Genetic markers for aroma in cannabis and related methods

Abstract

Genetic markers for aroma in Cannabis are provided. In some embodiments, the genetic markers may be indicative of fruity aroma profiles. In some embodiments, the genetic markers may be single nucleotide polymorphisms (SNPs). Related methods for testing Cannabis plants for such genetic markers and methods for producing Cannabis plants with or without specific aroma profiles using the genetic markers are also provided.

Description

GENETIC MARKERS FOR AROMA IN CANNABIS AND RELATED METHODSCROSS REFERENCE TO RELATED APPLICATION

[0001] The present disclosure claims priority to U.S. Provisional No. 63 / 580,064, filed September 1 , 2023, the entire content of which is herein incorporated by reference.TECHNICAL FIELD

[0002] The present disclosure relates to genetic markers for plant traits. More particularly, the present disclosure relates to genetic markers for aroma in cannabis and related methods.BACKGROUND

[0003] Cannabis refers to plants of the Cannabis genus. The Cannabis genus is generally understood to comprise one species, Cannabis sativa L., although some botanical authorities also recognize Cannabis indica and Cannabis ruderalis. Cannabis plant varieties or cultivars can be categorized as “drug-type” (i.e. (i.e., contains appreciable levels of the psychoactive cannabinoid tetrahydrocannabinol (“THC")) or “non-drug” (e.g., “hemp”).

[0004] Dried plant material from drug-type Cannabis plants is regularly consumed in a variety of ways including smoking, vaporization, and ingestion of prepared edible extractions. For the consumption of dried flower through vaporization or smoking, the aroma of the flower can have a significant impact on consumer experience. Desirable aromas in Cannabis include: sweet (fruity, berry, sugary), sour (citrus, lactic, cheesy), chemical (bitter, sharp, dank, gas), and earthy (vegetal, nutty, floral, woody) aromas.

[0005] Aroma can be a complex trait in plants that is a combination of subjective and objective qualities. In Cannabis, there are numerous chemical metabolites that contribute to the plant’s aroma. Research to date has largely focusedon Cannabis terpenes; however, terpenes may not be responsible for some of the most desirable aromas in Cannabis such as fruity or cheesy scents.

[0006] Accordingly, there remains a need to overcome the shortcomings of the prior art.SUMMARY

[0007] Genetic markers for aroma in Cannabis are provided. In some embodiments, the genetic markers may be indicative of fruity aroma profiles. In some embodiments, the genetic markers may be single nucleotide polymorphisms (SNPs). Related methods for testing Cannabis plants for such genetic markers and methods for producing Cannabis plants with or without specific aroma profiles using the genetic markers are also provided.

[0008] In some embodiments, a method of identifying a Cannabis plant having a desired allele at a polymorphic site associated with a specific aroma profile is provided. The method can comprise providing nucleic acid from the Cannabis plant; and testing the nucleic acid to determine the presence or absence of a variant at a polymorphic site.

[0009] In one aspect, there is provided a method comprising: providing nucleic acid from a Cannabis plant; and testing the nucleic acid to determine the presence or absence of a variant at a polymorphic site, wherein the polymorphic site is one of the following single nucleotide polymorphisms (SNPs): NC_044375.1_5442458; NC_044372.1_2504083; NC_044374.1_32889222; NC_044375.1_5207089;NC_044375.1_5074714; SNC_044375.1_5104580; SNC_044375.1_5204439; SNC-044375.1—5321892; SNC_044375.1_5470023; NC_044377.1_74617793; NC-044374.1_54892770; or another polymorphic site in linkage disequilibrium therewith.

[0010] In some embodiments, the polymorphic site is the polymorphic site is NC-044375.1_5442458 and the variant is a G at position 101 of SEQ ID NO: 1 , or aC in the complement thereof, the polymorphic site is NC_044372.1_2504083 and the variant is an A at position 101 of SEQ ID NO: 2, or a T in the complement thereof, the polymorphic site is NC_044374.1_32889222 and the variant is a T at position 101 of SEQ ID NO: 3, or an A in the complement thereof, the polymorphic site is NC_044375.1_5207089 and the variant is an A at position 101 of SEQ ID NO: 4, or a T in the complement thereof, the polymorphic site is NC_044375.1_5074714 and the variant is an A at position 101 of SEQ ID NO: 5, or a T in the complement thereof, the polymorphic site is SNC_044375.1_5104580 and the variant is an A at position 100 of SEQ ID NO: 6, or an T in the complement thereof, the polymorphic site is SNC_044375.1_5204439 and the variant is a C at position 100 of SEQ ID NO: 7, or a G in the complement thereof, the polymorphic site is SNC_044375.1_5321892 and the variant is a C at position 100 of SEQ ID NO: 8, or an G in the complement thereof, the polymorphic site is SNC_044375.1_5470023 and the variant is a G at position 100 of SEQ ID NO: 9, or a C in the complement thereof, the polymorphic site is NC_044377.1_74617793 and the variant is a T at position 101 of SEQ ID NO: 10, or an A in the complement thereof, or the polymorphic site is NC_044374.1_54892770 and the variant is a G at position 101 of SEQ ID NO: 11 , or an C in the complement thereof.

[0011] In some embodiments, the presence of the variant indicates at least one of: a high ethyl ester concentration in at least a portion of the Cannabis plant; and a fruity aroma profile. In some embodiments, the presence of the variant indicates at least one of: a high hexyl butanoate concentration in at least a portion of the Cannabis plant.

[0012] In some embodiments, the nucleic acid is genomic DNA. In some embodiments, the testing comprises nucleic acid amplification.

[0013] In some embodiments, the testing can be performed using allelespecification amplification, allele-specific probe hybridization, allele-specific primer extension, sequencing, 5' nuclease digestion, molecular beacon assay, oligonucleotide ligation assay, size analysis, single-stranded conformationpolymorphism analysis, denaturing gradient gel electrophoresis (DGGE), or a combination thereof.

[0014] In another aspect, there is provided a method for producing a Cannabis plant with or without a specific aroma profile, the method comprising identifying a first parent plant having a desired allele at a polymorphic site associated with the specific aroma profile, crossing the first parent plant with a second parent plant to produce F1 progeny, and identifying a first F1 progeny plant that has the desired allele at the polymorphic site and / or that has or does not have the specific aroma profile.

[0015] In some embodiments, the polymorphic site can be one of the following single nucleotide polymorphisms (SNPs): NC_044375.1—5442458, NC-044372.1_2504083, NC_044374.1 _32889222, NC_ 044375.1— 5207089, NC-044375.1_5074714, SNC-044375.1—5104580, SNC— 044375.1— 5204439, SNC_044375.1_5321892, SNC-044375.1_5470023, NC_ 044377.1— 74617793, NC-044374.1_54892770, or another polymorphic site in linkage disequilibrium therewith.

[0016] In some embodiments, at least one of the first parent plant and the first F1 progeny plant are identified as having the desired allele using any embodiment of the method disclosed herein.

[0017] In some embodiments, the method further comprises: identifying a second F1 progeny plant with the desired allele and crossing the first F1 progeny plant and the second F1 progeny plant to produce F2 progeny; and identifying a first F2 progeny plant that has the desired allele at the polymorphic site and / or that has or does not have the specific aroma profile.

[0018] In some embodiments, the method further comprises: self-pollinating the first F1 progeny plant to produce F2 progeny; and identifying a first F2 progeny plant that has the desired allele at the polymorphic site and / or that has or does not have the specific aroma profile.

[0019] In some embodiments, the method further comprises: backcrossing the first F1 progeny plant with the first parent plant to produce F2 progeny; and identifying a first F2 progeny plant that has the desired allele at the polymorphic site and / or that has or does not have the specific aroma profile.

[0020] In another aspect, there is provided a method for producing a plant with or without a specific aroma profile, the method comprising identifying a first parent plant having a desired allele at a polymorphic site associated with the specific aroma profile, crossing the first parent plant with a second parent plant to product F1 progeny; crossing a first F1 progeny plant to produce F2 progeny, wherein the first F1 progeny plant is crossed to one of: a second F1 progeny plant, the first parent plant, the second parent plant, and itself, and identifying a first F2 progeny plant that has the desired allele at the polymorphic site and / or that has or does not have the specific aroma profile.

[0021] In some embodiments, the polymorphic site can be one of the following single nucleotide polymorphisms (SNPs): NC_044375.1_5442458, NC-044372.1_2504083, NC_044374.1_32889222, NC_044375.1_5207089,NC-044375.1_5074714, SNC_044375.1_5104580, SNC_044375.1_5204439,SNC-044375.1—5321892, SNC_044375.1_5470023, NC_044377.1_74617793, NC_044374.1_54892770, or another polymorphic site in linkage disequilibrium therewith.

[0022] In some embodiments, at least one of the first parent plant and the first F2 progeny plant are identified as having the desired allele using any embodiment of the method disclosed herein.

[0023] In another aspect, there is provided a method for producing a Cannabis plant, comprising crossing a Cannabis plant produced according to any embodiment of the methods disclosed herein to a second Cannabis plant having at least one other desired trait.

[0024] In another aspect, there is provided a Cannabis plant or a plant cell produced according to any embodiment of the methods disclosed herein. In some embodiments, the plant or plant cell is a seed or a seed cell. In another aspect, there is provided a Cannabis plant material obtained from the Cannabis plant or plant cell disclosed herein. In some embodiments, the cannabis plant material comprises dried flower. In another aspect, there is provided a Cannabis-derived product generated from the Cannabis plant material disclosed herein, wherein the Cannabis-derived product is one or more of an extract, a resin, a concentrate, an isolate, and an oil. In another aspect, there is provided a composition comprising the Cannabis-derived product disclosed herein, wherein the composition is in the form of a pharmaceutical product, a veterinary product, a natural health product, a dietary supplement, a food product, a beverage product, a cosmetic product, a topical product, a tincture, and / or a vaporizable product for use with a vaporizer.

[0025] In another aspect, there is provided a method for altering levels of a chemical marker in a Cannabis plant, plant cell, or plant tissue, the method comprising expressing, over-expressing, down-regulating, or silencing in theCannabis plant, plant cell, or plant tissue, one or more nucleic acid sequences in linkage disequilibrium with one of the following single nucleotide polymorphisms(SNPs): NC_044375.1_5442458, NC_044372.1_2504083,NC_044374.1_32889222, NC_044375.1_5207089, NC_044375.1_5074714,SNC-044375.1_5104580, SNC_044375.1_5204439, SNC_044375.1_5321892, SNC-044375.1_5470023, NC_044377.1_74617793, NC_044374.1_54892770, wherein the expression, over-expression, down-regulation, or silencing is in comparison to a similar Cannabis plant, plant cell, or plant tissue grown under similar conditions but without the expressing, over-expressing, down-regulating, or silencing of the nucleic acid sequence.

[0026] In another aspect, there is provided a method for altering levels of a chemical marker in a Cannabis plant, plant cell, or plant tissue, comprising mutating one or more genes in the Cannabis plant, plant cell, or plant tissue, and using one ofthe following single nucleotide polymorphisms (SNPs): NC_044375.1_5442458,NC_044372.1_2504083, NC_044374.1_32889222, NC-044375.1_5207089,NC_044375.1—5074714, SNC_044375.1_5104580, SNC— 044375.1— 5204439,SNC_044375.1_5321892, SNC-044375.1_5470023, NC_ 044377.1— 74617793,NC-044374.1_54892770, or another polymorphic site in linkage disequilibrium therewith to select for Cannabis plants, plant cells, or plant tissues containing a mutant or variant of a gene associated with altered expression of the chemical marker.

[0027] Other aspects and features of the present disclosure will become apparent, to those ordinarily skilled in the art, upon review of the following description of specific embodiments of the disclosure.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Some aspects of the disclosure will now be described in greater detail with reference to the accompanying drawings.

[0029] Figure 1 is a schematic of Cannabis aroma profiles, according to some embodiment.

[0030] Figure 2 is a flowchart of an example method, according to some embodiments.

[0031] Figures 3A is a flowchart of an example method for producing a Cannabis plant with a desired aroma profile, according to some embodiments.

[0032] Figure 3B is a flowchart of another example method for producing a Cannabis plant with a desired aroma profile, according to some embodiments.

[0033] Figure 4 is a volcano plot representing significantly different metabolic feature concentrations between C. sativa flower with a complex aroma profile (upper left section) and a fruity aroma profile (upper right section). The x-axis represents the Log2 fold-change and the y-axis represents the logio p-value.

[0034] Figure 5 is a volcano plot representing significantly different metabolic feature concentrations between C. sativa flower with a complex aroma profile (upper left section) and a dank aroma profile (upper right section). The x-axis represents the Log2 fold-change and the y-axis represents the logio p-value.

[0035] Figure 6 is an example of a correlation matrix used to identify metabolic features associated with specific aroma profiles. Aroma profiles are shown sorted by most to least correlating variables for fruity aroma plants. Correlations use the Pearson r- value.

[0036] Figure 7 is a graph showing ethyl butyrate concentrations over time during plant growth. The grey background represents long day (18h) growth conditions while the white background represents short day (12h) conditions used to induce flowering. The arrow labeled “A” denotes the time-point where a “fruity” aroma was first observed.

[0037] Figure 8 is a Manhattan plot of single nucleotide polymorphisms (SNPs) associated with ethyl ester concentrations among 465 C. sativa cultivars.

[0038] Figures 9A to 9C are box plots of ethyl ester concentrations between alleles of the SNPs NC_044372.1_2504083, NC_044374.1_32889222, and NC_044375.1_5442458, respectively.

[0039] Figure 10 is a Manhattan plot of SNPs associated with hexyl butanoate concentrations among 465 C. sativa cultivars.

[0040] Figure 11 is a box plot of hexyl butanoate concentrations between SNP NC_044375.1_5207089 alleles.

[0041] Figure 12 is a box plot of hexyl butanoate concentrations between SNP NC_044375.1_5074714 alleles.

[0042] Figure 13 is a Manhattan plot of SNPs from a genome wide association study (GWAS) identifying SNPS significantly associated with methyl anthranilate.

[0043] Figure 14 is FAMD analysis of F2 population segregating for fruity aroma.

[0044] Figure 15 is FAMD analysis of cannabis diversity (CanD) population.

[0045] Figure 16 is a set of boxplots depicting the concentration and distribution of chemical markers measured by genetic markers. Compound concentrations are the mean of sample peak height divided by anhydrous weight of sample flower. Y axis of plots B) and D) are shown in Iog10 scale for easier interpretation.

[0046] Figure 17 is a set of bar graphs depicting genetic markers associated with fruity aromatic compounds and the perceived aromas reported in a F2 population relative to marker allele.

[0047] Figure 18 is a set of boxplots depicting the concentration and distribution of methyl anthranilate measured by genetic markers in a Cannabis diversity (CanD) population.

[0048] Figure 19 is a set of bar graphs depicting genetic markers associated with methyl anthranilate concentration and the perceived aromas reported in a Cannabis diversity (CanD) population relative to marker allele.DEFINITIONS

[0049] As used herein and in the appended claims, the singular forms of “a”, “an” and “the" include plural referents unless the context clearly dictates otherwise.

[0050] “Allele” or “variant” as used herein refer to alternative nucleic acid sequences at a genetic locus, such as a polymorphic site. As Cannabis has a diploid genome, there are two copies of every genetic locus. A plant that is “homozygous” has the same allele at both copies of the locus, whereas a plant that is “heterozygous" has one allele at one copy of the locus and a different allele at the other copy.

[0051] “Aroma profile” as used herein refers to an overall olfactory impression provided by a Cannabis plant or any part thereof.

[0052] “Cannabis” as used herein is inclusive of all species and varieties falling within the genus Cannabis, whether Cannabis sativa, Cannabis indica, or Cannabis ruderalis, and whether or not the variety is “drug” (i.e. , contains appreciable levels of the psychoactive cannabinoid tetrahydrocannabinol, “THC") or “non-drug” (i.e., contains less than about 0.2% or 0.3% by dry weight of THC, e.g., “hemp”).

[0053] “Cultivar" and “variety” are used interchangeably herein to refer to a group of plants that is substantially distinct, stable, and uniform in its characteristics when propagated. It will be understood to a person skilled in the art that individual plants of a given variety or cultivar may be genetically similar but may not be genetically identical.

[0054] “Genetic marker” as used herein refers to a polymorphic site that is associated with a phenotype or trait such that inheritance of particular allele of the genetic marker is indicative of the presence or absence of the phenotype / trait.

[0055] “Genotype” as used herein refers to the nucleic acid sequence at one or more loci in an individual plant. The genotype of a SNP is the specific nucleotide at the SNP locus.

[0056] “Linkage" as used herein is refers to the co-inheritance of alleles at two polymorphic sites due to the proximity of the polymorphic sites on the same chromosome.

[0057] “Linkage disequilibrium” as used herein refers to a situation where two alleles of respective polymorphic sites segregate in a non-random manner i.e., have a recombination frequency of less than 50%.

[0058] “Nucleotide sequence”, “polynucleotide sequence”, “nucleic acid” or “nucleic acid molecule" as used herein refers to a DNA or RNA polymer which can besingle or double stranded and optionally contains synthetic, non-natural, or altered nucleotide bases capable of incorporation into the DNA or RNA polymer. For a given nucleotide sequence, the acronyms A, C, G, and T are used to refer to adenine, cytosine, guanine, and thymine, respectively. The “complement" of a given DNA sequence is the sequence of the opposing strand based on nucleotide pairing rules (i.e., A pairs with T and C pairs with G).

[0059] “Plant material" as used herein refers to any portion or combination of portions of a Cannabis plant including but not limited to seeds, stems, stalks, leaves, flowers, roots, whether fresh or dry, or whether intact, cut, or comminuted.

[0060] “Polymorphism” or a “polymorphic site” as used herein refers to a position within a given genomic region at which variation exists within a population. A “polymorphism” or “polymorphic site” is the occurrence of two or more forms (alleles) at a position in the genome within a population, in such frequencies that the presence of the rarest of the forms cannot be explained by mutation alone. Polymorphic sites have at least two alleles, each occurring at a frequency of greater than 1 %. Polymorphic sites may occur in both coding regions and noncoding regions of genes or in intergenic regions. Polymorphic sites may involve a single nucleotide polymorphism or may involve an insertion or deletion (“indel”), or rearrangement.

[0061] “Primer” as used herein refers to an oligonucleotide capable of annealing to a nucleic acid template and providing a 3’ end that serves as a substrate for extension by a polymerase or reverse transcriptase. As used herein, “forward primer” and “reverse primer” are relative terms to refer to primers that anneal to opposite strands of the nucleic acid template. A “primer set” refers to a combination of a forward primer and a reverse primer for the same nucleic acid template.

[0062] “Polymerase chain reaction” or “PCR” as used herein refers to in vitro amplification of at least one target nucleic acid sequence by repeated cycles of denaturation and polymerase-mediated extension of primers. The PCR process is conducted via repeated heating and cooling steps referred to as “thermal cycling”. A“thermocycler” or “PCR machine” refers to an apparatus that performs thermal cycling.

[0063] A “single nucleotide polymorphism” (“SNP”) as used herein refers to a polymorphism that involves a variation at a single nucleotide position.DETAILED DESCRIPTION

[0064] Generally, the present disclosure provides genetic markers for aroma profiles in Cannabis and related methods for testing and breeding Cannabis plants using the genetic markers.

[0065] In some embodiments, the aroma profile may be generated by the Cannabis plant as a whole. In other embodiments, the aroma profile may be generated by one or more specific parts or tissues of the Cannabis plant. For example, the aroma profile may be generated by floral tissue of the Cannabis plant. The term “floral tissue” in this context refers to at least one of flowers, flower buds, trichome heads, trichomes, and / or combinations thereof.

[0066] In some embodiments, the aroma profile may be categorized based on its similarity to other known aromas. For example, the aroma profile may be categorized by one of the primary categories provided in Figure 1 , namely: “floral”, “greens”, “fruit” or “fruity”, “dank”, “sharp”, “gas”, or “earthy”. Optionally, the aroma profile may be further categorized by one of the secondary categories in Figure 1 . For example, an aroma profile categorized as “fruity” can be further categorized as “berries”, “tropical”, “banana”, and “grape”.

[0067] Different Cannabis plants may have different aroma profiles resulting from differences in concentrations of one or more chemical compounds that provide a specific aroma (hereafter referred to as “aroma compounds”). In some embodiments, one or more of the aroma compounds may be a metabolite. In some embodiments, the metabolite may be a secondary metabolite. In other embodiments,the aroma compound may be any other chemical compound present in a Cannabis plant.

[0068] Each aroma profile may therefore be the result of varying concentrations of aroma compounds that together contribute to the overall aroma profile. In some embodiments, one or more aroma compounds may have a greater impact on the overall aroma profile than others and thus concentrations of such compounds within a given Cannabis plant (or a specific part or tissue thereof) may be indicative of a specific aroma profile. Hereafter, an aroma compound, or combination of aroma compounds, that is indicative of a specific aroma profile will be referred to as a “chemical marker” of that aroma profile. The chemical marker may be present throughout the Cannabis plant or may be primarily (or only) present in a specific part or tissue such as the floral tissue.

[0069] In some embodiments, a chemical marker of a fruity aroma profile may comprise one or more ethyl esters, including ethyl isovalerate, ethyl butyrate, ethyl 3- methylcrotonoate, or a combination thereof. As discussed in the Examples below, concentrations of these chemical markers in Cannabis floral tissue were found to correlate with their respective aroma profiles.

[0070] It will be understood that multiple chemical compounds may contribute to a given aroma profile and, thus, a given aroma profile may have multiple chemical markers. The chemical markers disclosed herein are not exhaustive of the possible markers for a given chemical profile and embodiments are not limited to the specific chemical markers disclosed herein.

[0071] In some embodiments, a genetic marker may be identified for a given aroma profile. In some embodiments, the genetic marker may be identified for a given chemical marker and, thus, for a given aroma profile. In some embodiments, the genetic marker may comprise an SNP. In some embodiments, the genetic marker can be one of the SNPs in Table 1 below:TABLE 1

[0072] All sequences in Table 1 are shown in the 5’ to 3’ direction. The SNP locations use the chromosome numbering of the cs10 genome for the CBDRx cultivar, for example version GCF_900626175.2. However, it will be understood by a person skilled in the art that different Cannabis genome assemblies use different chromosome numbering systems and the same SNPs may be present in other reference genomes at different chromosome numbers.

[0073] SNP1 , SNP2, and SNP3 may each be used as a genetic marker for ethyl ester concentrations in at least a portion of a Cannabis plant. In some embodiments, these SNPs may be genetic markers for ethyl ester concentrations in the floral tissue of the Cannabis plant. The ethyl esters may include ethyl isovalerate, ethyl butyrate, and ethyl 3-methylcrotonoate. Ethyl esters can create a “fruity” aroma and, thus, these SNPs may be used as genetic markers of a “fruity” aroma profile in Cannabis.

[0074] Without being limited by theory, as discussed in the Examples below, SNP1 is at a locus that contains two genes annotated as shikimate O- hydroxycinnamoyl transferase genes (LOC115719745 and LOC115721021 ). Shikimate O-hydroxycinnamoyl transferase enzymes can catalyze the formation of acid esters from alcohol and CoA precursors and, therefore, may be involved in ethylester biosynthesis in Cannabis flowers. Variation at SNP1 may therefore affect expression of these genes, which in turn may affect the production of ethyl esters. Alternatively, SNP1 may be in linkage disequilibrium with one or more other genetic variants that affect expression of the LOC1 15719745 and LOC115721021 genes. SNP2 and SNP3 are also hypothesized to be associated with genes that contribute to ethyl ester biosynthesis or may be in linkage disequilibrium with one or more other genetic variants that affect expression of relevant genes.

[0075] In other embodiments, the genetic marker may be another polymorphic site (e.g., another SNP) in linkage disequilibrium with one of the SNPs in Table 1 .

[0076] The presence or absence of a particular variant at a given SNP may be indicative of the associated chemical marker and aroma profile. In some embodiments, the variant may be: a G at position 101 in SEQ ID NO: 1 or a C in the complement sequence thereof (SNP1 ); an A at position 101 in SEQ ID NO: 2 or a T in the complement sequence thereof (SNP2); or a T at position 101 in SEQ ID NO: 3 or an A in the complement sequence thereof (SNP3). In these embodiments, the presence of the variant may be indicative of high ethyl ester concentrations, including ethyl isovalerate, ethyl butyrate, and ethyl 3-methylcrotonoate. The presence of the variant may therefore also be indicative of a fruity aroma profile. Conversely, the absence of these variants (i.e., the presence of the reference nucleotide or a different nucleotide at the SNP locus) may be indicative of lower ethyl ester concentrations and the absence (or reduced strength or intensity) of the fruity aroma profile.

[0077] As discussed in more detail below, the genetic markers disclosed herein can be used for testing and selective breeding of Cannabis plants. The genetic markers may be used to select for a desired aroma profile and / or to select against an undesired aroma profile. In some embodiments, applications of the markers and methods disclosed herein can include marker assisted selection to improve rare fruity aromas in cannabis cultivars through breeding, masking of unappealing aromas with more desirable fruity aromas, creating novel fruity aromas by making crosses with parents having different classes of fruity compounds, for example in someembodiments, making a cross with a parent having high concentration of ethyl esters with a parent with high concentrations of methyl anthranilate.

[0078] Figure 2 is a flowchart of an example method 200 for testing Cannabis plants, according to some embodiments.

[0079] At block 202, nucleic acid from a Cannabis plant may be provided. The term “provide” in this context refers to extracting, obtaining, or otherwise acquiring the nucleic acid. In some embodiments, the nucleic acid may be genomic DNA. In other embodiments, the nucleic acid may be RNA, cDNA, or any other suitable nucleic acid that contains the genetic marker of interest.

[0080] In some embodiments, Cannabis plant material may be provided and the nucleic acid may be extracted therefrom. The plant material may comprise a whole Cannabis plant or may comprise a portion thereof, such as at least a portion of the floral tissue, leaves, stems, roots, or a combination thereof. In other embodiments, the Cannabis plant material may comprise one or more seeds. The nucleic acid may be extracted using any suitable extraction technique including, but not limited to, organic extraction, silica column-based extraction, magnetic separation, anion exchange technology, salting out, and / or cesium chloride density gradients. In other embodiments, the nucleic acid may be provided in a purified form, having been previously extracted from Cannabis plant material.

[0081] At block 204, the nucleic acid may be tested to determine the presence or absence of a variant at a polymorphic site. In some embodiments, the polymorphic site may be a SNP. In some embodiments, the SNP may be one of SNPs 1-3 in Table 1 and the variant may be the respective variant for that SNP as listed in Table 1 and discussed above. In other embodiments, the polymorphic site may be a polymorphic site (e.g., another SNP) in linkage disequilibrium with one of the SNPs in Table 1 .

[0082] The variant may be in one or both copies of the endogenous genomic DNA sequence. In other words, the Cannabis plant may be heterozygous or homozygous for the variant.

[0083] In some embodiments, testing the nucleic acid comprises nucleic acid amplification, e.g., amplification carried out by polymerase chain reaction (PCR). Nucleic acid amplification alone may detect the presence or absence of a given variant (e.g., using allele-specific primers) or may be done as a preliminary step for another analytical method.

[0084] The testing may performed using any suitable technique in the art for detecting a variant at a polymorphic site including, but not limited to, allele-specific amplification, allele-specific probe hybridization, allele-specific primer extension, sequencing, 5' nuclease digestion, molecular beacon assay, oligonucleotide ligation assay, size analysis, single-stranded conformation polymorphism analysis, denaturing gradient gel electrophoresis (DGGE), or a combination thereof.

[0085] In some embodiments, the nucleic acid may be tested to determine the presence or absence of respective variants at two or more polymorphic sites, such as the variants of two or more of the SNPs in Table 1 . The two or more polymorphic sites may be tested simultaneously or sequentially. In some embodiments, the two or more polymorphic sites may be tested in the same assay, for example, using multiplex PCR. In other embodiments, the two or more polymorphic sites may be tested in separate assays.

[0086] Thus, the method 200 may be used to determine the genotype of a Cannabis plant for a given genetic marker. The testing may also determine if the plant is heterozygous or homozygous for a specific variant. Such testing may be used to predict the aroma profile for a Cannabis plant. For example, seeds or young plants may be screened for a particular variant associated with a desired aroma profile in their floral tissue, without the requirement of growing the plants to the flowering stage. The method 200 may also be used to identify plants having (or not having) a particular variant to aid in selective breeding methods, such as the methods 300 and 350 of Figures 3A and 3B discussed below.

[0087] Figure 3 is a flowchart of an example method 300 for producing a Cannabis plant with or without a specific aroma profile. The aroma profile may be any of the profiles discussed above.

[0088] At block 302, a first parent plant having a desired allele at a polymorphic site associated with the specific aroma profile may be identified. In some embodiments, the polymorphic site may be one of the SNRs in Table 1. In embodiments in which it is desired to select for the specific aroma profile associated with the polymorphic site, the desired allele may be one of the variants listed in Table 1 and discussed above. In embodiments in which it is desired to select against that aroma profile, the desired allele may be the reference allele in the genomic context sequence of Table 1 or any other desired allele at a given polymorphic site. The first parent plant may be homozygous or heterozygous for the desired allele at the given polymorphic site.

[0089] In some embodiments, the first parent plant may be identified as having the desired allele by performing the steps of the method 200 as discussed above. In other embodiments, the first parent plant may be identified by any other suitable method. In yet other embodiments, the genotype of the first parent plant at the polymorphic site may have been previously determined and the step of identifying the first parent plant may comprise selecting a plant with the desired allele.

[0090] At block 304, the first parent plant may be crossed with a second parent plant to produce F1 progeny. The second parent plant may be a plant that has a different aroma profile from the first parent plant. For example, the second parent plant may be a plant of a different cultivar or variety than the first parent plant. The second parent plant may be selected based on one or more other desired traits (e.g. , autoflowering, desired height, early or late maturity, disease resistance, cannabinoid and / or terpene content, etc.). In some embodiments, the second parent plant may be identified as not having the desired allele at the given polymorphic site. In other embodiments, the genotype of the second parent plant at the polymorphic site may not be known.

[0091] The first parent plant may be female and the second parent plant may be male (or vice versa) and the first and second parent plants may be crossed by pollinating the female plant with pollen from the male plant using any known crosspollination technique. The F1 seed may then be collected, germinated and grown to produce one or more F1 progeny plants. The F1 progeny plants may be grown to a suitable size and state of maturity prior to the steps of block 306 below.

[0092] At block 306, a first F1 progeny plant may be identified that has the desired allele at the polymorphic site. The first F1 progeny plant may be identified as having the desired allele by performing the steps of the method 200 or by any other suitable method. In some embodiments, a plurality of F1 progeny plants may be screened to identify one or more plants that have the desired allele. The proportion of F1 progeny plants having the desired allele will be based on whetherthe first parent plant is homozygous or heterozygous for the desired allele. In embodiments in which the first parent plant is homozygous, all of the F1 progeny plants are expected to have the desired allele. In embodiments in which the first parent plant is heterozygous for the desired allele, 50% of the F1 progeny plants are expected to have the desired allele. In either case, the F1 progeny plants with the desired allele would all be heterozygous.

[0093] Alternatively, or additionally, the first F1 progeny plant may be identified as having or not having the specific aroma profile, in embodiments in which a single copy of the desired allele is sufficient to determine the aroma profile. In these embodiments, the F1 progeny plant(s) may be grown and allowed to flower to assess the floral tissue for the specific aroma profile. In some embodiments, the first F1 progeny plant may be identified based on its aroma profile, followed by testing to confirm that the F1 progeny plant has the desired allele (or vice versa).

[0094] The method 300 may further comprise using the first F1 progeny plant to produce one or more F2 progeny plants and identifying at least one F2 progeny plant that has the desired allele at the polymorphic site and / or that has (or does not have) the specific aroma profile. The F2 progeny plants may be tested for thepresence of the desired allele using the method 200 or any other suitable method. The F2 progeny plants may be heterozygous or homozygous for the desired allele at the polymorphic site. In some embodiments, one or more homozygous plants may be selected for further breeding and / or for clonal propagation.

[0095] Various breeding techniques may be used to produce the F2 progeny. In some embodiments, the F2 progeny may be produced by identifying a second F1 progeny plant that has the desired allele and / or has or does not have the specific aroma profile and crossing the first F1 progeny plant with the second F1 progeny plant (“sib-crossing").

[0096] In other embodiments, the F2 progeny may be produced by selfpollinating (“selfing”) the first F1 progeny plant. Cannabis plants are typically dioecious, with separate male and female plants. However, selfing can be accomplished by artificially reversing the sex of flowers from female to male (or vice versa) on some branches. For example, silver thiosulfate treatment may be used to masculinize some branches of a female plant to provide male flowers with pollen that can be used to pollinate female flowers of the same plant. In other embodiments, any other suitable technique may be used to reverse the sex of flowers on one or more branches of a Cannabis plant. In other embodiments, the F2 progeny may be selfed using any other suitable method.

[0097] In yet other embodiments, the F2 progeny may be produced by backcrossing the first F1 progeny plant to the first parent plant or the second parent plant. Backcrossing may utilize the same first or second parent plant as was used to produce the F1 progeny or a genetic clone or another plant of the same cultivar. In some embodiments, several generations of backcrossing to the second parent may be done in order to produce Cannabis plants that are genetically similar or substantially genetically identical to the second parent plant but with the addition of the desired allele from the first parent plant. In this manner, a desired aroma profile from one Cannabis cultivar can be introduced into another Cannabis cultivar or an undesired aroma profile can be removed from a given Cannabis cultivar.

[0098] In some embodiments, a combination of the breeding techniques discussed above may be used. As one example, the first F1 progeny plant may be backcrossed to the first or second parent plant to produce BC1 (backcross 1 ) progeny, followed by selfing of one of the BC1 plants or sib-crossing of two BC1 plants with one another.

[0099] Although the breeding steps in method 300 above refer to a “first" F1 progeny plant, it will be understood that this is for simplicity only and any number of plants may be crossed to produce additional progeny.

[0100] Figure 3B is a flowchart of an alternative method 350 for producing a Cannabis plant with or without a specific aroma profile.

[0101] At block 352, a first parent plant having a desired allele at a polymorphic site associated with the specific aroma profile may be identified. At block 354, the first parent plant may be crossed with a second parent plant to produce F1 progeny. The steps at blocks 352 and 354 may be similar to the steps of blocks 302 and 304 of the method 300 as described above.

[0102] At block 356, a first F1 progeny plant is crossed to produce F2 progeny. Unlike the method 300, the first F1 progeny plant may not be identified as having the desired allele at the polymorphic site prior to breeding. To produce the F2 progeny, the first F1 progeny plant may be crossed with one of: a second F1 progeny plant (i.e., sib-crossing), the first parent plant or the second parent plant (i.e., backcrossing), or itself (i.e., selfing) to produce F2 progeny.

[0103] At block 358, a first F2 progeny plant is identified that has the desired allele and / or that has (or does not have) the specific aroma profile. The steps at block 358 may be similar to the steps at block 306 of the method 300 as described above.

[0104] The method 350 may be used, for example, in circumstances where the first parent plant is homozygous for the desired allele and thus all F1 progeny are assumed to be heterozygous for the desired allele. In these embodiments, genetictesting may only be performed on the F2 progeny to identify F2 progeny that are homozygous for the desired allele. Alternatively, or additionally, the F2 progeny may be grown to a stage at which their aroma profile can be assessed (e.g., flowering) to identify F2 plants with or without the specific aroma profile. In some embodiments, plants identified as being homozygous (and / or plants identified as having or not having the specific aroma profile) may be selected for further breeding and / or for clonal propagation.

[0105] Also provided herein is a method comprising crossing a Cannabis plant produced by any embodiment of the methods 300 or 350 described above with a second Cannabis plant having at least one other desired trait. The Cannabis plant may be an F1 plant, F2 plant, or any subsequent generation of plants produced by backcrossing, selfing, sib-crossing or any other suitable breeding technique. The other desired trait may be another aroma profile (or absence of another aroma profile) or any other desired trait including, but not limited to, autoflowering, desired height, early or late maturity, disease resistance, cannabinoid and / or terpene content, etc. The F1 progeny from the crossing of the Cannabis plant and the second Cannabis plant may then be selfed, back-crossed, sib-crossed, or bred in any other suitable manner to produce F2 progeny and subsequent generations of plants having both the desired aroma profile and the other desired trait.

[0106] Also provided herein is a Cannabis plant or a plant cell produced by any embodiment of the selective breeding methods described above, including F1 plants, F2 plants, and any subsequent generation of plants produced by backcrossing, selfing, sib-crossing or any other suitable breeding technique. In some embodiments, the Cannabis plant may be in the form of a seed and the plant cell may be a seed cell. In other embodiments, the Cannabis plant may be a plant at any other stage of development and the plant cell may be any cell in any plant tissue at any development stage.

[0107] Also provided herein is plant material obtained from the Cannabis plant or plant cell. In some embodiments, the plant material comprises dried flower. In otherembodiments, the plant material may comprise seeds produced by the Cannabis plant or plant cell.

[0108] Also provided herein is a Cannabis-derived product generated from the plant material. The term “Cannabis-derived product” in this context may refer to any substance comprising, or produced from, Cannabis plant material. In some embodiments, the Cannabis-derived product may comprise one or more of an extract, a resin, a concentrate, an isolate, and an oil. In some embodiments, the Cannabis- derived product may further comprise a carrier oil. In some embodiments, the carrier oil comprises a medium-chain triglyceride (MCT) oil (e.g., coconut oil, vegetable oil, olive oil, etc.).

[0109] Also provided herein is a composition comprising the Cannabis-derived product. In some embodiments, the composition may be in the form of a pharmaceutical product, a veterinary product, a natural health product, a dietary supplement, a food product, a beverage product, a cosmetic product, a topical product, a tincture, and / or a vaporizable product for use with a vaporizer.

[0110] In some embodiments, the composition may further comprise at least one pharmaceutically and / or nutritionally acceptable excipient. Non-limiting examples of suitable excipients include fillers, binders, carriers, diluents, stabilizers, lubricants, glidants, coloring agents, flavoring agents, coatings, disintegrants, preservatives, sorbents, sweeteners and any other pharmaceutically or nutritionally acceptable excipient. In other embodiments, the composition may further comprise any other ingredient or combination of ingredients.

[0111] Methods are also provided for altering levels of a chemical marker in a Cannabis plant, plant cell, or plant tissue. The chemical marker may be any of the chemical markers discussed above, including one or more ethyl esters and / or hexyl butyrate. In other embodiments, the chemical marker may be any other suitable chemical marker. Altering levels of the chemical marker may alter the aroma profile.

[0112] In some embodiments, the method may comprise expressing or overexpressing in the Cannabis plant, plant cell, or plant tissue, one or more nucleic acid sequences in linkage disequilibrium with one of the following single nucleotide polymorphisms (SNPs): NC_044375.1_5442458; NC_044372.1_2504083;NC_044374.1_32889222, wherein the expression or over-expression is in comparison to a similar Cannabis plant, plant cell, or plant tissue grown under similar conditions but without the expressing or over-expressing of the nucleic acid sequence. The nucleic acid sequence may be one of the candidate genes associated with the SNPs disclosed herein or any other suitable sequence. In some embodiments, the nucleic acid sequence is expressed or over-expressed by introduction of an expression construct carrying the nucleic acid sequence. In other embodiments, the nucleic acid sequence may be expressed or over-expressed by any suitable means. Expression or over-expression of a given nucleic acid sequence may increase levels of the associated chemical marker, thereby producing (or increasing the intensity of) a specific aroma profile.

[0113] In other embodiments, the method may comprise down -regulating or silencing in the Cannabis plant, plant cell, or plant tissue, one or more nucleic acid sequences in linkage disequilibrium with one of the following single nucleotide polymorphisms (SNPs): NC_044375.1_5442458; NC_044372.1_2504083;NC_044374.1_32889222, wherein the down-regulating or silencing is in comparison to a similar Cannabis plant, plant cell, or plant tissue grown under similar conditions but without the down -regulating or silencing of the nucleic acid sequence. The nucleic acid sequence may be one of the candidate genes associated with the SNPs disclosed herein or any other suitable sequence. In some embodiments, the nucleic acid sequence may be down -regulated or silenced using RNA interference (RNAi) techniques. In other embodiments, the nucleic acid sequence may be down-regulated or silenced using any other suitable means. Down-regulation or silencing of a given nucleic acid sequence may decrease levels of the associated chemical marker, thereby reducing or eliminating a specific aroma profile.

[0114] In other embodiments, the method may comprise mutating one or more genes in the Cannabis plant, plant cell, or plant tissue, and using one of the following single nucleotide polymorphisms (SNPs): NC_044375.1_5442458; NC_044372.1_2504083; NC_044374.1_32889222 to select for Cannabis plants, plant cells, or plant tissues containing a mutant or variant of a gene associated with altered expression of the chemical marker. The gene may be one of the candidate genes associated with the SNPs disclosed herein or any other suitable gene. Mutation may be used to increase or decrease levels of the associated chemical marker depending on the nature of the mutation.

[0115] Without any limitation to the foregoing, the genetic markers and methods are further described by way of the following examples. However, it is to be understood that these examples are for illustrative purposes only and should not be used to limit the scope of the present disclosure in any manner.EXAMPLESEXAMPLE 1 - Sensory categorization of C. sativa flower

[0116] Using a population of 465 genetically distinct Cannabis cultivars, each plant was grown in triplicate to the mature flower stage and flowers were harvested and dried. Dried flower samples from each cultivar were then assessed for their aroma using the primary aroma categories presented in Figure 1 (i.e., floral, greens, fruit, dank, sharp, gas, or earth).

[0117] Each cultivar was assigned a primary aroma category by an untrained sensory panel consistent of 4-7 individuals. After each panelist assigned a sensory category to the individual cultivar, all answers were compiled and a score for each aroma category was assigned, based on the percentage of aroma category agreement among panelists. For example, if two panelists classified the cultivar as “sharp” and three panelists classified the cultivar as “greens”, the cultivar was assigned a score of 40 for sharp and 60 for greens. Any cultivar with a score above 50 in a primary aroma category was assigned an aroma group of that category. If thecultivar did not have a score of above 50 in any category it was assigned a “complex” grouping. The percentage distribution of aroma categories for screened genotypes is presented in Table 2 below:TABLE 2EXAMPLE 2 - Non-targeted metabolomic analysis of aroma molecules

[0118] Fresh flowers from the 465 unique cultivars were homogenized and subjected to a solvent extraction prior to chemical analysis. Sample data was collected using a non-targeted analytical method collecting mass spectral information following gas chromatography separation. Total ion chromatograms were deconvoluted and analytical features were assigned. The integrated data-matrix was normalized by sample weight and internal standard response to allow for relative quantitation between samples. Approximately 300,000 quantitative data-points were collected. The assembled data matrix was subjected to multivariate statistical analysis using the published webtool MetaboAnalyst™ 5.0 (https: / / www.metaboanalyst.ca / ). Analysis of Variance (ANOVA) tests revealed statistically important analytical features for fruity and cheesy aromas (see Figures 4 and 5, respectively). Pearson correlation analysis generated a correlation matrix that was curated to identify the most highly correlated analytical features with individual aroma categories (Figure 6).EXAMPLE 3 - Identification of chemical markers of specific aromas

[0119] Metabolic features (i.e., metabolites / chemical compounds) were identified based on spectral library searching, retention index comparison, and authentic chemical standards. Aroma characteristics of individual metabolites were assessed, where feasible, with a GC-MS-ODP (gas chromatography - mass spectroscopy - olfactory detection port) unit and directly assessed via the sensory port following chromatographic separation. Across 465 unique genotypes, several chemical compounds were found to be commonly associated with fruity aroma in cannabis. Several ethyl esters including ethyl isovalerate, ethyl 3-methylcrotonoate, and ethyl butyrate correlated with fruity aroma. The sensory databases from Flavornet and Goodscents (http: / / flavornet.org / , http: / / thegoodscentscompany.com / ) identify these esters has having fruity aromas. Authentic pure chemicals were also used to confirm the fruity aroma of these molecules.

[0120] In addition, time course trials were conducted measuring ethyl butyrate concentrations over time during C. sativa plant growth (Figure 7). Ethyl butyrate concentrations became detectable upon induction of flowering and a “fruity” aroma was first observed shortly thereafter. The results of these trials confirm that ethyl butyrate contributes to the fruity aroma profile.

[0121] The relative quantification of each of these molecules was used to compare to the genetic data discussed below.EXAMPLE 4 - Identification of genetic markers for the identified chemical markers of aroma

[0122] To identify genetic markers associated with specific chemical markers and their corresponding aroma profiles, Genome Wide Association Studies (GWAS) were conducted using Single Nucleotide Polymorphism (SNP) data from 457 of the 465 cultivars from which aroma and chemical profile data had been obtained. In total, the genomic data collection contained 6,991 ,320 SNP calls per cultivar across the Cannabis genome. GWAS was performed using both General Linear Models (GLM) and Mixed Linear Models (MLM) in TASSEL5 (Bradbury et al., 2007; Zhang et al.,2010). The first three principal components (PCs) were used to account for population structure (Q-matrix) within all models. For MLM models, the kinship matrix (K-matrix) was generated with TASSLE5 and calculated using centered Identity-By-State (IBS) (Endelman and Jannink, 2012).Example 4.1 - Identification of genetic markers for ethyl ester concentrations

[0123] Loci in three separate locations were identified that can be used as genetic markers of total ethyl ester concentrations. Loci were identified using Manhattan plots (Figure 8) and significance values. Total ethyl ester concentrations were determined by summing the area • gFW-1of ethyl isovalerate, ethyl butyrate, and ethyl 3-methylcrotonoate. GWAS (genome wide association studies) analysis of total ethyl ester concentrations identified a single major locus (NC_044375.1_5442458; SNP1 in Table 1 ), with two minor loci: NC_044372.1_ 2504083 (SNP2) and NC_044374.1_32889222 (SNP3). The relationship between variants of SNP1 , SNP2, and SNP3 and total ethyl ester concentrations are shown in Figures 9A-9C, respectively.

[0124] The major loci contains two genes annotated as shikimate O- hydroxycinnamoyl transferase genes (LOC1 15719745 and LOC115721021 ). The shikimate O-hydroxycinnamoyl transferase enzymes can catalyze the formation of acid esters from alcohol and CoA precursors and are a reasonable candidate as a biosynthetic enzyme for ethyl ester formation (Bizzio et aL, 2022).

[0125] For a different chemical marker, Figure 10 is a Manhattan plot which depicts SNPs associated with hexyl butanoate concentrations among 465 C. sativa cultivars. Figure 1 1 is a box plot of hexyl butanoate concentrations between SNP NC-044375.1_5207089 alleles and Figure 12 is a box plot of hexyl butanoate concentrations between SNP NC_044375.1_5074714 alleles.EXAMPLE 5 - Validation of genetic and chemical markers

[0126] Three plant populations of different composition and origin were chosen for validation of genetic markers: A) a large cannabis diversity population (CanD, n = 465) composed of elite production cultivars, landraces, and exotic lines, B) a segregating population (n=84) obtained by selfing a genotype with fruity aroma marker association with fruity compounds and fruity aroma sensory attributes in this population demonstrate its use and applicability in cannabis breeding, and C) a diversity population (n=79), independently generated from the CanD population use to identify the aroma markers. Marker association in this independent diversity population can support the applicability of the markers in a broad range of genetic backgrounds in Cannabis.Example 5.1 - Chemical analysis

[0127] Flower samples from cannabis plants of different populations can be subjected to a chemical analysis.

[0128] Liquid metabolite extraction: A 2 g sample of cannabis can be transferred into a sterile 50mL Falcon tube. Each sample can be extracted with 40 mL of a methanokchloroform (9:1 ) solvent mixture, along with steel beads and homogenized in a GenoGrinder™ tissue homogenizer for 3 min at 1500 rpm. After homogenization, 1 mL of the extract can be transferred to a microcentrifuge tube and centrifuged at 15,000 rpm for 3min to remove any particulate material. The clear extract can be used to make a 5x dilution with methanol and the internal standard 4- Methyl-2-pentanol (1 ug * mL'1) was added before analysis.

[0129] GC instruments: Head space Solid Phase Microextraction (HS-SPME) analysis can be conducted on an Agilent™ 5977B GC / MSD with a 7890 GC system using Selected Ion Monitoring (SIM). The Multiple Reaction Monitoring (MRM) analysis can be conducted on an Agilent™ 7010B GC / MS / MS with a 7890B GC system.

[0130] GC methods: GC-MS-HS-SPME: Approximately 1 g of dry flower sample can be added to a 20 mL glass headspace vial. The sample can be incubatedat 60°C for 5 minutes before SPME absorption. The SPME fibre (DVB / C- WR / PDMS / 10) can be conditioned for 5min at 250°C before being inserted into the vial, where absorption can take place for 12min. The sample can be desorbed in the front inlet for 2 min, and the fibre conditioned for a further 1min before returning to the home position. Separation can be conducted using an Agilent DB-Wax column, with helium as a carrier gas. A split flow of 10:1 can be used. The temperature gradient can start at 35°C and can proceed at a flow of 10 mL*min-1 until a final temperature of 200°C is reached. The front inlet temperature can be maintained at 250°C. Results can be evaluated using Single Ion Monitoring (SIM), with peak height as the response, normalized by the anhydrous sample weight (Table 3).TABLE 3: Selected Ion Monitoring (SIM) ions and retention times of selected cannabis aroma compound

[0131] GC-MS / MS: Separation of the liquid metabolite sample can be conducted using an Agilent™ HP-5ms Ultra Inert column, with helium as a carrier gas. A split flow of 15:1 can be used, with an injection volume of 1 uL. The temperature gradient can start with an isocratic hold at 40°C and can proceed at a flow of 10mL*min-1with subsequent isocratic holds at 90°C for 3min,100°C for 3 min and 150°C for 2min, until a final temperature of 300°C is reached. The front inlet temperature can be maintained at 250°C. Analysis can be conducted using MultipleReaction Monitoring (MRM). Results can be quantitated using a 9-point calibration curve with a mixture of analytical standards (Table 4) ranging from 0.04ug / mL to 5ug / mL and normalized to a w / w% result.TABLE 4: Multiple Reaction Monitoring (MRM) transitions and retention times of selected cannabis aroma compoundsExample 5.2 - Sensory analysis

[0132] Aroma sensory analysis can be performed by an untrained panel of people following the Breeder Sensory test guidelines provided by Hagenguth et al., (2022). A cannabis aroma lexicon (Table 5) can be developed and assessments canbe performed with 3.5 g of dry flower stored in plastic containers used to sell cannabis. Assessors can open the containers, sniff the headspace for two to three seconds and qualify the aroma of the sample according to the categories in Table 5. Assessors can assign only one aroma category for each sample. The final aroma category of a sample can be assigned by majority if 50% or more of the assessors agreed. If there is no agreement between assessors in the aroma of a sample, a “complex" aroma category can be assigned. Comments on secondary aromas, if present, can be captured separately as free text for each sample.TABLE 5: Aroma lexicon for cannabis dry flowerAroma 1aryDescriptionCategoriesFruity Berries (blueberry, cherry, strawberry), Tropical Fruits (guava, passionfruit), Tree Fruits (apples, pears, peaches), and Bananas. Does not include Citrus fruits.Sharp Citrus Fruits (orange, lemon, lime, grapefruit), Pine / Evergreens,Aromatic Herbs (thyme, basil, rosemary, sage, oregano)Floral Lavender, Rose, and Potpourri.Greens Leafy (fresh cut grass, leaves), Melon (honeydew, watermelon).Dank Sweat / Body Odour, Lactic aromas (cheese, sour cream), Skunk, and Sulfurous aromas (garlic, rotten eggs).Earthy Soil, Fungus, Wood / Hay, and Baking (yeasts, breads, cakes).Gas Gasoline / Diesel, Solvents, and Cleaners.

[0133] Factor Analysis of Mixed Data (FAMD) can be conducted on both an F2 population segregating for fruity aroma and a diversity population using chemistry and sensory data. Analysis can be conducted in R, using the FactoMineR™ package (Le et al., 2008). Continuous variables can be scaled to unit variance. Results of some embodiments of FAMD are discussed below.Example 5.3 - Results

[0134] In some embodiments, markers can be further validated using a variety of techniques. See Table 6 for some examples of markers that have underwent further validation.

[0135] Chemometrics and aroma sensory analysis - To validate the identified associations between concentrations of volatile compounds in cannabis dry flower and the aroma sensory attributes of the flower in previous GWAS aroma studies, factor analysis of mixed data (FAMD) can be performed in an F2 population segregating for fruity aroma and in an independent diversity population with a range of different aromas.

[0136] F2 Population segregating for fruity aroma - FAMD analysis in a F2 population can be performed using all detected compounds in GC-MS-HS-SPME in a non-targeted approach to simultaneously confirm the associations discovered in a CanD population and to potentially identify new compounds associated with aromas. In some embodiments, 204 aromatic compounds were identified in a headspace analysis.

[0137] Referring now to Figure 14, FAMD analysis of an F2 population segregating for fruity aroma is depicted. In Figure 14A, quantitative (aromatic compounds) and qualitative (aroma) variables are plotted to first two principal dimensions. Figure 14B shows a correlation circle plot with quantitative variables on principal dimensions 1 and 2. Figure 14C depicts aroma categories plotted on principal dimensions 1 and 2. The “contrib" scale depicts the level of contribution each aroma category has to the principal dimensions. Figure 14D is set up the same as Figure 15C, but with individual genotypes plotted with their corresponding reported aroma.

[0138] Figure 14 shows principal dimensions 1 and 2 accounting for 31.3% and 14.0% of total variance observed respectively. The FAMD variables graph in Figure 14a shows two main variable clusters. The first group includes hexyl esters,octyl acetate, methyl anthranilate, and terpenes myrcene, limonene, linalool, and beta-pinene among other compounds. This variable group is projected higher on principal dimension 1 compared to 2, with limonene and beta-pinene having the highest quality of representation, measured by cos2, indicating that these two variables are well projected onto dimension 1 . The second cluster contains variables for ethyl esters, terpenes humulene and caryophyllene, and the aroma categorical variable. Ethyl esters ethyl isovalerate, ethyl isobutyrate, and ethyl 3-methylcrotonate show the highest quality of representation on dimension 2, as measured by the cos2. Cluster 2 projects higher on principal dimension 2 compared to 1.

[0139] The correlations of aromatic compounds measured within a F2 population are shown highlighted in red in Figure 14b. Variables found to cluster together in Figure 14a are positively correlated with each other in Figure 14b, with the exception of humulene and caryophyllene, which are negatively correlated to the ethyl esters along principal dimension 2. Hexyl esters and most terpenes are positively correlated to principal dimension 1 , while ethyl esters are positively correlated to principal dimension 2. The near 90° angle formed between these two vector groups indicates that the hexyl ester plus terpenes (group 1 ) and ethyl esters (group 2) are uncorrelated.

[0140] Figure 14c shows the projections of aroma categories on the first two principal dimensions. The fruity category scores high on both principal dimension 1 and 2. Contribution and cos2 metrics for the fruity category scored high for both dimensions, with dimension 2 being the highest. All other aroma categories were not well projected on the first two principal dimensions. These results are expected for an F2 population obtained by selfing a plant with strong fruity aroma. The positive projection of the fruity aroma category on principal dimension 1 indicates that the aromatic compounds highly correlated to that dimension (group 1) contribute to perceived fruity aroma. However, it is primarily the ethyl esters of group 2 that are contributing the most to fruity aroma, with the fruity category best represented by principal dimension 2, similarly to ethyl esters. Projecting each individual F2 plantson the same dimensions can show that those reported as having fruity aroma are concentrated in quadrant 1 (positive for both principal dimensions) with a higher score on principal dimension 2 than 1 (Figure 14d).

[0141] In summary, the results indicated that the aroma compounds identified in a GWAS analysis in our CanD diversity population can be associated with fruity aroma in an F2 population, with compounds being positively correlated to principal dimension 1 , along with the fruity aroma category. However, it is principal dimension 2 that best represents fruity aroma, with ethyl esters being highly positively correlated to this dimension in contrast to all other compounds investigated. Taken together, while aromatic compounds belonging to group 1 do contribute to fruity aroma, it is the likely that ethyl esters that are the main drivers of perceived fruity aroma in this population.

[0142] Independent diversity validation population - To further validate the results from a F2 population, a FAMD analysis can be performed on an independent diversity population composed of mostly unrelated cannabis genotypes with a large diversity of aromas. An extended list of volatile compounds with known aroma impact can be measured using GC-MS / MS using a targeted approach. FAMD analysis using 18 volatile compounds and 7 aroma categories (Figure 15a) can show two orthogonal vector groups of correlated variables (Figure 15b). The first group primarily can include hexyl esters and positively correlate to principal dimension 1 . The second group can be largely composed of ethyl ester compounds and strongly correlate to principal dimension 2. AS with the FAMD results in the F2 population, the fruity aroma category can score high on the principal dimension associated with ethyl esters (Figure 15c). Genotypes perceived as having fruity aromas can also score higher along principal dimension 2 (Figure 15d).

[0143] Figure 15 depicts a FAMD analysis of cannabis diversity (CanD) population. In Figure 15A, quantitative (aromatic compounds) and qualitative (aroma) variables are plotted to first two principal dimensions. Figure 15B is a correlation circle plot with quantitative variables on principal dimensions 1 and 2. In Figure 15C, aromacategories are plotted on principal dimensions 1 and 2. “Contrib” scale depicts the level of contribution each aroma category has to the principal dimensions. Figure 15D is set up the same as Figure 15C, but with individual genotypes plotted with their corresponding reported aroma.

[0144] The results observed in the independent diversity population support observations in the F2 population, where two main groups of correlated aromatic compounds were observed, one composed primarily of ethyl esters, and the other hexyl esters. Genotypes with fruity aromas in a diversity population can be largely correlated with having high amounts of ethyl esters, as can be observed in a F2 segregating population. However, while some genotypes with fruity aroma in a F2 population were associated with the hexyl ester group of aromatic compounds, this was not observed in the diversity panel, where genotypes with fruity aroma were associated with ethyl ester compounds only.

[0145] Ethyl and Hexyl Esters - Genetic markers can be developed to target SNRs in a genomic region found to be significantly associated with high amounts of ethyl 3-methyl crotonoate, ethyl butyrate, ethyl crotonoate, and hexyl butyrate, as determined from previous GWAS studies (for example, in chromosome 2). In some embodiments, seven markers can be developed to target a region of interest in a genome and validate their association to high levels of 4 ethyl esters and 2 hexyl esters known to contribute to fruity aromas and found within cannabis.

[0146] Referring now to Figure 16 shows the results of each marker’s allele call and mean aromatic compound concentration when genotyping a F2 population segregating for fruity aroma. All genetic markers can be found to be associated with aromatic compound concentration, with an A2 / A2 variant associated with high levels of each compound. Figure 16 depicts genetic markers associated with fruity compound concentrations measured in F2 population segregating for fruity aroma. Boxplots depict mean concentration and distribution for A) Hexyl butyrate, B) Ethyl Isobutyrate, C) Ethyl Butyrate, D) Ethyl Isovalerate, E) Ethyl 3-methylcrotonate, F) Hexyl Propionate measured by 7 genetic markers: MR278, MR283, MR289, MR298,MR301 , 1 HB, and 2HB. Compound concentrations are the mean of sample peak height divided by anhydrous weight of sample flower. A1 / A1 represents the low compound concentration homozygous allele, A1 / A2 the intermediate compound concentration heterozygous allele, and A2 / A2 the high compound concentration homozygous allele. The Y axis of plots B) and D) are shown in Iog10 scale for easer interpretation.

[0147] To determine if a genetic marker’s ability to predict the concentrations of fruity aromatics translated to perceived fruity aroma in cannabis flower, the number genotypes classified as having fruity aromas in the sensory analysis of a F2 population, and their respective allele call for all seven genetic markers can be examined (Figure 17). For example, markers MR283, MR289, MR298, MR301 , and 1 HB, genotypes called with the homozygous A2 allele (associated with higher aromatic compound concentrations) were more likely to be perceived as having fruity aromas compared to genotypes called with the homozygous A1 allele. Genotypes with heterozygous calls were found to be less likely perceived as fruity than the homozygous A2 allele, but more likely than the homozygous A1 allele. For markers MR278 and HB2, several plants homozygous A1 were perceived as fruity, however this was still less than the number of plants perceived as fruity genotyped as homozygous A2. Taken together, these results indicate that genetic markers associated with aromatic compound concentration can also predict the perceived fruity aroma in cannabis dry flower.

[0148] Methyl Anthranilate (MA) GWAS - Genome Wide Association Analysis can be performed using a concentration of MA in flower in a CanD diversity population and may not reveal a strong associations in a Cannabis genome, for example, see Figure 13. Given the absence of significant GWAS peaks for MA, a cannabis genome can be manually scanned for gene annotations related to know methyl anthranilate biosynthesis genes in other plants. A significant region of chromosome NC_044377 with a bases in the 75,000,000 region was suggestive of a methyl anthranilate biosynthesis related gene. Manual review of the region identified the genes:L0C1 15725687, which is annotated as benzoate carboxyl methyltransferase and LOC1 15724678, also annotated as a benzoate carboxyl methyltransferase. The region contains two genes annotated as methylxanthosine synthase-like. All four genes may contribute, in part, to the biosynthesis of methyl anthranilate, with gene variants responsible for variable concentrations in Cannabis. Within the same chromosomal region (74215869:75428547) the genetic marker SNC_044377.1_74617793 is presented in Figure 18A, which shows the relationship between allelic variants (for example in A1 and A2) and methyl anthranilate concentration. The same SNP can also appear to have a relationship with overall fruity aroma as show in Figure 19B.

[0149] Two genetic markers, MA1 and MA2, tracking methyl anthranilate abundance were validated on a cannabis diversity panel. In some cases, such as Figure 18 and Table 6, MA1 and MA2 can be referred to as 1 MA and 2MA respectively.

[0150] A GWAS analysis using a CanD diversity population can indicate that SNPs two genomic regions (Chr 5 and 6) were significantly associated methyl anthranilate concentration. Two marker assays developed to target each region were tested and shown to track methyl anthranilate concentrations. Marker MA1 showed the homozygous A1 allele associated with higher methyl anthranilate concentrations, the heterozygous A1 / A2 allele was associated with intermediate concentrations and to the homozygous A2 allele with low concentrations. The difference in methyl anthranilate concentration between C and T nucleotides in the MA1 marker can be significant with a p-value of <0.001. For marker 2MA, genotypes with the heterozygous A1 / A2 allele were associated with high methyl anthranilate concentrations, while the A2 / A2 homozygous allele was associated with low MA concentrations, and the homozygous A1 / A1 allele with an intermediate concentration.

[0151] Referring now to Figure 17, genetic markers associated with fruity aromatic compounds and the perceived aromas reported in a F2 population relative to marker allele. The A1 allele can be associated to low concentrations of fruitycompounds, while the A2 allele is associated with higher concentrations. Referring to Figure 18, methyl anthranilate marker results used to genotype Cannabis diversity population are shown for A) marker MA1 , and B) marker MA2. In some embodiments, an A1 allele tracks higher methyl anthranilate concentration, while A2 allele tracks lower concentrations.

[0152] Whether or not MA marker alleles could also predict the fruity aroma perceived in dry flower can be investigated. For marker MA1 , more genotypes can be perceived as having fruity aromas for plants genotyped as homozygous A1 allele compared to heterozygous and homozygous A2 plants. For the MA2 marker, most plants with fruity aroma were heterozygous A1 / A2 with its higher methyl anthranilate concentration. Referring to Figure 19, genetic markers associated with methyl anthranilate concentration and the perceived aromas reported in cannabis diversity population relative to marker allele. A) marker MA1 , and B) marker MA2.TABLE 6 Validated markers in F2 population and Diversity population

[0153] Although a few embodiments have been shown and described, it will be appreciated by those skilled in the art that various changes and modifications can be made to those skilled in the art that various changes and modifications can be made to these embodiments without changing or departing from their scope, intent or functionality. The terms and expressions used in the preceding specification have been used herein as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding equivalents of the features shown and described or portions thereof.References:The following documents are incorporated by reference herein:Plumb et al., 2022. The nose knowns: Aroma, but not THC mediates the subjective effects of smoked and vaporized cannabis flower. Psychoactives, 1 , 70-86.March et al., 2015. Structure-odour relationships reviewed in the postgenomic era. Flavour and Fragrance Journal. 30: 342-361Booth, J., Page, J., Bohlmann, J. 2017. Terpene synthases from Cannabis sativa. PLOS One. 12: e0173911Journal of Dairy Science Vol. 85, No. 7, 2002Song, J., Forney, C. 2008. Flavour volatile production and regulation in fruit. Canadian Journal of Plant Science. 88: 537-550Oswald et al., 2021 . Identification of a new family of prenylated volatile sulfur compounds in cannabis revealed by comprehensive two-dimensional gas chromatography. ACS Omega. 6: 31667-31676Oswald et al., 2023. Minor, Nonterpenoid Volatile Compounds Drive the Aroma Differences of Exotic. ACS Omega, doi: 10.1021 / acsomega.3c04496M. ESPINO-DIAZ et al.: Biochemistry of Apple Aroma, Food Technol. Biotechnol. 54 (4) 375-394 (2016)Moaz et al., 2018. Concealed ester formation and amino acid metabolism to volatile compounds in table grape (Vitis vinifera L.) berries. Plant Science. 274: 223-230Cannabis sativa genome assembly cs10 - NCBI - NLM (nih.gov)Bradbury, P. J., Zhang, Z., Kroon, D. E., Casstevens, T. M., Ramdoss, Y., & Buckler, E. S. (2007). TASSEL: Software for association mapping of complex traits in diverse samples. Bioinformatics, 23(19), 2633-2635. https: / / doi.org / 10.1093 / bioinformatics / btm308Endelman, J. B., & Jannink, J.-L. (2012). Shrinkage estimation of the realized relationship matrix. G3: Genes, Genomes, Genetics, 2(11), 1405-1413. https: / / doi.Org / 10.1534 / g3.112.004259Zhang, Z., Ersoz, E., Lai, C. Q., Todhunter, R. J., Tiwari, H. K., Gore, M. A., ... & Buckler, E. S. (2010). Mixed linear model approach adapted for genome-wide association studies. Nature genetics, 42(4), 355-360.Bizzio, L., Tieman, D., Munoz, P. 2022. Branched-chain volatiles in fruit: a molecular perspective. Frontiers in Plant Science. 12: 814138Hagenguth J., et al. 2022. Breeders ’ Sensory Test : A new tool for early selection in breeding for tomato ( Solanum lycopersicum ) flavour. Plant Breed 141 : 96-107Le, S., Josse, J., Husson, F. (2008) FactoMineR: An R Package for Multivariate Analysis. J Stat Softw 25: 1-18

Claims

CLAIMS:

1. A method of identifying a Cannabis plant having a desired allele at a polymorphic site associated with a specific aroma profile, the method comprising: providing nucleic acid from the Cannabis plant; and testing the nucleic acid to determine the presence or absence of a variant at a polymorphic site.

2. The method of claim 1 , wherein the polymorphic site is one of the following single nucleotide polymorphisms (SNPs): NC_044375.1_5442458; NC_044372.1_ 2504083; NC_044374.1_32889222; NC_044375.1_5207089; NC_044375.1_5074714; SNC_044375.1_5104580; SNC_044375.1_5204439;SNC-044375.1_5321892; SNC_044375.1_5470023; NC_044377.1_74617793; NC_044374.1_54892770; or another polymorphic site in linkage disequilibrium therewith.

3. The method of claim 2, wherein:- the polymorphic site is NC_044375.1_5442458 and the variant is a G at position 101 of SEQ ID NO: 1 , or a C in the complement thereof;- the polymorphic site is NC_044372.1_2504083 and the variant is an A at position 101 of SEQ ID NO: 2, or a T in the complement thereof;- the polymorphic site is NC_044374.1_32889222 and the variant is a T at position 101 of SEQ ID NO: 3, or an A in the complement thereof;- the polymorphic site is NC_044375.1_5207089 and the variant is an A at position 101 of SEQ ID NO: 4, or a T in the complement thereof;- the polymorphic site is NC_044375.1_5074714 and the variant is an A at position 101 of SEQ ID NO: 5, or a T in the complement thereof;- the polymorphic site is SNC_044375.1_5104580 and the variant is an A at position 100 of SEQ ID NO: 6, or an T in the complement thereof;- the polymorphic site is SNC_044375.1_5204439 and the variant is a C at position 100 of SEQ ID NO: 7, or a G in the complement thereof;- the polymorphic site is SNC_044375.1_5321892 and the variant is a C at position 100 of SEQ ID NO: 8, or an G in the complement thereof;- the polymorphic site is SNC_044375.1_5470023 and the variant is a G at position 100 of SEQ ID NO: 9, or a C in the complement thereof;- the polymorphic site is NC_044377.1_74617793 and the variant is a T at position 101 of SEQ ID NO: 10, or an A in the complement thereof; or- the polymorphic site is NC_044374.1_54892770 and the variant is a G at position 101 of SEQ ID NO: 11, or an C in the complement thereof.

4. The method of claim 3, wherein the presence of the variant indicates at least one of: a high ethyl ester concentration in at least a portion of the Cannabis plant; and a fruity aroma profile.

5. The method of any one of claims 1 to 4, wherein the nucleic acid is genomic DNA.

6. The method of any one of claims 1 to 5, wherein the testing comprises nucleic acid amplification.

7. The method of any one of claims 1 to 6, wherein the testing is performed using allele-specification amplification, allele-specific probe hybridization, allele-specific primer extension, sequencing, 5' nuclease digestion, molecular beacon assay, oligonucleotide ligation assay, size analysis, single-stranded conformation polymorphism analysis, denaturing gradient gel electrophoresis (DGGE), or a combination thereof.

8. A method for producing a Cannabis plant with or without a specific aroma profile, comprising:- identifying a first parent plant having a desired allele at a polymorphic site associated with the specific aroma profile;- crossing the first parent plant with a second parent plant to produce F1 progeny; and- identifying a first F1 progeny plant that has the desired allele at the polymorphic site and / or that has or does not have the specific aroma profile.

9. The method of claim 8 wherein the polymorphic site is one of the following single nucleotide polymorphisms (SNPs): NC_044375.1_5442458;NC_044372.1_2504083; NC_044374.1_32889222; NC_044375.1_5207089; NC_044375.1_5074714; SNC_044375.1_5104580; SNC_044375.1_5204439;SNC_044375.1—5321892; SNC_044375.1_5470023; NC_044377.1_74617793; NC_044374.1_54892770; or another polymorphic site in linkage disequilibrium therewith.

10. The method of either one of claim 8 or 9, wherein at least one of the first parent plant and the first F1 progeny plant are identified as having the desired allele using the method of any one of claims 1 to 7.

11. The method of any one of claims 8 to 10, further comprising: identifying a second F1 progeny plant with the desired allele and crossing the first F1 progeny plant and the second F1 progeny plant to produce F2 progeny; and identifying a first F2 progeny plant that has the desired allele at the polymorphic site and / or that has or does not have the specific aroma profile.

12. The method of any one of claims 8 to 10, further comprising: self-pollinating the first F1 progeny plant to produce F2 progeny; andidentifying a first F2 progeny plant that has the desired allele at the polymorphic site and / or that has or does not have the specific aroma profile.

13. The method of any one of claims 8 to 10, further comprising: backcrossing the first F1 progeny plant with the first parent plant to produce F2 progeny; and identifying a first F2 progeny plant that has the desired allele at the polymorphic site and / or that has or does not have the specific aroma profile.

14. A method for producing a plant with or without a specific aroma profile, comprising:- identifying a first parent plant having a desired allele at a polymorphic site associated with the specific aroma profile;- crossing the first parent plant with a second parent plant to produce F1 progeny;- crossing a first F1 progeny plant to produce F2 progeny, wherein the first F1 progeny plant is crossed to one of: a second F1 progeny plant, the first parent plant, the second parent plant, and itself; and- identifying a first F2 progeny plant that has the desired allele at the polymorphic site and / or that has or does not have the specific aroma profile.

15. The method of claim 14, wherein the polymorphic site is one of the following single nucleotide polymorphisms (SNPs): NC_044375.1_5442458;NC_044372.1_2504083; NC_044374.1_32889222; NC_044375.1_5207089; NC_044375.1_5074714; SNC_044375.1_5104580; SNC_044375.1_5204439;SNC-044375.1—5321892; SNC_044375.1_5470023; NC_044377.1_74617793; NC-044374.1_54892770; or another polymorphic site in linkage disequilibrium therewith.

16. The method of either one of claim 14 or 15, wherein at least one of the first parent plant and the first F2 progeny plant are identified as having the desired allele using the method of any one of claims 1 to 7.

17. A method for producing a Cannabis plant, comprising crossing a Cannabis plant produced according to the method of any one of claims 8 to 16 to a second Cannabis plant having at least one other desired trait.

18. A Cannabis plant, plant cell, seed, or seed cell produced according to the method of any one of claims 8 to 17.

19. A method for altering levels of a chemical marker in a Cannabis plant, plant cell, or plant tissue, comprising expressing, over-expressing, down-regulating, or silencing in the Cannabis plant, plant cell, or plant tissue, one or more nucleic acid sequences in linkage disequilibrium with one of the following single nucleotide polymorphisms (SNPs): NC_044375.1_5442458; NC_044372.1_2504083;NC-044374.1_32889222; NC_044375.1_5207089; NC_044375.1_5074714;SNC_044375.1_5104580; SNC_044375.1_5204439; SNC_044375.1_5321892; SNC-044375.1_5470023; NC_044377.1_74617793; NC_044374.1_54892770; wherein the expression, over-expression, down-regulation, or silencing is in comparison to a similar Cannabis plant, plant cell, or plant tissue grown under similar conditions but without the expressing, over-expressing, down-regulating, or silencing of the nucleic acid sequence.

20. A method for altering levels of a chemical marker in a Cannabis plant, plant cell, or plant tissue, comprising mutating one or more genes in the Cannabis plant, plant cell, or plant tissue, and using one of the following single nucleotide polymorphisms (SNPs): NC_044375.1_5442458; NC_044372.1_2504083;NC_044374.1_32889222; NC_044375.1_5207089; NC_044375.1_5074714;SNC_044375.1_5104580; SNC_044375.1_5204439; SNC_044375.1_5321892;SNC_044375.1—5470023; NC_044377.1_74617793; NC_044374.1_54892770; or another polymorphic site in linkage disequilibrium therewith to select for Cannabisplants, plant cells, or plant tissues containing a mutant or variant of a gene associated with altered expression of the chemical marker.

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