WO2024044692A2 - Cannabis résistant au mildiou poudreux - Google Patents

Cannabis résistant au mildiou poudreux Download PDF

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Publication number
WO2024044692A2
WO2024044692A2 PCT/US2023/072827 US2023072827W WO2024044692A2 WO 2024044692 A2 WO2024044692 A2 WO 2024044692A2 US 2023072827 W US2023072827 W US 2023072827W WO 2024044692 A2 WO2024044692 A2 WO 2024044692A2
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plant
seq
resistance gene
cannabis sativa
gene encodes
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PCT/US2023/072827
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English (en)
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WO2024044692A3 (fr
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Jordan ZAGER
Paul MIHALYOV
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Dewey Scientific
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Publication of WO2024044692A3 publication Critical patent/WO2024044692A3/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/02—Flowers
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular markers
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/12—Processes for modifying agronomic input traits, e.g. crop yield
    • A01H1/122—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • A01H1/1245—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance
    • A01H1/1255—Processes for modifying agronomic input traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, e.g. pathogen, pest or disease resistance for fungal resistance
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/28—Cannabaceae, e.g. cannabis

Definitions

  • the present invention relates to powdery mildew resistance genes in Cannabis sativa L. and especially to powdery mildew resistant Cannabis sativa L. plants.
  • the present invention further relates to methods for obtaining the present powdery mildew resistant Cannabis sativa L. plants and the use of the present genes for providing powdery mildew resistance in Cannabis sativa L.
  • Cannabis sativa commonly known as hemp or marijuana
  • CBDA cannabidiolic acid
  • THCA cannabinoids tetrahydrocannabinolic acid
  • CBDA cannabidiolic acid
  • One priority should be identifying genetic markers and genes linked to naturally occurring disease resistance, thereby accelerating the development of varieties which are less dependent on the use of pesticides in disease management.
  • the present invention addresses the widespread need for collaborative germplasm improvement strategies, including fast routes (single-cross) for mapping genetic resistance to powdery mildew, one of the most prevalent fungal diseases in cannabis.
  • Powdery mildew is a common term for several taxa of plant pathogenic fungi, including fungi from the genus Golovinomyces, and has been reported on both marijuana and industrial hemp-type C. sativa. Powdery mildew represents a significant limitation to cannabis production, especially in greenhouse systems. Symptoms of powdery mildew include patchy white mycelial colonies that develop on foliage and flowers, contributing to reduced plant vigor. Infection can result in a reduction of end-use quality. The economic impact of powdery mildew on cannabis production is significant.
  • Fungicides may be used to manage powdery mildew, but are accompanied by uncertainties regarding compliance, efficacy, and consumer response. Regulatory agencies throughout the world have been slow to adopt new guidelines for the use of agrochemicals on cannabis, creating a backlog of research needed to safely apply pesticides. Furthermore, efficacy data for the use of pesticides against powdery mildew in cannabis is lacking. Alternative strategies to mitigate powdery mildew include strategic environmental control, the use of high-powered UV-C lamps, and application of plant-growth promoting rhizobacteria, yet powdery mildew continues to represent one of the most prominent biological diseases in both field and greenhouse settings. Thus, the identification and characterization of powdery mildew resistance genes are of vital importance to the growth and sustainability of the cannabis industry.
  • the present application provides an isolated Cannabis sativa L. plant that is resistant to powdery mildew, wherein the plant comprises one or more powdery mildew resistance genes, and wherein the plant produces flowers with greater than 22% tetrahydrocannabinolic acid (THCA) (wt/wt). In some embodiments, the isolated plant produces flowers with greater than 23%, 24%, or 25% THCA (wt/wt).
  • THCA tetrahydrocannabinolic acid
  • the resistance gene is located on Chromosome 2 of GenBank assembly accession no. GCA_900626175.2. In some embodiments, the resistance gene encodes a protein in the NB-ARC-LRR family. In some embodiments, the resistance gene encodes a transcript comprising a polynucleotide at least 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 1-28.
  • the one or more resistance gene encodes one or more transcripts comprising a polynucleotide selected from the group consisting of SEQ ID NOs: 1-28. In some embodiments, the resistance gene encodes a transcript comprising a polynucleotide at least 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% identical to a polynucleotide selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO:27. In some embodiments, the resistance gene encodes a transcript comprising SEQ ID NO: 6. In some embodiments, the resistance gene encodes a transcript comprising SEQ ID NO: 27.
  • the one or more resistance genes encodes one or more proteins comprising at least one polypeptide that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a polypeptide selected from the group consisting of SEQ ID NOs: 29-56.
  • the resistance gene encodes a protein comprising a polypeptide selected from the group consisting of SEQ ID NOs: 29-56.
  • the resistance gene encodes a protein comprising a polypeptide at least 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% identical to a polypeptide selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO: 55. In some embodiments, the resistance gene encodes a protein comprising a polypeptide selected from the group consisting of SEQ ID NOs: 34 and 55. In some embodiments, the resistance gene encodes a protein comprising a polypeptide of SEQ ID NO: 34. In some embodiments, the resistance gene encodes a protein comprising a polypeptide of SEQ ID NO: 55. In some embodiments, the plant comprises more than one resistance gene.
  • the plant comprises two, three, or four resistance genes.
  • the resistance gene allele is a dominant allele.
  • the plant is homozygous for the resistance gene allele.
  • the plant is heterozygous for the resistance gene allele.
  • the plant is resistant to infection by fungi of the genus Golovinomyces.
  • the plant is resistant to infection by fungi of the species Golovinomyces ambrosiae.
  • the plant produces at least 41g per plant of harvestable floral material. In some embodiments, the plant produces at least 42g, 43g, 44g, 45g, 46g, or 47g per plant of harvestable floral material. In some embodiments, the harvestable floral material comprises flowers and/or flower buds. In some embodiments, the plant produces harvestable material comprising flowers and/or flower buds, leaves, stalks, and seeds. In some embodiments, a plant part, tissue, or cell of the isolated Cannabis sativa L. plant is claimed. In some embodiments, the plant cell is a non-regenerable cell. In some embodiments, the isolated plant part further comprises harvestable material comprising flowers and/or flower buds, leaves, stalks, and seeds.
  • the plant has increased lateral branching as compared to a control Cannabis sativa L. plant. In some embodiments, the plant has at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% more lateral branches as compared to a control Cannabis sativa L. plant. In some embodiments, the isolated powdery mildew resistant Cannabis sativa L. plant has an increased number of top of the canopy flowers as compared to a control Cannabis sativa L. plant. In some embodiments, the isolated powdery mildew resistant Cannabis sativa L.
  • the plant has at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% more top of the canopy flowers as compared to a control Cannabis sativa L. plant.
  • the isolated powdery mildew resistant Cannabis sativa L. plant has an increased number of nodes when compared to a control Cannabis sativa L. plant.
  • the plant has increased flower site stacking as compared to a control Cannabis sativa L. plant.
  • the control Cannabis sativa L. plant is a naturally- occurring powdery mildew resistant plant.
  • the control Cannabis sativa L. plant is from the ‘PNW39’ population of plants.
  • Several embodiments relate to a method of selecting a Cannabis sativa L. plant that is resistant to powdery mildew, comprising crossing a first Cannabis sativa L. plant resistant to powdery mildew to a second Cannabis sativa L. plant to produce a population of offspring Cannabis sativa L. plants; genotyping the offspring population of Cannabis sativa L. plants for the presence of one or more powdery mildew resistance genes; and selecting an offspring Cannabis sativa L. plant on the basis of the genotyping, wherein the selected offspring Cannabis sativa L. plant is resistant to powdery mildew.
  • the plant produces flowers with greater than 22% tetrahydrocannabinolic acid (THCA) (wt/wt).
  • THCA tetrahydrocannabinolic acid
  • the selected offspring Cannabis sativa L. plant produces flowers with greater than 23%, 24%, or 25% THCA (wt/wt).
  • the resistance gene is located on Chromosome 2 of GenBank assembly accession no. GCA_900626175.2.
  • the resistance gene encodes a protein in the NB-ARC-LRR family.
  • the resistance gene encodes a transcript comprising a polynucleotide at least 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 1-28. In some embodiments, the resistance gene encodes a transcript comprising a polynucleotide selected from the group consisting of SEQ ID NOs: 1-28.
  • the resistance gene encodes a transcript comprising a polynucleotide at least 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% identical to a polynucleotide selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 27. In some embodiments, the resistance gene encodes a transcript comprising a polynucleotide selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 27. In some embodiments, the resistance gene encodes a transcript comprising SEQ ID NO: 6. In some embodiments, the resistance gene encodes a transcript comprising SEQ ID NO: 27.
  • the one or more resistance genes encodes one or more proteins comprising at least one polypeptide that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to one or more of SEQ ID NOs: 29-56.
  • the resistance gene encodes a protein comprising a polypeptide selected from the group consisting of SEQ ID NOs: 29-56.
  • the resistance gene encodes a protein comprising a polypeptide at least 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% identical to a polypeptide selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO:55. In some embodiments, the resistance gene encodes a protein comprising a polypeptide selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO: 55. In some embodiments, the resistance gene encodes a protein comprising a polypeptide of SEQ ID NO: 34. In some embodiments, the resistance gene encodes a protein comprising a polypeptide of SEQ ID NO: 55.
  • the second Cannabis sativa L. plant is not resistant to powdery mildew.
  • the selected offspring Cannabis sativa L. plant produces at least 41g per plant of harvestable floral material.
  • the selected offspring Cannabis sativa L. plant produces at least 42g, 43g, 44g, 45g, 46g, or 47g per plant of harvestable floral material.
  • the selected offspring Cannabis sativa L. plant has increased lateral branching as compared to the first Cannabis sativa L. plant.
  • the selected offspring Cannabis sativa L. plant has increased flower site stacking as compared to the first Cannabis sativa L. plant.
  • FIG. 1 shows susceptible and resistant phenotypes revealed by experiments with heavy disease pressure imposed through plant maturity.
  • the left image shows a susceptible phenotype with dense mycelial growth (pictured: individual #45 from the population “PNW39” x “Jumping Jack”).
  • the right image shows a resistant phenotype absent of any powdery mildew colonies (pictured: individual #49 from the population “PNW39” x “Jumping Jack”).
  • FIG. 2 shows a linkage map describing locations of selected markers on chromosome 2 (based on “CBDRx” chromosome naming). Resistance gene PM1 co-localizes with SNP markers LH3804, LH31156, and LH17304 on chromosome 2.
  • FIGS. 3A-3B show allelic discrimination plots from SNP assay PM_LH3804. Black dots represent individuals with the susceptible phenotype and gray dots represent individuals with the resistant phenotype.
  • FIG. 3A shows an allelic discrimination plot using the parents and 89 progeny from “PNW39” x “Jumping Jack”.
  • FIG. 3B shows an allelic discrimination plot using 96 Fl progeny from “PNW39” x “Dwyl337”.
  • the present disclosure relates to isolated Cannabis sativa L. plants that are resistant to infection with powdery mildew.
  • the term ‘and/or’ as used in a phrase such as "A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).
  • An ‘isolated plant’ describes a plant that is not a naturally- occurring strain or ecotype, but was deliberately bred with human intervention.
  • ‘Powdery mildew’ is a fungal disease that is capable of infecting numerous species of plants, including Cannabis sativa L. Infection results in a layer of white mildew growing on the surface of the leaves of the plant, and may inhibit growth of the plant as well as reduce yield and quality of harvestable material.
  • Powdery mildew is caused by infection with fungi of the family Erysiphaceae, including the following genera: Arthrocladiella, Blumeria, Brasiliomyces Bulbomicrosphaera, Bulbouncinula, Caespitotheca, Cystotheca, Erysiphe (Oidium), Golovinomyces, Eeveillula (Oidiopsis), Medusosphaera, Microsphaera, Neoerysiphe, Oidium, Phyllactinia ( Ovulariopsis ), Pleochaeta, Podosphaera, Sawadaea, Setoerysiphe, Sphaerotheca, Typhulochaeta, Uncinula, and Uncinuliella.
  • fungi of the family Erysiphaceae including the following genera: Arthrocladiella, Blumeria, Brasiliomyces Bulbomicrosphaera, Bulbouncinula,
  • Powdery mildew on Cannabis has been reportedly caused by infection with fungi of the genera Podosphaera and Golovinomyces (see Ocamb, “Hemp (Cannabis sativa)-Po N Qvy Mildew”, Pacific Northwest Plant Disease Management Handbook, revised March 2023).
  • infection could be caused by the following species: G. adenophorae, G. ambrosiae, G. americanus, G. andinus, G. arabidis, G. artemisiae, G. asperifolii, G. asperifoliorum, G. asterum, G. biocellatus, G. bolayi, G.
  • a plant that is ‘resistant to powdery mildew’ identifies a plant that will not be infected with powdery mildew, even when exposed to high disease pressure, i.e., when grown surrounded by plants that are infected with powdery mildew.
  • a ‘powdery mildew resistance gene’ identifies a gene or an allele of a gene that confers resistance to infection with powdery mildew to a plant.
  • a ‘dominant’ allele identifies an allele of a gene that confers a given phenotype when one or more copies of the allele are present in the organism.
  • a dominant powdery mildew resistance allele confers resistance to powdery mildew to an organism when the genome of the organism comprises one or more copies of the dominant powdery mildew resistance allele.
  • heterozygous identifies a locus in the genome at which more than one distinct allele is present.
  • homozygous identifies a locus in the genome at which no more than one distinct allele is present.
  • THCA is defined as an abbreviation for tetrahydrocannabinolic acid.
  • the present disclosure provides plants that are resistant to powdery mildew while also having high levels of THCA.
  • THCA is the most abundant non-psychoactive cannabinoid found in cannabis, and is a precursor to tetrahydrocannabinol (THC), an active component of cannabis.
  • Cannabinoids are compounds that can interact with the vertebrate endocannabinoid system by binding to cannabinoid receptors. Other cannabinoids include but are not limited to tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN).
  • THCA tetrahydrocannabinolic acid
  • the isolated plant produces flowers with greater than 23%, 24%, or 25% THCA (wt/wt).
  • Cannabinoid levels can be quantified with High Performance Liquid Chromatography - Diode-Array Detection (HPLC-DAD).
  • HPLC-DAD High Performance Liquid Chromatography - Diode-Array Detection
  • a flower is ground and suspended in Methanol (HPLC-grade).
  • the sample is vortexed for 15 minutes at maximum speed, then incubated in methanol at room temperature for 1 hour.
  • the supernatant is filtered through a 0.22pm PTFE filter, then diluted 20-fold in methanol before injection on the HPLC.
  • the liquid sample is pressurized and passed through a column filled with solid adsorbent material. Different chemicals within the sample will adhere to the column at different strengths, resulting in these different chemicals reaching the end of the column after different amounts of time.
  • the chemical composition of a solution can be determined and quantification of a particular chemical performed against known reference standards and a calibration curve.
  • ‘Harvestable material’ includes flowers and/or flower buds, leaves, stalks, seeds, oils, and roots of a plant. ‘Harvestable floral material’, as used herein, refers to flowers and/or flower buds. In some embodiments, the plant produces at least 41g per plant of harvestable floral material. In some embodiments, the plant produces at least 42g, 43g, 44g, 45g, 46g, or 47g per plant of harvestable floral material. In some embodiments, the harvestable floral material comprises flowers and/or flower buds. In some embodiments, the plant produces harvestable material comprising flowers and/or flower buds, leaves, stalks, and seeds.
  • a plant ‘part’, ‘tissue’, or ‘cell’ refers to any portion of the vegetative or reproductive parts of the plant, including both roots and aerial portions of the plant, including but not limited to, seed (including mature seed and immature seed); a plant cutting; a plant cell; a plant cell culture; or a plant organ (e.g., pollen, embryos, flowers, fruits, shoots, trichomes, buds, leaves, roots, stems, and explants).
  • a plant tissue or plant organ may be a seed, protoplast, callus, or any other group of plant cells that is organized into a structural or functional unit.
  • a plant cell or tissue culture may be capable of regenerating a plant having the physiological and morphological characteristics of the plant from which the cell or tissue was obtained, and of regenerating a plant having substantially the same genotype as the plant.
  • Regenerable cells in a plant cell or tissue culture may be embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, roots, root tips, silk, flowers, kernels, ears, cobs, husks, or stalks.
  • some plant cells are not capable of being regenerated to produce plants and are referred to herein as “non-regenerable” plant cells.
  • a plant part, tissue, or cell of the isolated Cannabis sativa L. plant is claimed.
  • the plant cell is a non-regenerable cell.
  • the isolated plant part further comprises harvestable material comprising flowers and/or flower buds, leaves, stalks, and seeds.
  • ‘Lateral branching’ refers to the phenomenon in which lateral buds off of the main stem of a plant produce outgrowths from the main stem. These lateral buds each have their own apical meristem and can produce their own flowers. ‘Increased lateral branching’ as used herein indicates that a plant or population of plants has at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% more lateral branches when compared to another plant or plant population.
  • top of the canopy flowers refer to flowers deriving from the terminal bud, the flowering site on a female cannabis plant where flowers grow together tightly. This is also known as a ‘cola’. Healthy plants typically form one main cola from the center of their structure and smaller colas on the outside of the plant. Top of the canopy flowers generally display increased trichome density, are richer in cannabinoids and terpenes, are denser, and have increased size when compared to flowers that develop from lateral buds.
  • ‘Increased top of the canopy flowers’ indicates that a plant or population of plants has at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% more ‘top of the canopy flowers’ when compared to another plant or plant population.
  • a ‘node’ refers to a site on a plant stem from which lateral buds and leaves originate.
  • ‘Flower site stacking’ refers to the shortening of internode length on a plant, resulting in increased number of nodes, increased number of flowers, and increased yield. ‘Increased flower site stacking’, as used herein, indicates a plant or population of plants that has at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% more nodes when compared to another plant or plant population.
  • control plant refers to reference plant to which an isolated plant is compared.
  • the control plant is a naturally-occurring Cannabis sativa L. plant.
  • the control plant is a naturally-occurring powdery mildew-resistant Cannabis sativa L. plant.
  • the control plant is from the ‘PNW39’ population of plants.
  • the method includes genotyping the offspring population of Cannabis sativa L. plants for the presence of one or more resistance genes.
  • Several embodiments relate to a method of selecting a Cannabis sativa L. plant that is resistant to powdery mildew, comprising crossing a first Cannabis sativa L. plant resistant to powdery mildew to a second Cannabis sativa L. plant to produce a population of offspring Cannabis sativa L. plants; genotyping the offspring population of Cannabis sativa L.
  • the selected offspring Cannabis sativa L. plant produces flowers with greater than 22% tetrahydrocannabinolic acid (THCA) (wt/wt). In some embodiments, the selected offspring Cannabis sativa L. plant produces flowers with greater than 23%, 24%, or 25% THCA (wt/wt). In some embodiments, the resistance gene is located on Chromosome 2 of GenBank assembly accession no. GCA_900626175.2.
  • the resistance gene encodes a protein in the NB-ARC-LRR family. In some embodiments, the resistance gene encodes a transcript comprising a polynucleotide at least 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 1-28. In some embodiments, the resistance gene encodes a transcript comprising a polynucleotide selected from the group consisting of SEQ ID NOs: 1- 28.
  • the resistance gene encodes a transcript comprising a polynucleotide at least 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% identical to a polynucleotide selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 27. In some embodiments, the resistance gene encodes a transcript comprising a polynucleotide selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 27. In some embodiments, the resistance gene encodes a transcript comprising SEQ ID NO: 6. In some embodiments, the resistance gene encodes a transcript comprising SEQ ID NO: 27.
  • the resistance gene encodes a protein comprising a polypeptide that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a polypeptide selected from SEQ ID NOs: 29-56. In some embodiments, the resistance gene encodes a protein comprising a polypeptide selected from the group consisting of SEQ ID NOs: 29-56.
  • the resistance gene encodes a protein comprising a polypeptide at least 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% identical to a polypeptide selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO: 55. In some embodiments, the resistance gene encodes a protein comprising a polypeptide selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO: 55. In some embodiments, the resistance gene encodes a protein comprising a polypeptide of SEQ ID NO: 34. In some embodiments, the resistance gene encodes a protein comprising a polypeptide of SEQ ID NO: 55.
  • the second Cannabis sativa L. plant is not resistant to powdery mildew.
  • the selected offspring Cannabis sativa L. plant produces at least 41g per plant of harvestable floral material.
  • the selected offspring Cannabis sativa L. plant produces at least 42g, 43g, 44g, 45g, 46g, or 47g per plant of harvestable floral material.
  • the selected offspring Cannabis sativa L. plant has increased lateral branching as compared to the first Cannabis sativa L. plant.
  • the selected offspring Cannabis sativa L. plant has increased flower site stacking as compared to the first Cannabis sativa L. plant.
  • Genotyping refers to process of determining which allele or alleles is/are present in the genome of the plant at one or more given loci. Genotyping involves extracting a nucleic acid such as DNA or RNA, then performing common laboratory procedures to identify the present alleles, including but not limited to such techniques as polymerase chain reaction (PCR), restriction enzyme digest assays, restriction fragment length polymorphism identification (RFLPI), random amplified polymorphic detection (RAPD), amplified fragment length polymorphism detected (AFLPD), allele specific oligonucleotide (ASO) probes, microarrays, hybridization techniques, single nucleotide polymorphism (SNP) genotyping chips, Sanger sequencing, and next-generation sequencing.
  • PCR polymerase chain reaction
  • RFLPI restriction fragment length polymorphism identification
  • RAPD random amplified polymorphic detection
  • AFLPD amplified fragment length polymorphism detected
  • ASO allele specific oligonucleotide
  • kits for identifying a Cannabis sativa L. plant that is resistant to powdery mildew comprising genotype the plant for the presence of a powdery mildew resistance gene, wherein the powdery mildew resistance gene encodes a transcript comprising a polynucleotide selected from the group consisting of SEQ ID NOs: 1-28, and/or encodes a polypeptide that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a polypeptide selected from SEQ ID NOs: 29-56.
  • the resistance gene encodes a transcript comprising a polynucleotide at least 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 1-28. In some embodiments, the resistance gene encodes a transcript comprising a polynucleotide selected from the group consisting of SEQ ID NOs: 1-28.
  • the resistance gene encodes a transcript comprising a polynucleotide at least 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% identical to a polynucleotide selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 27. In some embodiments, the resistance gene encodes a transcript comprising a polynucleotide selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 27. In some embodiments, the resistance gene encodes a transcript comprising SEQ ID NO: 6. In some embodiments, the resistance gene encodes a transcript comprising SEQ ID NO: 27.
  • the resistance gene encodes a protein comprising a polypeptide that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a polypeptide selected from SEQ ID NOs: 29-56. In some embodiments, the resistance gene encodes a protein comprising a polypeptide selected from the group consisting of SEQ ID NOs: 29-56.
  • the resistance gene encodes a protein comprising a polypeptide at least 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% identical to a polypeptide selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO:55. In some embodiments, the resistance gene encodes a protein comprising a polypeptide selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO: 55. In some embodiments, the resistance gene encodes a protein comprising a polypeptide of SEQ ID NO: 34. In some embodiments, the resistance gene encodes a protein comprising a polypeptide of SEQ ID NO: 55.
  • nucleic acid sequences in the text of this specification are provided in the 5’ to 3’ direction when read from left to right.
  • a given DNA sequence is understood to define a corresponding RNA sequence which is identical to the DNA sequence except for replacement of the thymine (T) nucleotides of the DNA with uracil (U) nucleotides.
  • T thymine
  • U uracil
  • a given first polynucleotide sequence whether DNA or RNA, further defines the sequence of its exact complement (which can be DNA or RNA), i.e., a second polynucleotide that hybridizes perfectly to the first polynucleotide by forming Watson-Crick base-pairs.
  • polynucleotide commonly refers to a DNA or RNA molecule containing multiple nucleotides and generally refers both to ‘oligonucleotides’ (a polynucleotide molecule of 18-25 nucleotides in length) and longer polynucleotides of 26 or more nucleotides.
  • polypeptide as used herein, commonly refers to a protein molecule containing multiple amino acids.
  • Percent (%) identity of a sequence is defined as the percentage of nucleotides or amino acid residues in a candidate sequence that are identical or homologous with the nucleotides or amino acid residues in the polynucleotide or polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity.
  • Homology between polynucleotides is determined based on a substitution matrix, such as is described in: Smith and Waterman (1981); Needleman and Wunsch (1970); Pearson and Lipman (1988); Higgins and Sharp (1988); Higgins and Sharp (1989); Corpet et al., (1988); Huang et al., (1992); and Pearson et al., (1994).
  • Altschul et al., (1994) presents a detailed consideration of sequence alignment methods and homology calculations.
  • Homology between different amino acid residues is determined based on a substitution matrix, such as the BLOSUM (B LOcks Substitution Matrix alignment).
  • Determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
  • NCB 1 Basic Local Alignment Search Tool (BLAST) (Altschul et al., 1990) is available from several sources, including the National Center for Biotechnology Information (NCB1, Bethesda, MD) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx.
  • NCB1 Basic Local Alignment Search Tool
  • the plant is resistant to infection by fungi of the family Erysiphaceae, including genera Arthrocladiella, Blumeria, Brasiliomyces Bulbomicrosphaera, Bulbouncinula, Caespitotheca, Cystotheca, Erysiphe (Oidium), Golovinomyces, Leveillula ( Oidiopsis ), Medusosphaera, Microsphaera, Neoerysiphe, Oidium, Phyllactinia (Ovulariopsis), Pleochaeta, Podosphaera, Sawadaea, Setoerysiphe, Sphaerotheca, Typhulochaeta, Uncinula, and Uncinuliella.
  • fungi of the family Erysiphaceae including genera Arthrocladiella, Blumeria, Brasiliomyces Bulbomicrosphaera, Bulbouncinula, Caespitotheca, Cysto
  • the Cannabis sativa L. plant is resistant to infection by fungi of the genus Golovinomyces.
  • the isolated plant is resistant to infection by fungi of the species G. adenophorae, G. ambrosiae, G. americanus, G. andinus, G. arabidis, G. artemisiae, G. asperifolii, G. asperifoliorum, G. asterum, G. biocellatus, G. bolayi, G. brunneopunctatus, G. calceolariae, G. californicus, G. caulicola, G. chrysanthemi, G. cichoracearum, G.
  • the plant is resistant to infection by fungi of the species Golovinomyces ambrosiae.
  • the plant has one or more powdery mildew resistance genes. In some embodiments, the plant has one powdery mildew resistance gene. In some embodiments, the plant has more than one powdery mildew resistance gene. In some embodiments, the plant has two, three, or four powdery mildew resistance genes. In some embodiments, the one or more powdery mildew resistance gene is located on Chromosome 2 of GenBank assembly accession no. GCA_900626175.2. In some embodiments, the resistance gene encodes a protein in the NB-ARC-LRR family.
  • the resistance gene encodes a transcript comprising a polynucleotide at least 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 1-28.
  • the one or more resistance gene encodes one or more transcripts comprising a polynucleotide selected from the group consisting of SEQ ID NOs: 1-28.
  • the resistance gene encodes a transcript comprising a polynucleotide at least 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% identical to a polynucleotide selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO:27. In some embodiments, the resistance gene encodes a transcript comprising SEQ ID NO: 6. In some embodiments, the resistance gene encodes a transcript comprising SEQ ID NO: 27.
  • the one or more resistance genes encodes one or more proteins comprising at least one polypeptide that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a polypeptide selected from the group consisting of SEQ ID NOs: 29-56.
  • the resistance gene encodes a protein comprising a polypeptide selected from the group consisting of SEQ ID NOs: 29-56.
  • the resistance gene encodes a protein comprising a polypeptide at least 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% identical to a polypeptide selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO: 55. In some embodiments, the resistance gene encodes a protein comprising a polypeptide selected from the group consisting of SEQ ID NOs: 34 and 55. In some embodiments, the resistance gene encodes a protein comprising a polypeptide of SEQ ID NO: 34. In some embodiments, the resistance gene encodes a protein comprising a polypeptide of SEQ ID NO: 55. In some embodiments, the plant comprises more than one resistance gene.
  • the plant comprises two, three, or four resistance genes.
  • the resistance gene allele is a dominant allele.
  • the plant is homozygous for the resistance gene allele.
  • the plant is heterozygous for the resistance gene allele.
  • the powdery mildew resistance gene encodes an NB-LRR family disease resistance protein. In some embodiments, the powdery mildew resistance gene encodes a disease resistance protein in InterPro family IPR044974. In some embodiments, the one or more powdery mildew resistance genes encodes a protein comprising a leucine-rich repeat domain (accession no. C134836 as defined by NCBI Conserved Domain Database; and/or IPR032675 as defined by InterProScan). In some embodiments, the one or more powdery mildew resistance genes encodes a protein comprising an NB-ARC domain (accession no.
  • the one or more powdery mildew resistance genes encodes a protein comprising an RX-CC-like coiled-coil domain (accession no. cdl4798 as defined by NCBI Conserved Domain Database; and/or IPR038005 as defined by InterProScan). In some embodiments, the one or more powdery mildew resistance genes encodes a protein comprising both an RX-CC-like coiled-coil domain and an NB-ARC domain.
  • the one or more powdery mildew resistance genes encodes a protein comprising both an RX-CC-like coiled-coil domain and a leucine-rich repeat domain. In some embodiments, the one or more powdery mildew resistance genes encodes a protein comprising both a leucine-rich repeat and an NB-ARC domain. In some embodiments, the one or more powdery mildew resistance genes encodes a protein comprising a leucine-rich repeat, an RX-CC-like coiled-coil domain, and an NB-ARC domain. In some embodiments, the one or more powdery mildew resistance genes encodes a protein comprising a PLN03150 domain (accession no.
  • the one or more powdery mildew resistance genes encodes a protein comprising a PLN00113 domain (accession no. PLN00113 as defined by NCBI Conserved Domain Database).
  • the isolated powdery mildew resistant Cannabis sativa L. plant has flowers that contain more than 22%, 23%, 24%, 25%, or 26% THCA (wt/wt). In some embodiments, the isolated powdery mildew resistant Cannabis sativa L. plant has flowers that contain on average more than 22%, 23%, 24%, 25%, or 26% THCA (wt/wt). [0050] In some embodiments, the invention provides an isolated plant resistant to powdery mildew which also has increased yield of harvestable floral material over a control Cannabis sativa L. plant.
  • the plant yields at least 41g, 42g, 43g, 44g, 45g, 46g, or 47g of harvestable floral material per plant.
  • the plant provides harvestable floral material comprising flowers and/or flower buds.
  • the plant provides harvestable material comprising flowers and/or flower buds, leaves, stalks, seeds, oils, and roots.
  • this invention provides a plant part, tissue, or cell from a powdery mildew resistant Cannabis sativa L. plant, including any portion of the vegetative or reproductive parts of the plant, including both roots and aerial portions of the plant, including but not limited to, a cell, protoplast, embryo, pollen grain, ovule, flower, leaf, stem, cotyledon, hypocotyl, meristematic cell, rootstock, root, root tip, pistil, anther, shoot tip, shoot, fruit, seed, and petiole.
  • the isolated powdery mildew resistant Cannabis sativa L. plant has increased lateral branching over a naturally-occurring powdery mildew resistant Cannabis sativa L. plant.
  • the isolated powdery mildew resistant Cannabis sativa L. plant has at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% more lateral branches when compared to a control Cannabis sativa L. plant.
  • the isolated powdery mildew resistant Cannabis sativa L. plant has an increased number of ‘top of the canopy flowers’ when compared to a control Cannabis sativa L. plant. In some embodiments, the isolated powdery mildew resistant Cannabis sativa L. plant has at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% more ‘top of the canopy flowers’ when compared to a control Cannabis sativa L. plant.
  • the isolated powdery mildew resistant Cannabis sativa L. plant has increased flower site stacking when compared to a control Cannabis sativa L. plant. In some embodiments, the isolated powdery mildew resistant Cannabis sativa L. plant has at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50% more nodes when compared to a control Cannabis sativa L. plant.
  • Certain embodiments of the present invention relate to methods for selecting a Cannabis sativa L. plant resistant to powdery mildew, comprising crossing a first Cannabis sativa L. plant resistant to powdery mildew to a second Cannabis sativa L. plant to produce a population of offspring Cannabis sativa L. plants; genotyping the offspring population of Cannabis sativa L. plants for the presence of one or more powdery mildew resistance genes; and selecting an offspring Cannabis sativa L. plant that is resistant to powdery mildew.
  • the second Cannabis sativa L. plant is not resistant to powdery mildew.
  • the method includes selecting an offspring Cannabis sativa L.
  • the selected offspring Cannabis sativa L. plant produces flowers with greater than 22% tetrahydrocannabinolic acid (THCA) ( t/wt). In some embodiments, the selected offspring Cannabis sativa L. plant produces flowers with greater than 23%, 24%, or 25% THCA (wt/wt).
  • the resistance gene is located on Chromosome 2 of GenBank assembly accession no. GCA_900626175.2. In some embodiments, the resistance gene encodes a protein in the NB-ARC-LRR family.
  • the resistance gene encodes a transcript comprising a polynucleotide at least 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% identical to a polynucleotide selected from the group consisting of SEQ ID NOs: 1-28.
  • the sequence of the resistance gene encodes one or more transcripts comprising a polynucleotide selected from the group consisting of SEQ ID NOs: 1-28.
  • the resistance gene encodes a transcript comprising a polynucleotide at least 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% identical to a polynucleotide selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 27. In some embodiments, the resistance gene encodes a transcript comprising a polynucleotide selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 27. In some embodiments, the resistance gene encodes a transcript comprising SEQ ID NO: 6. In some embodiments, the resistance gene encodes a transcript comprising SEQ ID NO: 27.
  • the powdery mildew resistance gene encodes a protein comprising one or more polypeptides selected from the group consisting of SEQ ID NOs: 29-56.
  • the one or more powdery mildew resistance genes encodes a protein comprising a sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to one or more of SEQ ID NOs: 29-56.
  • the resistance gene encodes a protein comprising a polypeptide selected from the group consisting of SEQ ID NOs: 29-56.
  • the resistance gene encodes a protein comprising a polypeptide at least 75% identical, 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% identical to a polypeptide selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO:55. In some embodiments, the resistance gene encodes a protein comprising a polypeptide selected from the group consisting of SEQ ID NO: 34 and SEQ ID NO: 55. In some embodiments, the resistance gene encodes a protein comprising a polypeptide of SEQ ID NO: 34. In some embodiments, the resistance gene encodes a protein comprising a polypeptide of SEQ ID NO: 55.
  • the one or more powdery mildew resistance genes encodes an NB-LRR family disease resistance protein.
  • the one or more powdery mildew resistance genes encodes a protein comprising a leucine-rich repeat (accession no. 034836 as defined by NCBI Conserved Domain Database; and/or IPR032675 as defined by InterProScan), an NB-ARC domain (accession no. pfam00931 as defined by NCBI Conserved Domain Database; and/or IPR002182 as defined by InterProScan), and/or an RX-CC-like coiled-coil domain (accession no.
  • the one or more powdery mildew resistance genes encodes a protein comprising a PLN03150 domain (accession no. PLN03150 as defined by NCBI Conserved Domain Database).
  • the selected offspring Cannabis sativa L. plant produces at least 41g per plant of harvestable floral material. In some embodiments, the selected offspring Cannabis sativa L. plant produces at least 42g, 43g, 44g, 45g, 46g, or 47g per plant of harvestable floral material. In some embodiments, the selected offspring Cannabis sativa L.
  • the selected offspring Cannabis sativa L. plant has increased lateral branching as compared to the first Cannabis sativa L. plant.
  • the selected offspring Cannabis sativa L. plant has at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% more top of the canopy flowers as compared to the first Cannabis sativa L. plant.
  • the selected offspring Cannabis sativa L. plant has increased flower site stacking as compared to the first Cannabis sativa L. plant.
  • Example 1 Identification of a powdery mildew resistant plant
  • Example 2 A single dominant gene is likely associated with disease resistance
  • Plants were maintained through maturity (flowering) to confirm the integrity of resistance in all phenological stages. Plants were scored as “resistant” or “susceptible” since intermediate phenotypes were not apparent. Fifty-five clonally propagated individuals from the “PNW39” x “Jumping Jack” population were re-infected to provide replication and validation of initial scores. Thus, segregating phenotypic resistance was observed in three separate trials spanning two genetic backgrounds.
  • Table 1 Chi-squared tests for goodness of fit among each mapping population.
  • a total of 39,801 SNP markers were curated for the “PNW39” x “Jumping Jack” population, expressed as genotypes “AA,” “AB,” and “BB.”
  • markers were removed: (i) those with >10% missing data, (ii) those with monomorphic allele scores, and (iii) markers with no “AB” genotypes, as all polymorphic loci in this population structure should have heterozygous clusters.
  • Markers were characterized as type B3.7 (ab x ab if both parental genotypes were heterozygous, then saved for linkage map construction if the progeny segregated in the expected ratio of 1:2: 1 (aa:ab:bb) according to a chi-squared test. This resulted in a final total of 1,889 high-quality type B3.7 SNP markers.
  • the first stage of genetic mapping in Fl populations includes generating separate linkage maps for each heterozygous parent; thus, the remaining markers were characterized as type DI.10 (ab x aa) or D2.15 (aa x ab). Markers were discarded if the progeny failed to reflect 1: 1 segregation according to a chi-squared test. This resulted in a total of 3,779 type DI.10 markers and 4,220 type D2.15 markers.
  • This marker bin (130.1/145.3 cM, or 95.39/96.15 Mb) placed locus LH3804 in a distal position on the long arm of chromosome 2 of “CBDRx” (accession no. LR213632.1, formerly chromosome 6, accession no. UZAU01000623.1) (FIG. 2).
  • the physical locations of the probe sequences for the additional markers on the linkage map largely corroborate their placement on “CBDRx” chromosome 2, except for proximal regions with low marker density.
  • This SNP assay was used to test progeny from both segregating populations in 10 pL qPCR reactions containing: 5 pL TaqPathTM ProAmpTM Master Mix (Applied Biosystems), 0.5 pL of the SNP Genotyping Assay (20x), IpL of template DNA, and water.
  • Samples were run in a QuantStudioTM 5 (Applied Biosystems) using the “Fast” and “Genotyping” protocol with the following cycling conditions: a pre-read at 60°C for 30 seconds; 95°C for 5 minutes; 40 cycles of 95°C for 5 seconds followed by 60°C for 30 seconds; and a post-read at 60°C for 30 seconds. Genotyping calls were made automatically by the QuantStudioTM 5 software and manually checked for accuracy.
  • the qPCR SNP genotyping assay PM_LH3804 classified Fl progeny from the “PNW39” x “Jumping Jack” population in two distinct clusters representing the susceptible (AA) and resistant (AG) individuals (FIG. 3A). The SNP genotyping assay also confirmed that the resistant parent “PNW39” was heterozygous for the resistance allele. None of the individuals were classified as “GG,” in accordance with hypothesized inheritance patterns. [0075] PM_LH3804 assay results from the “PNW39” x “Dwyl337” cross were unexpected insofar as a third genotype (representing “GG”) was indicated in the SNP analysis (FIG. 3B) despite homozygous resistance being unlikely in this population. All susceptible progeny were scored as “AA,” but two genotype clusters represented the resistant progeny (19 individuals with “AG” and 30 individuals with “GG”) (FIG. 3B). Despite the anomaly no recombinants were observable in this second population.
  • Example 4 The powdery mildew resistance gene lies within a cluster of R genes
  • Table 2 Annotated resistance-like genes on chromosome 2 of the Cannabis sativa “CBDRx” genome surrounding the location of the LH3804 locus.
  • Hybrid cultivars are both resistant to powdery mildew and have significantly higher yield and concentrations of THCA than the parental “PNW39” cultivar
  • This Example demonstrates that hybrid cultivars of Cannabis sativa L. plants derived from crossing a resistant “PNW39” with a susceptible second parent are resistant to powdery mildew and have higher THCA concentrations than their “PNW39” parent.
  • Two cultivars of progeny of “PNW39” and a susceptible second parent were assayed for THCA content, yield, and resistance to powdery mildew.
  • Both “CB” and “WS” were found to be resistant to infection by powdery mildew. These values are all represented in Table 3.
  • Table 3 Quantification of THCA and yield from resistant parent and progeny cultivars.
  • Transcriptomic analysis of nine powdery mildew-resistant cultivars was performed using flower tissue from material during the 7 th week of the flowering cycle, with 3 replicates of each cultivar assayed on HiSeq4000, resulting in 30x genome coverage. At the time of sampling, plants were not under disease pressure from G. ambrosiae. Two assembly approaches were used: i) a genome-guided assembly using reads from all assayed cultivars (“the consensus”), and ii) de novo transcriptome assembly for each of the nine cultivars assayed. Blastn was used to search for the marker LH3804 in the consensus transcriptome sequence, in order to identify putative powdery mildew resistance genes.
  • SEQ ID NOs: 1-28 were identified as putative transcripts from powdery mildew resistance genes, encoding polypeptides SEQ ID NOs: 29-56.
  • Statistically significant blast results returned putative R genes belonging to two families: LRR-like proteins, or leucine-rich repeat-like proteins, and RPP8-like proteins, or RECOGNITION OF PERONOSPORA PARASITICA 8-like proteins, consistent with the location of LH3804 in the CslO genome.
  • the identified sequences from the consensus were then blasted against the CslO genome to confirm their location on the tail end of chromosome 2 of the CslO genome.
  • nucleotide sequences SEQ ID NOs: 6 and 27 were translated into amino acid sequences SEQ ID NOs: 34 and 55 respectively using the ExPasy translation tool, and SEQ ID NOs: 34 and 55 were then analyzed using the InterProScan online protein domain classification tool.
  • SEQ ID NO: 6 encodes the 960 amino acid SEQ ID NO: 34, containing a C- terminal RX-like coiled coil domain, a central NB-ARC (nucleotide binding adaptor shared by APAF-1, R proteins, and CED-4 domain, and an N-terminal Leucine-rich-repeat (LRR) domain. These domains make up a classic structure that places the protein encoded by this transcript as a plant disease resistance protein in the NB-ARC-LRR protein family IPR044974.
  • NB-ARC nucleotide binding adaptor shared by APAF-1
  • R proteins and CED-4 domain
  • LRR N-terminal Leucine-rich-repeat
  • the predicted protein shows a high level of homology to the Arabidopsis resistance protein RPP8, which is knopwn to function in the hypersensitive response to multiple biotrophic pathogens, including pathogens of the lifestyle employed by Golovinomyces ambrosiae.
  • SEQ ID NO: 27 encodes the 937 amino acid SEQ ID NO: 55, containing the same predicted domains as SEQ ID NO: 34 and belonging to the same protein family, IPR044974.
  • BLAST searches using SEQ ID NO: 34 reveal a high level of homology to Arabidopsis disease resistance protein CAR1 (CEL Activated Resistance- 1), which is known to function in a similar manner to RPP8 yet is a distinct gene.
  • CAR1 CEL Activated Resistance- 1
  • transcripts from SEQ ID NOs: 1-28 were found to be partial transcripts of either SEQ ID NO: 6 or SEQ ID NO: 27, or were expressed at levels much higher or lower than expected for an NB-ARC-LRR protein.
  • the genes of chromosome 2 that produce the transcripts of SEQ ID NOs: 6 and 27 were the primary candidates for PM1, genes for producing resistance to powdery mildew.

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Abstract

La présente invention concerne une plante de Cannabis sativa L isolée qui est résistante au mildiou poudreux et a des taux élevés d'acide tétrahydrocannabinolique. La résistance est conférée par des gènes de résistance au mildiou poudreux ; des produits de transcription et protéines obtenus à partir de gènes de résistance au mildiou poudreux sont fournis. L'invention concerne également des plantes résistantes au mildiou poudreux isolées présentant des rendements accrus, une ramification latérale accrue, un empilement de sites de fleurs accru et/ou un nombre accru de fleurs de sommet de la canopée. L'invention concerne en outre des procédés de fourniture et de sélection d'une plante de Cannabis sativa L qui est résistante au mildiou poudreux.
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