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

[0001] The present Application for Patent claims priority to Provisional Application No. 63/235,309 entitled “AUTO-FLOWERING CANNABIS WITHOUT UNDESIRABLE AGRONOMIC OR COMPOSITION TRAITS” filed August 20, 2021, which is hereby expressly incorporated by reference herein.

BACKGROUND

Field

[0002] The present invention provides autoflower Cannabis (Cannabis saliva) plants without the undesirable agronomic or composition traits generally associated with the autoflower trait. The present invention also provides methods of making such plants and methods of using such plants to produce additional autoflower Cannabis plants.

Background

[0003] “Autoflower” or “day-length neutral” Cannabis varieties are those that transition from a vegetative growth stage to a flowering stage based upon age, rather than length-of day. In contrast, most varieties of Cannabis in commercial use transition to the flowering stage based upon the plant’s perception of day length, such that the plants flower according to the seasonal variation in day length rather than the age of the plant.

[0004] The autoflower trait in Cannabis plants allows for a more consistent crop in terms of growth, yield, and harvest times as compared with day-length sensitive Cannabis varieties. In outdoor Cannabis cultivation, the availability of elite autoflower Cannabis varieties would expand the latitude and planting dates for productive Cannabis cultivation.

SUMMARY

[0005] Embodiments of the invention relate to a plant having an Autoflower (AF) Value Phenotype. Other embodiments of the invention relate to a method of plant breeding to develop plants with an Autoflower Value Phenotype. In some embodiments, the method can include: (a) providing a first parent plant, having a phenotype defined as a Value Phenotype, wherein the Value Phenotype comprises at least one trait of interest; (b) providing a second parent plant, having an autoflower phenotype; (c) crossing the first and second parent plants; (d) recovering progeny from the crossing step; (e) identifying one or more loci on chromosome 1 for which the first and second parent plants are polymorphic such that, for each such polymorphic locus, there exists a first-parent allele and a different second-parent allele; (f) screening individuals of the progeny for presence of (1) at least one autoflower allele (2a) presence of one or more first-parent alleles; and/or (2b) absence one or more second-parent alleles, wherein plants meeting criteria (1) and (2) are designed as desirable progeny; (g) selecting the desirable progeny; (h) conducting further breeding steps using the desirable progeny in one or more of subsequent crosses selected from any of (i) a self-cross of a desirable progeny individual; (ii) a cross between different desirable progeny individuals; (iii) a cross between a desirable progeny individual and the first parent plant; and/or (iv) a cross between a desirable progeny individual and a plant having the Value Phenotype that is not the first parent plant; (i) repeating steps f, g, and h until at least one plant having an Autoflower Value Phenotype is obtained. In some embodiments, step f employs one or more markers from Table 1.

[0006] In some embodiments, the method can include providing a first daylength-sensitive parent plant, having a phenotype defined as a Value Phenotype, wherein the Value Phenotype comprises at least one desirable agronomic or composition trait; providing a second parent plant, having an Autoflower phenotype or known or suspected of carrying an autoflower allele; genotyping the first and second parent plants with at least one marker from Table 1 and at least one marker from Table 2; identifying markers from Table 1 and Table 2 for which the first and second parent plants have different alleles (referred to as polymorphic markers); crossing the first and second parent plants; recovering progeny from the crossing step; screening the progeny for presence of (1) at least one autoflower allele using at least one polymorphic marker from Table 1 or a marker having comparable correlation with presence of an autoflower allele to those listed in Table 1 and (2) presence of at least one allele from the second parent using at least one polymorphic marker from Table 2 or a marker correlated to one or more markers from Table 2 or a marker having comparable correlation with presence of a Value Trait to those listed in Table 2; wherein plants meeting criteria (1) and (2) are designated as “Desirable Progeny;” selecting Desirable Progeny, wherein cells of said Desirable Progeny comprise at least one autoflower allele; conducting further breeding steps using the Desirable Progeny crossed with plants either (a) having the Value Phenotype (including but not limited to backcrossing to the Value Phenotype parent); or (b) crossed with other Desirable Progeny plants (including but not limited to self-crossing, sibling crossing, and crossing between different generations of Desirable Progeny plants); repeating the screening, selecting and conducting steps until at least one plant having an Autoflower Value Phenotype is obtained. In some embodiments, the progeny can be screened for presence of at least one autoflower allele using a marker having at least 60, 70, 80, 90, or about 100% correlation with presence of the autoflower allele.

[0007] In some embodiments, the autoflower allele can be an allele of (a) a mutated pseudoresponse regulator (PRR) protein gene; (b) a mutated binding partner of a PRR protein; (c) a mutated member of a PRR protein or DNA complex; or (d) a non-PRR gene involved in circadian rhythm or daylength sensing; and wherein the presence of the autoflower allele contributes to or results in an autoflower phenotype in combination with at least one value phenotype.

[0008] In some embodiments, 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 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.

[0009] The invention relates to employing natural breeding schemes assisted by molecular screening that permits to develop autoflower Cannabis plants that do not have the undesirable traits typically associated with autoflower in Cannabis plants.

[0010] Some embodiments of the invention relate to a plant or plant part produced by any of the methods described herein. Some embodiments of the invention relate to harvested material from the plant. Some embodiments of the invention relate to a product made from the harvested material. In some embodiments, the product can include an extract or concentrate. In some embodiments, the extract or concentrate can be selected from hash, live resin, cured resin, rosin, oil, shatter, wax, crumble, and any combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 is a schematic view of the pedigree used in an Example as described.

[0012] FIG. 2 is a schematic view of haplotype-blocks.

[0013] FIG. 3 shows results of a Quantitative Trait Locus (QTL) scan. DETAILED DESCRIPTION

[0014] Day-length neutral (autoflower) Cannabis varieties typically express less desirable phenotypic characteristics than day-length sensitive Cannabis varieties. For example, lower cannabinoid content, leafy inflorescences, and a limited aroma profile are commonly associated with day-length neutral varieties and tend to produce an inferior finished product. There is significant interest in breeding Cannabis to develop autoflower varieties that otherwise have desirable genotypes or phenotypes. Such breeding typically involves a cross of a first, day-length sensitive (photoperiod) parent plant having a desired phenotype (referred to herein as a “Value Phenotype”) with a second parent plant having an autoflower phenotype, whatever other traits it may have. For purposes of this disclosure, a plant expressing all of the desirable features of a given first parent, the Value Phenotype, but in an autoflower form, can be referred to as an “Autoflower Value Phenotype” plant.

[0015] Any plant with a “modulated day-length sensitivity phenotype” 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 of day-length sensitivity, or increase of day-length sensitivity.

[0016] In some embodiments, the Value Phenotype can include at least one trait selected from one or more of: high THCA accumulation; specific cannabinoid ratio(s); a composition of terpenes and/or other aroma active and 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; modulators of the flowering time phenotype that increase or decrease maturation time; flower biomass yield and composition; crude oil yield and composition; resistance to botrytis, powdery mildew, fusarium, pythium, cladosporium, alternaria, spider mites, broad mites, russet mites, aphids, nematodes, caterpillars, hop latent viroid (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.

[0017] The invention relates to one or more molecular markers and marker-assisted breeding of autoflower Cannabis plants. Detection of a marker and/or other linked marker can be used to identify, select, and/or produce plants having the autoflower phenotype and/or to eliminate plants from breeding programs or from planting that do not have the autoflower phenotype. The molecular marker can be utilized to indicate a plant’s possession of an autoflower allele well before the trait can morphologically or functionally manifest in the plant, and also when the plant is heterozygous for the autoflower allele and therefore would never display the autoflower phenotype. Specifically, in the context of breeding to develop Autoflower Value Phenotype varieties, a molecular marker correlating strongly with the autoflower trait can permit very early testing of progeny of a cross to identify those progeny that possess one or more autoflower alleles and discard those individuals that do not. This permits shifting the allele frequency of any plants remaining in the breeding pool, after such screening, to eliminate any plants that do not have at least one autoflower allele. In some embodiments of the invention, the analysis can be capable of distinguishing between individuals that are homozygous for the autoflower allele versus those that are heterozygous. In such situations, it can be advantageous to discard any heterozygous individuals.

[0018] The relatively consistent expression, in autoflower Cannabis plants, of multiple phenotypic traits that are less desirable than day-length sensitive plants suggests that at least some of these traits tend to co-segregate with the autoflower trait. The recent confirmation that the autoflower gene is located on chromosome 1, combined with the prior observations of various undesirable traits being common in autoflower plants suggests that genes affecting such traits may also be on chromosome 1.

[0019] The term “linkage drag” refers to the (usually undesirable) effects of genes linked to the genes or quantitative trait locus (QTL) a breeder wishes to introgress into a different genetic background. For examples, if a desirable QTL for trait X lies close to an undesirable gene affecting trait Y, breaking the linkage drag - that is, separating the “good” QTL from the “bad”, can be a very beneficial step in the breeding intended to achieve the introgression.

Linkage Drag

[0020] When plant breeding introduces a desired gene (“target gene”) from a donor parent to improve a cultivar for a specific trait, other genes closely linked to the target gene are also typically carried from the donor parent to the recipient cultivar. The undesired alleles of non-target genes from the donor parent, because of their close linkage with the target gene, often persist even after multiple backcrosses. The persistent non-target genes often reduce the fitness or desirability of the backcross progeny— a phenomenon known as linkage drag. Molecular makers offer a tool in which the amount of donor DNA can be monitored during each backcross generation, in order to reduce linkage drag.

[0021] It is well known that efforts to introgress the AF trait into other cultivars of Cannabis result in progeny that are not as phenotypically desirable as the original photoperiod parent. This can be attributed to linkage drag. Accordingly, the markers of the present invention can be used to monitor and minimize linkage drag as plants are crossed and backcrossed in efforts to introgress AF into Value Phenotype recipient plants.

[0022] Inheritance patterns from crosses of AF and photoperiod parents indicate that AF is determined by a recessive allele of a single gene. The markers of the present invention define a region of chromosome 1 in which this single AF locus resides. The region defined by these markers comprises 98 transcripts, according to Cannabis sativa cslO RefSeq assembly accession: GCF_900626175.2 (Assembly [Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2012 -2022 Jan 24. Accession No. GCF_900626175.2, cslO; Available from: www <dot> ncbi <dot> nlm <dot> nih <dot> gov <slash> assembly <slash> GCF_900626175.2). Disclosed herein are genes and positions within the segment of the chromosome defined by the markers. Thus, given that only one gene controls the AF trait, many or all of the other genes listed herein can contribute to linkage drag, to some degree. The invention includes a breeding protocol capable of introgressing the AF gene into a Value Phenotype recipient parent, while leaving most or all of the other genes listed in Table 1 behind, will result in an improved AF Value Phenotype cultivar. [0023] One object of the present invention is to identify and provide tools and techniques for breaking linkage drag between the desirable autoflower trait and the undesirable traits that are commonly found in autoflower plants. The more effectively the linkage drag can be broken, the more quickly and/or completely the autoflower trait can be introgressed into a more desirable genetic background of a plant having a Value Phenotype, by breeding autoflower plants with Value Phenotype plants. Another object of the present invention is to provide plants and plant parts that combine the AF phenotype with one or more Value Phenotypes, resulting in Cannabis plants that can be cultivated and harvested more efficiently than day-length sensitive Cannabis plants, but that are substantially free of the undesirable traits commonly present in AF Cannabis plants.

[0024] Since the autoflower trait behaves as a simple recessive trait, then it would be predicted that Fl progeny of a cross of a Value Phenotype plant with an autoflower plant will be heterozygous for the autoflower trait and will therefore display no autoflower behavior. The Fl progeny will likely also show a reduction or elimination of the Value Phenotype. This is especially true in a situation in which the traits that characterize the Value Phenotype are many and have complex inheritance.

[0025] The F2 would be predicted to be 'A autoflower but with high variability of the traits of the Value Phenotype parent. Further breeding of autoflower progeny can eventually result in an Autoflower Value Phenotype population.

[0026] The Value Phenotype can include but need not be limited to at least one of the traits listed in paragraph [0008] in a desirable form.

[0027] The invention relates to molecular markers and marker-assisted breeding of autoflower Cannabis plants. The molecular markers can be utilized to indicate the presence of an autoflower allele in a plant's genome well before the trait can morphologically or functionally manifest in the plant, and also when the plant is heterozygous for the autoflower allele and therefore would not display the autoflower phenotype. Specifically, in the context of breeding to develop Autoflower Value Phenotype plants, a molecular marker correlating strongly with the autoflower trait can permit very early testing of progeny of a cross or a self-fertilization to identify those progeny that have one or more autoflower alleles and discard those individuals that have none. In some embodiments of the invention, the molecular markers can be utilized to distinguish between individuals that have two autoflower alleles and are therefore homozygous for the autoflower allele, and individuals that have one and only one autoflower allele and are therefore heterozygous for the autoflower allele. In such situations, it can be advantageous to discard any heterozygous individuals.

[0028] Markers within the autoflower locus can be selected from Table 1. See Example 8.

[0029] Several exemplary polymorphic markers located in regions of chromosome 1 suitable for reduction of linkage drag around the autoflower locus are provided in Table 2. AF and Value Phenotype parents in a given cross can be genotyped for these or other polymorphic markers on chromosome 1 to identify which loci are actually polymorphic as to the two parents in the cross. At any locus with an allele pair, if the AF parent has one allele and the Value Phenotype parent has the other allele in the pair, the alleles at such locus are then identified as a “Useful Allele Pair.” Progeny of a given cross can be screened for one or more Useful Allele Pairs to confirm individual progeny with desirable recombinations of chromosome 1. Such progeny would carry the autoflower allele of the AF parent but with a reduced number of other chromosome 1 alleles of the AF parent.

[0030] In some embodiments, the markers can be defined by their position on chromosome 1, in various ways, for example, in terms of physical position or genetic position. In some embodiments, the markers can be defined by their physical position on chromosome 1, expressed as the number of base pairs from the beginning of the chromosome to the marker (using CS10 as the reference genome). In some embodiments, the markers can be defined by their genetic position on chromosome 1, expressed as the number of centimorgans (a measure of recombination frequency) from the beginning of the chromosome to the marker. In other embodiments, a marker can be defined based upon its location within a given QTU

[0031] The present invention provides an improved Cannabis plant exhibiting at least one Value Phenotype combined with an autoflower phenotype. The present invention further provides methods for producing the aforementioned improved Cannabis plant using marker-assisted breeding to select for recombinations around the autoflower locus, permitting efficient introgression of the autoflower trait into a Value Phenotype genetic background.

[0032] To produce the improved Cannabis plant, undesirable traits associated with the autoflower phenotype are selected against. Selection against undesirable traits associated with autoflower controlled by genes or QTUs on chromosomes 2 to 9 or X/Y is rather straightforward due to the random reassortment of chromosomes during meiosis. However, selection against undesirable traits associated with autoflower controlled by genes or QTLs on chromosome 1 is more difficult as it requires to select for recombinations between the autoflower locus and genes or QTLs of traits such as traits listed in paragraph [0008], involved in the Value Phenotype located on either side of the autoflower locus on chromosome 1. The closer the genes or QTLs of traits involved in the Value Phenotype to the autoflower locus, the less frequent the recombinations between the autoflower locus and genes or QTLs of such traits.

[0033] Selection against undesirable traits associated with autoflower controlled by genes or QTLs on chromosome 1 can be done using markers that permit the identification of the parental origin of alleles in a progeny. Such markers permit the identification and selection of recombinations between the autoflower locus and genes or QTLs of traits involved in the Value Phenotype, located on chromosome 1. For genes or QTLs of traits involved in the Value Phenotype are located very close to the autoflower locus, recombinations are very scarce, yet detectable and selectable with markers such as markers from Table 2.

[0034] A "plant" as used herein refers to any plant at any stage of development, particularly a seed plant. The term "plant" can include the whole plant and, in some embodiments, can include or refer to any viable parts or derivatives thereof, such as plant cells, seeds, plant protoplasts, plant cell tissue culture from which Cannabis plants can be regenerated, plant callus or calli, meristematic cells, microspores, embryos, immature embryos, pollen, ovules, anthers, fruit, flowers, leaves, cotyledons, pistil, seeds, roots, root tips, and the like.

[0035] 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 can 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.

[0036] 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, embryos at various stages of development, leaves, roots, root tips, anthers, meristematic cells, microspores, flowers, cotyledons, pistil, fruit, seeds, or any combination thereof. [0037] The term "plant material" or "plant part" as 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.

[0038] The term "plant organ" as used herein refers to a distinct and/or visibly structured and differentiated part of a plant such as but not limited to a root, stem, leaf, flower, flower bud, embryo, or the like.

[0039] 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, 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.

[0040] As used herein, the terms "progeny" or "progenies" refer in a non- limiting manner to offspring or descendant plants. According to certain embodiments, the terms "progeny" or "progenies" refer to plants developed or grown or produced from the disclosed or deposited cells and/or seeds as detailed herein. The grown plants preferably have the desired traits of the disclosed or deposited cells and/or 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.

[0041] 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 true taxonomic term.

[0042] 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.

[0043] Additional breeding methods that, in some embodiments, can be combined with marker- assisted breeding are known to those of ordinary skill in the art and include, e.g. , methods discussed in Chahal and Gosal (Principles and procedures of plant breeding: biotechnological and conventional approaches, CRC Press, 2002, ISBN 084931321X, 9780849313219); Taji et al. (In vitro plant breeding, Routledge, 2002, ISBN 156022908X, 9781560229087); Richards (Plant breeding systems, Taylor & Francis US, 1997, ISBN 0412574500, 9780412574504); Hayes (Methods of Plant Breeding, Publisher: READ BOOKS, 2007, ISBN1406737062, 9781406737066); each of which is incorporated by reference in its entirety. The Cannabis genome has been sequenced (Bakel et al., The draft genome and transcriptome of Cannabis sativa, Genome Biology, 12(10):R102, 2011 ). Molecular makers for Cannabis plants are described in Datwyler et al. (Genetic variation in hemp and marijuana (Cannabis sativa L.) according to amplified fragment length polymorphisms, J Forensic Sci. 2006 March; 51(2):371-5.); Pinarkara et al., (RAPD analysis of seized marijuana (Cannabis sativa L.) in Turkey, Electronic Journal of Biotechnology, 12(1), 2009), Hakki et al., (Inter simple sequence repeats separate efficiently hemp from marijuana (Cannabis sativa L.), Electronic Journal of Biotechnology, 10(4), 2007); Gilmore et al. (Isolation of microsatellite markers in Cannabis sativa L. (marijuana), Molecular Ecology Notes, 3(1): 105-107, March 2003); Pacifico et al., (Genetics and marker- assisted selection of chemotype in Cannabis sativa L.), Molecular Breeding (2006) 17:257-268); and Mendoza et al., (Genetic individualization of Cannabis sativa by a short tandem repeat multiplex system, Anal Bioanal Chem (2009) 393:719-726); each of which is herein incorporated by reference in its entirety.

[0044] Additional breeding methods that can be used in certain embodiments of the invention, can be found, for example in, U.S. Patent No. 10441617B2, which is fully incorporated by reference herein.

[0045] 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 SENSITIVE CANNABIS PLANTS, GENES, MARKERS AND BREEDING,” filed April 30, 2021; U.S. Provisional Application No. 63/188,682, entitled “AUTOFLOWER CANNABIS PLANTS WITH VALUE PHENOTYPES,” filed May 14, 2021; PCT Application No. US2022/70402, entitled “MARKER- ASSISTED BREEDING IN CANNABIS PLANTS,” filed January 28, 2022; PCT Application No. US2022/70696, entitled “VALUE-PHENOTYPED AUTOFLOWER CANNABIS PLANTS,” filed February 17, 2022; PCT Application No. US2022/71972, entitled “MODULATED DAY-LENGTH SENSITIVE CANNABIS PLANTS, GENES, MARKERS, AND BREEDING,” filed April 28, 2022; and PCT Application No. US2022/72295, entitled “MODIFIED AUTOFLOWER CANNABIS PLANTS WITH VALUE PHENOTYPES,” filed May 12, 2022. The entire contents of each of the forgoing are fully incorporated by reference herein.

[0046] Some embodiments of the invention relate to a plant or plant part produced by any of the methods disclosed herein. Some embodiments relate to harvested material from the plant. Some embodiments relate to a product made from the harvested material. Other embodiments of the invention relate to a Cannabis extract material produced by the methods disclosed herein. The extract material can include a shatter, a crumble, a budder, an oil (e.g., butane hash oil, propane hash oil), nug run concentrate, CO2 concentrate, rosin, trim run, live resin, sap, dry sift, ice water hash, full melt, wax, pull and snap, fraction, THC, CBD, or any other product resulting from an extraction, for example an extraction of hydraulic press operation applied to plant, or a synthetic material that includes a Cannabis derivative.

Table 1: Markers within the AF locus

Table 2: SNP Marker Sequence List

Table 3: Agronomic and Composition Traits Genes of Interest EXAMPLES

Example 1 QTL Detection / Mapping

[0047] A QTL analysis of an auto-flowering (AF) trait was conducted using an F2 pedigree with 192 progeny samples. A single categorical phenotype was measured on the progeny. The phenotype shows a recessive segregation pattern, expressed in approximately 25% of the samples. QTL analysis identified a single locus in perfect correlation with the trait, consistent with the recessive model.

Example 2 Sequencing

[0048] Parents were deep-sequenced and progeny were skim- sequenced. Genotypes were imputed and HBs defined. These blocks were tested for association with the autoflower trait.

[0049] Sequencing depth varied as follows: 173 samples at 2x coverage, 20 samples at 8x coverage, and a parental line at 30x coverage. The sequencing data for 192 progeny samples passed required QC standards and were used in the QTL analysis. Figure 1 shows a schematic view of the pedigree including the sequencing depth (note that only one parental line, Banana OG, was sequenced in the analysis):

Example 3

Analysis Pipeline / Haplotype Inference

[0050] CS10 assembly from NCBI, version: GCA_900626175.2 (https ://www <dot> ncbi. ilm.nih <dot>..goy/assesiiMy/GCF..900626175.2) was used as a reference genome. Chrom-X was changed to Chrom-10 due to technical reasons but no other change to the reference was made.

[0051] All samples from Example 2 were mapped to the reference genome followed by a Variant- Calling pipeline using Genome Analysis Toolkit (GATK) and in-house tools to process the Skim-Seq data optimally. After variant filtration, a total of 45,573 SNPs were selected for the next stage. The Variant-Calling procedure was followed by a haplotype- inference algorithm that infers the 2 haplotypes in the Fl generation (A and B). The segregating genotypes in the progeny were inferred for each sample at each location along the genome. The 3 possible genotypes are designated as follows: AA, AB, and BB.

[0052] The basic genotyping unit is the HB, defined as a segment between consecutive recombination events in any of the progeny samples. Within HBs, there are no recombination events, and all markers (SNPs) could be used to measure sample genotypes. See a schematic view of the HBs in Figure 2.

Example 4 QTL Analysis

[0053] A QTL scan was performed by regressing the phenotype on the genotype at each HB. A significant QTL was declared if a model including the genotype was substantially better than a model without the genotype using a likelihood-ratio test. A threshold of FDR < 0.01 was used to declare significant results. (FDR = false discovery rate)

[0054] Assuming a categorical (Yes/No) phenotype, a genome-wide scan using logistic regression was implemented. The result is presented in the figure and tables below. The figure shows the FDR values on a log scale for each chromosome on each of the HBs. The horizontal line indicates a significance threshold (FDR of 0.01). Figure 3 shows a single QTL peak on Chromosome 1 that is highly significant, along with a minor peak on Chromosome 10.

Example 5

Confidence Interval

[0055] The table below shows the Confidence Interval (CI) around the peak. This interval can be the suggested region for generating markers for the QTL.

Table 4

Example 6

Effect Size [0056] Below is a summary count of the phenotype values per genotype at the peak on Chromosome 1. Note that there is a perfect match between the phenotype and genotype at this location. The peak on Chromosome 10 is tagged as a false positive since the phenotype/genotype correlation on Chromosome 1 is perfect.

Table 5

Example 7 SNP Markers

[0057] An SNP set was generated to be used as markers for the QTL locus. This SNP set was generated under the assumption that the phenotype is recessive and the causative haplotype is found in a homozygous state in the relevant progeny samples (Phenotype=l). The marker set is provided in Table 1.

Example 8 SNP Markers

[0058] Table 1 shows SNP markers for the segregating allele (/.<?. , the BB genotype) at the QTL locus were selected based on the following criteria:

At least lOObp flanking region with no other variant

GC content between 30-70%

Scored well within the haplotype inference algorithm

[0059] The data contain the following attributes for each SNP:

Chrom/Pos: Coordinates relative to CS10 reference genome

Ref/ Alt: The reference and alternative alleles relative to CS10 reference genome. Marker_Allele: The allele linked with the B haplotype

Flanking sequences around the SNP allele GC content of the flanking sequences Example 9

Haplotype Blocks file

[0060] The HB and the sample genotype within each block are provided.

[0061] A file containing the location of each HB detected in the analysis together with the assigned genotype of each sample was obtained. The genotypes were coded as characters with the following schema:

• AA: Homozygous for A allele

• BB: Homozygous for B allele

• AB: Heterozygous

[0062] Note that the A and B alleles are arbitrary and bear no relation to the reference/alternative alleles found in the variant-calling analysis.

[0063] The file format is depicted below for a few selected samples (the complete table holds 1293 entries).

Table 6 Example 10

Phenotypic correlation between AF and agronomic or composition (value trait) performance

Varieties extracted for commercial production were evaluated for different traits including: total cannabinoid concentration, total THC concentration, total terpene concentration (as mg/g of dry matter) and oil yield as % of fresh frozen biomass. Autoflower varieties showed significantly lower cannabinoid, THC, and terpene concentrations, as well as oil yield when compared with the daylength sensitive varieties. Sample descriptions for total concentration of cannabinoids, THC, terpenes, and oil yield percent.

Table 7

[0064] These results clearly show the relationship between auto-flowering/daylength sensitivity and economically important traits in Cannabis sativa. The auto-flowering characteristic is always/generally associated with lower values of these economically important traits than daylength sensitivity. Because of the genetic structure of these two groups of materials - being selfed progenies of auto-flowering x daylength sensitive segregating crosses - this observation is strong evidence for the existence of negative genetic linkage between the AF allele at the auto-flower locus and agronomically and economically desirable traits. Breaking such negative linkage will require specific processes, including the use of specific markers outside of yet closely flanking the AF locus. Example 11 Breeding for Improved AF Materials

[0065] A number of crosses are made between AF lines and PP materials (clones) with the objective of developing AF lines with agronomic and composition (value trait or traits) performance similar to that of the PP parent. Large (several hundred) F2 populations are developed and screened for the presence of the AF allele using an SNP previously found to be diagnostic of AF. Plants homozygous for the AF allele are selected. The selected plants are phenotyped for flowering behavior to confirm their being AF. They are also phenotyped for composition traits, based on which a further selection step is carried out. F2 plants with positive results as to all selection criteria are self-fertilized to generate F3 seed. F3 families are phenotyped for agronomic and composition traits, and selected on the basis of their performance. A number of plants from each selected family are selfed to generate the following generation. This process is followed for a number of generations, up to the F7 generation in a number of cases. All materials from F3 and beyond always show the AF phenotype. All, however, also show performance levels significantly lower than day-length sensitive materials for one or more agronomic or composition traits (value traits).

[0066] Without wishing to be bound by a particular theory, the difficulty in recovering an agronomically- or compositionally acceptable C. sativa plant with AF is most likely the result of linkage drag of undesirable traits from the AF sources.

Example 12

Association Mapping of AF and Agronomic and Composition Traits (value traits)

[0067] A set of 267 Cannabis sativa materials, including heterozygous clones and inbred families (F3’s and F4’s) were selected to form a diverse association mapping (AM) panel. The AM panel consisted of materials with a wide range of flowering behavior, terpenes, maturity, and other agronomic traits.

[0068] These materials were phenotyped in 2020 for a number of traits including daylength sensitivity (AF or photo), days to maturity, CBD, THC, and a set of terpene profiles.

[0069] All materials were genotyped with 600 SNPs and used for the Genome-Wide Association Study (GWAS) analysis. [0070] Data Analysis: AM based on mixed linear model (MLM) with population structure as a covariate was conducted using TASSEL, a JAVA based open-source software for linkage and association analysis (Bradbury et al., 2007).

[0071] Results: The AF locus was mapped to chromosome 1 at position 19,988,827 bp (as positions are established in the cslO reference genome). Significant associations for different terpene profiles and maturity were identified on chromosome 1 as well as other chromosomes. More detail on the mapping of this locus is provided in U.S. Provisional Application No. 63/182,725, entitled “MODULATED DAY-LENGTH SENSITIVITY CANNABIS PLANTS, GENES, MARKERS AND BREEDING,” filed April 30, 2021 and PCT Application No. US2022/71972, entitled “MODULATED DAY-LENGTH SENSITIVE CANNABIS PLANTS, GENES, MARKERS, AND BREEDING, filed on April 28, 2022.

[0072] Significant marker trait associations were used to assign co-segregating or adjacent significant markers into QTL intervals. Markers with the most significant p-values were extracted as representative markers for each marker trait association. Some of the loci were detected for multiple traits, so all those were combined under one QTL interval. The most significant QTLs were positioned based on physical position against the CslO Genome Assembly (GCA_900626175.2).

QTL regions significantly associated with terpene profiles and days to maturity (p.MLM

< 0.001), and linked to the AF locus, on chromosome 1:

Table 8 [0073] GWAS revealed the existence of loci involved in agronomic and composition traits (value traits) linked to the AF locus on chromosome 1 , and where the AF allele is in repulsion phase with favorable alleles for these agronomic and composition traits (that is the AF allele and unfavorable alleles for agronomic and composition traits are carried by one of the two homologous copies of chromosome 1, while the day length-sensitive allele and unfavorable alleles for agronomic and composition traits are carried by the other homologous copy of chromosome 1. As a result, AF and unfavorable alleles for agronomic and composition traits are generally inherited together. Breaking this undesirable inheritance relationship between AF and favorable alleles for agronomic and composition traits requires being able to select very infrequent recombination events that may occur between the AF locus and linked loci involved in agronomic and composition traits. Selecting such infrequent recombination events would require the screening of very large numbers of individual plants. Such recombination events are practically impossible to observe phenotypically on individual plants. Therefore, the most and possibly only effective approach to select such desirable recombination events is through the use of the markers located between the AF locus and neighboring agronomic and composition trait loci, as illustrated herein.

Example 13 Evidence for Linkage between AF Locus and Loci involved in Agronomic and Composition Traits (value traits)

[0074] Genes of interest for agronomic and composition traits including Abiotic Stress Response, Autoflower, Defense Response, Flowering, Plant Development and Terpene Synthesis were identified and categorized based on functionality and gene ontology descriptions. The selected genes of interest were placed relative to the markers identified in the AM.

[0075] For the sake of simplification genes were grouped into gene intervals. Some of these gene intervals included multiple genes involved in multiple traits. These gene intervals were positioned based on physical position against the CslO Genome Assembly (GCA_900626175.2).

Table 9: Genes linked with AF locus on chromosome 1

Example 14

Development and Use of Markers to Break Unfavorable Associations between the AF Phenotype and other value traits

[0076] Based on the evidence for linkage between AF locus and loci involved in agronomic and composition traits, markers were developed to enable the breaking of unfavorable linkage between the AF phenotype and other value traits. The use of such markers allows for selection of recombination events between the AF locus and other loci involved in other value traits, on chromosome 1, where the AF locus is located.

[0077] These markers were grouped into marker intervals for simplification purposes. See table below. The AF locus is exemplary.

Table 10: Marker intervals and number of markers in each region:

[0078] As used herein “upstream” of the AF locus can be defined by: Any individual marker or group of markers within MI2 (alone or together with one or more markers from within Mil), can be used to select for recombination between the AF locus and QTLs located within QTI1 (QTI = QTL Interval) and beyond (all the way to the end of the short arm of Chromosome 1), and therefore to break unfavorable associations between the AF phenotype and all value traits explained by those QTLs.

[0079] Any individual marker or group of markers within MI3 (alone or together with one or more markers from within Mil, MI2, or Mil and MI2), can be used to select for recombination between the AF locus and QTLs located within QTI2 or QTI1 and beyond (all the way to the end of the short arm of Chromosome 1), and therefore to break unfavorable associations between the AF phenotype and all value traits explained by those QTLs.

[0080] As used herein “downstream” of the AF locus can be defined by: Any individual marker or group of markers within MI4 (alone or together with one or more markers from within MI5, MI6, MI7, MI5 and MI6, MI5 and MI7, MI6 and MI7, or MI5 and MI6 and MI7), can be used to select for recombination between the AF locus and QTLs located within QTI3, QTI4 or QTI5 and beyond (all the way to the end of the long arm of Chromosome 1), and therefore to break unfavorable associations between the AF phenotype and all value traits explained by those QTLs.

[0081] Any individual marker or group of markers within MI5 (alone or together with one or more markers from within MI6, MI7, or MI6 and MI7), can be used to select for recombination between the AF locus and QTLs located within QTI4 or QTI5 and beyond (all the way to the end of the long arm of Chromosome 1), and therefore to break unfavorable associations between the AF phenotype and all value traits explained by those QTLs.

[0082] Any individual marker or group of markers within MI6 (alone or together with one or more markers from within MI7), can be used to select for recombination between the AF locus and QTLs located within QTI5 and beyond (all the way to the end of the long arm of Chromosome 1), and therefore to break unfavorable associations between the AF phenotype and all value traits explained by those QTLs.

[0083] As used herein “upstream” and “downstream” of the AF locus can be defined by:

[0084] Any combination of one of the above “upstream” and one of the above “downstream” processes can be used to select for recombinations simultaneously on both sides of the AF locus, and therefore to break unfavorable associations between the AF phenotype and all value traits explained by the respective QTLs. [0085] 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.

[0086] 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.

[0087] 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.

[0088] 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 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. [0089] 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-claimed element essential to the practice of the application.

[0090] 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. [0091] 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 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.

[0092] 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.