WO2022241461A1 - Modified autoflower cannabis plants with value phenotypes - Google Patents

Abstract

Provided herein are modified Cannabis plants exhibiting a modified day-length sensitivity phenotype and at least one Value Phenotype such as architecture, determinant growth, cannabinoid content, terpene content, aromatics or flavor molecules profile, disease resistance, biomass yield, time to maturity, etc., compared with wild type Cannabis. More particularly, plants produced by manipulating genes controlling day-length sensitivity are provided.

Description

MODIFIED AUTOFLOWER CANNABIS PLANTS WITH VALUE PHENOTYPES

Claim of Priority under 35 U.S.C. §119

[0001] The present Application for Patent claims priority to Provisional Application No.

63/188,682 entitled “AUTOFLOWER CANNABIS PLANTS WITH VALUE PHENOTYPES” filed May 14, 2021, the entirety of which is hereby expressly incorporated by reference herein.

BACKGROUND

Field

[0002] The present disclosure relates to conferring and improving Value Phenotypes in

Cannabis plants. More particularly, the current invention pertains to producing modulated day-length sensitive Cannabis plants with a Value Phenotype by manipulating genes controlling day-length sensitivity.

Background

[0003] Cannabis is one of the oldest domesticated plants with evidence of being used by a vast array of ancient cultures. It is thought to have originated from central Asia being then spread by humans to China, Europe, the Middle East, and the Americas. Thus, Cannabis has been bred by many different cultures for various uses such as food, fiber, and medicine since the dawn of agricultural societies. In recent decades, the illegality of Cannabis limited breeding to a relatively small cadre of dedicated enthusiasts who were largely self-trained and whose freedom to collaborate and publish their work was severely limited. With recent legalization of Cannabis in many jurisdictions, there is a growing need for the implementation of new and advanced Cannabis breeding techniques and programs. This will streamline the long process of classical breeding and accelerate reaching new and genetically improved Cannabis varieties for fiber, food, and medicinal products. Developing and implementing molecular biology tools to support breeders will allow creation of new traits and developing new combinations of desired traits.

1 [0004] Much of the current breeding of Cannabis continues to be done by the community of independent breeders and small farms that carried on this work prior to legalization. There are only very limited molecular tools that are as yet available in Cannabis breeding.

SUMMARY

[0005] It is one object of the present invention to disclose a modified Cannabis plant exhibiting a modified day-length sensitivity phenotype and at least one Value Phenotype such as architecture, determinant growth, cannabinoid content, terpene content, aromatics or flavor molecules profile, disease resistance, biomass yield, time to maturity, etc. compared with wild type Cannabis, wherein said modified plant comprises at least one mutated pseudoresponse regulator (PRR) protein gene, such as CsPRR37 or any of its binding partners, members of its protein or DNA complex, or any other gene involved in circadian rhythm or photoperiod sensing, resulting in a new day-length sensitivity phenotype in combination with at least one other value phenotype. It can be especially useful to directly modify a plant when, in the alternative, obtaining the natural variation through traditional breeding schemes results in linkage drag of negative, undesirable traits such as a high ratio of leaf to floral organ identity in flowering tissues or disadvantageous alleles contributing negatively to any of the above-mentioned traits.

[0006] Embodiments of the invention relate to a modified Cannabis plant exhibiting a modulated day-length sensitivity phenotype and at least one Value Phenotype. In some embodiments, the modified Cannabis plant can include at least one mutated pseudoresponse regulator 37 (CsPRR37) gene or a non-functional variant thereof with a mutation.

[0007] In some embodiments, the non- functional variant has at least 75% sequence identity to the CsPRR37 or the CsPRR37 nucleotide sequence.

[0008] In some embodiments, the mutation can be introduced using mutagenesis, small interfering RNA (siRNA), microRNA (miRNA), artificial miRNA (amiRNA), DNA introgression, endonucleases or any combination thereof.

[0009] In some embodiments, the mutation can be introduced using targeted genome modification.

2 [0010] In some embodiments, the mutation can be introduced using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR- associated (Cas) gene (CRISPR/Cas), Transcription activator-like effector nuclease (TALEN), Zinc Finger Nuclease (ZFN), meganuclease or any combination thereof.

[0011] In some embodiments, the Cas gene can be Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, CaslO, CastlOd, Casl2, Casl3, Casl4, CasX, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csnl, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpfl, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Cszl, Csxl5, Csfl, Csf2, Csf3, Csf4, Cul966 or any combination thereof.

[0012] In some embodiments, the mutated CsPRR37 gene is a CRISPR/Cas9- induced heritable mutated allele.

[0013] In some embodiments, the mutation is a missense mutation, nonsense mutation, insertion, deletion, indel, substitution or duplication.

[0014] In some embodiments, the insertion or the deletion produces a gene comprising a frameshift.

[0015] In some embodiments, the plant is homozygous for a CsPRR37 mutated gene. In some embodiments, the plant is heterozygous for a CsPRR37 mutated gene.

[0016] In some embodiments, the mutation is in the coding region of said allele, a mutation in the regulatory region of said allele, or an epigenetic factor.

[0017] In some embodiments, the mutation is a silencing mutation, a knockdown mutation, a knockout mutation, a loss of function mutation or any combination thereof.

[0018] In some embodiments, the mutation is generated in planta. In some embodiments, the mutation is generated in planta via introduction of a construct comprising a sequence corresponding to a gRNA sequence listed in Tables 1-2.

[0019] In some embodiments, the gRNA sequence includes a 3 ’ NGG Protospacer Adjacent Motif (PAM).

[0020] In some embodiments, the construct is introduced into the plant cells via Agrobacterium infiltration, virus-based plasmids for delivery of the genome editing molecules or mechanical insertion such as polyethylene glycol (PEG) mediated DNA transformation, electroporation, or gene gun biolistics.

3 [0021] In some embodiments, the plant has decreased expression levels of CsPRR37 gene.

[0022] In some embodiments, the sequence of the expressed CsPRR37 gene is 50% identical to the native CsPRR37 gene or a non-functional variant thereof.

[0023] In some embodiments the Value Phenotype can be: high THCA accumulation; specific cannabinoid ratio(s); a composition of terpenes and/or other aroma active or aromatic molecules; monoecy or dioecy (enable or prevent hermaphroditism); branchless or branched architectures with specific height to branch length ratios or total branch length; determinant growth; time to maturity; high flower to leaf ratios that enable pathogen resistance through improved airflow; high flower to leaf ratios that maximize light penetration and flower development in the vertical canopy space; a finished plant height that enables tractor farming inside high tunnels; a finished plant height and flower to leaf ratio that maximizes light penetration all the way to the ground but minimizes total plant height; trichome size; trichome density; advantageous flower structures for oil or flower production (flower diameter length, long or short intemodal spacing distance, flower-to-leaf determination ratio (leafiness of flower); metabolites that provide enhanced properties to finished oil products (oxidation resistance, color stability, cannabinoid and terpene stability); specific variants affecting cannabinoid or aromatic molecule biosynthetic pathways; modulators of the flowering time phenotype that increase or decrease maturation time; flower biomass yield and composition; flower crude oil yield and composition; resistance to botrytis, powdery mildew, fusarium, pythium, cladosporium, alternaria, spider mites, broad mites, russet mites, aphids, nematodes, caterpillars, HLVd or any other Cannabis pathogen or pest of viral, bacterial, fungal, insect, or animal origin; propensity to host specific beneficial and/or endophytic microflora; heavy metal composition in tissues; specific petiole and leaf angles and lengths; and/or the like.

[0024] Some embodiments of the invention relate to a Cannabis plant, plant part or plant cell as disclosed herein wherein the plant does not comprise a transgene.

[0025] Some embodiments of the invention relate to a plant part, plant cell or plant seed of a plant disclosed herein.

[0026] Some embodiments of the invention relate to a tissue culture of regenerable cells, protoplasts or callus obtained from a modified Cannabis plant as disclosed herein.

4 [0027] Some embodiments of the invention relate to a method for producing a modified

Cannabis plant exhibiting a modified day-length sensitivity phenotype and at least one Value Phenotype compared with wild type Cannabis. In some embodiments, the method can include genetically modifying at least one Cannabis CsPRR gene.

[0028] Some embodiments of the invention relate to a method for producing a modified

Cannabis plant exhibiting a modified day-length sensitivity phenotype and at least one Value Phenotype compared with wild type Cannabis by targeted genome modification. The method can include the steps of genetically introducing a loss of function mutation in at least one Cannabis CsPRR gene.

[0029] Some embodiments of the invention relate to an isolated nucleotide sequence having at least 75% sequence identity to a native CsPRR37 nucleotide coding sequence wherein the native CsPRR37 protein is not expressed.

[0030] Some embodiments of the invention relate to an isolated amino acid sequence having at least 75% sequence similarity to a native CsPRR37 amino acid sequence wherein a protein having the amino acid sequence does not have the function of the native protein.

[0031] Some embodiments of the invention relate to a use of any of the nucleotide sequences as set forth in Tables 1 and 2, and any combination thereof, for targeted genome modification of Cannabis CsPRR37 allele.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 depicts an alignment of the CsPRR37 mRNA to the CCA_PP_PRR37 de novo assembled PRR37 gene sequence along with the predicted protein sequence translation.

[0033] Figure 2 depicts an alignment of the CsPRR37 mRNA to the CCA_AF_PRR37 de novo assembled PRR37 gene sequence along with the predicted protein sequence translation.

[0034] Figure 3 depicts an alignment of the CsPRR37 mRNA to the Finola_PRR37 de novo assembled PRR37 gene sequence along with the predicted protein sequence translation.

[0035] Figure 4 depicts an alignment of the CsPRR37 mRNA to the LowRyder_PRR37 assembled PRR37 gene sequence along with the predicted protein sequence translation.

[0036] Figure 5 depicts an alternative alignment of the CsPRR37 mRNA to the Finola_PRR37 de novo assembled PRR37 gene sequence along with the predicted protein sequence translation.

5 DETAILED DESCRIPTION

[0037] The present invention provides a modified Cannabis plant exhibiting at least one Value Phenotype compared with wild type Cannabis, wherein said modified plant comprises at least one altered autoflower gene. The present invention further provides methods for producing the aforementioned modified Cannabis plant using genome editing or other genome modification techniques.

[0038] Embodiments of the invention relate to genome editing such as the CRISPR/Cas system in order to create cultivated Cannabis plants with day-length-insensitivity. Day- length insensitivity is also referred to as an “autoflower” trait or phenotype. Plant improvement using genome editing allows a precise and significantly shorter process in order to achieve these goals with a much higher success rate.

[0039] Legal limitations and traditional breeding techniques significantly limit the success of generating new and improved Cannabis varieties fit for intensive agriculture.

[0040] In addition, in many cases, Cannabis growers use Cannabis strains that were bred for indoor cultivation and are now using those for their greenhouse operations. This situation is obviously not ideal and can cause many logistical issues for growers. For example, since Cannabis plants require short days for the induction of flowering, growers install darkening curtains in the greenhouse to control day length for the plants. This artificial darkening can be cumbersome and can result in increased humidity in the greenhouse, thus creating optimal conditions for fungal pathogens to spread and thrive. These conditions may induce some growers to intensively use fungicides to control pathogen populations. With strict regulatory constraints in place across the legalized states, these conditions pose a great challenge for sustainable Cannabis production and consumer health.

[0041] In order to generate a consistent product, many Cannabis growers use vegetative propagation (cloning or tissue culture). However, in much of conventional agricultural, genetic stability of field crops and vegetables is maintained by using FI hybrid seeds. These hybrids are generated by crossing homozygous or inbred parental lines.

[0042] Use of hybrid seeds for propagation is likely to be widely adopted as well in the Cannabis industry. In addition, breeding for basic agronomic traits that are completely lacking in currently available Cannabis varieties (with an emphasis on day length

6 insensitivity and compact growth habit) will significantly increase growers’ productivity. This will allow growing and supplying high quality flower and other plant material for the Cannabis industry.

[0043] In general, plant flowering is triggered by seasonal changes in day length. However, day-length sensitivity in crops limits their geographical range of cultivation, and thus modification of the photoperiod response can be beneficial for their efficiency of production.

[0044] The present invention provides modulated day-length sensitive Cannabis plants with at least one Value Phenotype. The Value Phenotype can include at least one trait selected from: high THCA accumulation; specific cannabinoid ratio(s); a composition of terpenes and/or other aroma active or aromatic molecules; monoecy or dioecy (enable or prevent hermaphroditism); branchless or branched architectures with specific height to branch length ratios or total branch length; determinant growth; time to maturity; high flower to leaf ratios that enable pathogen resistance through improved airflow; high flower to leaf ratios that maximize light penetration and flower development in the vertical canopy space; a finished plant height that enables tractor farming inside high tunnels; a finished plant height and flower to leaf ratio that maximizes light penetration all the way to the ground but minimizes total plant height; trichome size; trichome density; advantageous flower structures for oil or flower production (flower diameter length, long or short internodal spacing distance, flower-to-leaf determination ratio (leafiness of flower); metabolites that provide enhanced properties to finished oil products (oxidation resistance, color stability, cannabinoid and terpene stability); specific variants affecting cannabinoid or aromatic molecule biosynthetic pathways;

7 modulators of the flowering time phenotype that increase or decrease maturation time; flower biomass yield and composition; flower crude oil yield and composition; resistance to botrytis, powdery mildew, fusarium, pythium, cladosporium, alternaria, spider mites, broad mites, russet mites, aphids, nematodes, caterpillars, HLVd or any other Cannabis pathogen or pest of viral, bacterial, fungal, insect, or animal origin; propensity to host specific beneficial and/or endophytic microflora; heavy metal composition in tissues; specific petiole and leaf angles and lengths; and/or the like.

[0045] In some embodiments, the current invention discloses the generation of non- transgenic Cannabis plants with day-length-insensitivity, using genome editing technology such as, for example, CRISPR/Cas9. The generated mutations can be introduced into elite or locally adapted Cannabis lines rapidly, with relatively minimal effort and investment.

[0046] For example, a photoperiod Cannabis plant with a Value Phenotype not normally present in an autoflower Cannabis plant can be genetically modified to produce a day- length sensitive Cannabis plant with the Value Phenotype.

[0047] Genome editing is an efficient and useful tool for increasing crop productivity, and there is particular interest in advancing manipulation of domestication genes in Cannabis wild species, which often have undesirable characteristics.

[0048] Genome-editing technologies, such as the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) (CRISPR- Cas9) can provide opportunities to address these deficiencies, with the aims of increasing quality and yield, improve adaptation and expand geographical ranges of cultivation. In some embodiments, a mutation can be introduced using mutagenesis, small interfering RNA (siRNA), microRNA (miRNA), artificial miRNA (amiRNA), DNA introgression, antisense DNA, zinc finger nucleases, transcription activator- like effector nucleases (TALENS), endonucleases, or the like or any combination thereof.

[0049] In some embodiments, a mutation can be introduced using targeted genome modification.

8 [0050] In some embodiments, a mutation can be introduced using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated (Cas) gene (CRISPR/Cas), Transcription activator-like effector nuclease (TALEN), Zinc Finger Nuclease (ZFN), meganuclease, or the like, or any combination thereof.

[0051] In some embodiments, the Cas gene can be Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, CaslO, CastlOd, Casl2, Casl3, Casl4, CasX, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csnl, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpfl, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Cszl, Csxl5, Csfl, Csf2, Csf3, Csf4, Cul966, or the like, or any combination thereof.

[0052] It is another object of the present invention to disclose the modified Cannabis plant as defined in any of the above, wherein the mutated gene can be a CRISPR/Cas9- induced heritable mutated allele.

[0053] Precise editing of an autoflower protein in wild Cannabis species, as disclosed by the present invention, can serve as a step towards generating commercially cultivable lines, without causing an associated linkage drag on other useful traits. The autoflower protein can be a pseudoresponse regulator (PRR) protein or a protein that interacts with a PRR protein or a protein that interacts with a protein in a PRR protein complex or a protein upstream or downstream of a signal transduction pathway of PRR.

[0054] To that end, guide RNAs (gRNAs) can be designed for each of the target genes identified in Cannabis to induce mutations in CsPRR37 through genome editing.

[0055] A point mutation in CsPRR37 can modify the day-length sensitivity of a photoperiod Cannabis plant. As used herein, a plant with modulated day-length sensitivity can be defined as a plant that demonstrates a different sensitivity to day length than wild type plants. For example, the phenotype can include an autoflower phenotype, attenuation or complete absence of day-length sensitivity, or increase of day-length sensitivity.

[0056] “”As used herein the term “corresponding” generally means similar, analogous, like, alike, akin, parallel, identical, resembling, or comparable. In further aspects it means having or participating in the same relationship (such as type or species, kind, degree, position, correspondence, or function). It further means related or accompanying.

9 In some embodiments of the present invention, it refers to plants of the same Cannabis species or strain or variety or to sibling plant, or one or more individuals having one or both parents in common.

[0057] A “plant” as used herein refers to any plant at any stage of development, particularly a seed plant. The term “plant” includes the whole plant or any parts or derivatives thereof, such as plant cells, seeds, plant protoplasts, plant cell tissue culture from which tomato plants can be regenerated, plant callus or calli, meristematic cells, microspores, embryos, immature embryos, pollen, ovules, anthers, fruit, flowers, leaves, cotyledons, pistil, seeds, seed coat, roots, root tips and the like.

[0058] The term “plant cell” used herein refers to a structural and physiological unit of a plant, comprising a protoplast and a cell wall. The plant cell may be in a form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, plant tissue, a plant organ, or a whole plant. In some embodiments where clear from context, a plant cell can refer to a protoplast without its cell wall.

[0059] The term “plant cell culture” as used herein means cultures of any culturable plant units such as, for example, protoplasts, regenerable cells, cell culture, cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes, and embryos at various stages of development, leaves, roots, root tips, anthers, meristematic cells, microspores, flowers, cotyledons, pistil, fruit, seeds, seed coat or any combination thereof.

[0060] The term “plant material” or “plant part” used herein refers to leaves, stems, roots, root tips, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, seed coat, cuttings, cell or tissue cultures, or any other part or product of a plant or a combination thereof.

[0061] A “plant organ” as used herein means a distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower, flower bud, or embryo.

[0062] The term “plant tissue” as used herein means a group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture, meristematic cells, and any group of plant cells organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of,

10 any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.

[0063] As used herein, the term “progeny” or “progenies” refers in a non-limiting manner to offspring or descendant plants. According to certain embodiments, the term “progeny” or “progenies” refers to plants developed or grown or produced from the disclosed or deposited seeds as detailed herein. The grown plants preferably have the desired traits of the disclosed or deposited seeds, e.g., loss-of-function mutation in at least one PRR gene like CsPRR37 or a member of its protein or DNA complex. In some embodiments the mutation is a silencing mutation, a knockdown mutation, a knockout mutation, a loss of function mutation or any combination thereof.

[0064] The term “Cannabis” refers hereinafter to a genus of flowering plants in the family Cannabaceae. Cannabis is an annual, dioecious, flowering herb that, by some taxonomic approaches, includes, but is not limited to three different species, Cannabis sativa, Cannabis indica and Cannabis ruderalis. Other taxonomists argue that the genus Cannabis is monospecific, and use sativa as the species name. The genus Cannabis is inclusive of hemp, which term is a legal or functional classification rather than being a taxonomic term.

[0065] Cannabis plants produce a group of chemicals called cannabinoids. Cannabinoids, terpenoids, and other compounds are secreted by glandular trichomes that occur most abundantly on the floral calyxes and bracts of female Cannabis plants.

[0066] As used herein the term “genetic modification” refers hereinafter to genetic manipulation or modulation, which is the direct manipulation of an organism’s genes using biotechnology. It also refers to a set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species, targeted mutagenesis and genome editing technologies to produce improved organisms. According to main embodiments of the present invention, modified Cannabis plants with Value Phenotypes are generated using genome editing. This technique enables to achieve in planta modification of specific genes that relate to and/or control the flowering time and plant architecture in the Cannabis plant. In other embodiments, the invention also provides other approaches to achieving Value Phenotypes by targeting at least one PRR gene such as CsPRR37 or any of its binding partners, members of its protein complex, or any other gene involved in circadian rhythm or photoperiod sensing. These

11 approaches can include but are not limited to genetic transformation, gene editing, base editing, gene silencing, and mutagenesis.

[0067] The term “genome editing”, or “genome/genetic modification” or “genome engineering” generally refers hereinafter to a type of genetic engineering in which DNA is inserted, deleted, modified, or replaced in the genome of a living organism. Unlike previous genetic engineering techniques that randomly insert genetic material into a host genome, genome editing targets the insertions to site-specific locations.

[0068] It is within the scope of the present invention that the common methods for such editing use engineered nucleases, or “molecular scissors”. These nucleases create site- specific double strand breaks (DSBs) at desired locations in the genome. The induced double-strand breaks are repaired through nonhomologous end-joining (NHEJ) or homologous recombination (HR), resulting in targeted mutations (“edits”). Families of engineered nucleases used by the current invention can include, but are not limited to: meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TAFEN), and the clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system.

[0069] Reference is now made to exemplary genome editing terms used by the current disclosure:

[0070] Genome Editing Glossary

Cas = CRISPR-associated genes Indel = insertion and/or deletion

Cas9, Csn = CRISPR-associated protein containing two nuclease domains, that is programmed by small RNAs to cleave DNA

NHEJ = Non-Homologous End Joining

PAM = Protospacer- Adjacent Motif

RuvC = an endonuclease domain named for an £ coli protein involved in DNA repair crRNA = CRISPR RNA dCAS9 = nuclease-deficient Cas9 sgRNA - single guide RNA

DSB = Double-Stranded Break tracrRNA, trRNA - trans-activating crRNA gRNA = guide RNA

TALEN = Transcription- Activator Like Effector Nuclease

12 HDR = Homology-Directed Repair

HNH = an endonuclease domain named for characteristic histidine and asparagine residues

ZFN = Zinc-Finger Nuclease

[0071] According to specific aspects of the present invention, the CRISPR and Cas genes can be used for generating genome modification in targeted genes in the Cannabis plant. The functions of CRISPR and Cas genes are essential in adaptive immunity in select bacteria and archaea, enabling the organisms to respond to and eliminate invading genetic material. These repeats were initially discovered in the 1980s in E. coli. Without wishing to be bound by theory, reference is now made to a type of CRISPR mechanism, in which invading DNA from viruses or plasmids is cut into small fragments and incorporated into a CRISPR locus comprising a series of short repeats (around 20 bps). The loci are transcribed, and transcripts are then processed to generate small RNAs (crRNA, namely CRISPR RNA), which are used to guide effector endonucleases that target invading DNA based on sequence complementarity.

[0072] According to further aspects of the invention, Cas protein, such as Cas9 (also known as Csnl) can be required for gene silencing. Cas9 participates in the processing of crRNAs, and is responsible for the destruction of the target DNA. Cas9’s function in both of these steps relies on the presence of two nuclease domains, a RuvC-like nuclease domain located at the amino terminus and a HNHlike nuclease domain that resides in the mid-region of the protein. To achieve site-specific DNA recognition and cleavage, Cas9 is complexed with both a crRNA and a separate trans-activating crRNA (tracrRNA or trRNA), that is partially complementary to the crRNA. The tracrRNA is required for crRNA maturation from a primary transcript encoding multiple pre-crRNAs. This occurs in the presence of RNase III and Cas9.

[0073] Without wishing to be bound by theory, during the destruction of target DNA, the HNH and RuvC-like nuclease domains cut both DNA strands, generating double- stranded breaks (DSBs) at sites defined by a 20-nucleotide target sequence within an associated crRNA transcript. The HNH domain cleaves the complementary strand, while the RuvC domain cleaves the noncomplementary strand.

13 [0074] It is further noted that the double- stranded endonuclease activity of Cas9 can also requires that a short-conserved sequence, (2-5 nts) known as protospacer-associated motif (PAM), follows immediately 3 of the crRNA complementary sequence.

[0075] According to further aspects of the invention, a two-component system can be used by the current invention, combining trRNA and crRNA into a single synthetic single guide RNA (sgRNA) for guiding targeted gene alterations.

[0076] It is further within the scope that Cas9 nuclease variants include wild-type Cas9, Cas9D10A and nuclease-deficient Cas9 (dCas9).

[0077] The term "meganucleases" as used herein refers hereinafter to endodeoxyribonucleases characterized by a large recognition site (double- stranded DNA sequences of 12 to 40 base pairs); as a result this site generally occurs only once in any given genome. Meganucleases are therefore considered to be the most specific naturally occurring restriction enzymes.

[0078] The term "protospacer adjacent motif’ or "PAM" as used herein refers hereinafter to a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. PAM is a component of the invading virus or plasmid, but is not a component of the bacterial CRISPR locus. PAM is an essential targeting component which distinguishes bacterial self from non-self DNA, thereby preventing the CRISPR locus from being targeted and destroyed by nuclease.

[0079] The term "Next-generation sequencing" or "NGS" as used herein refers hereinafter to massively parallel, high- throughput or deep sequencing technology platforms that perform sequencing of millions of small fragments of DNA in parallel. Bioinformatics analyses are used to piece together these fragments by mapping the individual reads to the reference genome. The term "gene knockdown" as used herein refers hereinafter to an experimental technique by which the expression of one or more of an organism's genes is reduced. The reduction can occur through genetic modification, i.e., targeted genome editing or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript. The reduced expression can be at the level of RNA or at the level of protein. It is within the scope of the present invention that the term gene knockdown also refers

14 to a loss of function mutation and /or gene knockout mutation in which an organism's genes is made inoperative or nonfunctional.

[0080] The term "gene silencing" as used herein refers hereinafter to the regulation of gene expression in a cell to prevent the expression of a certain gene. Gene silencing can occur during either transcription or translation. In certain aspects of the invention, gene silencing is considered to have a similar meaning as gene knockdown. When genes are silenced, their expression is reduced. In contrast, when genes are knocked out, they are completely not expressed. Gene silencing may be considered a gene knockdown mechanism since the methods used to silence genes, such as RNAi, CRISPR, or siRNA, generally reduce the expression of a gene by at least 70% but do not completely eliminate it.

[0081] The term "loss of function mutation" as used herein refers to a type of mutation in which the altered gene product lacks the function of the wild-type gene. A synonyms of the term included within the scope of the present invention is null mutation.

[0082] The term "microRNAs" or "miRNAs" refers hereinafter to small non-coding RNAs that have been found in most of the eukaryotic organisms. They are involved in the regulation of gene expression at the post-transcriptional level in a sequence specific manner. MiRNAs are produced from their precursors by Dicer-dependent small RNA biogenesis pathway. MiRNAs are candidates for studying gene function using different RNA-based gene silencing techniques. For example, artificial miRNAs (amiRNAs) targeting one or several genes of interest is a potential tool in functional genomics.

[0083] The term "in planta" means in the context of the present invention within the plant or plant cells. More specifically, it means introducing CRISPR/Cas complex into plant material comprising a tissue culture of several cells, a whole plant, or into a single plant cell, without introducing a foreign gene or a mutated gene. It also used to describe conditions present in a non-laboratory environment (e.g., in vivo).

[0084] The term "earliness" refers hereinafter to early flowering and/or rapid transition from the vegetative to reproductive stages, or reduced “time to initiation of flowering” and more generally to earlier completion of the life-cycle.

[0085] The term “reduced flowering time” as used herein refers to time to production of first inflorescence. Such a trait can be evaluated or measured, for example, with reference to the number of leaves produced prior to appearance of the first inflorescence.

15 [0086] The term “harvest index” can be herein defined as the total yield per plant weight.

[0087] The term “day length” or “day length sensitivity” as used in the context of the present invention generally refers to photoperiodism, which is the physiological reaction of organisms to the length of day or night. Photoperiodism can also be defined as the developmental responses of plants to the relative lengths of light and dark periods. Plants are classified under three groups according to the photoperiods: short-day plants, long- day plants, and day-neutral plants. Photoperiodism affects flowering by inducing the shoot to produce floral buds instead of leaves and lateral buds. It is within the scope of the present invention that Cannabis is included within the short-day facultative plants. The Cannabis plants of the present invention are genetically modified so as to exhibit loss of day-length sensitivity, which is highly desirable agronomical trait enabling enhanced yield of the cultivated crop.

[0088] The term “determinate” or “determinate growth” as used herein refers to plant growth in which the main stem ends in an inflorescence or other reproductive structure (e.g., a bud) and stops continuing to elongate indefinitely with only branches from the main stem having further and similarly restricted growth. It also refers to growth characterized by sequential flowering from the central or uppermost bud to the lateral or basal buds. It further means naturally self-limited growth, resulting in a plant of a definite maximum size.

[0089] The term “indeterminate” or “indeterminate growth” as used herein refers to plant growth in which the main stem continues to elongate indefinitely without being limited by a terminal inflorescence or other reproductive structure. It also refers to growth characterized by sequential flowering from the lateral or basal buds to the central or uppermost buds.

[0090] The term “orthologue” as used herein refers hereinafter to one of two or more homologous gene sequences found in different species.

[0091] The term “functional variant” or "functional variant of a nucleic acid or amino acid sequence" as used herein, for example with reference to CsPRR37 refers to a variant of a sequence or part of a sequence which retains the biological function of the full non variant allele (e.g., CsPRR37 allele) and hence has the activity of CsPRR37 expressed gene or protein. A functional variant also comprises a variant of the gene of interest encoding a polypeptide which has sequence alterations that do not affect function of the

16 resulting protein, for example, in non-conserved residues. Also encompassed is a variant that is substantially identical, i.e., has only some sequence variations, for example, in non-conserved residues, to the wild type nucleic acid or amino acid sequences of the alleles as shown herein, and is biologically active

[0092] The term “non-functional variant” as used herein is in contrast to the term “functional variant” and means a variant that does not retain the biological function of the non- variant allele.

[0093] The term “variety” or “cultivar” used herein means a group of similar plants that by structural features and performance can be identified from other varieties within the same species.

[0094] The term “allele” used herein means any of one or more alternative or variant forms of a gene or a genetic unit at a particular locus, all of which alleles relate to one trait or characteristic at a specific locus. In a diploid cell of an organism, alleles of a given gene are located at a specific location, or locus (loci plural) on a chromosome. Alternative or variant forms of alleles may be the result of single nucleotide polymorphisms, insertions, inversions, translocations or deletions, or the consequence of gene regulation caused by, for example, by chemical or structural modification, transcription regulation or post-translational modification/regulation. An allele associated with a qualitative trait may comprise alternative or variant forms of various genetic units including those that are identical or associated with a single gene or multiple genes or their products or even a gene disrupting or controlled by a genetic factor contributing to the phenotype represented by the locus. According to further embodiments, the term “allele” designates any of one or more alternative forms of a gene at a particular locus. Heterozygous alleles are two different alleles at the same locus. Homozygous alleles are two identical alleles at a particular locus. A wild type allele is a naturally occurring allele.

[0095] As used herein, the term “locus” (loci plural) means a specific place or places or region or a site on a chromosome where for example a gene or genetic marker element or factor is found. In specific embodiments, such a genetic element is contributing to a trait.

[0096] As used herein, the term “homozygous"” refers to a genetic condition or configuration existing when two identical or like alleles reside at a specific locus, but are

17 positioned individually on corresponding pairs of homologous chromosomes in the cell of a diploid organism.

[0097] In specific embodiments, the Cannabis plants of the present invention comprise homozygous configuration of at least one of the mutated CsPRR37.

[0098] Conversely, as used herein, the term “heterozygous” means a genetic condition or configuration existing when two different or unlike alleles reside at a specific locus, but are positioned individually on corresponding pairs of homologous chromosomes in the cell of a diploid organism.

[0099] As used herein, the phrase “genetic marker” or “molecular marker” or

"biomarker" refers to a feature in an individual's genome e.g., a nucleotide or a polynucleotide sequence that is associated with one or more loci or trait of interest. In some embodiments, a genetic marker is polymorphic in a population of interest, or the locus occupied by the polymorphism, depending on context. Genetic markers or molecular markers include, for example, single nucleotide polymorphisms (SNPs), indels (i.e. insertions deletions), simple sequence repeats (SSRs), restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNAs (RAFDs), cleaved amplified polymorphic sequence (CAPS) markers, Diversity Arrays Technology (DArT) markers, and amplified fragment length polymorphisms (AFLPs) or combinations thereof, among many other examples such as the DNA sequence per se. Genetic markers can, for example, be used to locate genetic loci containing alleles on a chromosome that contribute to variability of phenotypic traits. The phrase “genetic marker” or “molecular marker” or “biomarker” can also refer to a polynucleotide sequence complementary or corresponding to a genomic sequence, such as a sequence of a nucleic acid used as a probe or primer.

[00100] As used herein, the term “germplasm” refers to the totality of the genotypes of a population or other group of individuals (e.g. , a species). The term "germplasm" can also refer to plant material; e.g., a group of plants that act as a repository for various alleles. Such germplasm genotypes or populations include plant materials of proven genetic superiority; e.g., for a given environment or geographical area, and plant materials of unknown or unproven genetic value; that are not part of an established breeding population and that do not have a known relationship to a member of the established breeding population.

18 [00101] The terms “hybrid”, “hybrid plant” and “hybrid progeny” used herein refers to an individual produced from genetically different parents (e.g., a genetically heterozygous or mostly heterozygous individual).

[00102] As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins, it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. The term further refers hereinafter to the amount of characters which match exactly between two different sequences. Hereby, gaps are not counted and the measurement is relational to the shorter of the two sequences.

[00103] It is further within the scope that the terms "similarity" and "identity" additionally refer to local homology, identifying domains that are homologous or similar (in nucleotide and/or amino acid sequence). Bioinformatics tools such as BLAST, SSEARCH, FASTA, and HMMER calculate local sequence alignments which identify the most similar region between two sequences. For domains that are found in different sequence contexts in different proteins, the alignment should be limited to the homologous domain, since the domain homology is providing the sequence similarity captured in the score. According to some aspects the term similarity or identity further includes a sequence motif, which is a nucleotide or ami no- acid sequence pattern that is widespread and has, or is conjectured to have, a biological significance. Proteins can have a sequence motif and/or a structural motif, a motif formed by the three- dimensional arrangement of amino acids which may not be adjacent.

[00104] As used herein, the terms "nucleic acid", "nucleic acid sequence", "nucleotide",

"nucleic acid molecule" or "polynucleotide" are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), natural occurring, mutated, synthetic DNA or RNA molecules, and analogs of the DNA or RNA generated using nucleotide analogs. It can be single stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes,

19 anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products. These terms also encompass a gene. The term "gene", "allele" or "gene sequence" is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes can include introns and exons as in the genomic sequence, or can include only a coding sequence as in cDNAs, and/or can include cDNAs in combination with regulatory sequences. Thus, according to the various aspects of the invention, genomic DNA, cDNA, or coding DNA can be used. In one embodiment, the nucleic acid is cDNA or coding DNA.

[00105] The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and refer to amino acids in a polymeric form of any length, linked together by peptide bonds.

[00106] According to other aspects of the invention, a "modified" or a "mutant" or

“altered” plant or gene is a plant or gene that has been altered compared to the naturally occurring wild type (WT) plant or gene. As used herein, a “modified”, “mutant” or “altered” plant or gene is a result of human intervention and is not intended to apply any naturally occurring mutation or mutant. Specifically, the endogenous nucleic acid sequences of each of the CsPRR37 homologs in Cannabis have been altered compared to wild type sequences using mutagenesis and/or genome editing methods as described herein. This causes inactivation of the endogenous CsPRR37 and thus disables CsPRR37 function. Such plants have a modified day length sensitivity and show Value Phenotypes. Therefore, the modified day length sensitivity phenotype is conferred by the presence of at least one mutated CsPRR37 gene in the Cannabis plant genome which has been specifically targeted using genome editing technique.

[00107] It should be noted that nucleic acid sequences of wild type alleles are designated using capital letters namely PRR37. Mutant prr37 nucleic acid sequences use non capitalization. Cannabis plants of the invention are modified plants compared to wild type plants which comprise and express a mutant Csprr37 allele.

[00108] It is further within the scope of the current invention that prr37 mutations that downregulate or disrupt functional expression of the wild-type PRR37 sequence respectively, can be recessive, such that they are complemented by expression of a wild- type sequence.

20 [00109] It is further noted that a wild type Cannabis plant is a plant that does not have a mutant prr37 allele.

[00110] Main aspects of the invention involve targeted mutagenesis methods, specifically genome editing, and exclude embodiments that are solely based on generating plants by traditional breeding methods. In a further embodiment of the current invention, as explained herein, the improved domestication at least one trait is not due to the presence of a transgene.

[00111] In some embodiments, the invention relates to mutant Cannabis lines with mutations inactivating a CsPRR37 homoeoallele which confer heritable Value Phenotype(s). In this way no functional CsPRR37 protein is made. Thus, the invention relates to these mutant Cannabis lines and related methods.

[00112] It is further within the scope of the present invention that breeding Cannabis cultivars with mutated prr37 allele enables the mechanical harvest of the plant. According to a further aspect of the present invention, loss of PRR37 function results in a modified day-length sensitivity when compared with WT Cannabis.

[00113] The work described herein has important implications: CRISPR/Cas9 or other molecular tools can be used to create heritable mutations in pathway family members that result in desirable phenotypic effects.

[00114] To identify other targets for day-length modification without negative effects on productivity, homologues of CsPRR37 can be identified in Cannabis, and targeted using guide RNAs.

[00115] According to a further embodiment of the present invention, CRISPR-Cas9- induced null prr37 mutations in Cannabis modify day-length sensitivity.

[00116] It is further within the scope of the present invention that targeting PRR37 homologs and/or other autoflower genes may allow immediate customization of day- length sensitivity in Cannabis elite germplasm.

[00117] The loss of function mutation can be a deletion or insertion ("indels") with reference the wild type CsPRR37 allele sequence. The deletion can include 1-20 or more nucleotides, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18 or 20 nucleotides or more in one or more strand. The insertion can include 1-20 or more nucleotides, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18 or 20 or more nucleotides in one or more strand.

21 [00118] The plant of the invention includes plants wherein the plant is heterozygous for one or more of the mutations that can affect day-length sensitivity. Heterozygous carries of valuable alleles are thus within the scope of the invention. In a preferred embodiment however, the plant is homozygous for the mutation(s). Progeny that is also homozygous can be generated from these plants according to methods known in the art.

[00119] It is further within the scope that variants of a particular CsPRR37 nucleotide or amino acid sequence according to the various aspects of the invention will have at least about 50%-99%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 92%, 94%, 95%, 96%, 97%, 98% or 99% or more sequence identity to that particular non-variant CsPRR37 nucleotide sequence of the CsPRR37 allele. Sequence alignment programs to determine sequence identity are well known in the art.

[00120] Also, the various aspects of the invention encompass not only a CsPRR37 nucleic acid sequence or amino acid sequence, but also fragments thereof. By "fragment" is intended a portion of the nucleotide sequence or a portion of the amino acid sequence and hence of the protein encoded thereby. Fragments of a nucleotide sequence can encode protein fragments that retain the biological activity of the native protein, in this case Value Phenotype.

[00121] According to a further embodiment of the invention, the herein newly identified

Cannabis CsPRR37 locus have been targeted using the double sgRNA strategy.

[00122] According to further embodiments of the present invention, DNA introduction into the plant cells can be done by Agrobacterium infiltration, virus based plasmids for delivery of the genome editing molecules and mechanical insertion of DNA (PEG mediated DNA transformation, biolistics, etc.).

[00123] In addition, it is within the scope of the present invention that the Cas9 protein is directly inserted together with a gRNA (ribonucleoprotein- RNPs) in order to bypass the need for in vivo transcription and translation of the Cas9+gRNA plasmid in planta to achieve gene editing.

[00124] It is within the scope of the present invention that the usage of CRISPR/Cas system for the generation of Cannabis plants with at least one Value Phenotype, allows the modification of predetermined specific DNA sequences without introducing foreign DNA into the genome by GMO techniques. According to one embodiment of the present invention, this is achieved by combining the Cas nuclease (e.g., Cas9, Cpfl and the like)

22 with a predefined guide RNA molecule (gRNA). The gRNA is complementary to a specific DNA sequence targeted for editing in the plant genome and which guides the Cas nuclease to a specific nucleotide sequence. The predefined gene specific gRNAs are cloned into the same plasmid as the Cas gene and this plasmid is inserted into plant cells. Insertion of the aforementioned plasmid DNA can be done, but not limited to, using different delivery systems, biological and/or mechanical, e.g., Agrobacterium infiltration, vims based plasmids for delivery of the genome editing molecules and mechanical insertion of DNA (PEG mediated DNA transformation, biolistics, etc.).

[00125] It is further within the scope of the present invention that upon reaching the specific predetermined DNA sequence, the Cas9 nuclease cleaves both DNA strands to create double stranded breaks leaving blunt ends. This cleavage site is then repaired by the cellular non homologous end joining DNA repair mechanism resulting in insertions or deletions which eventually create a mutation at the cleavage site. For example, a deletion form of the mutation consists of at least 1 base pair deletion. As a result of this base pair deletion the gene coding sequence is disrupted and the translation of the encoded protein is compromised either by a premature stop codon or disruption of a functional or structural property of the protein. Thus, DNA is cut by the Cas9 protein and re-assembled by the cell’s DNA repair mechanism.

[00126] It is further within the scope that a modulated day-length sensitive Cannabis plant with a Value Phenotype is herein produced by generating gRNA with homology to a specific site of predetermined genes in the Cannabis genome i.e., PRR37 gene, sub cloning this gRNA into a plasmid containing the Cas9 gene, and insertion of the plasmid into the Cannabis plant cells. In this way a site-specific mutation in the PRR37 gene is generated thus effectively creating non-active molecules, resulting in modified day length sensitivity and a Value Phenotype.

[00127] Further information used in the invention can be found in U.S. Provisional

Application No. 63/142,906, entitled “MARKER-ASSISTED BREEDING IN CANNABIS PLANTS,” filed January 28, 2021; U.S. Provisional Application No. 63/150,381, entitled “VALUE-PHENOTYPED AUTOFLOWER CANNABIS PLANTS,” filed February 17, 2021; U.S. Provisional Application No. 63/182,725, entitled “MODULATED DAY-LENGTH SENSITIVITY CANNABIS PLANTS, GENES, MARKERS AND BREEDING,” filed April 30, 2021; International

23 Application No. PCT/US2022/070402, entitled “MARKER- ASSISTED BREEDING IN CANNABIS PLANTS,” filed January 28, 2022; International Application No. PCT/US 2022/070696 , entitled “VALUE-PHENOTYPED AUTOFLOWER CANNABIS PLANTS,” filed February 17, 2022; and International Application No. PCT/US 2022/071972, entitled “MODULATED DAY-LENGTH SENSITIVE CANNABIS PLANTS, GENES, MARKERS, AND BREEDING,” filed April 28, 2022. The entire contents of each of the forgoing are fully incorporated by reference herein.

[00128] In order to understand the invention and to see how it can be implemented in practice, embodiments will now be described, by way of non-limiting example only, with reference to the following examples.

EXAMPLES

Example 1

Designing and synthesizing gRNA molecules corresponding to the sequence targeted for editing, i.e., sequence of CsPRR37

[00129] It is noted that the editing event is preferably targeted to a unique restriction site sequence to allow easier screening for plants carrying an editing event within their genome. According to some aspects of the invention, the nucleotide sequence of the gRNAs can be completely compatible with the genomic sequence of the target gene. Therefore, for example, suitable gRNA molecules can be constructed for different CsPRR37 homologues of different Cannabis strains.

[00130] Reference is now made to the following tables presenting gRNA molecules targeted for silencing CsPRR37.

Table 1 lists guide sequence directed to exon 1

24

25

Table 2 lists guide sequence directed to exon 2

26

[00131] Tables 1 and 2 are merely exemplary of guide sequences that can be used to modify the protein. Other guide sequences can be designed and used as would be standard in the art.

[00132] The above gRNA molecules are cloned into suitable vectors and their sequence is verified. In addition, different Cas9 versions are analyzed for optimal compatibility between the Cas9 protein activity and the gRNA molecule in the Cannabis plant.

[00133] The efficiency of the designed gRNA molecules is validated by transiently transforming Cannabis tissue culture. A plasmid carrying a gRNA sequence together with the Cas9 gene is transformed into Cannabis protoplasts. The protoplast cells are grown for a short period of time and then are analyzed for existence of genome editing events. The positive constructs are subjected to a stable transformation protocol into Cannabis plant tissue for producing genome edited Cannabis plants.

Example 2

Transforming Cannabis plants using Agrobacterium or biolistics (gene gun) methods

[00134] For Agrobacterium and bioloistics, a DNA plasmid carrying (Cas9 + gene specific gRNA) is used. A vector containing a selection marker, Cas9 gene and relevant gene specific gRNAs is constructed. For biolistics, Ribonucleoprotein (RNP) complexes carrying (Cas9 protein + gene specific gRNA) are used. RNP complexes are created by mixing the Cas9 protein with relevant gene specific gRNAs.

[00135] According to some embodiments of the present invention, transformation of various Cannabis tissues is performed using particle bombardment of:

DNA vectors

Ribonucleoprotein complex (RNPs)

27 According to further embodiments of the present invention, transformation of various Cannabis tissues was performed using Agrobacterium tumefaciens by:

Regeneration-based transformation Floral-dip transformation Seedling transformation

[00136] Transformation efficiency by A. tumefaciens is compared to the bombardment method by transient GUS transformation experiment. After transformation, GUS staining of the transformants is performed.

[00137] According to further embodiments of the present invention, alternative transformation tools are used in Cannabis, including, but not limited to:

Protoplast PEG transformation Extend RNP use

Directed editing screening using fluorescent tags Electroporation

Example 3

Regeneration in tissue culture and selection of positive transformants [00138] When transforming DNA constructs into the plant, antibiotics are used for selection of positive transformed plants. Once regenerated plants appear in tissue culture, DNA is extracted from leaf sample of the transformed plant and PCR is performed using primers flanking the edited region. PCR products are then digested with enzymes recognizing the restriction site near the original gRNA sequence. If editing events occurred, the restriction site will be disrupted and the PCR product will not be cleaved. No editing event will result in a cleaved PCR product.

[00139] Screening for CRISPR/Cas9 gene editing events is performed by at least one of the following analysis methods:

Restriction Fragment Length Polymorphism (RFLP)

Next Generation Sequencing (NGS)

PCR fragment analysis

Fluorescent-tag based screening

High resolution melting curve analysis (HRMA)

[00140] The analysis includes the following steps: 1) Amplicon is isolated from two exemplified Cannabis strains by primers flanking the sequence of the gene of interest

28 targeted by the predesigned sgRNA. 2) RNP complex is incubated with the isolated amplicon. 3) The reaction mix is then loaded on agarose gel to evaluate Cas9 cleavage activity at the target site.

Example 4

Multiple Sequence Alignment of a photoperiodic PRR37 peptide (CCA_PP_PRR37_AA) and two autoflowering PRR37 peptides (CCA_AF_PRR37_AA and Finola_PRR37_AA).

[00141] The photoperiodic PRR37 has a functional psREC domain while the two autoflowering haplotypes produce truncated proteins that are disrupted inside the psREC domain. The CCA_AF_PRR37 haplotype contains a mutation in the splice site at the end of the second exon. Retention of the second intron during pre-mRNA processing results in a truncated protein. The Finola haplotype contains a 1 base pair deletion in the first exon. The frame shift mutation induces a STOP codon with the first exon, resulting in a truncated protein.

Example 5

Multiple Sequence Alignment of the psREC domain of PRR proteins in thale cress, pinus pinaster, Japanese rice, and long-grained rice [00142] Sequence analysis of PRR proteins in various species demonstrates certain domains whose disruption can result in an autoflower phenotype. These include the psREC domain, (https: <slash> <slash> www <dot>ncbi <dot>nlm <dot>nih <dot>gov <slash> Structure <slash> cdd <slash> cddsrv <dot>cgi?uid=381120 [00143] Other PRR-proteins are also targeted for disruption, including PRR9 (APRR9),

PRR7 (APRR7), PRR5 (APRR5), PRR3 (APRR3), and PRR1 (APRR1). The psREC domain is involved in protein-protein interactions.

Example 6

Multiple Sequence alignment of CCT domain.

[00144] The CCT domain is a short motif is found in a number of plant proteins. It is rich in basic amino acids and is called a CCT motif after Co, Col and Tocl. The CCT motif is about 45 amino acids long and contains a putative nuclear localization signal within the second half of the CCT motif. Tocl mutants have been identified in this region. It is

29 known that this domain, which is near the C-terminus, can be disrupted and also result in reduced photoperiod sensitivity.

Example 7

[00145] Three prr37 gene sequences were de novo assembled using a query CsPRR37 genomic sequence as bait to capture all aligning reads from >30x coverage WGS. One prr37 gene sequence (LowRyder) was anchored to CsPRR37 due to low read depth.

[00146] A bioinformatics pipeline was written to assemble high-quality haplotypes for gene sequences of a particular accession. DNA sequencing reads generated from the accession are aligned to a reference genome. A query gene sequence is also aligned to a reference genome with gmap aligner. Sequencing reads that align within 2,500 base pairs of the query gene sequence are collected. Next, all of the sequencing reads are searched for 27 letter words that are shared with the query gene sequence. Sequencing reads containing matches are added to the collection if they are not already present. The collection of reads are assembled with a de bruijn graph.

[00147] CCA_PP_PRR37 (SEQ ID NO. AAA) is a haplotype of a photoperiod sensitive individual from the F2 segregating population used in the original mapping experiment. CCA_AF_PRR37 (SEQ ID NO. AAB) is a haplotype of a day length neutral (autoflowering) individual from the F2 segregating population used in the original mapping experiment. Finola and EowRyder are two WGS samples of known autoflowering Cannabis.

[00148] SEQ ID NO. AAA

>CCA_PP_PRR37

TTGGGTGATTGTTTAATTGGTGTTAAGAGCTTTTTATCGTTCAGTCTGAACCT

ATCGTCT ATGTTC AC AT AG AGTTT A AT ATGTTGGCTT ATA A AGTT ACT A AGT

TAGGTCTTGGATTAGATAATGAGTTGGTTTGGAATAGTTCTCTACTTTTCTTG

TGAACTTATTTGTTTTGTATTCTCTGTTTTATCCAATGAAGGACAAGTTATTT

TTTTAAGAAAAAAAAAAACTTATTCATTATTCAACTCTTACTTCACCACAGA

TTTATCTTATTTTTATATGCATTTATTAAAATTTTAAATTAATTTTTTATTTTC

TGAATAATTTGATCATACTGACTTTTCAAATAAAATCTTCCAAGTATAAAAA

C ACTGTT AAA A AT A A AT AA AT A A ATA A AT AA A AGGTTA A A ACCTTTA AGCT

CCTACTTTTGAAGTCATGCCAAATACAGTCCAGCTTAATTATTATTCTTTCTT

30 TTTTCTTACAACTTTTTCGTCACGAGCATATTAGTATTTAGTTTGTGATGAAA AAACAAATACATAGCCGACAACAAATTTCCAGTCCAAATTTAACTTCTCTTT TATTTTATTTTCAGAAGATATGTGTATTTAGAAATTTAAAGAAAATTCATCC TTACAAAAGAAAAAAAAAATTATCATTTCTTCAAAAGCTGCGAAAGTGGAA TTTATCTTCTTAGTTTAAATAAAAAATTTAGATTTTTTTTTTCTAAATTTATA GCG ATTA A AG ATTGGT ACC AG ACCT A A A ATT AT ATTTT A ACT A ATT ACT ATT _{ATTTTTATTAATTAAAATAATAATAATAAATATGGTGCAAATGCAAACGTGA} ATGAATAAATTTTGGAAGAAGGAGAGAGAGATTTATATTTATCCAGCAGTA GTAGACTAGTGGAAATGGAAAACAAAACATAGAGGAAAGGGACACGTGGA AGTGTGGTAGCCAAGTGAAAGGGCCCCTTCATGCTGACCCGATATTCTGTAT CAAAATGGATGGCCTCAACTACCTCTTCGTAAAACGCGACCGAATTTCGTTT CCAGCCTTACACACGTGGACACCAAACTCACCACAACATCATTTCAACACTA CCCAATAGTCCAACGCCAACTCAGCATTTATTTCTTATACTTGATTTTACCAT CTTCTCCCTCCCCTCAACCTCCACCCCCTTCACCTTCACCTGAACACTAACAA CAACGATTCAACAACATATTCCAGAAAAAGACATTGGTATGCCGTTGCTAA TACCATTTTAACCAATACATTTTCTTTCTTTTAATAATAATAATAATGGAGAT CATGTAATCTTTTTTAAGCCAAAATAAAAGAGGTCATAAAAGGCATTGCCTT TCTTCTTTCTTTTCTCTCGTCCTTTTTATACTGCAACTTTAGCCCAATATGGTT CAGCCCATCACACTCTCTGGTCAGCTTCTTTCTCTATTTCTCTAACTCATCAA TGGAAATAATCTCCTATTTCTATCACCACCCTGTAAATCTTTGAATCCAAAC TTTTGAGTTTTCTCTGTACAATGTTACCATATCATAATCTATGAAAACTCTTT GGTACGTGAGTTTTTCAGTTTATTTAATTGAAAAACCTTCTGTTAATGTTTCT CTACGACTGAACTCTCTGCAGATATATATAACCAGAGAAGCACAGTACTTG ATTGGCTGGAAAAAGCTTCAGAATGATATATATACACAAAAGCAAGTTTGA TATTTCAAGAAACAACACAAGAAGAAGGAAAGAAGAAAGGTAAACAGGAA AAGAGTTTCTTCAACCATCCATTCAAGTTAAAATGATCATCTAGTCATGAAA TAATAATATTACACCCACTTAAAGTGATTTATTGAGAGAAGGCTCTATTCTT TTTGAGGGTTTATCACCTTCTATTTAGTAGAATTGTAGTACTTAATCCTAGCA C AGCCGTTGGC AT ATT ACTTAT AC ACCTTC A AT ATTA AT AC AGTCCC ATAT A TTCAATCATAAAAGCTCTGTAACTATCTACTGCAAACGCACCACATCTGCAT CAAGGATTCTAATGGCTCCTGTTATGAAATTGGTTCAGGTTCGTAATTTATG CCTTTTCCCTTGTTTTCATTCATTCCCTTTCTAATAAATTGTCCTCGAATTCGG

31 GGTCTTTGTTCTGCCAGTTTTTTTCTACTCTGATATCTCACCTGAAAAAGTAA

AAATTAATGCTTCAGGTCAGCAATATGGAACTTCAAAGGAAACAGATCAAT

TATTTATATTTCAGGGAAACAATTTTCTGTTCTTTTCCTTTTTTTTTTTCCCAA

AAAACTTCATACTAACTGGATCCCAAAGATGATGGTTCAAATGTGTAGTGTT

GAAAATGAAACTTCCTTCTTTCAGACTCCTACTTGTTTGTTTCCATGATTTAC

TTCTTAGAGAAAATAAGGGGGGTGATTTTCTGTTTACCTTTTTCTCAAAGCA

TCTTTCTTGCCTCTTCTGGCGGAATCTTTTCAAAGCAGCTTCTCTTTGTGCAA

ATCGATTTCCATCCGATTCACTTCCTTCAATGGTTTTCCCTCTTCCAGTAACC

ACACTCTCACTTTCTGTCTTTTTCCCCCTGGAGTTTGAAGCATTGGTGCTTCT

CTTTTGTCCGTTGCTACCATGGTTACTACCTGACCCACTCCTGTTGAGACTGT

GATT ACT AATATTGCCTTCCATGGGTGCTCTCAATGCATTGGATGAACCACA

CTGTGAAGCATCTGCAGCCATAGTTTTTGTAAAACAAATCAACATCGATGAA

AAGATATTATGAAGTGGTATGTGGCCTTTGCTTGTGTCTTATTTGAATTAATT

ACTGAGGCATCATATTCGAGTATTTACCTTGAATAATAGTCTGAAGGGAGGC

TTGGCCATTTTGCACTTGTTGGAAACCAGTGTTTGATTGGAGTTTGATTGTG

GATTTGTTTGTAGGCCTCTCTGCAAAAGCCACTTGTTTGGTAAAAGCATTAT

TCGTAGTGGATCCCATGTCATTATTGTTGCTACTACCATTGGAACCCTGGTT

GGGAGGTGTACCATTTGAATCAGATTGCAAATTTGGGATTGATTCTGTTTTT

GCTGCATCTGAGCTATTATCTAGAGGAGAACAGCTTCCTATGTTCCCTGTTG

GAGCTTGGTTTGCAGTTGAAACAATGTTATACCTGCATGGTAATGGGACTTA

GTT AT ATTCTT AA ATTT AG AGTT A ATTGTGGTTG AGC ATGTTT AT ATC A AC AC

AAAACATTTCTATACCTTGAAAAAGCTGAAATATCGGAATGTCTCAAAACA

TTTCGATCGTGTGCGCTGGTTCCAGTATCTTGGATATCTCTTGGCCTCTTTAA

AATGAGCTCAAAAAAAGGTGTTTCTTTATTCTCATGGTTGGCATTATCTCTC

ATGCAGGCAGCCTTGGAGAGTTCGTTTGGGACATCAAGAACTCCACTTTCCA

TGTGAGGATCAATACTATAATTGGAGACACCCATCAGATCAACAGCTTGGG

TCTTTTCTGCACTCGGTTCTTCATTGTTGAGGTCTAATTCCCTTTTCTCCTGCT

CCTGATCATCTTTCTTTGAGTTCAGTTCAGAACATTTATCTTTATTAGTACCT

TCCATGTTGGTCAGCCCTTTTTCACTTGTGTCTTCAAGAAGGCTAGGTAATCC

TATTTTCAAGTCTTTTCCCATGACTTTTTTGTCTAAACAATTTAAATAAATCA

TAACTTAGAGAATGATCGAGAATGGAAGAAAGTTAAAAAAAAAAAAGATG

TTAGGAAAATTTGCCTACCAAGCTCATCATTATGTGGGCGTGGTGTTACTGT

32 TGCATTTTCAGGTACCCAGTTGTTTCCAAAGGCTTCTTGCCTTGGATGATTGA

CCTGATGATTAGTGCTATCAGGAAGATCAGCAAACTGCTCCCACGACACTG

CCTGAGGGCTGTCAACTTCTGCTCTCTTTGTCCAAGAGCTCTGTAAAAGCTC

AGTAGTAAGTTTTCATTCTTTTTCTTGTTTTCTTATCTTGCTAATTATCATTCC

TTCTCCACTTACATATACATAAAAGTATTAAAAACTCTTACATGAATATTCC

TCCACCAAGATCAAATGAGTAATATGCAAACATGGTAAATAGATATATGGG

TGCTGTGATAGACAAATTCCAAACTAGGTGTGTGGAAACTAACATAATTAT

AGTAGTTTCTTGATACATAAAACATAAAACTTCATGTGGAAACTAACATTTC

GCTAATATTTCTGTAAATACAAAGACATAAAACTTCATTCCCACCAAAAAA

AAAAAAAACAAAACTCTTTCATGAAATTTAGTGAAATACCTGTGTTCCACTG

TCACTTTCATTCCTGAAATTTAGACCAATGCTGTCAGTATCATCCTCATCATT

GCTGCCACTGTTGTTGTCTGAATGTTCCACAGTTCTTGACTTTAAAGGCTTTT

CAATCCATATACCACTTTCACTTCCACTATTACTAGACTGATGAAAATATGA

AAAATTAATGCACAACTCGAGCAAATTTCTGTGTTTAATGGTATTATGTCAA

TTAAGTGATGTTTGCATTACATAAAACATATTTTTTATCAAGCAAAGATTGA

AGCCATTTTTTTGTTTGTTTTCTTTGGTATAGTTCGACAATTGGTGCTTAATG

AAAAACAAAAATGCATAAATGAGAAGAAACCTGAACCTCAAGGTGAATTG

GTCCATGATATAATAAATCAAATAGATTGCAACTCACAATGTGGCATTTTCT

CCAAACATGTTGCCAAAGGTTTTTCAGCTCATTCTTTCGAATAGGTTTCACT

AAAAAGTCAACGGCACCTTTCGATAAACACTTAAAGACCATACTCCTAGAA

TCATGAGAAGACATCACTGTGAAAACACAAACACAAACACACACAATAAAT

GTATT AGTT A A A AGT ATA A ATTAC A A A AAG AT ACT AG AG A AAA A AGT ATT A

TTTT AA ATCC A AA AT A A AGGA A AT AT ACTC ACT AATT AC AGGG ATGTCCTT G

CATGTTTTTTTGCTCATGATCTTGCCTAGAAGACCAATACCAGATAAACAGG

GCATGACTACCTCAGTCAAAACGAGATCAACATCTGTCACAAGATCTTCTAA

GACTTTCCAAGCTTGTCTGCCATTTTCTACAGCTGTAACTGCCAATATGAGA

ATACATTGAATAATTGTTAGTTTATTGAGGGAACCAATAAAATTCTAGTTTT

G ACTTTT AGG ATGT A A ACT A A ATC AA AGCTTCTGGCTA AC ATTT ATGT A AT A

TTATACCAAGCAGTTTGCGTTGGAAATAAAACATGGTCAAATAAAATAACA

ATATTTAAATAGCTTACCATAACTTGAACACATATATGAAAAATGATGATGA

AGAAAGCATGACATAAATATAGCGACTGAATGTCTAATTATCATAGCTAGA

AAGTAAGATGTCAATTGCCAACAGGTCTGGAGCAACATGTTTTTCTTTTCGA

33 G A AT ATC AT ACTTAT A ACC AGGTT ATCTTT AG A AGGT AC AC AT A A AC AT ACT

CTACAAACTAAGTAACCCACTGTAGTATCCTCAAGAATGGTTCTATGTCAAA

ATCTAAGCTAGGTCAAGAAATTGTACTTGACCCTTCATGAACGAAATGACTA

AAAAACTTCCCGCTAGACTATAAATATGAACATCAAAATAGCACAAATAGA

CACGTGAAGCACCAGAGTATAGGAACTGTTGTTAAAATGTAGTGTTAAAAC

A A AAG A A A AT A A AT A AGT A AC A A A A ACT AGTTT A ATAGTTTTC AGC ATTTC

TGATAGGAACTCAGAAAAAGAGCGCTGCCACAAGATCAGGGGATCAGTCTA

GAAATGTGTGTAATTAGATGGTGGAAAATAACATTTCAGAAATAAGATCTA

CTTATATTAAACTAAAACAAGCTTTAGTCTAATAATAGTGCCATGCTCACCT

TACTTGTTTGTTTTTTATTCTTTTGATTCGTTTTCTGTTTGTTATATCTTTCTTT

GGAGGGCGGGGGAGGGATTGAACCCTTGACCTTCTCTACAAGAGGTGAGGT

GTGGATCAGCGAGGCTAATCCTAAGACCACTCTTTCTAGGTTATGGTTAGTG

ACAACAACTATTAGACCATGGATATTATTACTCGTAGTGGAGCAAATAAAC

ATGGTTTCCTTTCATATCATATAACATCAAGTTTATTCATAAGATCTACTACA

GTGTTTACTTTAAAAGTCATCTCTGTCAATAATCAAACATAAAGCCATTAAC

ATAGAGTGTATACTTCTGCATTAGAGAAATGCAATGATCATTTCATGTTGCT

GAAACGTACTGACCTTCGTAGCCACAATTTCTTAGTAGCGCGCTGACAATAT

GGCGAGTTGAGTCATCATTTTCCACCAATAAAACCTTTAGCGACCTGAAAGC

TAGGAACCTCTCCCAGCGAACCAAATGTCCTTGAGGCTGTTGCTGAGACCTC

CGCTGGCCAGAATGACTCCTCTCCAGAGCCTGTACTGTTCCAATCTCCCCAT

TGCTGACATGTTGTTGCACATCTTCATTGATCCTAGATTCATGTTCCTCTGAG

AGCCCTTGGCGTTCTCTCGTAACCCTCTCCCTTCTTTCTTTTTTCCCATCTTGA

ATCCGGTGATTCAGCTCAGTTAGCTCATCATTGGTTACGGGAGCTTTATTAT

TCATCTGGACCATTTTCATCAACCAGATTCAATAAGATCAATGATGAGAAGG

GTAATGGATAGAGCTGGCGAGAAAGCTGATCCACAAGAGTTCTTGCACATC

CCGTTGTGACAAAAATACAGGCTCAAGTAAAACAGAAAGCCCCTTCACAGA

TCCTCTACAAAAGACAAATAATAAGTACAAATATATTGCTATCAGAAACCA

ATAAGAAGAATAAGGTGGATAGCATATGTGTGTGTCTGTGTGTTCTGATCAA

AGTCCCAATAGCATAAAATGAGGAAAAGTTGAATTAGACACAACAAATAAA

AGGGAAGGAAATGATTATGATCCTTTTCTATTCCCATATCTCTACTACTTTCC

CACTATCAATCTATATTGCGATCAAAAGTATACAAGGTATGTAGCACATATA

AATTTGACCGTAGCTTTTTTTGATGCATATAACAGTATAATTAACAAAAACT

34 GAACCCAGATAATGGACATAAAAAATAACAGAAGCACACCTATATGGTTAA

ATAAGAATAAAAAAGTTCCTGAATATAAAGTTCATATCAAACTATCTTTTGA

TATAGAGATAATAACAGCATCATGATATACCAAACTCATGAATCTTCAAAC

AATTAACAAGTGTGTAAGACTTTAATCATGCATATTCAACTCAATTAAAACT

TATAGGGTTTGCTAATCTATGGTGAGAAAGAGGTTCTTTGGTAGATTTCAGA

ACACTTAATTCCAAAGGTCATTCTCCTTTTCATATCATCAAGAAGTTAAGAA

ACACTTCAATTATCCGAGATAAAATATATATTAATTTATGAATTTATAGAAT

ACATGCAAACAATTTAAAGAGCGGGAAGAGAAGGAAAGCCAAATAATTCA

GTAAATACAAATATATTAGATATATACATATTTTTTTTCATTTTTCAGTGAAT

GAGTGAGTAGTACTCGGAAACACTATACTGTGAAAAATCAAATACTCTCGT

TCCTTCATTATAACATTTTTCCCGTTGTGTTCCTTTAAAATTTTCTTAACAGC

CAAACAAAAGATGAAGAAACAAATAAGGAAAAGAAAATCAACATTGAGCA

AAAAGTTGCTACTTTATACAAAGTGAGAATACCTCTCTCTCAAAACTTCATT

CGAACTTGGATGATAATCGCCAATGGTGGAATAAAGTTCAAAAGTTAGAAA

CCAGAGGTTTTTCTTTTTTTTTTTCCTATGAAGTTAATTTTTTTTTTTAAAAAA

AATTATTTATTTTTTTCTGTAGAGTTTGAGAAATCTCAGTGAGTTTTGTACCA

_{TTTTTTTTTTCCAAATTATATTTAAAAAAAAAAATTGAAAAGAAAAATTATT}

GGTAGAGAAAGAAGGAAGGTTGGAAAGGTAAATGGAAGCGATGGATATAT

GATATAAATGAGAAAAAAGATATTAGAATTGTCCCTACAAAGTTCTAAATG

GAGCGAATAAAGGTTGCCACGTAGGACGCCACGTAAGAGGAAAATGTTCTA

GTGCGCTCCAATATCTATAGCTGGGCTGTGAATTGTTTACCGCGGATCCAGA

AGCGATCCGTCAGATCCGTATTCGACACGTGGCGTTCTCTCTGATGAGTAAA

AGGTTTCCCAAAGGAAGTGGCTGCAAATCCTTTCTATTTTCGAAGCCACTTT

CGCTTTTCCATCCACAACCACACCTACTTTTGTTCTTCCCATACCAATTATTA

ATAATATAATATAATATAATATAATACAAATGAGTTCCACACCATCAAATTT

ATATCATGCCTGAATCAATTTAGGTCTTTTTTAAGGAAATTATCTTTTACATT

TGT AT ACT AT AT ATT ATT A ATATTTTT ATGTT AG AT ATT A A AAT A A AAG A A A

ATAAAAAAGATAGAATATTTAGTATTTTCCTTTTCTTTTTATGCTTTTTTTAT

ACACTTAGTAAAAATGGAGAAGTCTTTTCTCTCTTTTTGTAGGCGAGAATAC

TTCAGAGTTTTCTTCTGATTTAGTTTTTATTATTTTATTTATAGTGTATTCGAT

T AT ATTTTGA ATTC A ATTT AATTTGC AT GATAGCTT A A AGTT ATTCT A A AAC A

AATTGGATCAGTTATTTTAGATTGATTACTTTATAAATATTAAATTATTTAAT

35 ATT A ATT AT A A A AT ATTTTTTTTTACATCATT A A A AT CACTTAAATAT A A A AT

ATAAAATATTCTTTTTAAATTCTCCAACAATAAAGTAAAATAAATTATTATT

CTACATCTCTAACAAT A A ATT C A A AT A A AT AAACAAAAAT A AC A AT A A A AG

A A A A A A A AT AAAATTATATGTATAAAGAGGAGATT GAT AT A GTTTT AG ATT

ATGTTTTATTTTGTATATAAAATATATATTTGATATATAAAATTTTAAAAATA

TTTGTATTATGTATTAGTTTTGTCTAATTACTTTAATGTATTTATAACTTTTAA

ATAAATATAAATAAATGTAGATTTAATTATGTTGACTATATTTGCATGTAAA

AAAAATAAACAATCTGTACTTTGTAAATTGCATTAGATTATGTGGATTGAAT

TTGATTAAGTTCTTTATGAATAGCACTAAGAACAACTAGTATTTTTTTTAAA

AAAAAAATAAAAAACTAAATACTACT

CCA_AF_PRR37 (SEQ ID NO AAB)

AACTCAATTCAAACTGCAATTTATATTGCAATTTACGTAACAACTCAGATA

AC A ACTT A A ATCGT A A A A AT A AT A AT A A A A A A A A A A AT CAAAAAGTAGTA

TATGTGATGATTTTCCTTTATAATATTTAATAAATTATATAATAATATTGAC

AAATCATATTTTTAATTTGTTTTATTGTCAATATTTTTTTATATATATATATT

TAAGTGATTTTAATGATGTAAAAAAAAATATTTTATAATTAATATTAAATA

ATTTAATATTTATAAAGTAATCAATCTAAAATAACTGATCCAATTTGTTTTA

G AAT A ACTTT A AGCT ATC ATGC AA ATTA A ATTG A ATTC AA A AT AT A ATCG A

AT AC ACT AT A A AT A A A AT A AT A A A AACT A A ATC AG A AG A A A ACTCTG A AG

TATTCTCGCCTACAAAAAGAGAGAAAAGACTTCTCCATTTTTACTAAGTGT

ATAAAAAAAGCATAAAAAGAAAAGGAAAATACTAAATATTCTATCTTTTTT

ATTTTCTTTTATTTTAATATCTAACATAAAAATATTAATAATATATAGTATA

CAAATGTAAAAGATAATTTCCTTAAAAAAGACCTAAATTGATTCAGGCATG

ATATAAATTTGATGGTGTGGAACTCATTTGTATTATATTATATTATATTATA

TTATTAATAATTGGTATGGGAAGAACAAAAGTAGGTGTGGTTGTGGATGGA

AAAGCGAAAGTGGCTTCGAAAATAGAAAGGATTTGCAGCCACTTCCTTTGG

GAAACCTTTTACTCATCAGAGAGAACGCCACGTGTCGAATACGGATCTGAC

GGATCGCTTCTGGATCCGCGGTAAACAATTCACAGCCCAGCTATAGATATT

GGAGCGCACTAGAACATTTTCCTCTTACGTGGCGTCCTACGTGGCAACCTTT

ATTCGCTCCATTTAGAACTTTGTAGGGACAATTCTAATATCTTTTTTCTCATT

TATATCATATATCCATCGCTTCCATTTACCTTTCCAACCTTCCTTCTTTCTCT

36 ACCAATAATTTTTCTTTTCAATTATTTTTTTTTAAATATAATTTGGAAAAAA

AAAATGGTACAAAACTCACTGAGATTTCTCAAACTCTACAGAAAAAAATA

AATAATTTTTTTTAAAAAAAAAAAATTAACTTCATAGGAAAAAAAAAAAG

AAAAACCTCTGGTTTCTAACTTTTGAACTTTATTCCACCATTGGCGATTATC

ATCCAAGTTCGAATGAAGTTTTGAGAGAGAGGTATTCTCACTTTGTATAAA

GTAGCAACTTGTTGCTCAATGTTGATTTTCTTTTCCCTATTTGTTTCTTCATC

TTTTGTTTGGCTGTTAAGAAAATTTTAAAGGAACACAACGGGAAAAATGTT

ATAATGAAGGAACGAGAGTATTTGATTTTTCACAGTATAGTGTTTCCGAGT

ACT ACTC ACTC ATTC ACTG A A A A AT GA A A A A A A A AT ATGT AT AT ATGT AAT

ATATTTGTATTTACTGAATTATTTGGCTTTCCTTCTCTTCCCGCTCTCTAAAT

TGTTTGCATGTATTCTATAAATTCATAAATTAATATATATTTTATCTCGGAT

AATTGAAGTGTTTTTTAACGTTTTGATGAAATGAAAAGGAAAATGACCTTT

GGAATTAAGTGTTCTGAAATCTTCCAAAGAACCTCTTTCTCACCATATATTA

GC A A ACCCT ATA AGTTTT A ATTG AGTTG A AT ATGC ATG ATT A AAGTCTT AC

ACACTTGTTAATTGTTTGAAGATTCATGAGTTTGGTATATCATGATGCTGTT

ATTATCTCTATATCAAAAGATGGTTTGATATGAACTTTATATTCAGGAACTT

TTTTATTCTTATTTAACCATATAGGTGTGCTTCTGTTATTTTTTATGTCCATT

ATCTGGGTTCAGTTTTTGTTAATTATACTGTTATATGCATCAAAAAAAGCTA

CGGTCAAATTTATATGTGCTACATACCTTGTATACTTTTGATCGCAATATAG

ATTGATAGTGGGAAAGTAGTAGAGATATGGGAATAGAAAAGGATCATAAT

CATTTCCTTCCCTTTTATTTGTTGTGTCTAATTCAACTTTTCCTCATTTTATGC

TATTGGGACTTTGATCAGAACACACAGACACACACATATGCTATCCACCTT

ATTCTTCTTATTGGTTTCTGATAGCAATATATTTGTACTTATTATTTGTCTTT

TGTAGAGGATCTGTGAAGGGGCTTTCTGTTTTACTTGAGCCTGTATTTTTGT

CACAACGGGATGTGCAAGAACTCTTGTGGATCAGCTTTCTCGCCAGCTCTA

TCCATTACCCTTCTCATCATTGATCTTATTGAATCTGGTTGATGAAAATGGT

CCAGATGAATAATAAAGCTCCCGTAACCAATGATGAGCTAACTGAGCTGA

ATCACCGGATTCAAGATGGGAAAAAAGAAAGAAGGGAGAGGGTTACGAG

AGAACGCCAAGGGCTCTCAGAGGAACATGAATCTAGGATCAATGAAGATG

TGCAACAACATGTCAGCAATGGGGAGATTGGAACAGTACAGGCTCTGGAG

AGGAGTCATTCTGGCCAGCGGAGGTCTCAGCAACAGCCTCAAGGACATTTG

GTTCGCTGGGAGAGGTTCCTAGCTTTCAGGTCGCTAAAGGTTTTATTGGTG

37 GAAAATGATGACTCAACTCGCCATATTGTCAGCGCGCTACTAAGAAATTGT

GGCTACGAAGGTCAGTACGTTTCAGCAACATGAAATGATCATTGCATTTCT

CTTATGCAGAAGTATACACTCTATGTTAATGGCTTTATGTTTGATTATTGAC

GGAGATGACTTTTAAAGTAAACACTGTAGTAGATCTTATGAATAAACTTGA

TGTTATATGATATGAAAGGAAACCATGTTTATTTGCTCCACTACGAGTAAT

AATATCCATGGTCTAATAGTTGTTGTCACTAACCATAACCTAGAAAGAGTG

GTCTTAGGATTAGCCTCGCTGATCCACACCTCACCTCTTGTAGAGAAGGTC

AAGGGTTCAATCCCTCCCCCGCCCTCCAAAGAAAGATATAACAAACAGAA

AACGAATCAAAAGAATAAAAAACAAACAAGTAAGGTGAGCATGGCACTAT

TATTAGACTAAAGCTTGTTTTAGTTTAATATAAGTAGATCTTATTTCTGAAA

TGTTATTTTCCACCATCTAATTACACACATTTCTAGACTGATCCCCTGATCT

TGTGGCAGCGCTCTTTTTCTGAGTTCCTATCAGAAATGCTGAAAACTATTAA

ACT AGTTTTT GTT ACTT ATTT ATTTTCTTTTGTTTT A AC ACT ACATTTTAACA

ACAGTTCCTATACTCTGGTGCTTCACGTGTCTATTTGTGCTATTTTGATGTTC

ATATTTATAGTCTAGCGGGAAGTTTTTTAGTCATTTCGTTCATGAAGGGTCA

AGTACGATTTCTTGACCTAGCTTAGATTTTGACATAGAACCATTCTTGAGGA

TACTACAGTGGGTTACTTAGTTTGTAGAGTATGTTTATGTGTACCTTCTAAA

GATAACCTGGTTATAAGTATGATATTCTCGAAAAGAAAAACATGTTGCTCC

AGACCTGTTGGCAATTGACATCTTACTTTCTAGCTATGATAATTAGACATTC

AGTCGCTATATTTATGTCATGCTTTCTTCATCATCATTTTTCATATATGTGTT

CAAGTTATGGTAAGCTATTTAAATATTGTTATTTTATTTGACCATGTTTTATT

TCC A ACGC A A ACTGCTTGGT AT A AT ATT AC ATA A ATGTT AGCC AG AAGCTT

TGATTTAGTTTACATCCTAAAAGTCAAAACTAGAATTTTATTGGTTCCCTCA

ATAAACTAACAATTATTCAATGTATTCTCATATTGGCAGTTACAGCTGTAG

AAAATGGCAGACAAGCTTGGAAAGTCTTAGAAGATCTTGTGACAGATGTTG

ATCTCGTTTTGACTGAGGTAGTCATGCCCTGTTTATCTGGTATTGGTCTTCT

AGGCAAGATCATGAGCAAAAAAACATGCAAGGACATCCCTGTAATTATTG

AGTATATTTCCTTTATTTTGGATTTAAAATAATACTTTTTTCTCTAGTATCTT

TTTGTAATTTATACTTTTAACTAATACATTTATTGTGTGTGTTTGTGTTTGTG

TTTTCACAGTGATGTCTTCACATGATTCTAGGAGTATGGTCTTTAAGTGTTT

ATCGAAAGGTGCCGTTGACTTTTTAGTGAAACCTATTCGAAAGAATGAGCT

GAAAAACCTTTGGCAACATGTTTGGAGAAAATGCCACATTGTGAGTTGCAA

38 TCTATTTGATTTATTATATCATGGACCAATTCACCTTGAGGTTCAGGTTTCT

TCTCATTTATGCATTTTGTTTTTCATTAAGCACCAATTGTCGAACTATACCA

AAGAAAACAAACAAAAAAATGGCTTCAATCTTTGCTTGATAAAAAATATGT

TTT ATGT A ATGC A A AC ATC ACTT A ATTG AC ATA AT ACC ATT AA AC AC AG A A

ATTTGCTCGAGTTGTGCATTAATTTTTCATATTTTCATCAGTCTAGTAATAG

TGGAAGTGAAAGTGGTATATGGATTGAAAAGCCTTTAAAGTCAAGAACTGT

GGAACATTCAGACAACAACAGTGGCAGCAATGATGAGGATGATACTGACA

GCATTGGTCTAAATTTCAGGAATGAAAGTGACAGTGGAACACAGGTATTTC

ACTAAATTTCATGAAAGAGTTTTGTTTTTTTTTTTTGGTGGGAATGAAGTTT

TATGTCTTTGTATTTACAGAAATATTAGCGAAATGTTAGTTTCCACATGAAG

TTTTATGTTTT ATGT ATC A AG A A ACT ACT AT AATT ATGTT AGTTTCC AC AC A

CCTAGTTTGGAATTTGTCTATCACAGCACCCATATATCTATTTACCATGTTT

GCATATTACTCATTTGATCTTGGTGGAGGAATATTCATGTAAGAGTTTTTAA

TACTTTT ATGT AT ATGT AAGTGGAGAAGGAATGATAATTAGCAAGATAAGA

AAACAAGAAAAAGAATGAAAACTTACTACTGAGCTTTTACAGAGCTCTTGG

ACAAAGAGAGCAGAAGTTGACAGCCCTCAGGCAGTGTCGTGGGAGCAGTT

TGCTGATCTTCCTGATAGCACTAATCATCAGGTCAATCATCCAAGGCAAGA

AGCCTTTGGAAACAACTGGGTACCTGAAAATGCAACAGTAACACCACGCC

CACATAATGATGAGCTTGGTAGGCAAATTTTCCTAACATCTTTTTTTTTTTT

AACTTTCTTCCATTCTCGATCATTCTCTAAGTTATGATTTATTTAAATTGTTT

AGACAAAAAAGTCATGGGAAAAGACTTGAAAATAGGATTACCTAGCCTTC

TTGAAGACACAAGTGAAAAAGGGCTGACCAACATGGAAGGTACTAATAAA

GATAAATGTTCTGAACTGAACTCAAAGAAAGATGATCAGGAGCAGGAGAA

AAGGGAATTAGACCTCAACAATGAAGAACCGAGTGCAGAAAAGACCCAAG

CTGTTGATCTGATGGGTGTCTCCAATTATAGTATTGATCCTCACATGGAAAG

TGGAGTTCTTGATGTCCCAAACGAACTCTCCAAGGCTGCCTGCATGAGAGA

TAATGCCAACCATGAGAATAAAGAAACACCTTTTTTTGAGCTCATTTTAAA

GAGGCCAAGAGATATCCAAGATACTGGAACCAGCGCACACGATCGAAATG

TTTTGAGACATTCCGATATTTCAGCTTTTTCAAGGTATAGAAATATTTTGTG

TTGATATAAACATGCTCAATAACAATTAACTCTAAATTTAAGAATATAACT

AAGTCCCATTACCATGCAGGTATAACATTGTTTCAACTGCAAATCAAGCTC

CAACAGGGAACATAGGGAGCTGTTCTCCTCTAGATAATAGCTCAGATGCAG

39 CAAAAACAGAATCAATCCCAAATTTGCAATCTGATTCAAATGGTACACCTC CCAACCAGGGTTCCAATGGTAGTAGCAACAATAATGACATGGGATCCACTA CGAATAATGCTTTTACCAAACAAGTGGCTTTTGCAGAGAGGCCTACAAACA AATCCACAATCAAACTCCAATCAAACACTGGTTTCCAACAAGTGCAAAATG GCCAAGCTTCCCTTCAGACTATTATTCAAGGTAAATATTCGAATATGATGC CT A AGT A ATT A ATTCT A AT A AG AC AC A AGC A A AGGCC AC ATACC ACTTC AT AATAACTTTTCATCGATGTTGATTTGTTTTACAAAAACTATGGCAGCAGATG CTTCACAGTGTGGTTCATCCAATGCATTGAGAGCACCCATGGAAGGCAATA TTAGTAATCACAGTCTCAACAGGAGTGGGTCAGGTAGTAACCATGGTAGCA ACGGACAAAAGAGAAGCACCAATGCTTCAAACTCCAGAGGGAAAAAGACA GAAAGTGAGAGTGTGGTTACTGGAAGAGGGAAAACCATTGAAGGAAGTGA ATCGGATGGAAATCGATTTGCACAAAGAGAAGCTGCTTTGAAAAGATTCCG CCAGAAGAGGCAAGAAAGATGCTTTGAGAAAAAGGTAAACAGAAAATCAC CCCCCTTATTTTCTCTAAGAAGTAAATCATGGAAACAAACAAGTAGGCGTC TGAAAGAAGGAAGTTTCATTTTCAACACTACACATTTGAACCATCATCTTT GGGATCCAGTTAGTATGAAGCTTTTTGGGAAAAAAAAAAGGAAAAGAACA GAAAATTGTTTCCCTGAAATATAAATAATTGATCTGTTTCCTTTGAAGTTCC ATATTGCTGACCTGAAGCATTAATTTTTACTTTTTCAGGTGAGATATCAGAG TAGAAAAAAACTGGCAGAACAAAGACCCCGAATTCGAGGACAATTTATTA GAAAGGGAATGAATGAAAACAAGGGAAAAGGCATAAATTACGAACCTGA ACCAATTTCATAACAGGAGCCATTAGAATCCTTGATGCAGATGTGGTGCGT TTGCAGTAGATAGTTACAGAGCTTTTATGATTGAATATATGGGACTGTATT AATATTGAAGGTGTATAAGTAATATGCCAACGGCTGTGCTAGGATTAAGTA CTACAATTCTACTAAATAGAAGGTGATAAACCCTCAAAAAGAATAGAGCCT TCTCTCAATAAATCACTTTAAGTGGGTGTAATATTATTATTTCATGACTAGA TGATCATTTTAACTTGAATGGATGGTTGAAGAAACTCTTTTCCTGTTTACCT TTCTTCTTTCCTTCTTCTTGTGTTGTTTCTTGAAATATCAAACTTGCTTTTGT GTATATATATCATTCTGAAGCTTTTTCCAGCCAATCAAGTACTGTGCTTCTC TGGTTATATATATCTGCAGAGAGTTCAGTCGTAGAGAAACATTAACAGAAG _{GTTTTTCAATTAAATAAACTGAAAAACTCACGTACCAAAGAGTTTTCATAG} ATTATGATATGGTAACATTGTACAGAGAAAACTCAAAAGTTTGGATTCAAA GATTTACAGGGTGGTGATAGAAATAGGAGATTATTTCCATTGATGAGTTAG

40 AGAAATAGAGAAAGAAGCTGACCAGAGAGTGTGATGGGCTGAACCATATT

GGGCTAAAGTTGCAGTATAAAAAGGACGAGAGAAAAGAAAGAAGAAAGG

CAATGCCTTTTATGACCTCTTTTATTTTGGCTTAAAAAAGATTAAATGATCT

CCATTATTATTATTATTAAAAGAAAGAAAATGTATTGGTTAAAATGGTATT

AGCAACGGCATACCAATGTCTTTTTCTGGAATATGTTGTTGAATCGTTGTTG

TTAGTGTTCAGGTGAAGGTGAAGGGGGTGGAGGTTGAGGGGAGGGAGAAG

ATGGTAAAATCAAGTATAAGAAATAAATGCTGAGTTGGCGTTGGACTATTG

GGTAGTGTTGAAATGATGTTGTGGTGAGTTTGGTGTCCACGTGTGTAAGGC

TGGAAACGAAATTCGGTCGCGTTTTACGAAGAGGTAGTTGAGGCCATCCAT

TTTGATACAGAATATCGGGTCAGCATGAAGGGGCCCTTTCACTTGGCTACC

ACACTTCCACGTGTCCCTTTCCTCTATGTTTTGTTTTCCATTTCCACTAGTCT

ACTACTGCTGGATAAATATAAATCTCTCTCTCCTTCTTCCAAAATTTATTCA

TTCACGTTTGCATTTGCACCATATTTATTATTATTATTTTAATTAATAAAAAT

AATAGTAATTAGTTAAAATATAATTTTAGGTCTGGTACCAATCTTTAATCGC

TAT A A ATTT AG AA A AA A A A A ATCT A A ATTTTTT ATTT A A ACT A AG A AGAT A

AATTCCACTTTCGCAGCTTTTGAAGAAATGATAATTTTTTTTTTCTTTTGTAA

GGATGAATTTTCTTTAAATTTCTAAATACACATATCTTCTGAAAATAAAATA

AAAGAGAAGTTAAATTTGGACTGGAAATTTGTTGTCGGCTATGTATTTGTTT

TTTCATCACAAACTAAATACTAATATGCTCGTGACGAAAAAGTTGTAAGAA

AAAAGAAAGAATAATAATTAAGCTGGACTGTATTTGGCATGACTTCAAAA

GTAGG AGCTT A AAGGTTTT A ACCTTTTATTT ATTT ATTT ATTT ATTTTTA AC A

GTGTTTTTATACTTGGAAGATTTTATTTGAAAAGTCAGTATGATCAAATTAT

TC AG AAA AT A A AAA ATT A ATTT A A A ATTTT AAT A A ATGCCT AT A A A AAT A A

GATAAATCTGTGGTGAAGTAAGAGTTGAATAATGAATAAGTTTTTTTTTTCT

TAAAAAAATAACTTGTCCTTCATTGGATAAAACAGAGAATACAAAACAAA

TAAGTTCACAAGAAAAGTAGAGAACTATTCCAAACCAACTCATTATCTAAT

CC A AG ACCT A ACTTAGT AACTTT ATA AGCC A AC AT ATT A A ACTCTATGTG A

ACATAGACGATAGGTTCAGACTGAACGATAAAAAGCTCTTAACACTAATTA

AA

41 > Finola| QK VJ 02000031.1 :228502-

237689_Cannabis_sativa_cultivar_Finola_000030F,_whole_genome_shotgun_sequenc e (SEQ ID NO AAC)

AAAAAAAAGACTAGTTGCTCTTAGGGCTATTCATAAAGTGCTTGATCAAAT

TTAATCTGCACAATCTATAATGCAATTCACAAAATACGGATTGGTTATTTTT

TCACATGCAAAAATAGCCAACATTATTAAATCCGCATTTATTTATATTTATT

A AA AGTT ATA A AT AT ATT AT AAT ATTTT A A AGC A ATTA A AC A A A ACT A AT A

CAT A AT ACAAATTTTGT A A A ATTTT AT AT ATC A AT AT AT ATTTTATACAAAA

TAAAACATAATTTATACAATATCAATCTCTTCTTTTTACATATAATTTTATTT

TCTTCCTTTGTGACTTTTGTTTATTTACTTTAATTTGGTTAGAGACATAGAAT

AAT AATTCCTTTTACGTTGTTGTTGGAAAATTTAAAAGAAT ATTTT ATGTTT

TATATTTAAGTGATTTTGAT GAT GT A A A A A A AT ATTTTT A ATT A ATTTT AAA

TAATTTATATTTAAAAAATAATCAATTTAAAATAATTGATCTAATTCGCCCT

AGAATGAATTTAAGCTATCGTGCAAATTAAATTAGATTCAAAATAAACTAA

ATCGGATAAAAACTCCTAATTGTTTTTGCCCAAAAGAAGGAAAAGACTTCT

CC ATTTTT ACT A AGT GT AT AG A AAA A AC ATA A A A AG AAA AGG AAA AT ACT

AAATATTCTATCTTTTTATTTTCTTT ATTTT AAT ATTTAACATAAAAAT ATTA

AT AT ATAGT AT AA A AATGT AA AAG AT A ATTTCCTT AA A A A AG ACCT A A ATT

GATTCAGGCATGATATAAATTTGATGGTGTGGAACTCATTTGTATTATATTT

ATTATTATTAATAATTGCAATGGGAAGAACAAAAGTAGGTGTGGTTGTGGA

TGGAAAAGCGAAAGTGGCTTCGAAAATAGAAAGGATTTGCAGCCACTTCC

TTTGGGAAACCTTTTACTCATCAGAGAGAACGCCACGTGTCGAATACGGAT

CTGACGGATCGCTTCTGGATCCGCGGTAAACAATTCACAGCCCAGCTATAG

ATATTGGAGCGCACTAGAACATTTTCCTCTTACGTGGCGTCCTACGTGGCA

ACCTTTATTCGCTCCATTTAGAACTTTGTAGGGACAATTCTAATATCTTTTTT

CTCATTTATATCATATATCCATCGCTTCCATTTACCTTTCCAACCTTCCTTCT

TTCTCTACCAATAATTTTTCTTTTCAATTATTTTTTTAAAATATAATTTGGAA

AAAAAAAATGGTACAAAAACTCACTGAGATTTCTCAAACTCTACAGAAAA

TATAAATAATTTTTTTAAAAAAAAAAATTAACTTCATAGGAAAAAAAAAAA

GAAAAACCTCTGGTTTCTAACTTTTGAACTTTATTCCACCATTGGCGATTAT

CATCCAAGTTCGAATGAAGTTTTGAGAGAGAGGTATTCTCACTTTGTATAC

AGTAGCAACTTGTTGCTCAATGTTGATTTTCTTTTCCCTATTTGTTTCTTCAT

42 CTTTTGTTTGGCTGTTAAGAAAATTTTAAAGGAACACAACGGGAAAAATGT

TATAATGAAGGAACGAGAGTATTGATTTTTCACAGCATAGTGTTTCCGAGT

ACTACTCACTCATTCACTGAAAAATGAAAAAAAAAAATATGTATATATGTA

ATATATTTGTATTTACTGAATTATTTGGCTTTCCTTCTCTTCCGCTCTCTAAA

TTGTTTGCATGTATTCTATAATTCATAAATTAATATATATTTTATCTCGGAT

AATTGAAGTGTTTCTTAACGTCTTGATGATATGAAAAGGAAAATGACCTTT

GGAATTAAGTGTTCTGAAATCTACCAAAGAACCTCTTTCTCACCATATATTA

GC A A ACCCT ATA AGTTTT A ATTG AGTTG A AT ATGC ATG ATT A AAGTCTT AC

ACACTTGTTAATTGTTTGAAGATTCATGAGTTTGGTATATCATGATGCTGTT

ATTATCTTTCTATCAAAAGATAGTTTAATATGAACTTTTTTATTCTTATTTAA

TC ATAT AGGTTTTTG AAT A A ATTTGTACTT CTGTT ATTTTTT ATGTCC ATT AT

CTGGGTTCAGTTTTTGTTAATTATACTGTTATATGCATCAAAAAAAGCTACG

GTCAAATTTATATGCTACATACCTTGTATACTTTCGATCTCAATATAGATTG

ATAGTGGGAAAGTAGTAGAGATATGGGAATAGAAAAGGATCATAATCATT

TCCTTCCCTTTTATTTGTTGTGTCTAATTCAACTTTTCCTCATTTTATGCTATT

GGGACTTTGATCAGAACACACAGGCACACACATATACTATCCTCCTTATTC

TTCTTATTGGTTTCTGATAGCAATATATCTGTACTTATTATTTGTCTTTTGTA

GAGGATCTGTGAAGGGGCTTTCTCTTTTACTTGAGCCTGTATTTTTGTCACA

ACGGGATGTGCAAGAACTCTTGTGGATCAGCTTTCTCGCCAGCTCTATCCA

TTACCCTTCTCATCATTGATCTTATTGAATCTGGTTGATGAAAATGGTCCAG

ATGAATAATAAAGCTCCCGTAACCAATGATGAGCTAACTGAGCTGAATCAC

CGGATTCAAGATGGGAAAAAAGAAAGAAGGGAGAGGGTTACGAGAGAAC

GACAAGGGCTCTCAGAGGAACATGAATCTAGGATAATGAAGATGTGCAAC

AACATGTCAGCAATGGGGAGATTGGAACAGTACAGGCTCTGGAGCGGAGT

CATTCTGGCCAGCGGAGGTCTCAGCAACAGCCTCAAGGCATTTGGTTCGCT

GGGAGAGGTTCCTAGCTTTCAGGTCGCTAAAGGTTTTATTGGTGGAAAATG

ATGACTCAACTCGCCATATTGTCAGCGCGCTACTAAGAAATTGTGGCTACG

AAGGTCAGTACGTTTCAGCAACATGAAATGATCATTGCATTTCTCTAATGC

AGAAGTATACACTCTATGTTAATGGCTTTATGTTTGATTATTGACGGAGATG

ACTTTTAAAGTAAACACTGTAGTAGATCTTATGAATAAACTTGATGTTATAT

GATATGAAAGGAAACCATATTTATTTGCTCCACTCCACTACGAGTAATAAT

ATCCATGGTCTAATAGTTGTTGTCACTAACCATAACCTAGAAAGAGTGGTC

43 TTAGGATTAGCCTCGCTGATCCACACCTCACCTCTTGTAGAGAAGGTCAAG

GGTTTAATCCCTCCCCCGCCCTCCAAAGAAAGATATAACAAACAGAAAACG

AATCAAAAGAATAAAAAACAAACAAGTAAGGTGAGCAGCACTATTATTAA

CTAAAGCTTGTTTTAGTTTAATATAAGTAGATCTTATTTCTGAAATGTATTT

TCCACCATCTAATTACACGACATTTCTAGACTGATCCCCTGATCTTGTGGCA

GCGCTCTTTTTCTGAGTTCCTATCAGAAATGCTGAAAACTATTAAACTAGTT

TTTGTT ACTT ATTT ATTTTCTTTT ATTTTA AC ACT AC ATTTT A AC A AC AGTTC

CTATACTCTGGTGCTTCACGTGTCTATTTGTGCTATTTTGATGTTCATGGTTA

TAGTCTAGCGGGAAGTTTTTTAGTCATTTCGTTCATGAAGTGTCAAGTACGA

TTTCTTGAGCTAACTTAGATTTTGACCTAGAACCATTCTTGAGGATACTACA

GTGGGTTACTTAGTTTGTAGAGTATGCTTATGTGAATCTTCTAAAGATAAAC

TTGTTACAAGTATGATATTCTCGAAAAGAAAAACATGTTGCTCCAGACCTG

TTGGCAATTGACATCTTACTTTCTAGCTATGATAATTAGACATTCAGTCGCT

ATATTTATGTCATGCTTTCTTCATCATCATTTTTCATATATGTGTTCAAGTTA

TGGT AAGCTATTTAAATATTGTT ATTTT ATTTGACCATGTTTTATTTCCAACG

CAAACTGCTTGGTATAATATTACATAAATGTTAGCCAGAAGCTTTGATTTA

GTTTACATCCTAAAAGTCAAAACTAGAATTTTATTGGTTCCCTCAATAAACT

AACAATTATTCAATGTATTCTCATATTGGCAGTTACAGCTGTAGAAAATGG

CAGACAAGCTTGGAAAGTCTTAGAAGATCTTGTGACAGATGTTGATCTCGT

TTTGACTGAGGTAGTCATGCCCTGTTTATCTGGTATTGGTCTTCTAGGCAAG

ATCATGAGCAAAAAAACATGCAAGGACATCCCTGTGATTAGTGAGTATATT

TCCTTTACTTTATTTTCGATTTAAAAATATTTTTTTCTCTAGTATCTTTTTGTA

GTTGATACTTTTAACTAATACATTTATTGTGTGTGTTTGTGTTTTGTGTTTTC

ACAGTGATGTTCTCATGATTCTAGGAGTATGGTCTTTAAGTGTTTATCGAAA

GGTGCCGTTGACTTTTTAGTGAAACCTATTCGAAAGAATGAGCTGAAAAAC

CTTTGGCAACATGTTTGGAGAAAATGCCACATTGTGAGTTGCAATCTATTT

GATTTATTATATCATGGACCAATTCACCTTGAGGTTCAGGTTTCTTCTCATT

TATGCATTTTTTTTTTCAGTAAGCACCAATTGTCGAACTATACCAAGAAAAC

AAACAAAAAAAAATGGCTTCAATCTTTGCTTGATAAAAAATATGTTTTATG

TAATGCAAATATCACTTAATTGACATAATACCATTAAACACTGAAATTTGC

TCGAGTTGTGTATTAATCTTTCATATTTTCATCAGTCTAGTAATAGTGGAAG

TGAAAGTGGTATATGGATTGAAAAGCCTTTAAAGTCAAGAACTATGGAAC

44 ATTCAGACAACAACAGTGGCAGCAATGATGAGGATGACACTGACAGCATT

GGTCTAAATTTCAGGAATGAAAGTGACAGTGGAACACAGGTATTTCACTAA

ATTTCATGAAAGTTTTTTTTTTTTTTTTGGTGGGAATGAAGTTTTATGTCTTT

GTATTTACGGAAATATTAGCGAAATGTTAGTTTCCACATGAAGTTTTATGTT

TTATGTATCAAGAAGTACTATAAATATGTTAGTTTCCACACACCTAGTTTGG

AATTTGTCTATCACAGCACCCTATATACCGGTGACACAACATCAATCTATTT

ACCATGTTTGCATATTACTCATTTGATCTTGGTGGAGGAATATTCATGTAAG

AGTTTTT A AT ACTTTT ATGT AT ATTT A AGT GG AG A AGG A ATGAT A ATTAGC

AAGATAAGAAAACAAGAAAAAGAATGAAAACTTACTACTGAGCTTTTACA

GAGCTCTTGGACAAAGAGAGCAGAAGTTGACAGCCCACAGGCAGTGTCGT

GGGAGCAGTTTGCTGATCTTCCTGATAGCACTAATCATCAGGTCAATCATC

CAAGGCAAGAAGCCTTTGGAAACAACTGGGTACCTGAAAATCCAACAGTA

ACACCACGTCCACATAATGATGAGCTTGGTAGGCATTTCCTAACATCTTCTT

_{TTTTTTTTTAACTTTCTTCCATTCTCGATCATTCTCTAAGTTATGATTTATTTA}

AATTGTTTAGACAAAAAAGTCATGGGAAAAGACTTGAAAATAGGATTACC

TAGCCTTCTTGAAGACACAAGTGAAAAAGGGCTGACCAACATGGAAGGTA

CTAATAAAGATAAATGTTCTGAACTGAACTCAAAGAAAGATGATCAGGAG

CAGGAGAAAAGGGAATTAGACCTCAACAATGAAGAACCGAGTGCAGAAA

AGACCCAAGCTGTTGATCTGATGGGTGTCTCCAATTATAGTATTGATCCTCA

CATGGAAAGTGGAGTTCTTGATGTCCCAAACGAACTCTCCAAGGCTGCCTG

CATGAGAGATAATGCCAACCATGAGAATAAAGAAACACCTTTTTTTGAGCT

CATTTTAAAGAGGCCAAGAGATATCCAAGATACTGGAACCAGCGCACACG

ATCGAAATGTTTTGAGACATTCCGATATTTCAGCTTTTTCAAGGTATAGAAA

TGTTTTGTGTTGATGTAAACATGCTCAACCACAATTAATTCTAAATTTAAGA

ATATAACTAAGTCCCATTACCTTGCAGGTATAACATTGTTTCAACTGCAAAT

CAAGCTCCAACAGGGAACATAGGAAGCTGTTCTCCTCTAGATAATAGCTCA

GATGCAGCAAAAACAGAATCAATCCCAAATTTGCAATCTGATTCAAATGGT

ACACCTCCCAACCAGGGTTCCAATGGTAGTAGCAACAATAATGACATGGG

ATCCACTACGAATAATGCTTTTACCAAACAAGTGGCTTTTGCAGAGAGGCC

TACAAACAAATCCACAATCAAACTCCAATCAAACACTGGTTTCCAACAAGT

GCAAAATGGCCAAGCTTCCCTTCAGACTATTATTCAAGGTAAATATTCGAA

TATG ATGCCT A AGT A ATT A ATTCT A AT A AG AC AC AAGC AA AGGCC AC AT AC

45 CACTTCATAATATCTTTTCATCGATGTTGATTTGTTTTACAAAAACTATGGC

TGCAGATGCTTCACAGTGTGGTTCATCCAATGCATTGAGAGCACCCATGGA

AGGCAATATTAGTAATCACAGTCTCAACAGGAGTGGGTCAGGTAGTAACC

ATGGTAGCAACGGACAAAAGAGAAGCACCAATGCTTCAAACTCCAGAGGG

AAAAAGACAGAAAGTGAGAGTGTGGTTACTGGAAGAGGGAAAACCATTGA

AGGAAGTGAATCTGATGGAAATCGATTTGCACAAAGAGAAGCTGCTTTGA

AAAGATTCCGCCAGAAGAGGCAAGAAAGATGCTTTGAGAAAAAGGTAAAC

AGAAAATCACCCCCCTTATTTTCTCTAAGAAGTAAATCATGGAAACAAACA

AGTAGGAGTCTGAAAGAAGGAAGTTTCATTTTCAACACTACACATTTGAAC

CATCATCTTTGGGATCCAGTTAGTATGAAGCTTTTTGGGAAAAAAAAAAGG

AAAAGAACAGAAAATTGTTTCCCTGAAATATAAATAATTGATCTGTTTCCT

TTGAAGTTCCATATTGCTGACCTGAAGCATTAATTTTTACTTTTTCAGGTGA

GATATCAGAGTAGAAAAAAACTGGCAGAACAAAGACCCCGAATTCGAGGA

CAATTTATTAGAAAGGGAATGAATGAAAACAAGGGAAAAGGCATAAATTA

CGAACCTGAACCAATTTCATAACAGGAGCCATTAGAATCCTTGATGCAGAT

GTGGTGCGTTTGCAGTAGATAGTTACAGAGCTTTTATGATTGAATATATGG

GACTGTATTAATATTGAAGGTGTATAAGTAATATGCCAACGGCTGTGCTAG

GATTAAGTACTACAATTCTACTAAATAGAAGGTGATAAACCCTCAAAAAGA

ATAGAGCCTTCTCTCAATAAATCACTTTAAGTGGGTGTAATATTATTATTTC

ATGACTAGATGATCATTTTAACTTGAATGGATGGTTGAAGAAACTCTTTTCC

TGTTTACCTTTCTTCTTTCCTTCTTCTTGTGTTGTTTCTTGAAATATCAAACTT

GCTTTTGTGTATATATATCATTCTGAAGCTTTTTCCAGCCAATCAAGTACTG

TGCTTCTCTGGTTATATATATCTGCAGAGAGTTCAGTCGTAGAGAAACATT

AACAGAAGGTTTTTCAATTAAATAAACTGAAAAACTCACGTACGAAAGAG

TTTTCATAGATTATGATATGGTAACATTGTACAGAGAAAACTCAAAAGTTT

GGATTCAAAGATTTACAGGGTGGTGATAGAAATAGGAGATTATTTCCATTG

ATGAGTTAGAGAAATAGAGAAAGAAGCTGACCAGAGAGTGTGATGGGCTG

AACCATATTGGGCTAAAGTTGCAGTATAAAAAGGACGAGAGAAAAGAAAG

AAGAAAGGCAATGCCTTTTATGACCTCTTTTATTTTGGCTTAAAAAAGATTA

AATGATCTCCAATTATTATTATTATTAAAAGAAAGAAAATGTATTGGTTAA

AATGGTATTAGCAACGGCATACCAATGTCTTTTTCTGGAATCGTTGTTGTTA

GTGTTCAGGTGAAGGTGAAGGGGGTGGAGGTTGAGGGGAGGGAGAAGATG

46 GTAAAATCAAGTATAAGAAATAAATGCTGAGTTGGCGTTGGACTATTGGGT

AGTGTTGAAATGATGTTGTGGTGAGTTTGGTGTCCACGTGTGTAAGGCTGG

AAACGAAATTCGGTCGCGTTTTACGAAGAGGTAGTTGAGGCCATCCATTTT

GATACAGAATATCGGGTCAGCATGAAGGGGCCCTTTCACTTGGCTACCACA

CTTCCACGTGTCCCTTTCCTCTATGTTTTGTTTTCCATTTCCACTAGTCTACT

ACTGCTGGATAAATATAAATCTCTCTCTCCTTCTTCCAAAATTTATTCATTC

ACGTTTGCATTTGCACCATATTTATTATTATTATTTTAATTAATAAAAATAA

TAGTAATTAGTTAAAATATAATTTTGGTTCTGGTCACAATCTTTAATCGCTA

TAAATTTAGAAAGAAAAAAAAATCTAAATTTTTTATTTAAACTAAGAAGAT

AAATTCCACTTTCGCAGCTTTTGAAGAAATGATAATTTTTTTTTTTTTGTAA

GGAGAATTTCCTTTAAATTTCTAAATACACATATCTTCTGAAAATAAAATA

AAAGAGAAGTTAAATTTGGACTGGAAATTTGTTGTCGGCTATGTATTTGTTT

TTTCATCACAAACTAAATACTAATATGCTCGTGACGAAAAAGTTGTAAGAA

AAAAGAAAGAATAATAATTAAGCTGGACTGTATTTGGCATGACTTCAAAA

GTAGG AGCTT A AAGGTTTT A ACCTTTTATTT ATTT ATTT ATTT ATTTTTA AC A

GTGTTTTTATACTTGGAAGATTTTATTTGAAAAGTCAGTATGATCAAATTAT

TC AG AAA AT A A AAA ATT A ATTT A A A ATTT A AT A A AT ACCTAT A A A A AT A AG

ATAAATCTGTGGTGAAGTAAGAGTTGAAT AATGAAT AAGTTTTTTTTTTTTT

TAAATAATTTGTCCGAGAATACAAAACAAACAAGTTCACAAAAAAGTAGA

GAATTATTCCAAACCAACTCATTATTTAATCCAAGACCTAACTTAGTAACTT

TATAAGCCAACATATTAAACTCTATGTGAACATAGATGATAGGTTCGGACT

G AAG AT A A A AAGCTCTT AAC ACC AATT AA AC A ATC ATCC A

>LowRyder (SEQ ID NO AAD)

AACTCAATTCAAACTGCAATTTATATTGCAATTTACGTAACAACTCAGATA AC A ACTT A A ATCGT A A A A AT A AT A AT A A A A A A A A A A AT CAAAAAGTAGTA TATGTGATGATTTTCCTTTATAATATTTAATAAATTATATAATAATATTGAC A AATC AT ATTTTT AAGTGGTTTT ATTGTC A AT ATTTTTTT AT AT AT AT AT ATT TAAGTGATTTTAATGATGTAAAAAAAAATATTTTATAATTAATATTAAATA ATTT AAT ATTT AT AAAGTAATCAATCTAAAATAACTGATCCAATTTGTTTTA G AAT A ACTTT A AGCT ATC ATGC AA ATTA A ATTG A ATTC AA A AT AT A ATCG A AT AC ACT AT A A AT A A A AT A AT A A A AACT A A ATC AG A AG A A A ACTCTG A AG

47 TATTCTCGCCTACAAAAAGAGAGAAAAGACTTCTCCATTTTTACTAAGTGT

ATAAAAAAAGCATAAAAAGAAAAGGAAAATACTAAATATTCTATCTTTTTT

ATTTTCTTTTATTTTAATATCTAACATAAAAATATTAATAATATATAGTATA

CAAATGTAAAAGATAATTTCCTTAAAAAAGACCTAAATTGATTCAGGCATG

ATATAAATTTGATGGTGTGGAACTCATTTGTATTATATTATATTATATTATA

TTATTAATAATTGGTATGGGAAGAACAAAAGTAGGTGTGGTTGTGGATGGA

AAAGCGAAAGTGGCTTCGAAAATAGAAAGGATTTGCAGCCACTTCCTTTGG

GAAACCTTTTACTCATCAGAGAGAACGCCACGTGTCGAATACGGATCTGAC

GGATCGCTTCTGGATCCGCGGTAAACAATTCACAGCCCAGCTATAGATATT

GGAGCGCACTAGAACATTTTCCTCTTACGTGGCGTCCTACGTGGCAACCTTT

ATTCGCTCCATTTAGAACTTTGTAGGGACAATTCTAATATCTTTTTTCTCATT

TATATCATATATCCATCGCTTCCATTTACCTTTCCAACCTTCCTTCTTTCTCT

ACCAATAATTTTTCTTTTCAATTATTTTTTTTTAAATATAATTTGGAAAAAA

AAAATGGTACAAAACTCACTGAGATTTCTCAAACTCTACAGAAAAAAATA

AATAATTTTTTTTAAAAAAAAAAAATTAACTTCATAGGAAAAAAAAAAAG

AAAAACCTCTGGTTTCTAACTTTTGAACTTTATTCCACCATTGGCGATTATC

ATCCAAGTTCGAATGAAGTTTTGAGAGAGAGGTATTCTCACTTTGTATAAA

GTAGCAACTTGTTGCTCAATGTTGATTTTCTTTTCCCTATTTGTTTCTTCATC

TTTTGTTTGGCTGTTAAGAAAATTTTAAAGGAACACAACGGGAAAAATGTT

ATAATGAAGGAACGAGAGTATTTGATTTTTCACAGTATAGTGTTTCCGAGT

ACT ACTC ACTC ATTC ACTG A A A A AT GA A A A A A A A AT ATGT AT AT ATGT AAT

ATATTTGTATTTACTGAATTATTTGGCTTTCCTTCTCTTCCCGCTCTCTAAAT

TGTTTGCATGTATTCTATAAATTCATAAATTAATATATATTTTATCTCGGAT

AATTGAAGTGTTTTTTAACGTTTTGATGAAATGAAAAGGAAAATGACCTTT

GGAATTAAGTGTTCTGAAATCTTCCAAAGAACCTCTTTCTCACCATATATTA

GC A A ACCCT ATA AGTTTT A ATTG AGTTG A AT ATGC ATG ATT A AAGTCTT AC

ACACTTGTTAATTGTTTGAAGATTCATGAGTTTGGTATATCATGATGCTGTT

ATTATCTCTATATCAAAAGATGGTTTGATATGAACTTTATATTCAGGAACTT

TTTTATTCTTATTTAACCATATAGGTGTGCTTCTGTTATTTTTTATGTCCATT

ATCTGGGTTCAGTTTTTGTTAATTATACTGTTATATGCATCAAAAAAAGCTA

CGGTCAAATTTATATGTGCTACATACCTTGTATACTTTTGATCGCAATATAG

ATTGATAGTGGGAAAGTAGTAGAGATATGGGAATAGAAAAGGATCATAAT

48 CATTTCCTTCCCTTTTATTTGTTGTGTCTAATTCAACTTTTCCTCATTTTATGC

TATTGGGACTTTGATCAGAACACACAGACACACACATATGCTATCCACCTT

ATTCTTCTTATTGGTTTCTGATAGCAATATATTTGTACTTATTATTTGTCTTT

TGTAGAGGATCTGTGAAGGGGCTTTCTGTTTTACTTGAGCCTGTATTTTTGT

CACAACGGGATGTGCAAGAACTCTTGTGGATCAGCTTTCTCGCCAGCTCTA

TCCATTACCCTTCTCATCATTGATCTTATTGAATCTGGTTGATGAAAATGGT

CCAGATGAATAATAAAGCTCCCGTAACCAATGATGAGCTAACTGAGCTGA

ATCACCGGATTCAAGATGGGAAAAAAGAAAGAAGGGAGAGGGTTACGAG

AGAACGCCAAGGGCTCTCAGAGGAACATGAATCTAGGATCAATGAAGATG

TGCAACAACATGTCAGCAATGGGGAGATTGGAACAGTACAGGCTCTGGAG

AGGAGTCATTCTGGCCAGCGGAGGTCTCAGCAACAGCCTCAAGGACATTTG

GTTCGCTGGGAGAGGTTCCTAGCTTTCAGGTCGCTAAAGGTTTTATTGGTG

GAAAATGATGACTCAACTCGCCATATTGTCAGCGCGCTACTAAGAAATTGT

GGCTACGAAGGTCAGTACGTTTCAGCAACATGAAATGATCATTGCATTTCT

CTTATGCAGAAGTATACACTCTATGTTAATGGCTTTATGTTTGATTATTGAC

GGAGATGACTTTTAAAGTAAACACTGTAGTAGATCTTATGAATAAACTTGA

TGTTATATGATATGAAAGGAAACCATGTTTATTTGCTCCACTACGAGTAAT

AATATCCATGGTCTAATAGTTGTTGTCACTAACCATAACCTAGAAAGAGTG

GTCTTAGGATTAGCCTCGCTGATCCACACCTCACCTCTTGTAGAGAAGGTC

AAGGGTTCAATCCCTCCCCCGCCCTCCAAAGAAAGATATAACAAACAGAA

AACGAATCAAAAGAATAAAAAACAAACAAGTAAGGTGAGCATGGCACTAT

TATTAGACTAAAGCTTGTTTTAGTTTAATATAAGTAGATCTTATTTCTGAAA

TGTTATTTTCCACCATCTAATTACACACATTTCTAGACTGATCCCCTGATCT

TGTGGCAGCGCTCTTTTTCTGAGTTCCTATCAGAAATGCTGAAAACTATTAA

ACT AGTTTTT GTT ACTT ATTT ATTTTCTTTTGTTTT A AC ACT ACATTTTAACA

ACAGTTCCTATACTCTGGTGCTTCACGTGTCTATTTGTGCTATTTTGATGTTC

ATATTTATAGTCTAGCGGGAAGTTTTTTAGTCATTTCGTTCATGAAGGGTCA

AGTACGATTTCTTGACCTAGCTTAGATTTTGACATAGAACCATTCTTGAGGA

TACTACAGTGGGTTACTTAGTTTGTAGAGTATGTTTATGTGTACCTTCTAAA

GATAACCTGGTTATAAGTATGATATTCTCGAAAAGAAAAACATGTTGCTCC

AGACCTGTTGGCAATTGACATCTTACTTTCTAGCTATGATAATTAGACATTC

AGTCGCTATATTTATGTCATGCTTTCTTCATCATCATTTTTCATATATGTGTT

49 CAAGTTATGGTAAGCTATTTAAATATTGTTATTTTATTTGACCATGTTTTATT

TCC A ACGC A A ACTGCTTGGT AT A AT ATT AC ATA A ATGTT AGCC AG AAGCTT

TGATTTAGTTTACATCCTAAAAGTCAAAACTAGAATTTTATTGGTTCCCTCA

ATAAACTAACAATTATTCAATGTATTCTCATATTGGCAGTTACAGCTGTAG

AAAATGGCAGACAAGCTTGGAAAGTCTTAGAAGATCTTGTGACAGATGTTG

ATCTCGTTTTGACTGAGGTAGTCATGCCCTGTTTATCTGGTATTGGTCTTCT

AGGCAAGATCATGAGCAAAAAAACATGCAAGGACATCCCTGTAATTATTG

AGTATATTTCCTTTATTTTGGATTTAAAATAATACTTTTTTCTCTAGTATCTT

TTTGTAATTTATACTTTTAACTAATACATTTATTGTGTGTGTTTGTGTTTGTG

TTTTCACAGTGATGTCTTCACATGATTCTAGGAGTATGGTCTTTAAGTGTTT

ATCGAAAGGTGCCGTTGACTTTTTAGTGAAACCTATTCGAAAGAATGAGCT

GAAAAACCTTTGGCAACATGTTTGGAGAAAATGCCACATTGTGAGTTGCAA

TCTATTTGATTTATTATATCATGGACCAATTCACCTTGAGGTTCAGGTTTCT

TCTCATTTATGCATTTTGTTTTTCATTAAGCACCAATTGTCGAACTATACCA

AAGAAAACAAACAAAAAAATGGCTTCAATCTTTGCTTGATAAAAAATATGT

TTT ATGT A ATGC A A AC ATC ACTT A ATTG AC ATA AT ACC ATT AA AC AC AG A A

ATTTGCTCGAGTTGTGCATTAATTTTTCATATTTTCATCAGTCTAGTAATAG

TGGAAGTGAAAGTGGTATATGGATTGAAAAGCCTTTAAAGTCAAGAACTGT

GGAACATTCAGACAACAACAGTGGCAGCAATGATGAGGATGATACTGACA

GCATTGGTCTAAATTTCAGGAATGAAAGTGACAGTGGAACACAGGTATTTC

ACTAAATTTCATGAAAGAGTTTTGTTTTTTTTTTTTGGTGGGAATGAAGTTT

TATGTCTTTGTATTTACAGAAATATTAGCGAAATGTTAGTTTCCACATGAAG

TTTTATGTTTT ATGT ATC A AG A A ACT ACT AT AATT ATGTT AGTTTCC AC AC A

CCTAGTTTGGAATTTGTCTATCACAGCACCCATATATCTATTTACCATGTTT

GCATATTACTCATTTGATCTTGGTGGAGGAATATTCATGTAAGAGTTTTTAA

TACTTTT ATGT AT ATGT AAGTGGAGAAGGAATGATAATTAGCAAGATAAGA

AAACAAGAAAAAGAATGAAAACTTACTACTGAGCTTTTACAGAGCTCTTGG

ACAAAGAGAGCAGAAGTTGACAGCCCTCAGGCAGTGTCGTGGGAGCAGTT

TGCTGATCTTCCTGATAGCACTAATCATCAGGTCAATCATCCAAGGCAAGA

AGCCTTTGGAAACAACTGGGTACCTGAAAATGCAACAGTAACACCACGCC

CACATAATGATGAGCTTGGTAGGCAAATTTTCCTAACATCTTTTTTTTTTTT

AACTTTCTTCCATTCTCGATCATTCTCTAAGTTATGATTTATTTAAATTGTTT

50 AGACAAAAAAGTCATGGGAAAAGACTTGAAAATAGGATTACCTAGCCTTC

TTGAAGACACAAGTGAAAAAGGGCTGACCAACATGGAAGGTACTAATAAA

GATAAATGTTCTGAACTGAACTCAAAGAAAGATGATCAGGAGCAGGAGAA

AAGGGAATTAGACCTCAACAATGAAGAACCGAGTGCAGAAAAGACCCAAG

CTGTTGATCTGATGGGTGTCTCCAATTATAGTATTGATCCTCACATGGAAAG

TGGAGTTCTTGATGTCCCAAACGAACTCTCCAAGGCTGCCTGCATGAGAGA

TAATGCCAACCATGAGAATAAAGAAACACCTTTTTTTGAGCTCATTTTAAA

GAGGCCAAGAGATATCCAAGATACTGGAACCAGCGCACACGATCGAAATG

TTTTGAGACATTCCGATATTTCAGCTTTTTCAAGGTATAGAAATATTTTGTG

TTGATATAAACATGCTCAATAACAATTAACTCTAAATTTAAGAATATAACT

AAGTCCCATTACCATGCAGGTATAACATTGTTTCAACTGCAAATCAAGCTC

CAACAGGGAACATAGGGAGCTGTTCTCCTCTAGATAATAGCTCAGATGCAG

CAAAAACAGAATCAATCCCAAATTTGCAATCTGATTCAAATGGTACACCTC

CCAACCAGGGTTCCAATGGTAGTAGCAACAATAATGACATGGGATCCACTA

CGAATAATGCTTTTACCAAACAAGTGGCTTTTGCAGAGAGGCCTACAAACA

AATCCACAATCAAACTCCAATCAAACACTGGTTTCCAACAAGTGCAAAATG

GCCAAGCTTCCCTTCAGACTATTATTCAAGGTAAATATTCGAATATGATGC

CT A AGT A ATT A ATTCT A AT A AG AC AC A AGC A A AGGCC AC ATACC ACTTC AT

AATAACTTTTCATCGATGTTGATTTGTTTTACAAAAACTATGGCAGCAGATG

CTTCACAGTGTGGTTCATCCAATGCATTGAGAGCACCCATGGAAGGCAATA

TTAGTAATCACAGTCTCAACAGGAGTGGGTCAGGTAGTAACCATGGTAGCA

ACGGACAAAAGAGAAGCACCAATGCTTCAAACTCCAGAGGGAAAAAGACA

GAAAGTGAGAGTGTGGTTACTGGAAGAGGGAAAACCATTGAAGGAAGTGA

ATCGGATGGAAATCGATTTGCACAAAGAGAAGCTGCTTTGAAAAGATTCCG

CCAGAAGAGGCAAGAAAGATGCTTTGAGAAAAAGGTAAACAGAAAATCAC

CCCCCTTATTTTCTCTAAGAAGTAAATCATGGAAACAAACAAGTAGGCGTC

TGAAAGAAGGAAGTTTCATTTTCAACACTACACATTTGAACCATCATCTTT

GGGATCCAGTTAGTATGAAGCTTTTTGGGAAAAAAAAAAGGAAAAGAACA

GAAAATTGTTTCCCTGAAATATAAATAATTGATCTGTTTCCTTTGAAGTTCC

ATATTGCTGACCTGAAGCATTAATTTTTACTTTTTCAGGTGAGATATCAGAG

TAGAAAAAAACTGGCAGAACAAAGACCCCGAATTCGAGGACAATTTATTA

GAAAGGGAATGAATGAAAACAAGGGAAAAGGCATAAATTACGAACCTGA

51 ACCAATTTCATAACAGGAGCCATTAGAATCCTTGATGCAGATGTGGTGCGT TTGCAGTAGATAGTTACAGAGCTTTTATGATTGAATATATGGGACTGTATT AATATTGAAGGTGTATAAGTAATATGCCAACGGCTGTGCTAGGATTAAGTA CTACAATTCTACTAAATAGAAGGTGATAAACCCTCAAAAAGAATAGAGCCT TCTCTCAATAAATCACTTTAAGTGGGTGTAATATTATTATTTCATGACTAGA TGATCATTTTAACTTGAATGGATGGTTGAAGAAACTCTTTTCCTGTTTACCT TTCTTCTTTCCTTCTTCTTGTGTTGTTTCTTGAAATATCAAACTTGCTTTTGT GTATATATATCATTCTGAAGCTTTTTCCAGCCAATCAAGTACTGTGCTTCTC TGGTTATATATATCTGCAGAGAGTTCAGTCGTAGAGAAACATTAACAGAAG _{GTTTTTCAATTAAATAAACTGAAAAACTCACGTACCAAAGAGTTTTCATAG} ATTATGATATGGTAACATTGTACAGAGAAAACTCAAAAGTTTGGATTCAAA GATTTACAGGGTGGTGATAGAAATAGGAGATTATTTCCATTGATGAGTTAG AGAAATAGAGAAAGAAGCTGACCAGAGAGTGTGATGGGCTGAACCATATT GGGCTAAAGTTGCAGTATAAAAAGGACGAGAGAAAAGAAAGAAGAAAGG CAATGCCTTTTATGACCTCTTTTATTTTGGCTTAAAAAAGATTAAATGATCT CCATTATTATTATTATTAAAAGAAAGAAAATGTATTGGTTAAAATGGTATT AGCAACGGCATACCAATGTCTTTTTCTGGAATATGTTGTTGAATCGTTGTTG TTAGTGTTCAGGTGAAGGTGAAGGGGGTGGAGGTTGAGGGGAGGGAGAAG ATGGTAAAATCAAGTATAAGAAATAAATGCTGAGTTGGCGTTGGACTATTG GGTAGTGTTGAAATGATGTTGTGGTGAGTTTGGTGTCCACGTGTGTAAGGC TGGAAACGAAATTCGGTCGCGTTTTACGAAGAGGTAGTTGAGGCCATCCAT TTTGATACAGAATATCGGGTCAGCATGAAGGGGCCCTTTCACTTGGCTACC ACACTTCCACGTGTCCCTTTCCTCTATGTTTTGTTTTCCATTTCCACTAGTCT ACTACTGCTGGATAAATATAAATCTCTCTCTCCTTCTTCCAAAATTTATTCA TTCACGTTTGCATTTGCACCATATTTATTATTATTATTTTAATTAATAAAAAT AATAGTAATTAGTTAAAATATAATTTTAGGTCTGGTACCAATCTTTAATCGC TAT A A ATTT AG AA A AA A A A A ATCT A A ATTTTTT ATTT A A ACT A AG A AGAT A AATTCCACTTTCGCAGCTTTTGAAGAAATGATAATTTTTTTTTTCTTTTGTAA GGATGAATTTTCTTTAAATTTCTAAATACACATATCTTCTGAAAATAAAATA AAAGAGAAGTTAAATTTGGACTGGAAATTTGTTGTCGGCTATGTATTTGTTT TTTCATCACAAACTAAATACTAATATGCTCGTGACGAAAAAGTTGTAAGAA AAAAGAAAGAATAATAATTAAGCTGGACTGTATTTGGCATGACTTCAAAA

52 GTAGGAGCTTAAAGGTTTTAACCTTTTATTTATTTATTTATTTATTTTTAACA

GTGTTTTTATACTTGGAAGATTTTATTTGAAAAGTCAGTATGATCAAATTAT

TC AG AAA AT A A AAA ATT A ATTT A A A ATTTT AAT A A ATGCCT AT A A A AAT A A

GATAAATCTGTGGTGAAGTAAGAGTTGAATAATGAATAAGTTTTTTTTTTCT

TAAAAAAATAACTTGTCCTTCATTGGATAAAACAGAGAATACAAAACAAA

TAAGTTCACAAGAAAAGTAGAGAACTATTCCAAACCAACTCATTATCTAAT

CC A AG ACCT A ACTTAGT AACTTT ATA AGCC A AC AT ATT A A ACTCTATGTG A

ACATAGACGATAGGTTCAGACTGAACGATAAAAAGCTCTTAACACTAATTA

AA

Example 8

[00149] The following is an alignment of the CsPRR37 mRNA to the CCA_PP_PRR37 de novo assembled PRR37 gene sequence along with the predicted protein sequence translation. The psREC and CCT domains are annotated with ~ symbols underneath the sequence. (See Figure 1) It is noted that the full-length protein is translated and no variants are present which would create LOF. Both psREC and CCT domains are intact and the protein is spliced correctly and is not truncated.

>PRR37_CDS|XM_030632368.1:235-2406 PREDICTED: Cannabis sativa two- component response regulator-like PRR37 (LOCI 15705128), mRNA Paths (1):

Path 1: query 1..2172 (2172 bp) => genome CCA_PP_PRR37:6,690..2,057 (-4634 bp) cDNA direction: sense

Genomic pos: CCA_PP_PRR37:6,690..2,057 (- strand)

Number of exons: 9

Coverage: 100.0 (query length: 2172 bp)

Trimmed coverage: 100.0 (trimmed length: 2172 bp, trimmed region: 1..2172)

Percent identity: 99.9 (2169 matches, 2 mismatches, 0 indels, 1 unknowns)

Translation: 1..2172 (723 aa)

Amino acid changes:

Alignments:

53 Alignment for path 1 :

-CCA_PP_PRR37: 6690-6318 (1-373) 100% -> ...1166... 0.998, 0.980 -CCA_PP_PRR37 : 5151 -4990 (374-535) 100% -> ...118... 0.982, 0.998 -CCA_PP_PRR37:4871-4738 (536-669) 99% -> ...259... 0.994, 0.970 -CCA_PP_PRR37 :4478-4323 (670-825) 100% -> ...362... 0.995, 0.844 -CCA_PP_PRR37: 3960-3783 (826-1003) 100% -> ...89... 0.983, 0.923 -CCA_PP_PRR37:3693-3258 (1004-1439) 100% -> ...88... 0.996, 0.990 -CCA_PP_PRR37:3169-2853 (1440-1756) 100% -> ...121... 0.998, 0.947 -CCA_PP_PRR37 :2731 -2442 (1757-2046) 99% -> ...259... 0.996, 0.999 -CCA_PP_PRR37:2182-2057 (2047-2172) 100%

Example 9

[00150] The following is an alignment of the CsPRR37 mRNA to the CCA_AF_PRR37 de novo assembled PRR37 gene sequence along with the predicted protein sequence translation. (See Figure 2) The critical psREC and CCT domains are annotated with ~ symbols underneath the sequence. It is noted that a partial length protein is translated due to a splice site variant following the second coding sequence which is sufficient to create a Loss of Function allele. It is further noted that this truncation occurs within the psREC domain which is required for protein protein interaction. The CCT domain would otherwise be intact if the protein were spliced correctly.

>PRR37_CDS|XM_030632368.1:235-2406 PREDICTED: Cannabis sativa two- component response regulator-like PRR37 (LOCI 15705128), mRNA Paths (1):

Path 1: query 1..2172 (2172 bp) => genome CCA_AF_PRR37:2,363..6,992 (4630 bp) cDNA direction: sense

Genomic pos: CCA_AF_PRR37:2,363..6,992 (+ strand)

Number of exons: 9

Coverage: 100.0 (query length: 2172 bp)

Trimmed coverage: 100.0 (trimmed length: 2172 bp, trimmed region: 1..2172)

Percent identity: 99.8 (2166 matches, 5 mismatches, 0 indels, 1 unknowns)

54 Translation: 1..2172 (723 aa) Amino acid changes:

Alignments:

Alignment for path 1 :

+CCA_AF_PRR37:2363-2735 (1-373) 100% -> ...1166... 0.998, 0.980 +CCA_AF_PRR37:3902-4063 (374-535) 100% == ...118... 0.132, 0.998 +CCA_AF_PRR37:4182-4315 (536-669) 100% -> ...258... 0.994, 0.970 +CCA_AF_PRR37:4574-4729 (670-825) 100% -> ...360... 0.995, 0.844 +CCA_AF_PRR37:5090-5267 (826-1003) 100% -> ...89... 0.983, 0.923 +CC A_ AF_PRR37:5357-5792 (1004-1439) 100% -> ...88... 0.996, 0.990 +CC A_AF_PRR37:5881-6197 (1440-1756) 99% -> ...121... 0.998, 0.441 +CCA_AF_PRR37:6319-6608 (1757-2046) 99% -> ...258... 0.996, 0.999 +CC A_AF_PRR37 : 6867- 6992 (2047-2172) 100%

Example 10

[00151] The following is an alignment of the CsPRR37 mRNA to the Finola_PRR37 de novo assembled PRR37 gene sequence along with the predicted protein sequence translation. The critical psREC and CCT domains are annotated with ~ symbols underneath the sequence. (See Figure 3) It is noted that a partial length protein is translated due to an indel (deletion) variant between positions 2669-2670 in the Finola_PR37 nucleotide sequence causing a frameshift which creates an early stop codon leading to Loss of Function. It is further noted that this truncation occurs within the psREC domain which is required for protein protein interaction. The CCT domain alignment does not show variation in the coding sequences.

>PRR37_CDS|XM_030632368.1:235-2406 PREDICTED: Cannabis sativa two- component response regulator-like PRR37 (LOCI 15705128), mRNA

Paths (1):

Path 1: query 1..2172 (2172 bp) => genome 2, 521..7, 167 (4647 bp) cDNA direction: sense

55 Genomic pos: 2, 521..7, 167 (+ strand)

Number of exons: 9

Coverage: 100.0 (query length: 2172 bp)

Trimmed coverage: 100.0 (trimmed length: 2172 bp, trimmed region: 1..2172) Percent identity: 99.2 (2153 matches, 14 mismatches, 4 indels, 1 unknowns) Non-intron gaps: 3 openings, 4 bases in cdna; 0 openings, 0 bases in genome Translation: 562..2172 (536 aa)

Amino acid changes: M57V [729], P136A [966]

Alignments:

Alignment for path 1 :

2521-2891 (1-373) 98% -> ...1168... 0.998, 0.980 4060-4221 (374-535) 99% -> ...122... 0.982, 0.999 4344-4475 (536-669) 97% -> ...259... 0.994, 0.971 4735-4890 (670-825) 98% -> ...375... 0.995, 0.844 5266-5443 (826-1003) 98% -> ...88... 0.983, 0.923 5532-5967 (1004-1439) 100% -> ...88... 0.996, 0.998 6056-6372 (1440-1756) 99% -> ...121... 0.998, 0.947 6494-6783 (1757-2046) 98% -> ...258... 0.996, 0.999 7042-7167 (2047-2172) 100%

Example 11

[00152] The following is an alignment of the CsPRR37 mRNA to the LowRyder_PRR37 assembled PRR37 gene sequence along with the predicted protein sequence translation. The critical psREC and CCT domains are annotated with ~ symbols underneath the sequence. (See Figure 4) It is noted that a partial length protein is translated due to a splice site variant following the second coding sequence which is sufficient to create a Loss of Function (LOF) allele. It is further noted that this truncation occurs within the psREC domain which is required for protein protein interaction. It is further noted that this is the same LOF allele present in CCA_AF_PRR37. The CCT domain would otherwise be intact if the protein were spliced correctly. There is synonymous variation present in parts of the coding sequence.

56 >PRR37_CDS|XM_030632368.1:235-2406 PREDICTED: Cannabis sativa two- component response regulator-like PRR37 (LOCI 15705128), mRNA Paths (1):

Path 1: query 1..2172 (2172 bp) => genome LowRyder:2,363..6,992 (4630 bp) cDNA direction: sense

Genomic pos: LowRyder:2,363..6,992 (+ strand)

Number of exons: 9

Coverage: 100.0 (query length: 2172 bp)

Trimmed coverage: 100.0 (trimmed length: 2172 bp, trimmed region: 1..2172)

Percent identity: 99.8 (2166 matches, 5 mismatches, 0 indels, 1 unknowns)

Translation: 1..2172 (723 aa)

Amino acid changes:

Alignments:

Alignment for path 1 :

+LowRyder:2363-2735 (1-373) 100% -> ... 1166... 0.998, 0.980

+LowRyder:3902-4063 (374-535) 100% == ...118... 0.132, 0.998

+LowRyder :4182-4315 (536-669) 100% -> ...258... 0.994, 0.970

+LowRyder :4574-4729 (670-825) 100% -> ..360... 0.995, 0.844

+LowRyder:5090-5267 (826-1003) 100% -> ...89... 0.983, 0.923

+LowRyder:5357-5792

...88... 0.996, 0.990

+LowRyder:5881-6197 (1440-1756) 99% -> ...121... 0.998, 0.441

+LowRyder : 6319-6608 (1757-2046) 99% -> ...258... 0.996, 0.999

+LowRyder:6867-6992 (2047-2172) 100%

Example 12

[00153] The following is an alternative alignment of the CsPRR37 mRNA to the Finola_PRR37 de novo assembled PRR37 gene sequence along with the predicted protein sequence translation. The critical psREC and CCT domains are annotated with ~ symbols underneath the sequence. (See Figure 5) It is noted that a partial length protein is translated due to an indel (deletion) variant between positions 2669-2670 in the

57 Finola_PR37 nucleotide sequence causing a frameshift which creates an early stop codon leading to Loss of Function. It is further noted that this truncation occurs within the psREC domain which is required for protein protein interaction. It is further noted that in this alternative alignment, the translation starts at an ATG in the middle of the psREC domain, thereby missing the N-terminal portion of the protein containing part of the psREC domain, and leading to a truncated non-functional alternate peptide. The CCT domain alignment does not show variation in the coding sequences. There are synonymous and non-synonymous variants in the coding sequences outside of the psREC and CCT domains.

>PRR37_CDS|XM_030632368.1:235-2406 PREDICTED: Cannabis sativa two- component response regulator-like PRR37 (LOCI 15705128), mRNA Paths (1):

Path 1: query 1..2172 (2172 bp) => genome 2,521..7,167 (4647 bp) cDNA direction: sense

Genomic pos: 2,521..7,167 (+ strand)

Number of exons: 9

Coverage: 100.0 (query length: 2172 bp)

Trimmed coverage: 100.0 (trimmed length: 2172 bp, trimmed region: 1..2172)

Percent identity: 99.2 (2153 matches, 14 mismatches, 4 indels, 1 unknowns)

Non-intron gaps: 3 openings, 4 bases in cdna; 0 openings, 0 bases in genome Translation: 562..2172 (536 aa)

Amino acid changes: M57V [729], P136A [966]

Alignments:

Alignment for path 1 :

2521-2891 (1-373) 98% -> ...1168... 0.998, 0.980 4060-4221 (374-535) 99% -> ...122... 0.982, 0.999 4344-4475 (536-669) 97% -> ...259... 0.994, 0.971 4735-4890 (670-825) 98% -> ...375... 0.995, 0.844 5266-5443 (826-1003) 98% -> ...88... 0.983, 0.923 5532-5967 (1004-1439) 100% -> ...88... 0.996, 0.998 6056-6372 (1440-1756) 99% -> ...121... 0.998, 0.947

58 6494-6783 (1757-2046) 98% -> ...258... 0.996, 0.999 7042-7167 (2047-2172) 100%

[00154] The various methods and techniques described above provide a number of ways to carry out the application. Of course, it is to be understood that not necessarily all objectives or advantages described are achieved in accordance with any particular embodiment described herein. Thus, for example, those skilled in the art will recognize that the methods can be performed in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other objectives or advantages as taught or suggested herein. A variety of alternatives are mentioned herein. It is to be understood that some embodiments specifically include one, another, or several features, while others specifically exclude one, another, or several features, while still others mitigate a particular feature by including one, another, or several other features.

[00155] Furthermore, the skilled artisan will recognize the applicability of various features from different embodiments. Similarly, the various elements, features and steps discussed above, as well as other known equivalents for each such element, feature or step, can be employed in various combinations by one of ordinary skill in this art to perform methods in accordance with the principles described herein. Among the various elements, features, and steps some will be specifically included and others specifically excluded in diverse embodiments.

[00156] Although the application has been disclosed in the context of certain embodiments and examples, it will be understood by those skilled in the art that the embodiments of the application extend beyond the specifically disclosed embodiments to other alternative embodiments and/or uses and modifications and equivalents thereof.

[00157] In some embodiments, any numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the disclosure are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and any included claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters

59 should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the application are approximations, the numerical values set forth in the specific examples are usually reported as precisely as practicable.

[00158] In some embodiments, the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment of the application (especially in the context of certain claims) are construed to cover both the singular and the plural. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (for example, “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the application and does not pose a limitation on the scope of the application otherwise claimed. No language in the specification should be construed as indicating any non -cl aimed element essential to the practice of the application.

[00159] Variations on preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. It is contemplated that skilled artisans can employ such variations as appropriate, and the application can be practiced otherwise than specifically described herein. Accordingly, many embodiments of this application include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the application unless otherwise indicated herein or otherwise clearly contradicted by context.

[00160] All patents, patent applications, publications of patent applications, and other material, such as articles, books, specifications, publications, documents, things, and/or the like, referenced herein are hereby incorporated herein by this reference in their entirety for all purposes, excepting any prosecution file history associated with same, any of same that is inconsistent with or in conflict with the present document, or any of

60 same that may have a limiting effect as to the broadest scope of the claims now or later associated with the present document. By way of example, should there be any inconsistency or conflict between the description, definition, and/or the use of a term associated with any of the incorporated material and that associated with the present document, the description, definition, and/or the use of the term in the present document shall prevail.

[00161] In closing, it is to be understood that the embodiments of the application disclosed herein are illustrative of the principles of the embodiments of the application. Other modifications that can be employed can be within the scope of the application. Thus, by way of example, but not of limitation, alternative configurations of the embodiments of the application can be utilized in accordance with the teachings herein. Accordingly, embodiments of the present application are not limited to that precisely as shown and described.

61

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A modified Cannabis plant exhibiting a modulated day-length sensitivity phenotype and at least one Value Phenotype, wherein said modified Cannabis plant comprises at least one mutated pseudoresponse regulator 37 (CsPRR37) gene or a non functional variant thereof with a mutation.

2. The modified Cannabis plant according to claim 1, wherein said non functional variant has at least 75% sequence identity to said CsPRR37 or said CsPRR37 nucleotide sequence.

3. The modified Cannabis plant according to claim 1, wherein said mutation is introduced using mutagenesis, small interfering RNA (siRNA), microRNA (miRNA), artificial miRNA (amiRNA), DNA introgression, endonucleases or any combination thereof.

4. The modified Cannabis plant according to claim 1 , wherein said mutation is introduced using targeted genome modification.

5. The modified Cannabis plant according to claim 4, wherein said mutation is introduced using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR- associated (Cas) gene (CRISPR/Cas), Transcription activator-like effector nuclease (TALEN), Zinc Finger Nuclease (ZFN), meganuclease or any combination thereof.

6. The modified Cannabis plant according to claim 5, wherein said Cas gene is selected from the group consisting of Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9, CaslO, CastlOd, Casl2, Casl3, Casl4, CasX, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csnl, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Cpfl, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Cszl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966and any combination thereof.

7. The modified Cannabis plant according to claim 1, wherein the mutated CsPRR37 gene is a CRISPR/Cas9- induced heritable mutated allele.

62

8. The modified Cannabis plant according to claim 1, wherein said mutation is a missense mutation, nonsense mutation, insertion, deletion, indel, substitution or duplication.

9. The modified Cannabis plant of claim 8, wherein the insertion or the deletion produces a gene comprising a frameshift.

10. The modified Cannabis plant of claim 1, wherein said plant is homozygous for a CsPRR37 mutated gene.

11. The modified Cannabis plant of claim 1, wherein said plant is heterozygous for a CsPRR37 mutated gene.

12. The modified Cannabis plant according to claim 8, wherein said mutation is in the coding region of said allele, a mutation in the regulatory region of said allele, or an epigenetic factor.

13. The modified Cannabis plant according to claim 1, wherein said mutation is a silencing mutation, a knockdown mutation, a knockout mutation, a loss of function mutation or any combination thereof.

14. The modified Cannabis plant according to claim 1 wherein said mutation is generated in planta.

15. The modified Cannabis plant according to claim 1 wherein said mutation is generated in planta via introduction of a construct comprising a sequence corresponding to a gRNA sequence listed in Tables 1-2.

16. The modified Cannabis plant according claim 15 wherein said gRNA sequence comprises a 3 ’ NGG Protospacer Adjacent Motif (PAM).

17. The modified Cannabis plant according to claim 15 wherein said construct is introduced into the plant cells via Agrobacterium infiltration, virus-based plasmids for delivery of the genome editing molecules or mechanical insertion such as polyethylene glycol (PEG) mediated DNA transformation, electroporation or gene gun biolistics.

18. The modified Cannabis plant according to claim 1, wherein said plant has decreased expression levels of CsPRR37 gene.

19. The modified Cannabis plant according to claim 18, wherein the sequence of said expressed CsPRR37 gene is 50% identical to the native CsPRR37 gene or a non functional variant thereof.

63

20. The modified Cannabis plant according to claim 1, wherein the Value

Phenotype is selected from the group consisting of’ high THCA accumulation; specific cannabinoid ratio(s); a composition of terpenes and/or other aroma active or aromatic molecules; monoecy or dioecy (enable or prevent hermaphroditism); branchless or branched architectures with specific height to branch length ratios or total branch length; determinant growth; time to maturity; high flower to leaf ratios that enable pathogen resistance through improved airflow; high flower to leaf ratios that maximize light penetration and flower development in the vertical canopy space; a finished plant height that enables tractor farming inside high tunnels; a finished plant height and flower to leaf ratio that maximizes light penetration all the way to the ground but minimizes total plant height; trichome size; trichome density; advantageous flower structures for oil or flower production (flower diameter length, long or short intemodal spacing distance, flower-to-leaf determination ratio (leafiness of flower); metabolites that provide enhanced properties to finished oil products (oxidation resistance, color stability, cannabinoid and terpene stability); specific variants affecting cannabinoid or aromatic molecule biosynthetic pathways; modulators of the flowering time phenotype that increase or decrease maturation time; flower biomass yield and composition; flower crude oil yield and composition;

64 resistance to botrytis, powdery mildew, fusarium, pythium, cladosporium, altemaria, spider mites, broad mites, russet mites, aphids, nematodes, caterpillars, HLVd or any other Cannabis pathogen or pest of viral, bacterial, fungal, insect, or animal origin; propensity to host specific beneficial and/or endophytic microflora; heavy metal composition in tissues; specific petiole and leaf angles and lengths; and/or the like. 1. A Cannabis plant, plant part or plant cell according to claim 1 wherein said plant does not comprise a transgene. 2. A plant part, plant cell or plant seed of a plant according to claim 1. 3. A tissue culture of regenerable cells, protoplasts or callus obtained from the modified Cannabis plant according to claim 1. 4. A method for producing a modified Cannabis plant exhibiting a modified day- length sensitivity phenotype and at least one Value Phenotype compared with wild type Cannabis, said method comprises genetically modifying at least one Cannabis CsPRR gene. 5. A method for producing a modified Cannabis plant exhibiting a modified day- length sensitivity phenotype and at least one Value Phenotype compared with wild type Cannabis by targeted genome modification, said method comprises steps of genetically introducing a loss of function mutation in at least one Cannabis CsPRR gene. 6. An isolated nucleotide sequence having at least 75% sequence identity to a native CsPRR37 nucleotide coding sequence wherein the native CsPRR37 protein is not expressed. 7. An isolated amino acid sequence having at least 75% sequence similarity to a native CsPRR37 amino acid sequence wherein a protein having the amino acid sequence does not have the function of the native protein. 8. Use of any of the nucleotide sequences as set forth in Tables 1 and 2, and any combination thereof, for targeted genome modification of Cannabis CsPRR37 allele.

65