), DYT1 (SEQ ID NO:5), MYB35 (SEQ ID NO:6), MYB35 (SEQ ID NO:7), and MYB80 (SEQ ID NO:8), wherein the RNA nucleic acid molecules effectively silence expression of the at least one gene; and a delivery vehicle.

[0044] In some embodiments, the delivery vehicle is a nanoparticle. Representative nanoparticles include, without limitation, chitosan salts (e.g., CQAS), carbon dots, liposomes, clay nanosheets, and gold nanoparticles. In some embodiments, the delivery vehicle is conjugated to the RNA nucleic acid molecules. In some embodiments, the delivery vehicle is not conjugated to the RNA nucleic acid molecules.

[0045] In some embodiments, the article of manufacture further includes a linker.

[0046] In some embodiments, the article of manufacture is formulated as a spray. In yet another aspect, methods of preventing hermaphroditism (e.g., in females) or the viability in male flowers (e.g., in male hermaphrodites or biological males) in Cannabis plants is provided. Such methods typically include contacting Cannabis plants with a composition as described herein.

[0047] In some embodiments, the contacting is spraying. In some embodiments, the contacting is performed for about 10 seconds to about 60 seconds (per plant) at least one time a day. In some embodiments, the contacting is performed for about 10 seconds to about 60 seconds (per plant) a plurality of times each day.

[0048] In still another aspect, methods of producing male sterile Cannabis plants (e.g., hermaphroditic female or a biological male) are provided. Such methods typically include introducing a nucleic acid molecule into Cannabis cells to produce genetically-engineered Cannabis cells, wherein the nucleic acid molecule comprises a sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to one or more genes selected from AMS (SEQ ID NO: 1), bHLH91 (SEQ ID NO:2), TM6 (SEQ ID NON), STP6 (SEQ ID NON), DYT1 (SEQ ID NO:5), MYB35 (SEQ ID NO:6), MYB35 (SEQ ID NON), and MYB80 (SEQ ID NO:8) under direction of a promoter sequence; and regenerating a genetically engineered Cannabis plant from the genetically engineered Cannabis cells, where the genetically engineered Cannabis plant exhibits reduced or no expression from the one or more genes or reduced, altered, or lack of expression or function of the expressed protein.

[0049] In some embodiments, expression of the nucleic acid molecule triggers RNAi, thereby silencing expression of the at least one gene. In some embodiments, pollen produced by the genetically engineered Cannabis plants is non-viable. In some embodiments, the methos further include selecting for genetically engineered Cannabis plants.

[0050] In another aspect, methods of producing male sterile Cannabis plants (e.g., hermaphroditic female or a biological male) are provided. Such methods typically include introducing a nucleic acid molecule into Cannabis cells under conditions in which the genome of the Cannabis cells is edited to produce edited Cannabis cells, wherein the nucleic acid molecule has at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to one or more genes selected from AMS (SEQ ID NO: 1), bHLH91 (SEQ ID NON), TM6 (SEQ ID NON), STP6 (SEQ ID NON), DYT1 (SEQ ID NO:5), MYB35 (SEQ ID N0:6), MYB35 (SEQ ID N0:7), and MYB80 (SEQ ID N0:8); and regenerating an edited Cannabis plant from the edited Cannabis cells, where the edited Cannabis plant exhibits reduced or no expression from the one or more genes or reduced, altered, or lack of expression or function of the expressed protein.

[0051] In some embodiments, the nucleic acid molecule comprises one or more constructs comprising gene editing components for editing at least one of the genes selected from AMS (SEQ ID NO: 1), bHLH91 (SEQ ID NO:2), TM6 (SEQ ID NO:3), STP6 (SEQ ID NO:4), DYT1 (SEQ ID NO:5), MYB35 (SEQ ID NO:6), MYB35 (SEQ ID NO:7), and MYB80 (SEQ ID NO:8), wherein the gene editing reduces or knocks-out expression from the one or more genes or the expressed protein exhibits reduced, altered, or lack of function.

[0052] In some embodiments, the gene editing components are selected from CRISPR / Cas components, TALEN components, or the like.

[0053] In some embodiments, the pollen produced by the edited Cannabis plants is non-viable.

[0054] In yet another aspect, methods of making a Cannabis plant (e.g., hermaphroditic female or a biological male) male sterile are provided. Such methods typically include contacting Cannabis seeds with a mutagen to create mutant Cannabis seeds; producing mutant Cannabis plants from the mutant Cannabis seeds; and screening the mutant Cannabis plants for those having a mutation in one or more genes selected from AMS (SEQ ID NO: 1), bHLH91 (SEQ ID NO:2), TM6 (SEQ ID NO:3), STP6 (SEQ ID NO:4), DYT1 (SEQ ID NO:5), MYB35 (SEQ ID NO:6), MYB35 (SEQ ID NO:7), and MYB80 (SEQ ID NO:8), where the mutant Cannabis plants exhibit reduced or no expression from the one or more genes or reduced, altered, or lack of expression or function of the expressed protein.

[0055] In still another aspect, methods of evaluating a Cannabis plant for the propensity to trigger male flower formation (e.g., hermaphroditism) are provided. Such methods typically include determining the presence or absence of a variation in sequence (e.g., SNP, insertion, deletion, etc.) within one or more genes selected from AMS (SEQ ID NO: 1), bHLH91 (SEQ ID NO:2), TM6 (SEQ ID NO:3), STP6 (SEQ ID NO:4), DYT1 (SEQ ID NO:5), MYB35 (SEQ ID NO:6), MYB35 (SEQ ID NO:7), and MYB80 (SEQ ID NO:8), where the presence or absence in sequence variation within one or more of the genes is indicative of the propensity to trigger male flower formation (e.g., hermaphroditism) in the Cannabis plant. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions of matter belong. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of matter, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0056] DESCRIPTION OF DRAWINGS

[0057] FIGs. 1A-1D are schematics showing the dsRNA constructs for AMS (1A), bHLH91 (IB), TM6 (1C), and STP6 (ID).

[0058] FIG. 2 are schematics showing the experimental design for each cocktail.

[0059] FIG. 3 is an image of stained viable pollen from the Control Treatment group in the Cocktail 1 Experimental. The image shows almost 100% viable pollen from an ‘Original Bacio’ plant after being sprayed with 0.02% CQAS solution with no dsRNA (i.e., control). This pollen stain slide shows the male pollen produced by the female plant after STS application were fully viable as non-shriveled pollen grains with dark contents inside the grain indicate complete pollen viability (i.e., non-aborted). Pollen grains that look fully or largely empty without dark contents inside are not viable (i.e., aborted pollen).

[0060] FIG. 4 is an image of stained pollen from the Cocktail 1 Experimental Treatment group. The image shows nearly 100% non-viable pollen from ‘Original Bacio’ plant after application of the dsRNA — 0.02% CQAS solution. Effect of dsRNA Cocktail 1 on an ‘Original Bacio’ plant resulted in shriveled pollen grains without dark contents inside the grain, which indicates complete pollen inviability (i.e., aborted pollen). This image shows almost complete pollen abortion after four weeks of biweekly spraying. The pollen in this image was harvested three days after the final dsRNA spray.

[0061] FIG. 5 is an image of stained pollen from the Cocktail 2 Experimental Design Grow Tent prior to dsRNA — 0.02% CQAS solution application, representing the before application (i.e., control) conditions. Image is of 100% viable pollen from an ‘Original Bacio’ plant. Nonshriveled pollen grains with dark contents inside the grain indicate complete pollen viability (non-aborted). Pollen grains that look fully or largely empty without dark contents inside are not viable. This pollen stain slide shows the male pollen produced by the female plant after STS application but prior to Cocktail 2 dsRNA application were fully viable.

[0062] FIG. 6 is an image of stained pollen from the Cocktail 2 Experimental Design Grow Tent after dsRNA — 0.02% CQAS solution application. Effect of dsRNA — 0.02% CQAS Cocktail 2 solution on the same ‘Original Bacio’ plant as FIG. 5 showcasing a typical plant response posttreatment. This pollen sample was taken six days after a single application of Cocktail 2 dsRNA — 0.02% CQAS spray. Non-shriveled pollen grains with dark contents inside the grain indicate complete pollen viability (non-abortion), while pollen grains that look fully or largely empty without dark contents inside are not viable. The pollen grains in this image are all non- viable.

[0063] FIG. 7 is a graph showing pollen abortion rates in the presence of Cocktail 1 between the Control Treatment Grow Tent and the Experimental Treatment Grow Tent. The plants in the Experimental Treatment Grow Tent received a spray containing bHLH91 dsRNA and AMS dsRNA. Pollen abortion significantly increased in the Experimental Tent compared to the Control Tent. *, P<0.05.

[0064] FIG. 8 is a graph showing pollen abortion rates before and after application of Cocktail 2, which contains STP6 dsRNA and TM6 dsRNA. Pollen abortion significantly increased after spraying. *, P<0.05.

[0065] FIG. 9 is a graph showing the effect of Cocktail 1 (bHLH91 and AMS dsRNA) on AMS gene expression. AMS showed significantly reduced gene expression when sprayed with AMS dsRNA relative to control plants. Gene expression values are log-transformed. Statistical significance of ** indicates P<0.01.

[0066] FIG. 10 is a graph showing the effect of Cocktail 1 (bHLH91 and AMS dsRNA) on bHLH91 gene expression. bHLH91 showed significantly reduced gene expression when sprayed with bHLH91 dsRNA relative to control plants. Gene expression values are log-transformed. Statistical significance of ** indicates P<0.01.

[0067] FIG. 11 is a graph showing the effect of Cocktail 2 (TM6 and STP6 dsRNA) on STP6 gene expression. STP6 showed significantly reduced gene expression after being sprayed with STP6 dsRNA relative to expression before being sprayed. Gene expression values are log- transformed and analysis was paired. Statistical significance of ** indicates P<0.01 FIG. 12 is a graph showing the effect of Cocktail 2 (TM6 and STP6 dsRNA) on TM6 gene expression. TM6 showed significantly reduced gene expression after being sprayed with TM6 dsRNA in ‘Original Bacio’ relative to expression before being sprayed. Gene expression values are log-transformed and analysis was paired. Statistical significance of * indicates P<0.05., n.s. means not statistically significant.

[0068] FIG. 13-1 and 13-2 is a schematic pile-up showing the conserved and variable regions within the 8 common genotypes for AMS.

[0069] FIG. 14-1 and 14-2 is a schematic pile-up showing the conserved and variable regions within the 8 common genotypes for bHLH9I.

[0070] FIG. 15-1 and 15-2 is a schematic pile-up showing the conserved and variable regions within the 8 common genotypes for TM6.

[0071] FIG. 16-1 and 16-2 is a schematic pile-up showing the conserved and variable regions within the 8 common genotypes for STP6.

[0072] FIG. 17-1 and 17-2 is a schematic pile-up showing the conserved and variable regions within the 8 common genotypes for DYT1.

[0073] FIG. 18-1 and 18-2 is a schematic pile-up showing the conserved and variable regions within the 8 common genotypes for MYB35-15.

[0074] FIG. 19-1 and 19-2 is a schematic pile-up showing the conserved and variable regions within the 8 common genotypes for MYB35-16.

[0075] FIG. 20-1 and 20-2 is a schematic pile-up showing the conserved and variable regions within the 8 common genotypes for MYB80.

[0076] DETAILED DESCRIPTION

[0077] Plant male sterility refers to the failure of a plant to produce dehiscent anthers, functional pollen, and viable male gametes. In this disclosure, we identify key male reproductive genes in the genus Cannabis and demonstrate how silencing these genes creates male sterility by inhibiting functional pollen formation and the formation of viable male gametes in a Cannabis plant. Additionally, this document describes an anti-hermaphroditism spray for Cannabis plant cultivation and breeding management, and establishes methods to create male sterile, hermaphroditic resistant, and, or hermaphroditical stress-resistant Cannabis plants, plant lines or cultivars through the inactivation of these genes. Further, through the manipulation of these genes, this document describes a male-sterility-fertility restoration system in Cannabis, and also the use of one or more of these genes in marker-assisted breeding programs.

[0078] Genes Involved in Male Flower Production

[0079] Using methods described in this disclosure, eight genes were identified as having a role in the hermaphrodite phenotype and male reproductive development in Cannabis. The sequence of each gene from cslO are provided as follows: AMS (SEQ ID NO:1), bHLH91 (SEQ ID NO:2), TM6 (SEQ ID NO:3), STP6 (SEQ ID NO:4), DYT1 (SEQ ID NO:5), MYB35-15 (SEQ ID NO:6), MYB35-16 (SEQ ID NO:7) and / WIW (SEQ ID NO:8) (see Table 1). The eight genes are predicted to encode the proteins having the amino acid sequences shown in SEQ ID NO:9 (AMS), SEQ ID NO: 10 (bHLH91), SEQ ID NO: 11 (TM6), SEQ ID NO: 12 (STP6), SEQ ID NO:13 and 14 (DYT1), SEQ ID NO:15 (MYB35-15), SEQ ID NO: 16 (MYB35-16), and SEQ ID NO: 17 (MYB80). Brief summaries of each are provided below, with a focus on homologs in other plant species (i.e., orthologs) and / or paralogs, since, prior to this disclosure, these genes have not been identified or characterized in Cannabis.

[0080] Gene is used herein to refer to the nucleic acid sequences that, upon expression, produce a protein (i.e., the coding sequence), and also to non-expressed nucleic acid sequences (e.g., introns, 3’ and / or 5’ non-translated regions, etc.) and nucleic acid sequences that act as regulatory regions (e.g., promoters, enhancers, etc.). It would be appreciated that regulatory regions can act on a coding sequence from afar (e.g., up to 5 kb upstream and / or downstream of a coding sequence; see, for example, Chen et al., 2020, Nat. Commun., 11 :2472).

[0081] Each of the genes identified as being involved in male flower production was evaluated across almost 200 different publicly-available Cannabis accessions to examine the variation found within each gene in Cannabis. The gene sequence, including up to 5 kb upstream and downstream of the coding sequence for each individual were clustered into groups. A reference sequence representative of each cluster was chosen to represent the common genotype. Pairwise blast similarities were calculated between the reference and the other individuals in the cluster. Consensus sequences were identified for each gene that, based on sequence identity, represent the primary categories of variation. AMS (A borted Microspore)

[0082] The ABORTED MICROSPORE (AMS) gene in Arabidopsis is a transcription factor involved in the gene cascade related to anther and microspore development. AMS is a regulator of pollen wall formation and sporopollenin biosynthesis and is a homolog to TDR1 (TAPETUM DEGENERATION RETARDATIONl) in rice. The AMS gene sequence from Cannabis is shown in SEQ ID NO: 1, and the putative encoded amino acid sequence is shown in SEQ ID NO:9.

[0083] SEQ ID NOs: 81-88 represent the eight most common genotype sequences for AMS (see Table 2), and FIG. 13 is a schematic alignment of the eight most common genotype sequences for / IMS', depicting conserved regions and variable regions. bHLH91 (Basic Helix-Loop-Helix 91) bHLH91 is a transcription factor involved in developing pollen and tapetum in Arabidopsis. bHLH91 is a homolog to bHLHIO, bHLH89, and bHLH90 in Arabidopsis and to EAT1 in rice. The fact that Arabidopsis has multiple homologs to the Cannabis bHLH91 indicates that sex determination in Cannabis is different than in species like Arabidopsis . The bHLH91 gene sequence from Cannabis is shown in SEQ ID NO: 2, and the putative encoded amino acid sequence is shown in SEQ ID NO: 10.

[0084] SEQ ID NOs: 89-92 represent the four most common genotype sequences for bHLH91 (see Table 2), and FIG. 14 is a schematic alignment of the four most common genotype sequences for bHLH91, depicting conserved and variable regions.

[0085] TM6 (agamous-like MADS-box protein TM6)

[0086] TOMATO MADS BOX GENE6 (TM6) is paralogous to APETALA3 (AP3) in Arabidopsis. Different plant species express one or the other for petal or stamen development, where TM6 informs stamen development. The TM6 gene sequence from Cannabis is shown in SEQ ID NO:3, and the putative encoded amino acid sequence is shown in SEQ ID NO: 11.

[0087] SEQ ID NOs: 93-99 represent the seven most common genotype sequences for TM6 (see Table 2), and FIG. 15 is a schematic alignment of the seven most common genotype sequences for TM6, depicting the conserved and variable regions.

[0088] STP6 (Sugar Transport Protein 6)

[0089] Sugar Transport Protein6 (STP6) as well as other sugar transport proteins are mainly responsible for glucose uptake into the pollen grain in different plant species. In many species, STP6 is involved in the sugar supply of the germinating pollen or the growing pollen tube. The STP6 gene sequence from Cannabis is shown in SEQ ID NO:4, and the putative encoded amino acid sequence is shown in SEQ ID NO: 12.

[0090] SEQ ID NOs: 100-108 represent the nine most common genotype sequences for STP6 (see Table 2), and FIG. 16 is a schematic alignment of the nine most common genotype sequences for STP6, depicting the conserved and variable regions.

[0091] DYT1 (DYSFUNCTIONAL TAPETUM 1)

[0092] Arabidopsis DYT 1 (DYSFUNCTIONAL TAPETUM 1) is homologous with UNDEVELOPED TAPETUM1 (UDT1) in rice and Male Sterility32 (Ms32) in maize and is involved in anther development and male fertility. The DYT1 gene sequence from Cannabis is shown in SEQ ID NO:5, and the putative encoded amino acid sequence includes two isoforms shown in SEQ ID NOs: 13 and 14.

[0093] SEQ ID NOs: 109-115 represent the seven most common genotype sequences for DYT1 (see Table 2), and FIG. 17 is a schematic alignment of the seven most common genotype sequences for DYT1, depicting the conserved and variable regions.

[0094] TDF1 (Defective in Meristem Development and Function 1)

[0095] TDF1 (DEFECTIVE IN MERISTEM DEVELOPMENT AND FUNCTION 7) in Arabidopsis encodes a putative R2R3 MYB transcription factor that plays a role in tapetai differentiation and function and has homology to three different genes in Cannabis (2 MYB35 genes and 1 MYB80 gene). The fact that Cannabis has multiple homologs to the Arabidopsis TDF1 indicates that sex determination in Cannabis is more complex than in species like Arabidopsis. One of the MYB35 gene sequences from Cannabis (referred to herein as MYB ISIS)' is shown in SEQ ID NO:6, and the putative encoded amino acid sequence is shown in SEQ ID NO: 15; another MYB35 gene sequence from Cannabis (referred to herein as MYB35-16) is shown in SEQ ID NO:7, and the putative encoded amino acid sequence is shown in SEQ ID NO: 16; and the MYB80 gene sequence from Cannabis is shown in SEQ ID NO:8, and the putative encoded amino acid sequence is shown in SEQ ID NO: 17.

[0096] SEQ ID NOs: 116-119 represent the four most common genotype sequences for MYB 35- 15 (see Table 2), and FIG. 18 is a schematic alignment of the four most common genotype sequences for MYB 35- 15., depicting the conserved and variable regions. SEQ ID NOs: 120-124 represent the five most common genotype sequences for MYB35- 16 (see Table 2), and FIG. 19 is a schematic alignment of the five most common genotype sequences for MYB35-16, depicting the conserved and variable regions.

[0097] SEQ ID NOs: 125-127 represent the three most common genotype sequences for MYB80 (see Table 2), and FIG. 20 is a schematic alignment of the three most common genotype sequences for MYB80, depicting the conserved and variable regions.

[0098] Table 1. Gene Information a on Scaffold 9 in Sonr Pear Male, which corresponds to Chromosome X in cslO b on Scaffold 4 in Sour Pear Male, which corresponds to Chromosome 8 in cslO c includes 5 kbp upstream of the transcription start site d includes 5 kbp downstream of tire transcription stop site

[0099] Table 2. Common Genotype Sequences Among Cannabis Accessions a, the number of Accessions that fall within 95% sequence identity to the corresponding reference sequence, also referred to as a genotype sequence. b, see Table 8 for the Accession name corresponding to the indicated Accession number. e, see APPENDIX A for the sequences of the most common genotypes for AMS (i.e., reference sequences). d, see APPENDIX B for the sequences of the most common genotypes for bHLH91 (i.e., reference sequences). e, see APPENDIX C for the sequences of the most common genotypes for TM6 (i.e., reference sequences).

[0100] *, see APPENDIX D for the sequences of the most common genotypes for STP6 (i.e., reference sequences). g, see APPENDIX E for the sequences of the most common genotypes for DYT1 (i.e., reference sequences). h, see APPENDIX F for the sequences of the most common genotypes for MYB35-15 (i.e., reference sequences).

[0101] ', see APPENDIX G for the sequences of the most common genotypes for MYB35-16 (i.e., reference sequences). j, see APPENDIX H for the sequences of the most common genotypes for MYB80 (i.e., reference sequences).

[0102] The starting and ending positions indicated in Table 1 above for each gene are relative to the cslO Cannabis genome, as are the gene structure schematics shown in FIG. 1 A-1D. Intraspecific variation is seen in the chromosomal placement of these genes, depending on the genomic assembly of a specific Cannabis accession. For example, chromosome X in cslO corresponds to scaffold 9 in the Sour Pear genome in the LeafWorks genomic database and chromosome 8 in cslO corresponds to scaffold 4 in the Sour Pear genome. Also as described herein, there is variation within each gene across different Cannabis accession in introns as well as upstream and downstream regions, some characterized by large insertions. There are frequent small variations in gene start and stop locations and, occasionally, there is variation in the total number of exons. Internal gene regions are relatively conserved. See, for example, FIGs. 13-20. In addition to different chromosomal locations, it would be understood by a skilled artisan that the genes described herein (e.g., AMS, bHLH91, TM6, STP6, DYT1, MYB35-15, MYB35-16, MYB80) can have a different sequence from those provided herein (e.g., the nucleic acids shown in SEQ ID NOs: 1-8 and the proteins shown in SEQ ID NOs: 9-16). For example, representative AMS sequences that differ from SEQ ID NO: 1 are shown in SEQ ID NOs: 81-88 and correspond to the eight most common genotype sequences for AMS in Cannabis; representative bHLH91 sequences that differ from SEQ ID NO:2 are shown in SEQ ID NOs: 89- 92 and correspond to the four most common genotype sequences for bHLH91 in Cannabis; representative TM6 sequences that differ from SEQ ID NO:3 are shown in SEQ ID NOs: 93-99 and correspond to the seven most common genotype sequences for TM6 in Cannabis; representative STP6 sequences that differ from SEQ ID NON are shown in SEQ ID NOs: 100- 108 and correspond to the nine most common genotype sequences for STP6 in Cannabis; representative DYT1 sequences that differ from SEQ ID NO:5 are shown in SEQ ID NOs: 109- 115 and correspond to the seven most common genotype sequences for DYT1 in Cannabis; representative MYB35-15 sequences that differ from SEQ ID NO:6 are shown in SEQ ID NOs: 116-119 and correspond to the four most common genotype sequences for MYB35-15 in Cannabis; representative MYB35-16 sequences that differ from SEQ ID NO:7 are shown in SEQ ID NOs: 120-124 and correspond to the five most common genotype sequences for MYB35-16 in Cannabis; and representative MYB80 sequences that differ from SEQ ID NO:8 are shown in SEQ ID NOs: 125-127 and correspond to the three most common genotype sequences for MYB80 in Cannabis.

[0103] As used herein, an AMS nucleic acid sequence refers to any of SEQ ID NOs: 1 or 81-88, or a sequence having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to any of SEQ ID NOs: 1 or 81-88. As used herein, a bHLH91 nucleic acid sequence refers to any of SEQ ID NOs: 2 or 89-92, or sequences having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to any of SEQ ID NOs: 2 or 89-92. As used herein, an TM6 nucleic acid sequence refers to any of SEQ ID NOs: 3 or 93-99, or sequences having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to any of SEQ ID NOs: 3 or 93-99. As used herein, an STP6 nucleic acid sequence refers to any of SEQ ID NOs: 4 or 100-108, or sequences having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to any of SEQ ID NOs: 4 or 100- 108. As used herein, an DYT1 nucleic acid sequence refers to any of SEQ ID NOs: 5 or 109-115, or sequences having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to any of SEQ ID NOs: 5 or 109-115. As used herein, an MYB35-15 nucleic acid sequence refers to any of SEQ ID NOs: 6 or 116-119, or sequences having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to any of SEQ ID NOs: 6 or 116-119. As used herein, an MYB35-16 nucleic acid sequence refers to any of SEQ ID NOs: 7 or 120-124, or sequences having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to any of SEQ ID NOs: 7 or 120-124. As used herein, an MYB80 nucleic acid sequence refers to any of SEQ ID NOs: 8 or 125-127, or sequences having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to any of SEQ ID NOs: 8 or 125-127.

[0104] It would be understood that the differences in sequences can be described by percent sequence identity. In calculating percent sequence identity, two sequences are aligned and the number of identical matches of nucleotides or amino acid residues between the two sequences is determined. The number of identical matches is divided by the length of the aligned region (i.e., the number of aligned nucleotides or amino acid residues) and multiplied by 100 to arrive at a percent sequence identity value. It will be appreciated that the length of the aligned region can be a portion of one or both sequences up to the full-length size of the shortest sequence. It also will be appreciated that a single sequence can align with more than one other sequence and hence, can have different percent sequence identity values over different aligned regions. It is noted that the percent identity value is usually rounded to the nearest integer. For example, 78.1%, 78.2%, 78.3%, and 78.4% are rounded down to 78%, while 78.5%, 78.6%, 78.7%, 78.8%, and 78.9% are rounded up to 79%. It is also noted that the length of the aligned region is always an integer.

[0105] The alignment of two or more sequences to determine percent sequence identity can be performed using the algorithm described by Altschul et al. (1997, Nucleic Acids Res., 25:3389-3402) as incorporated into BLAST (basic local alignment search tool) programs, available at ncbi.nlm.nih.gov on the World Wide Web. BLAST searches can be performed to determine percent sequence identity between a nucleic acid and any other sequence or portion thereof aligned using the Altschul et al. algorithm. BLASTN is the program used to align and compare the identity between nucleic acid sequences, while BLASTP is the program used to align and compare the identity between amino acid sequences. When utilizing BLAST programs to calculate the percent identity between a sequence and another sequence, the default parameters of the respective programs generally are used.

[0106] It would be appreciated that conserved regions within genes remain similar or relatively unchanged across different species or within a group of related sequences, whereas variable regions exhibit differences or divergence between different species or within a group or population. Conserved regions often correspond to functionally important domains and can be used in phylogenetic analysis to understand relationships between species or organisms, while variable regions can be used for distinguishing or differentiating individuals.

[0107] Methods of Altering Gene Expression in Plants

[0108] Altering the expression of genes involved in male flower reproduction via a variety of gene silencing approaches allows one to alter and control male flower production in Cannabis during breeding and cultivation. Controlling expression or function of one or more of the genes involved in male reproductive development enables sterilization or restoration of male fertility in Cannabis plants, which, in turn, allows for the prevention of hermaphroditism in Cannabis plants and the creation of male sterile lines for development of hybrid Cannabis lines.

[0109] Altering gene expression of one or more of the Cannabis genes to result in non-viable pollen formation can eliminate the negative repercussions of male flower formation in the female Cannabis plant. In Cannabis, non-viable pollen cannot fertilize female flowers, and non-viable pollen protects flower harvests since flowers do not set seed in the absence of viable pollen. Non-viable pollen also avoids a loss in desirable metabolite production (e.g., cannabinoids and terpenes) that occurs upon pollen fertilization. Hence, the negative effects of Cannabis hermaphrodites are eliminated when pollen is non-viable (i.e., aborted).

[0110] Further, altering gene expression of one or more of the Cannabis genes involved in pollen formation can provide both abiotic and biotic stress tolerance. Since hermaphroditism in Cannabis can occur due to biological and environmental plant stress, silencing of male flower reproduction genes involved in hermaphroditism can provide resistance to the formation of hermaphroditic male flowers that occurs as a stress response. Thus, methods of preventing hermaphroditism in Cannabis plants and methods of making a male Cannabis plant or a hermaphroditic Cannabis plant sterile are described herein. Such methods typically involve silencing expression of one or more Cannabis genes involved in pollen formation (e.g., AMS, bHLH91, TM6, STP6, DYT1, MYB35, MYB35, and MYB80). Types of gene silencing in plants include but are not limited to transcriptional gene silencing (e.g., RNA-directed DNA methylation, genomic imprinting, paramutation, transposon silencing, transgene silencing, position effect) as well as post-transcriptional gene silencing (e.g., inhibitory or interfering RNA (RNAi), clustered regularly interspaced short palindromic repeats / Cas9 (CRISPR / Cas system) and the like, and nonsense-mediated decay).

[0111] RNAi is a gene silencing mechanism that relies on the homology between transcribed RNA and the target genes to cause post-transcriptional gene silencing. RNAi can be achieved with multiple types of small RNA (sRNA) such as microRNAs (miRNAs) and short interfering RNAs (siRNA) including, but not limited to, natural-antisense siRNA (nat-siRNA), trans-acting siRNA (ta-siRNA), heterochromatic siRNA (hc-siRNA), repeated-associated siRNAs (ra- siRNAs). In addition, double-stranded RNA (dsRNA) (e.g., short dsRNAs, long dsRNAs) can be used to achieve effective gene silencing. See, for example, Bennett et al., 2020, Front Plant Sci. 2020 Jun 12;11:816.

[0112] The methods of preventing hermaphroditism in Cannabis plants and of making a male Cannabis plant or a hermaphroditic Cannabis plant sterile that are described herein are made possible due to the identification of the relevant genes involved in male flower formation (e.g., AMS, bHLH91, 1M6, STP6, DYT1, MYB35, MYB35, and MYB80) and are not limited by the particular manner in which a gene is silenced. In other words, the transgenic and non-transgenic methods described herein, the gene editing methods described herein, and the mutagenesis described herein are intended to be representative and are not the only ways in which a gene can be silenced.

[0113] Spray-Induced Gene Silencing (SIGS)

[0114] The use of RNAi, via the use of double stranded RNA (dsRNA) foliar spray, to control gene expression creates a commercial product capable of giving breeders and cultivators the tools needed to alter gene expression without the need to develop transgenic plants. SIGS has shown up to 100% gene silencing. In this disclosure, we employed SIGS via dsRNA spraying as a representative method to demonstrate the silencing effect of specific genes involved in male flower formation and hermaphroditism in Cannabis.

[0115] With dsRNA spraying, foliar uptake and then cellular uptake occurs whereby double stranded RNA (dsRNA) of a targeted gene crosses the plant cell membrane and is taken up by plant cells. Although not intended to be bound by theory, once dsRNA enters the cytoplasm, plant RNAi machinery processes dsRNA into small interfering RNAs (siRNAs) that target and degrade the specific gene transcripts. These siRNAs target transcripts locally and are diffused via plant vasculature across the plant for comprehensive gene silencing.

[0116] Many barriers exist to achieve efficient foliar and cellular uptake including, but not limited to, the traversing of cell walls and membranes, dsRNA degradation on surfaces prior to uptake, leaf wettability and surface tension, environmental factors such as UV exposure, humidity, pH, dose, application method, stability of the molecule, wash-off, and the trichome barrier. These barriers can be overcome from spraying of “naked” dsRNA at high pressure, or with the addition of a delivery vehicle such as, without limitation, surfactants, chemical modification and / or nanoparticles including but not limited to chitosan salts like CQAS, carbon dots, liposomes, clay nanosheets, and gold nanoparticles. While application of naked dsRNA can result in effective gene silencing, the use of surfactants, chemical modification and / or nanoparticles can achieve longer-term gene silencing effects. Genes can be targeted separately or as mixtures with simultaneous application of different dsRNA molecules in a cocktail targeting different genes or genetic regions. dsRNA application can silence genes as early as 2 hours post-spray, with signal lasting for weeks or months. In some cases, gene silencing effects can last across generations. Doses can be effective with even a single, brief application, but multiple applications over time (e.g., intermittent spraying) or steady exposure (e.g., constant spraying, soaking) can ensure steady or constant gene silencing. Plants can be sprayed with a composition containing dsRNA for any length of time (e.g., about 10 seconds to about 60 seconds) once per day or more than once per day (e.g., twice a day; five times per day; ten times per day; a plurality of times; etc.).

[0117] Thus, a composition (e.g., an article of manufacture) is provided that includes one or more double-stranded RNA (dsRNA) molecules that are at least 10 nucleotides in length (e.g., at least 12, 15, 16, 17, 18, 19, 20, 25, 30, 50, or 100 nucleotides in length; e.g., at least 250, 500, 1000, 1500, 2000, 2500, or 3000 nucleotides or more in length) that are complementary or have at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to at least one of the genes described herein involved in male flower development (e.g., AMS, bHLH91, TM6, STP6, DYT1, MYB35, MYB35, and MYB80') (e.g., at least two of the genes, at least three of the genes, at least four of the genes, etc.). Upon uptake by the plant, the dsRNA molecules effectively silence expression of at least one of the target genes. It would be appreciated that, the dsRNA molecules can be essentially any length (e.g., from about 10 nucleotides in length up to the full length of the gene (e.g., the full length of the mRNA)) and can be directed to different portions of a gene (e.g., the promoter, the coding sequence with or without 5’ and / or 3’ untranslated regions, introns / splice sites, etc.).

[0118] As described herein, a composition including dsRNA molecules can be provided in an aqueous solution and formulated as a spray. Alternatively, a composition including dsRNA molecules can be provided in any number of other forms including, without limitation, dried or lyophilized, to be reconstituted by the ultimate or penultimate user. As an alternative to a spray, a composition including dsRNA molecules as described herein can be formulated for dipping, injecting, painting, and / or soaking.

[0119] Representative dsRNA nucleic acid molecules that are complementary to the AMS gene include any of SEQ ID NO:33-43, where each of the dsRNA nucleic acid molecules effectively silence expression of the AMS gene. Representative dsRNA nucleic acid molecules that are complementary to the bHLH91 gene include any of SEQ ID NOs:44-54, where each of the dsRNA nucleic acid molecules effectively silence expression of the bHLH91 gene. Representative dsRNA nucleic acid molecules that are complementary to the TM6 gene include any of SEQ ID NOs:55-65, where each of the dsRNA nucleic acid molecules effectively silence expression of the at TM6 gene. Representative dsRNA nucleic acid molecules that are complementary to the STP6 gene include any of SEQ ID NOs:66-80, where each of the dsRNA nucleic acid molecules effectively silence expression of the STP6 gene. See Table 3 below for the sequences of these representative dsRNA nucleic acid molecules.

[0120] When using SIGS, uptake of nucleic acids into plant cells can be facilitated or improved in the presence of a delivery vehicle. Without limitation, representative delivery vehicles include one or more surfactants, many different types of nanoparticles (e.g., chitosan salts (e.g., CQAS), carbon dots, liposomes, clay nanosheets, gold nanoparticles, etc.), biological vectors (e.g., viruses, bacteria (e.g., Agrobacterium spp.)), or can include buffers and / or chemical modifications. Thus, in addition to one or more dsRNAs, a composition (e.g., an article of manufacture) as described herein also can include one or more delivery vehicles (e.g., one or more surfactants, many different types of nanoparticles (e.g., chitosan salts (e.g., CQAS), carbon dots, liposomes, clay nanosheets, gold nanoparticles, etc.), biological vectors (e.g., viruses, bacteria (e.g., Agrobacterium spp.)), or can include buffers and / or chemical modifications). Depending upon the delivery vehicle, a composition (e.g., an article of manufacture) as described herein further can include one or more reagents or linkers (e.g., chemical, synthetic) for attaching or conjugating the dsRNA nucleic acid molecules to a delivery vehicle.

[0121] As described in more detail below, silencing of genes involved in male flower production was achieved with the creation and application of foliar sprays containing dsRNA directed to different combinations of four of the genes described herein. Since it is known that dsRNA exogenous sprays are effective individually but are more effective when applied as a mixture of dsRNA from different genes, two genes were grouped into different dsRNA sprays, bound to a nanoparticle that encourages plant uptake, and sprayed onto chemically-induced hermaphroditic females. For each spray, silencing of each individual gene was confirmed as was a significant reduction of pollen viability in treated hermaphroditic females, with instances of 100% pollen abortion and complete sterility.

[0122] Table 3. Representative Sequences _ _ * data presented in Examples

[0123] As described below in more detail, Cocktail 1 and Cocktail 2 demonstrated a 63% and 65% rate of pollen abortion, respectively, on average. No other observed phenotypic changes were observed in treatment plants; plants in all treatment groups remained healthy.

[0124] Transgenic Plants

[0125] Methods of genetically engineered plants to effectuate gene silencing are known in the art. As described herein, such methods can include genetically engineering plants to silence one or more of the genes involved in male flower production (e.g., AMS, bHLH91, TM6, STP6, DYT1, MYB35, MYB35, and MYB80).

[0126] Thus, methods of introducing nucleic acids into Cannabis cells are provided. Typically, nucleic acids having at least 80% sequence homology to any of the sequences shown in SEQ ID NOs: 1 or 81-88 (AMS),' SEQ ID NOs: 2 or 89-92 (bHLH9Ty, SEQ ID NOs: 3 or 93-99 (7M6); SEQ ID NOs: 4 or 100-108 (STP6y, SEQ ID NOs: 5 or 109-115 (DYTTy SEQ ID NOs: 6 or 116- 119 (MYB35-15),' SEQ ID NOs: 7 or 120-124 (MYB35-16),' or SEQ ID NOs: 8 or 125-127 (MYB80), or a portion thereof, or nucleic acids having a sequence that is complementary to any of the sequences shown in SEQ ID NOs: 1 or 81-88 (AMS),' SEQ ID NOs: 2 or 89-92 (bHLH91), SEQ ID NOs: 3 or 93-99 (7M6); SEQ ID NOs: 4 or 100-108 (STP6),' SEQ ID NOs: 5 or 109-115 (DYTTy, SEQ ID NOs: 6 or 116-119 (MYB35-15),' SEQ ID NOs: 7 or 120-124 (MYB35-16),' or SEQ ID NOs: 8 or 125-127 (MYB80), or a portion thereof, can be introduced into Cannabis cells. Introducing such a nucleic acid into Cannabis cells can effectively silence expression of the respective gene(s) in those cells or in a subsequently produced plant (e.g., a transgenic plant), and it would be understood that the methods described herein are not limited to any particular mechanism by which the gene silencing takes place.

[0127] Simply by way of example, transgenic plants are provided that express at least one nucleic acid (e.g., a transgene) at least 10 nucleotides in length (e.g., at least 12, 15, 16, 17, 18, 19, 20, 25, 30, 50, or 100 nucleotides in length; e.g., at least 250, 500, 1000, 1500, 2000, 2500, or 3000 nucleotides or more in length) having at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) to at least one of the nucleic acids described herein (e.g., any of SEQ ID NOs :1 or 81-88 (AMS); SEQ ID NOs: 2 or 89-92 (bHLH91); SEQ ID NOs: 3 or 93-99 (TM6); SEQ ID NOs: 4 or 100-108 (STP6); SEQ ID NOs: 5 or 109-115 (DYT1); SEQ ID NOs: 6 or 116-119 (MYB35-15); SEQ ID NOs: 7 or 120-124 (MYB35-16); or SEQ ID NOs: 8 or 125-127 (MYB80)). Upon transient or stable expression of the nucleic acid, the inhibitory or interfering RNA (RNAi) process is initiated, as a consequence of the expression leading to dsRNA, which ultimately results in the silencing of the corresponding endogenous nucleic acid. As described herein, such transgenic plants exhibit a greater amount of non-viable pollen relative to, e.g., a plant lacking or not expressing the nucleic acid.

[0128] Methods of introducing a nucleic acid (e.g., a transgene) into plant cells are known in the art and include, for example, Agrobacterinm-mediated transformation, agroinfiltration, particle bombardment, microinjection, polyethylene gly col-mediated transformation (e.g., of protoplasts), liposome-mediated DNA uptake, or electroporation. It would be understood that, in some instances, achieving stable or transient transformation is dependent upon the particular vector or construct used. Following stable transformation, transgenic plant cells can be regenerated into transgenic plants. It would be appreciated that transgenic plants can be screened for male flower development, viability of pollen, etc., compared to a corresponding plant not expressing or containing the nucleic acid.

[0129] Gene Editing

[0130] Any number of gene editing techniques can be used to specifically alter the sequence of one or more of the genes described herein involved in male flower development (e.g., AMS, bE[LH91, TM6, STP6, DYT1, MYB35, MYB35, and MYB80) in a Cannabis cell include, without limitation, gene replacement (e.g., using homologous recombination or homology-directed repair), gene editing, base editing, or prime editing.

[0131] In some cases, clustered regularly interspaced short palindromic repeat (CRISPR) / CRISPR-associated (Cas) nuclease (CRISPR / Cas) gene editing (e.g., therapeutic gene editing) techniques can be used to edit a gene (e.g., AMS, bHLH91, TM6, STP6, DYT1, MYB35, MYB35, and MYB80) present in a Cannabis cell. CRISPR / Cas molecules are components of a prokaryotic adaptive immune system that is functionally analogous to eukaryotic RNA interference, using RNA base pairing to direct nucleic acid cleavage resulting in double stranded breaks (DSBs) about 3-4 nucleotides upstream of a protospacer adjacent motif (PAM) sequence. Directing nucleic acid DSBs with the CRISPR / Cas system requires two components: a Cas nuclease, and a guide RNA (gRNA) targeting sequence directing the Cas to cleave a target DNA sequence. A CRISPR / Cas system can include any appropriate Cas nuclease. A number of Cas nucleases (e.g., Cas9, Cas 12) are known in the art.

[0132] In some cases, a TALEN system can be used (e.g., can be introduced into Cannabis cells) to edit the sequence of a gene (e.g., AMS, bHLH91, TM6, STP6, DYT1, MYB35, MYB35, and MYB80) in a Cannabis cell. For example, an engineered transcription activator-like (TAL) effector DNA binding domain targeting sequence can be fused to a nuclease to create a TALEN that can create nucleic acid double-stranded breaks (DSBs) at or near the sequence targeted by the TAL effector DNA binding domain. Directing nucleic acid DSBs with the TALEN system requires two components: a nuclease, and TAL effector DNA-binding domain directing the nuclease to a target DNA sequence. A TALEN system can include any appropriate nuclease.

[0133] Thus, methods of introducing (e.g., by transformation) one or more constructs that include gene editing components (e.g., TALEN technology, zinc-finger nuclease technology, CRISPR technology, or variations thereof (e.g., FANZOR, NICER, etc.)) into Cannabis cells are provided herein. Such methods enable at least one of the Cannabis genes selected from (e.g., AMS, bHLH91, TM6, STP6, DYT1, MYB35, MYB35, and MYB80) to be edited, so as to silence one or more of the genes involved in male flower production.

[0134] Mutagenesis

[0135] Methods of mutagenesis are known in the art, and methods of making a plant having a mutation in its genome are known in the art. Mutations can be random mutations or targeted mutations. For random mutagenesis, plant cells can be mutagenized using, for example, a chemical mutagen, ionizing radiation, or fast neutron bombardment. Representative chemical mutagens include, without limitation, nitrous acid, sodium azide, acridine orange, ethidium bromide, and ethyl methane sulfonate (EMS), while representative ionizing radiation includes, without limitation, x-rays, gamma rays, fast neutron irradiation, and UV irradiation. The dosage of the mutagenic chemical or radiation is determined experimentally for each type of plant tissue such that a mutation frequency is obtained that is below a threshold level characterized by lethality or reproductive sterility. The number of Mi generation seed or the size of Mi plant populations resulting from the mutagenic treatments are estimated based on the expected frequency of mutations. A mutation can be a point mutation, an insertion, a deletion, an inversion, a translocation, or combinations thereof.

[0136] One or more nucleotides within a gene can be mutated to alter the expression and / or function of the encoded polypeptide, relative to the expression and / or function of the corresponding wild type polypeptide. In addition, a mutation in a single nucleotide can create a stop codon, which would result in a truncated polypeptide and, depending on the extent of truncation, cause partial to complete loss-of-function. Further, a mutation in a promoter sequence can enhance, reduce or completely eliminate the gene expression in a plant comprising the mutation. A mutation in a promoter sequence can alter or eliminate the binding or recognition site of a transcription factor or of the polymerase enzyme, or a mutation in a promoter sequence can alter or eliminate the function of an enhancer, an activator or the like, or a repressor, a silencer or the like. Mutations in a promoter sequence can result in altered or absent transcription, or production of a less-than-functional or non-functional transcript. A less-than- functional or non-functional transcript can result from improper expression (e.g., expressed in the wrong place or at the wrong time), or from degradation of the transcript. Alternatively, a mutation in a promoter sequence may allow transcription to take place but may interfere with or eliminate the ability of the transcript to be translated into protein.

[0137] Mutations in a coding sequence can result in insertions of one or more amino acids, deletions of one or more amino acids, and / or non-conservative amino acid substitutions in the encoded polypeptide. Insertion or deletion of amino acids in a coding sequence, for example, can disrupt the conformation of the encoded polypeptide. Amino acid insertions or deletions also can disrupt sites important for recognition of a binding ligand or for activity of the polypeptide. It is known in the art that the insertion or deletion of a larger number of contiguous amino acids is more likely to render the gene product non-functional, compared to a smaller number of inserted or deleted amino acids. In addition, one or more mutations can change the localization of a polypeptide, introduce a stop codon to produce a truncated polypeptide, or disrupt an active site or domain (e.g., a catalytic site or domain, a binding site or domain) within the polypeptide. Non-conservative amino acid substitutions can replace an amino acid of one class with an amino acid of a different class. Non-conservative substitutions can make a substantial change in the charge or hydrophobicity of the gene product. Non-conservative amino acid substitutions can also make a substantial change in the bulk of the residue side chain, e.g., substituting an alanine residue for an isoleucine residue. Examples of non-conservative substitutions include a basic amino acid for a non-polar amino acid, or a polar amino acid for an acidic amino acid.

[0138] Thus, methods of exposing Cannabis cells to a mutagen are provided. After mutagenesis, plants having a mutation in one or more of the genes (e.g., AMS, bHLH91, TM6, STP6, DYT1, MYB35, MYB35, and MYB80) can be selected, where the mutation effectively silences expression of at least one of the genes.

[0139] Genetic Markers of Hermaphroditism

[0140] It would be appreciated that any of the genes described herein (e.g., AMS, bHLH91, TM6, STP6, DYT1, MYB35, MYB35, and MYB80}, or a particular domain therefrom (e.g., an activation domain) or a SNP or other type of variation, can be used, for example, as markers of hermaphroditism to predict male sterility or hermaphroditism resistance status of a plant or cultivar, to restore male functionality, or to create new plants or products.

[0141] It would be appreciated that any of the genes described herein (e.g., AMS, bHLH91, TM6, STP6, DYT1, MYB35, MYB35, and MYB80), or a particular domain therefrom (e.g., an activation domain) or a SNP or other type of variation, can be used, for example, in marker-assisted breeding. For example, DNA fingerprinting or similar technologies can be used in a marker- assisted breeding program to transfer or breed one or more variant alleles into other lines, varieties or cultivars, as known in the art. Progeny of the cross can be screened for the domain, SNP or other variation using methods described herein, and plants having the domain, SNP or other variation can be selected. For example, plants in the F2 or backcross generations can be screened using a marker developed from a sequence described herein or a fragment thereof, using any one of known techniques. Plants also can be screened for the domain, SNP or other variation, and those plants having the domain, SNP or other variation can be selected. Plants identified as possessing the domain, SNP or other variation and / or a desired phenotype can be backcrossed or self-pollinated to create a second population to be screened. Backcrossing or other breeding procedures can be repeated until the desired phenotype of the recurrent parent is recovered.

[0142] In accordance with the present invention, there may be employed molecular biology, microbiology, biochemical, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. The invention will be further described in the following examples, which do not limit the scope of the methods and compositions of matter described in the claims.

[0143] EXAMPLES

[0144] Example 1 — Identification of Pollen Formation Genes Using Differential Gene Expression and Co-Expression Analysis from Hermaphroditic, Male and Female Flowers

[0145] Plant Material and Sample Collection. Female flowers and natural (non-chemically induced) hermaphroditic flowers from the same plants were collected in three biological replicates per plant from the field and stored in RNAlater. The amount of hermaphroditic flower present on each plant was noted per plant. Natural hermaphroditic samples and paired female flowers from the same plant were selected for RNA extraction only from the plants expressing the most hermaphroditism. Separately, male flowers from a common garden experiment were collected in three biological replicates, flash frozen in liquid nitrogen, and stored at -80°C until processing.

[0146] RNA isolation and Sequencing'. RNA was extracted from floral tissue only (i .e., as much non-flower tissue (e.g., leaf) as possible was removed). RNA was isolated using Qiagen RNeasy Plant Mini Prep Kit following manufacturer's protocol. RNA was quantified using Thermo Fisher Qubit 4 (Waltham, Massachusetts, USA). CD Genomics (Shirley, New York, USA) prepared the samples for standard Poly-A enrichment and stranded cDNA library preparation with quality control performed using RIN scores from an Agilent 4200 Tapestation (Santa Clara, California, USA). Samples were barcoded and RNA-Seq was performed.

[0147] Sequencing Read Processing : Sequencing adapters and low-quality reads (Q score < 20) were removed from the raw sequencing reads using Trimmomatic software version 0.39 (Bolger et al. 2014) with parameters: “LEADING:20”, “TRAILING:20”, “SLIDINGWINDOW:4: 15”, “AVGQUAU20”, “MINLEN:35”. On average, 71.7 million reads were retained per sample for alignments. The remaining high-quality reads from each sample were aligned to the indexed CBDRx genome, also known as cslO genome (Grassa et al. 2021), using Hisat2 software with default parameters (Kim et al. 2019). The alignment files in BAM (binary alignment map) format were processed with Samtools software (Heng et al. 2009) to remove any unmapped reads, and the single mapped reads whose mate was not mapped. The remaining properly paired and aligned sequence reads from each sample were counted for individual genes in the Cannabis CBDRx (cslO) genome using HTSeq count software (Anders, Pyl and Huber, 2014). CslO is considered the most comprehensive reference genome available, but any Cannabis genome could be used with this pipeline.

[0148] Identification of Differentially Expressed Genes'. The gene-specific read counts from each sample were employed to identify differentially expressed genes between male flowers, female flowers and hermaphroditic flowers across multiple cultivars using DESeq2 package version 1.42.0 (Love, Huber and Anders 2014). The male, female, and hermaphroditic samples were compared pairwise (Herm vs. Female, Herm vs. Male, Male vs. Female) with a generalized linear model to obtain a p-value of significance and log2Fold change for each gene model across the tissues. The model used replicated data to normalize any expression variation due to sampling error. The expression statistics (p-value and log2Fold) were obtained for each gene in the CBDRx genome and the genes with a p-value of <0.05 and a minimum log2Fold threshold of 1.5 were identified as differentially expressed. The genes were designated as up-regulated and down-regulated in the hermaphroditic samples based on their relative normalized expression scores in the male or female samples and in the context of male vs female expression differences.

[0149] Identifying Co-Expression Modules for Hermaphroditism'. The normalized expression values obtained from DESeq2 were used to construct a co-expression network for all the annotated genes in the cslO genome using weighted correlation network analysis (WGCNA) R package (Langfelder and Horvath 2008). The co-expression modules were identified using the one-step network building and module detection method in the WGCNA software. WGCNA connects all data variables in the expression dataset to identify networks with similar expression patterns across samples. Initially, an unsigned topological overlap matrix was built to define a threshold for network detection, which led to a network threshold power of 9 to generate a coexpression network for hermaphroditic, male and female flower samples. In addition, parameters including a minimum module size equal to 30, and a branch merge cut height of 0.25 were used to identify the co-expression network modules. The resulting co-expression modules were visualized using heatmaps in R statistical software (Perry 2023) and in Cytoscape version 3.9.1 (Shannon et al. 2003). This analysis identified two specific modules that exhibited clear expression pattern differences in hermaphroditic samples from the male and female Cannabis samples.

[0150] Identifying Candidate Genes for Male Flower Reproduction. The differential expression analysis identified 2075, 3177, and 1436 significant (p < 0.05) differentially expressed genes (DEGs) between hermaphroditic vs. female, hermaphroditic vs. male, and female vs. male samples, respectively, across multiple genetic backgrounds. Out of the 2075 hermaphroditic vs. female DEGs, 1075 genes were upregulated in the hermaphroditic samples, and a maximum log2Fold change of 12.56 was observed. Similarly, 1591 DEGs showed upregulation in the hermaphroditic samples against the natural male samples with a maximum log2Fold change of 11.54. Collectively, this dataset of DEGs was called “LW-DEGs” for further reference.

[0151] Several DEGs were present in two different co-expression modules. These results highlight that hermaphroditic flower production and natural female flower production occurs through different pathways expressed in a concerted fashion. Moreover, this analysis provides a second layer of evidence to shortlist the candidate genes for hermaphroditic flower production.

[0152] These analyses identified 75 genes, whose expression was different in the hermaphroditic samples compared to the male and female samples. The annotations for these 75 genes were retrieved from the cslO genome annotations. If a gene was not annotated in the cslO genome, it was manually annotated using the Basic Local Alignment Search Tool (BLAST) tool in the National Center for Biotechnology Information (NCBI) database and the fgenesh software (Solovyev et al. 2006). These selected 75 genes were categorized into classes based on their annotated role in flowering or flowering related pathways.

[0153] Identification and Validation of Candidate Genes'. To further validate the expression differences of the 75 DEGs between hermaphroditic samples and male and female samples, we retrieved public RNA-Seq dataset from the male (M), female (F), and silver thiosulfate (STS) induced male (IM) samples, which are artificially produced hermaphroditic male flowers on female plants from Adal et al. (2021). The raw sequencing reads for these samples were downloaded from the NCBI Sequence Read Archive (SRA) database using the BioProject accession PRJNA669389, BioSample accessions SAMN16447877-SAMN16447891, and SRA accessions SRR12831863-SRR12831877. The raw read sequences were processed and analyzed to identify DEGs between induced hermaphroditic and male and female samples using the previously described methods. These DEGs are hereafter referred to as “Adal-DEGs”. In addition, we crosschecked DEGs from a similar study (McKay 2021) as an additional validation measure for gene expression differences observed in the LW-DEGs and Adal-DEGs.

[0154] Genes shared between the previously selected 75 LW-DEGs and Adal-DEGs were identified as candidate genes for further analysis. Based on these comparisons and an initial literature search of genes involved in flower formation, reproductive systems, pollen development, male sterility, and hermaphroditism in other species, a set of 10 candidate genes were selected for further analysis. Low or lack of expression of a gene in hermaphrodites and in males with relatively higher expression in females was confirmed for each gene to be retained as an acceptable candidate.

[0155] In addition to the top 10 selected genes, we performed a literature search to identify additional putative candidate genes in both LW-DEGs and Adal-DEGs datasets based on their function in male reproductive organs in other species. Any genes whose mutant showed phenotypic defects in the male reproductive parts in various species and which showed differential expression in either LW-DEGs or Adal-DEGs were added to the list of putative candidate genes. A list of 14 candidate genes was retained. We further filtered out candidate genes if a gene showed more than one match to any off-target regions in cs 10 or a genetically dissimilar Cannabis genome, Sour Pear. Additionally, genes exhibiting non-relevant or undesirable phenotypic effects outside of male reproductive function in other species were removed. Genes were kept for future analysis if they satisfied the following criteria: 1) genes were specifically expressed in a male flower, whether hermaphroditic or male 2) mutated genes expressed a desirable phenotype only in the male flower, and 3) genes exhibited a single BLAST hit within the Cannabis genome.

[0156] Example 2 — Identification of Relevant Genes and Evaluation of Genetic Variation Therein

[0157] A total of eight genes - AMS (ABORTED MICROSPORES), bHLH91 (Basic-loop-basic helix 91 - homolog to EAN1, ATM1, bHLHl 0^89 / 91), STP6 (SUGAR TRANSPORT PROTEIN 6), TM6 (Agamous-Like TOMATO MADS BOX GENE 6), DYT1 (DYSFUNCTIONAL TAPETUM 1), as well as MYB35-15, MYB35-16 and MYB80 (homologous to DEFECTIVE IN MERISTEM DEVELOPMENT AND FUNCTION 1) were retained from candidate gene analysis for functional validation. These genes were expressed at a higher level in the hermaphroditic and / or male flowers but largely minimally expressed or showed no expression in the female flowers, making RNAi an ideal approach to decrease their expression and test their involvement in male reproduction in Cannabis. Annotations for these genes were checked to confirm their identities and gene structure through NCBI nucleotide BLAST database and the fgenesh software (Solovyev et al. 2006).

[0158] To evaluate the genetic variation in the eight genes identified, we used whole genome sequences for 137 Cannabis accessions within the LeafWorks database. Trimmomatic software version 0.39 (Bolger et al. 2014) was used to filter out low quality read sequences using the following parameters: LEADING:20 TRAILING:20 SLIDINGWINDOW:4: 15 AVGQUAL:20 MINLEN:35.

[0159] The high-quality reads were aligned against the cslO genome using minimap2 software version 2.24 (Li, 2018). The alignment files were processed to remove PCR duplicates using Picard software version 2.27.4 (picard: Broad Institute, 2018). The resulting alignment files were used to call variants using “HaplotypeCaller”, “GenomicsDB Import”, and “GenotypeGVCFs” plugins in the GATK software version 4.3.0.0 (McKenna et al. 2010). The variants were retrieved in a Variant Call Format (VCF) file. The resulting VCF file was scanned based on the genomic coordinates for AMS, bHLH91, STP6, and TM6 genes to identify single nucleotide polymorphisms (SNPs) and Insertions / Deletions (INDELs) in the respective genes. Very little sequence variation was found in the genes, indicating they are conserved across many Cannabis accessions. Briefly, AMS had 8 SNPs, bHLH91 had 2 SNPs, STP6 had 5 SNPs, and TM6 had 1 SNP. See Table 4. In addition, a single 1 bp deletion in TM6 in a single cultivar, 680_S27, was identified.

[0160] Table 4. SNPs

[0161] We also conducted a population structure analysis to avoid potential issues in RNAi design due to specific clustering patterns in the population. The raw VCF file was processed to retain high quality variations for genetic structure analysis as follows: (1) the variants were filtered to remove any INDELs from the dataset; (2) the resulting variants were filtered to retain SNPs present in at least 90% of the samples and have minimum read depth of 5; (3) the remaining variants were further filtered using the minor allele frequency of 0.95. The final variants were used to perform a principal component (PC) analysis in the Tassel software (Bradbury et al. 2007). The first three PCs were plotted to visualize the genetic structure in the Cannabis population. This analysis revealed that a single accession, ‘Malawi Gold,’ was completely distinct from the remaining samples in the LeafWorks database. The remaining cultivars showed a random distribution pattern across the three principal components. A similar population structure analysis was conducted using the SNPs within a common genotype block spanning each gene of interest. The common genotype blocks were defined from the genome- wide SNP dataset using plink software (Purcell et al. 2007). The gene-specific PC plots mostly showed random distribution of samples across the three PCs, indicating that most of the Cannabis accessions in the LeafWorks database used in this analysis were suitable for RNAi experiments. A lack of distinct clustering of Cannabis accessions indicates that these Cannabis accessions lack specific population structure, which makes it possible to choose any Cannabis accession as a representative to do RNAi.

[0162] Example 3 — RNAi dsRNA Construction dsRNA Design'. The software, pssRNAit (Ahmed et al. 2020), was used to design the dsRNAs for each gene target. psRNATarget (Dai and Zhao 2011) was also used to look for off- target hits against the Cannabis transcriptome as well as look for off-target hits against Homo sapiens (i.e., humans). When evaluating different dsRNAs for each gene target, we assessed the potential number of siRNAs generated from a dsRNA molecule, total number of off-target hits, average siRNA per off target, average ‘expect’ score for off target hits, average siRNA efficiency, average target accessibility, and average RISC binding differential to decide which ones to pursue. Further, we investigated human off target hits and, if any dsRNA created multiple potential siRNAs that target human genes (i.e., that had a greater likelihood of eliciting a RNAi response for a human gene), these were removed as potential candidates.

[0163] Each gene had a single annotated transcript except for LOCI 15713370, which has five annotated transcripts in the cslO genome. During dsRNA design, we confirmed that siRNAs generated from potential dsRNA would bind to all annotated transcripts for target loci. Further, all annotated transcripts were taken into account in RTqPCR marker design to avoid confounding results. Information for dsRNA design can be found in Table 5. dsRNA for each of the four genes was synthesized by Creative Biogene Inc (Shirley, NY, USA). Product was synthesized using standard in vitro transcription (IVT) and lithium chloride for precipitation. Product passed quality inspection with clear appearance free from foreign particles, free of DNA, protein and nucleotide, OD260 / 280 at 2.0-2.1, and gel agarose band clean at the correct size. Final concentration was measured with a Nanodrop device (Thermo Fisher Scientific, Waltham, MA, USA). Schematics showing the design of the dsRNA molecules to each of the four genes are shown in FIG. 1 A (AMS); FIG. IB (bHLH91); FIG. 1C (TM6); FIG. ID (STP6). Table 5: Gene dsRNA and RTqPCR information, including gene name, Gene ID, length, primer / probe sequences, amplicon size, predicted number of siRNAs from dsRNA

[0164] Example 4 — Gene Expression Testing

[0165] RTqPCR Design'. We used FASTA and CDS sequences for each gene target for RTqPCR marker design. Markers were designed to span exons, thus gene expression evaluation was robust to DNA contamination and would ensure amplification of mRNA transcripts, not genomic DNA. Markers were designed using Primer3 v4.1.0 (Untergasser et al. 2012) and Geneious 2020.2.5 (Kearse et al. 2012). For a gene expression control reference marker, we selected EFlalpha for Cannabis based on work from Guo et al. (2018). We designed a probe to match primers published in Guo et al. (2018) using Primer3 v4.1.0 (Untergasser et al. 2012) (see Table 5).

[0166] RTqPCR testing and validation. Each marker was evaluated for PCR efficiency and expression levels of mRNA extracted from either male flowers or leaves. Note that the selected target genes are all putatively involved in hermaphroditic or male flower formation and / or pollen formation and, therefore, expression is expected to be minimal in RNA extracted from leaf material. Gene expression is expected to be higher in the flowers given their function. Leaf expression data was utilized as a control to show expression changes. The results are outlined below in Table 6.

[0167] Table 6. Gene PCR efficiency information.

[0168] *Note that Cq values for BHLH91 leaf RNA were always substantially lower than Cq values for male flower RNA. Example 5 — dsRNA Application

[0169] Plant Material Used. Cannabis cultivars used for each dsRNA application are listed in Table 7. The cultivar ‘Original Bacio’ was used to test the silencing effect of bHLH91 an&AMS in Cocktail 1. The cultivars ‘Original Bacio,’ ‘Velvet Grapes’ and ‘ Soap’ were used to test the silencing effect of TM6 and STP6 in Cocktail 2. These cultivars come from different lineages and were chosen to showcase the effectiveness in silencing across diverse genetic backgrounds.

[0170] Plant Cultivation Conditions. Young plants from clone cuttings from each cultivar (Table 7) were grown in replicate (i.e., 2 clones), potted in FoxFarm Happy Frog Potting Soil (Samoa, CA, USA) in 2-gallon pots. Plants were grown in MyHighGrow 3x3m grow tents (Perth Amboy, NJ, USA), each tent representing a different experimental treatment. The lights were set to create a 18 hour photoperiod during vegetative growth and a 12 hour photoperiod during flowering (see STS section below). Plants were fertilized with House & Garden Soil A + B Nutrients (Seacliff Park, South Australia) and General Hydroponics CALIMAGIC™ calcium-magnesium supplement (Santa Rosa, CA, USA) according to the manufacturer's instructions. Pests and pathogens were checked regularly, with Trifecta Crop Control (Williamsport, PA, USA) applied as needed.

[0171] Table 7: Cultivar Information for Cultivars used in RNAi Application Experiments

[0172] Hermaphrodite Creation'. To test if silencing of these genes would result in aborted pollen or not (i.e., viable), male flower production was induced in all experimental plant tents (FIG. 2). Female plants in which male flower production was required were sprayed with a Silver thiosulfate (STS) solution. STS induces male flower production via an endogenous hermaphroditic mechanism in Cannabis (Lubell and Brand 2018). STS solution was made by adding 3.79g NaS2O3 (STS) to 160 mL H2O, adding 0.69 g AgNCh to 40 mL H2O, mixing well and then mixing together for a total volume of 200 mL. The 200 mL solution was then diluted with 3 L FLO for a total volume of 3200 mL. STS spray was applied uniformly to each plant until the entire plant surface was covered and the solution was dripping in excess. The daily light cycle was changed to a 12-hour photoperiod on the day of the first application. Three additional STS spray treatments (five days, 11 days and 17 days) were applied after the first application to ensure hermaphroditism would occur. dsRNA Spray Creation. dsRNA were manufactured by Creative Biogene Inc (Shirley, NY, USA) and were validated using gel electrophoresis to verify desired fragment size. To create the spray solution, RNase activity was prevented by baking instruments at 150°C and covered with foil for three hours to maintain sterilization prior to use. The ventilation hood in which work was performed was sterilized by wiping down the surface and pipettes with RNase Away and applying UV lights prior to working to inactivate any RNase. Only RNase- free pipet tips were used.

[0173] In one of the many studies to evaluate nanoparticles for dsRNA efficacy, Xu et al. (2023) found that chitosan quaternary ammonium salt (CQAS) was the most effective at cellular uptake among all compared nanoparticle-dsRNAs complexes and naked-dsRNA. Further, when multiple types of nanoparticles are sprayed, CQAS exhibited consistent uptake in multiple plant species such as tobacco, tomato, and pepper. Thus, we used CQAS as a nanoparticle to aid dsRNA uptake. To make the CQAS solution, dsRNA was complexed with 0.02% CQAS using methods established by Xu et al. (2023) for RNAi application. 75% deacetylated CQAS was dissolved in 0.1 M sodium acetate buffer to produce a solution with a concentration of 0.02% (w / v). dsRNA was bound to 0.02% CQAS in 100 pL of Na2SO4 (0.25 mol / L). Immediately following one minute of incubation at 55°C, the solution was vigorously vortexed for 30 seconds to facilitate the formation of the dsRNA-CQAS complex. The mixture was allowed to sit for 30 minutes to ensure binding. A gel retention assay confirmed the complex creation was successful. Mixture was stored in a cool and dark place until use. Air exposure was minimized when loading and using the high-pressure air sprayer. dsRNA Spray Application-. Each spray application onto plants used an Iwata NEO CN Gravity Feed Dual Action Airbrush (Harrison, OH, USA) 5 mL high pressure sprayer airbrush (FIG. 2). One Cocktail 1 dsRNA spray applied 0.1 mg of dsRNA for bHLH91 and 0.1 mg of dsRNA for AMS in a 0.02% CQAS solution. One Cocktail 2 dsRNA spray applied 0.1 mg of dsRNA for STP6 and 0.1 mg of dsRNA for TM6 in a 0.02% CQAS solution. Control sprays applied 0.02% CQAS solution alone without dsRNA present. Plants were sprayed with equal volume no matter the treatment. Spray heads were not shared across tent treatments to prevent contamination. FIG. 2 describes the experimental spray design for each cocktail.

[0174] Cocktail 1 Experiments: Cocktail 1 Experimental and Control sprays were applied 34 days after the final STS treatment when females were producing hermaphroditic flowers throughout the plant. Plants were sprayed twice per week for four weeks in both the experimental and control tent with their respective spray types (FIG. 2). Gene expression of Cocktail 1 as compared to gene expression in the control tent was measured seven days after the first spray. Pollen abortion vs non-abortion rates from Cocktail 1 as compared to the control tent were measured by sampling dehisced pollen eight days after the final spray.

[0175] Cocktail 2 Experiments: The first dsRNA spray of Cocktail 2 occurred 34 days after the final STS application when females were producing hermaphroditic flowers throughout the plant. Control spray was applied three times before the dsRNA spray was applied once. Gene expression was measured three days before the application of Cocktail 2 dsRNA spray and six days after to see the effect of dsRNA on gene expression. Pollen abortion vs nonabortion rates from Cocktail 2 were measured by sampling dehisced pollen two days prior to Cocktail 2 application, then seven days after.

[0176] Gene Expression Analysis'. RTqPCR was performed to evaluate the effect of dsRNA application on target gene expression. Flower material from three branches on each experimental plant was collected and immediately stored in RNAlater for processing at a later date. RNA was extracted using Qiagen RNeasy Plant Kit (Hilden, Germany) modified to extract RNA from tissue stored in RNAlater and including on-column DNase treatment. Next, RNA was quantified using Qubit RNA HS assays (Waltham, MA, USA) and all RNA concentrations were standardized to a similar value. A NEB Luna Universal Probe One-Step RTqPCR kit (Ipswich, MA, USA) was used to perform RTqPCR experiments following the protocol of 55°C 30-min reverse transcription, 95°C three min denaturation, and 40 cycles of 95°C 10 seconds to 65°C one min, where hydrolysis probes were used to measure transcript abundance. EFl-alpha was used as an internal reference control as this is described to be the best for measuring gene expression in Cannabis sativa (Guo et al. 2018). We calculated relative gene expression using the standard 2-AA Cq method (Livak and Schmittgen 2001). We performed statistical analysis using the log transformed data and used a paired one-tailed t-test (TM6 and STP6) due to the paired nature of the before and after treatments for dsRNA Cocktail 2 and a standard one-tailed t-test (AMS and bHLH9F) between the control and experimental treatments for dsRNA Cocktail 1, as was statistically appropriate.

[0177] Pollen Abortion Rate Measurement. Pollen abortion rate was assessed by measuring the proportion of aborted and non-aborted pollen based upon Wizenberg et al. (2022). Briefly, dehisced pollen was shaken into a 1.5 mL microcentrifuge tube. Tubes were kept open up to 1 hour to dry before sealing and storage at 4°C before processing. A modified Alexander stain was prepared to stain pollen and test for abortion rates, following the protocol in Wizenberg et al. (2022). The following were combined: 10 mL of 95% alcohol, 1 mL of diluted malachite green (1% solution in 95% alcohol). 54.5 mL of distilled water, 25 mL of glycerol, 5 mL of diluted acid fuchsin (1% solution in distilled water), 0.5 mL of diluted orange G (1% solution in distilled water), 4 mL of glacial acetic acid.

[0178] To prepare samples for evaluation of abortion rates, each pollen sample tube was unsealed and a clean cotton swab was used to apply a small sample of pollen by streaking the swab on the center of a 75 mm x 25 mm glass microscope slide. Aliquots of 20 pL of the modified Alexander stain were pipetted directly onto the applied pollen sample. Next, the prepared slide was heated approximately 5-15 cm above a butane torch lighter for roughly 5 seconds then allowed to cool to room temperature. Lastly, a glass slide cover (25 mm x 25 mm) was placed on the cooled sample and incubated at room temperature for 24 hours.

[0179] Pollen abortion rates were estimated by counting aborted (stained blue) and nonaborted (stained purple) pollen grains, up to 100 grains total. Non-shriveled pollen grains with dark, purple contents inside the grain indicate complete pollen viability (non-abortion). Pollen grains that are blue, which look fully or largely empty inside, are not viable (aborted). To this end, a light microscope (AmScope Bl 00; Irvine, CA, USA) was used to perform vertical transects at lOx magnification, moving across the length and width of the 2 mm2 slide cover until approximately 100 grains were counted. The number of aborted and nonaborted grains were counted using two hand-held tally counters. Any burst pollen grains on each slide were noted but excluded from the total number of grains. Any clumped pollen grains were excluded from counts. The proportion of pollen that stained as aborted and nonaborted as percentages of the total sample were calculated.

[0180] To evaluate pollen abortion percentage differences and changes in gene expression from treatment with Cocktail 1, a one-tailed t-test was performed on plants sprayed with Cocktail 1 relative to plants sprayed with the Control spray. To evaluate pollen abortion percentage change in abortion rates and changes in gene expression from treatment with Cocktail 2, a paired one-tail t-test was performed on plants before and after exposure to Cocktail 2. All analyses were performed in JMP vl7 (Cary, NC, USA).

[0181] Example 6 — Pollen Abortion Rate Results

[0182] Both dsRNA cocktails, Cocktail 1 and Cocktail 2, demonstrated a strong negative effect on pollen viability with significantly higher pollen abortion percentages than the control samples after each cocktail application. No other visible changes were observed; plants in all treatments remained healthy.

[0183] The application of Cocktail 1 (bHLH91 and AMS dsRNA) resulted in a 74.63% difference in aborted pollen, rising to 62.89% aborted pollen on average in the Experimental Treatment from 28.72% aborted pollen in the Control Treatment (FIGs. 3-6) (t-ratio = -2.485; P -value = 0.0323). For an example image of pollen in control conditions for Cocktail 1, see FIG. 3. For an example image of pollen after spraying Cocktail 1, see FIG. 4. FIG. 7 is a graphical representation of pollen abortion rates in the presence of Cocktail 1.

[0184] Prior to spraying dsRNA Cocktail 2, the control conditions exhibited an average aborted pollen percentage of 39.36%, but, after dsRNA Cocktail 2 exposure, aborted pollen percentage increased to 65.45% on average (t-ratio = 2.250, P-value = 0.0271). Thus, Cocktail 2 demonstrated a percent increase of 66.29% in aborted pollen. For an example image of pollen in control conditions for Cocktail 2, see FIG. 5. For an example image of pollen after spraying Cocktail 2, see FIG. 6. FIG. 8 is a graphical representation of pollen abortion rates before and after exposure to Cocktail 2.

[0185] Example 7 — Gene Expression Results

[0186] A significant decrease in mRNA transcript levels was observed for target genes relative to control plants sprayed with control solution (FIGs. 9-12). RTqPCR analysis revealed that the level of AMS mRNAs were significantly lower in dsRNA-0.2% CQAS treated plants relative to control (FIG. 9, P<0.01) and a similar pattern was observed for mRNA levels of bHLH91 relative to controls (FIG. 10, P<0.01). Similarly, before and after comparisons for gene expression showed a statistically significant reduction of mRNA levels for STP6 across all cultivars tested (FIG. 11, P<0.01) and in a single cultivar (‘Original Bacio’) when looking at TM6 gene expression before and after spraying (FIG. 12, P<0.05).

[0187] Example 8 — Conclusions

[0188] Each of the four gene targets across Cocktails 1 and 2 demonstrated a statistically significant decrease in gene expression as compared to the control, and the application of the dsRNA resulted in statistically significantly higher abortion rates. Pollen abortion rates and, thus, pollen inviability increased by 29.6% from Cocktail 1 and 66.92% from Cocktail 2 on average. While gene expression of TM6 was statistically significantly reduced only in ‘Original Bacio’, gene expression c£STP6 was statistically significantly reduced across all cultivars.

[0189] Concentration, timing and method of application of these dsRNA can alter the effectiveness of the gene silencing and phenotypic expression. With the demonstration that these genes directly inhibit viable pollen formation, the silencing, eradication or manipulation of these genes will hinder or eliminate male flower reproduction in Cannabis. The spray method is effective at significantly reducing the impact of male pollen in Cannabis. Example 9 — Gene and Protein Sequences and Description of Variation Among Almost 200

[0190] Cannabis Accessions

[0191] Gene name: AMS

[0192] CBDRx assembly gene ID: LOCI 15706375

[0193] Number of Isoforms: GCF_029168945.1 6

[0194] Number of Indels: 4

[0195] Number of Exon SNPs: 100

[0196] Description of variation: many individuals have large ~2-3 kb insertions upstream and downstream of the start site for the AMS coding sequence. The AMS gene ends earlier for the CBDRx assemblies; exon 9, which is around -450 bp, is completely missing. In other individuals, exon 9 has one or more small -15 bp insertions.

[0197]

[0198]

[0199] g

[0200]

[0201]

[0202]

[0203]

[0204] Gene name: MYB35-16

[0205] CBDRx assembly gene ID: LOCI 15711316

[0206] Number of Isoforms: 1

[0207] Number of Indels: 2

[0208] Number of Exon SNPs: 141

[0209] Description of variation: The genomic region has individuals with large (e.g., -5 kb) insertions, which are adjacent to the gene start position, and some individuals with smaller insertions (e.g., -535 bp) in the downstream region. The large insertions make alignment difficult and add gaps in the exon only alignment. The CBDRx annotation is smaller than other assemblies; exon one starts -200 bp later than other assemblies and exon three ends -230 bp earlier.

[0210]

[0211]

[0212] Gene name: MYB80

[0213] CBDRx assembly gene ID: LOC115725196

[0214] Number of Isoforms: 1

[0215] Number of Indels: 3

[0216] Number of Exon SNPs: 50

[0217] Description of variation: The genomic region around the MYB80 gene is characterized by a few different insertions in the upstream regions. The MYB80 gene and the downstream regions are well conserved. The CBDRx annotation and the other NCBI assembly, pink pepper (GCF_029168945.1) have an additional exon at the beginning of the gene. This first exon starts -640 base pairs before the start position in other assemblies and ends -90 base pairs before the start position in other assemblies. The second exon in CBDRx is smaller and starts -365 base pairs later. The final and fourth exon ends -115 base pairs earlier than other assemblies.

[0218] Table 8. List of Cannabis Accessions

[0219] It is to be understood that, while the methods and compositions of matter have been described herein in conjunction with a number of different aspects, the foregoing description of the various aspects is intended to illustrate and not limit the scope of the methods and compositions of matter. Other aspects, advantages, and modifications are within the scope of the following claims.

[0220] Disclosed are methods and compositions that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that combinations, subsets, interactions, groups, etc. of these methods and compositions are disclosed. That is, while specific reference to each various individual and collective combinations and permutations of these compositions and methods may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular composition of matter or a particular method is disclosed and discussed and a number of compositions or methods are discussed, each and every combination and permutation of the compositions and the methods are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed.

[0221] Appendix B

[0222]

[0223]

[0224]

[0225] Appendix H

Claims

WHAT IS CLAIMED IS:

1. A composition comprising an RNA nucleic acid molecule, wherein the RNA nucleic acid molecule is at least 10 nucleotides in length and has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least one of the genes selected from: AMS, bHLH91, TM6, STP6, DYT1, MYB35, MYB35, and MYB80, wherein the RNA nucleic acid molecule effectively silences expression of the at least one gene, wherein the AMS gene comprises any of SEQ ID NOs: 1 or 81-88; wherein the bHLH9I gene comprises any of SEQ ID NOs: 2 or 89-92; wherein the TM6 gene comprises any of SEQ ID NOs: 3 or 93-99; wherein the STP6 gene comprises any of SEQ ID NOs: 4 or 100-108; wherein the DYT1 gene comprises any of SEQ ID NOs: 5 or 109-115; wherein the MYB35-15 gene comprises any of SEQ ID NOs: 6 or 116-119; wherein the MYB35-16 gene comprises any of SEQ ID NOs: 7 or 120-124; wherein the MYB80 gene comprises any of SEQ ID NOs: 8 or 125-127.

2. The composition of claim 1, wherein the composition comprises RNA nucleic acid molecules at least 10 nucleotides in length and having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least two of the genes selected from AMS, bHLH91, TM6, STP6, DYT1, MYB35, MYB35, and MYB80, wherein the RNA nucleic acid molecules effectively silence expression of the at least two genes.

3. The composition of claim 1 or claim 2, wherein the composition is an aqueous solution.

4. The composition of any one of claims 1, 2, or 3, wherein the composition is formulated as a spray.

5. The composition of any one of claims 1, 2, or 3, wherein the composition is dried / lyophilized.

6. The composition of any of the preceding claims, wherein the RNA nucleic acid molecules are double-stranded (ds) RNA nucleic acid molecules.

7. The composition of claim 6, wherein the dsRNA nucleic acid molecules are selected from any of SEQ ID NOs:33-43, wherein each of the dsRNA nucleic acid molecules effectively silence expression of the AMS gene.

8. The composition of claim 6, wherein the dsRNA nucleic acid molecules are selected from any of SEQ ID NOs:44-54, wherein each of the dsRNA nucleic acid molecules effectively silence expression of the bHLH91 gene.

9. The composition of claim 6, wherein the dsRNA nucleic acid molecules are selected from any of SEQ ID NOs:55-65, wherein each of the dsRNA nucleic acid molecules effectively silence expression of the at TM6 gene.

10. The composition of claim 6, wherein the dsRNA nucleic acid molecules are selected from any of SEQ ID NOs:66-80, wherein each of the dsRNA nucleic acid molecules effectively silence expression of the STP6 gene.

11. The composition of claim 1, wherein the composition further comprises a delivery vehicle.

12. The composition of claim 11, wherein the delivery vehicle is a nanoparticle.

13. An article of manufacture, comprising:RNA nucleic acid molecules, wherein the RNA nucleic acid molecules are at least 10 nucleotides in length and have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to at least one of the genes selected from AMS, bHLH91, TM6, STP6, DYT1, MYB35, MYB35, and MYB80, wherein the RNA nucleic acid molecules effectively silence expression of the at least one gene, wherein the AMS gene comprises any of SEQ ID NOs: 1 or 81-88; wherein the bHLPl91 gene comprises any of SEQ ID NOs: 2 or89-92; wherein the TM6 gene comprises any of SEQ ID NOs: 3 or 93-99; wherein the STP6 gene comprises any of SEQ ID NOs: 4 or 100-108; wherein the DYT1 gene comprises any of SEQ ID NOs: 5 or 109-115; wherein the MYB35-15 gene comprises any of SEQ ID NOs: 6 or 116-119; wherein the MYB35-16 gene comprises any of SEQ ID NOs: 7 or 120-124; wherein the MYB80 gene comprises any of SEQ ID NOs: 8 or 125-127; and a delivery vehicle.

14. The article of manufacture of claim 13, wherein the delivery vehicle is a nanoparticle.

15. The article of manufacture of claim 14, wherein the nanoparticle is selected from chitosan salts (e.g., CQAS), carbon dots, liposomes, clay nanosheets, and gold nanoparticles.

16. The article of manufacture of any one of claims 13-15, wherein the delivery vehicle is conjugated to the RNA nucleic acid molecules.

17. The article of manufacture of any one of claims 13-15, wherein the delivery vehicle is not conjugated to the RNA nucleic acid molecules.

18. The article of manufacture of any one of claims 13-17, further comprising a linker.

19. The article of manufacture of any one of claims 13-18, wherein the article of manufacture is formulated as a spray.

20. A method of preventing hermaphroditism (e.g., in females) or the viability in male flowers (e.g., in male hermaphrodites or biological males) in Cannabis plants, comprising contacting Cannabis plants with the composition of any of claims 1-12.21 . The method of claim 20, wherein the contacting is spraying.

22. The method of claim 20 or 21, wherein the contacting is performed for about 10 seconds to about 60 seconds (per plant) at least one time a day.

23. The method of claim 20 or 21, wherein the contacting is performed for about 10 seconds to about 60 seconds (per plant) a plurality of times each day.

24. A method of producing male sterile Cannabis plants (e.g., hermaphroditic female or a biological male), comprising: introducing a nucleic acid molecule into Cannabis cells to produce genetically-engineered Cannabis cells, wherein the nucleic acid molecule comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more genes selected from AMS, bHLH91, TM6, STP6, DYT1, MYB35, MYB35, and MYB80 under direction of a promoter sequence, wherein the AMS gene comprises any of SEQ ID NOs: 1 or 81-88; wherein the bHLH91 gene comprises any of SEQ ID NOs: 2 or 89-92; wherein the TM6 gene comprises any of SEQ ID NOs: 3 or 93-99; wherein the STP6 gene comprises any of SEQ ID NOs: 4 or 100-108; wherein the DYT1 gene comprises any of SEQ ID NOs: 5 or 109-115; wherein the MYB35-15 gene comprises any of SEQ ID NOs: 6 or 116-119; wherein the MYB35-16 gene comprises any of SEQ ID NOs: 7 or 120-124; wherein the MYB80 gene comprises any of SEQ ID NOs: 8 or 125-127; and regenerating a genetically engineered Cannabis plant from the genetically engineered Cannabis cells, wherein the genetically engineered Cannabis plant exhibits reduced or no expression from the one or more genes or reduced, altered, or lack of expression or function of the expressed protein.

25. The method of claim 24, wherein expression of the nucleic acid molecule triggers RNAi, thereby silencing expression of the at least one gene.

26. The method of claim 24 or claim 25, wherein pollen produced by the genetically engineered Cannabis plants is non-viable.

27. The method of any one of claims 24-26, further comprising selecting for genetically engineered Cannabis plants.

28. A method of producing male sterile Cannabis plants (e.g., hermaphroditic female or a biological male), comprising: introducing a nucleic acid molecule into Cannabis cells under conditions in which the genome of the Cannabis cells is edited to produce edited Cannabis cells, wherein the nucleic acid molecule has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to one or more genes selected from AMS, bHLH91, TM6, STP6, DYT1, MYB35, MYB35, and MYB80, wherein the / IMS' gene comprises any of SEQ ID NOs: 1 or 81-88; wherein the bHLH91 gene comprises any of SEQ ID NOs: 2 or 89-92; wherein the TM6 gene comprises any of SEQ ID NOs: 3 or 93-99; wherein the STP6 gene comprises any of SEQ ID NOs: 4 or 100-108; wherein the DYT1 gene comprises any of SEQ ID NOs: 5 or 109-115; wherein the MYB35-15 gene comprises any of SEQ ID NOs: 6 or 116-119; wherein the MYB35-16 gene comprises any of SEQ ID NOs: 7 or 120-124; wherein the MYB80 gene comprises any of SEQ ID NOs: 8 or 125-127; and regenerating an edited Cannabis plant from the edited Cannabis cells, wherein the edited Cannabis plant exhibits reduced or no expression from the one or more genes or reduced, altered, or lack of expression or function of the expressed protein.

29. The method of claim 28, wherein the nucleic acid molecule comprises one or more constructs comprising gene editing components for editing at least one of the genes selected from AMS, bHLH91, TM6, STP6, DYT1, MYB35, MYB35, and MYB80, wherein the gene editing reduces or knocks-out expression from the one or more genes or the expressed protein exhibits reduced, altered, or lack of function.

30. The method of claim 29, wherein the gene editing components are selected from CRISPR / Cas components, TALEN components, or the like.

31. The method of any one of claims 28-30, wherein the pollen produced by the edited Cannabis plants is non-viable.

32. A method of making a Cannabis plant (e.g., hermaphroditic female or a biological male) male sterile, comprising contacting Cannabis seeds with a mutagen to create mutant Cannabis seeds; producing mutant Cannabis plants from the mutant Cannabis seeds; and screening the mutant Cannabis plants for those having a mutation in one or more genes selected from AMS, bHLH91, TM6, STP6, DYT1, MYB35, MYB35, and MYB80, wherein the mutant Cannabis plants exhibit reduced or no expression from the one or more genes or reduced, altered, or lack of expression or function of the expressed protein.

33. A method of evaluating a Cannabis plant for the propensity to trigger male flower formation (e.g., hermaphroditism), comprising: determining the presence or absence of a variation in sequence within one or more genes selected from AMS, bHLH91, TM6, STP6, DYT1, MYB35, MYB35, and MYB80, wherein the presence or absence of sequence variation within one or more of the genes is indicative of the propensity to trigger male flower formation (e.g., hermaphroditism) in the Cannabis plant.

34. A method of obtaining a male sterile or hermaphroditic-resistant Cannabis plant, comprising: a) crossing a first Cannabis plant and a second Cannabis plant, wherein the first Cannabis plant exhibits male sterility or hermaphroditic-resistance; b) hybridizing genomic DNA from cells obtained from the cross or from cells of later filial generations of the cross with one or more markers within one or more genes selected from AMS, bHLH91, TM6, STP6, DYl’l, MYB35, MYB35, and MYB80 and; c) selecting a plant in which the genomic DNA hybridizes with one or more of the markers within one or more of the genes selected from AMS, bHLH91, TM6, STP6, DYT1, MYB35, MYB35, and MYB80 to obtain a male sterile or hermaphroditic-resistant plant.

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