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WO2021168396A1 - Marqueurs de détermination du sexe chez le cannabis et leur utilisation en sélection - Google Patents

Marqueurs de détermination du sexe chez le cannabis et leur utilisation en sélection Download PDF

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Publication number
WO2021168396A1
WO2021168396A1 PCT/US2021/018993 US2021018993W WO2021168396A1 WO 2021168396 A1 WO2021168396 A1 WO 2021168396A1 US 2021018993 W US2021018993 W US 2021018993W WO 2021168396 A1 WO2021168396 A1 WO 2021168396A1
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Prior art keywords
cannabis plant
seq
cannabis
nucleic acid
plant
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Inventor
Jaime SHERIDAN
Scott Smith
Richard LINCHANGCO
Eric Jackson
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Icaro Plant Science Inc
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Icaro Plant Science Inc
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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
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/28—Cannabaceae, e.g. cannabis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the present invention relates to the field of plant breeding and molecular biology and more specifically to methods and compositions for identifying male, female, and hermaphrodite Cannabis plants.
  • Cannabis is a genus of flowering plants that includes at least three species, Cannabis sativa, Cannabis indica , and Cannabis ruderalis. Cannabis plants have been cultivated for a variety of uses including making fibers (hemp), medicinal use and recreational drug use. Cannabis is also commonly known as marijuana.
  • Cannabis is normally dioecious, having female and male reproduction parts on separate plants. Plants containing both male and female flowers also exist and are referred to as monoecious, or hermaphrodite. Female flowers are characterized by pistils protruding from a calyx. The resinous glandular trichomes of the calyx are the primary site of cannabinoid synthesis. The ovaries are contained within the female calyx and, therefore, the calyx is site of seed development after fertilization by male pollen.
  • Female Cannabis plants are desirable for female floral biomass as well as the cannabinoids, terpenes, and other valuable compounds synthesized within the female floral tissue. While the male plants may be useful as pollinators, many breeders and growers find little use in maintaining male plants.
  • the presently disclosed subject matter relates generally to molecular markers associated with sex determination of Cannabis plants.
  • the disclosure relates to markers for distinguishing female plants, male plants, and hermaphrodite plants.
  • the sex-specific markers include CS 5987852 (SEQ ID NO: 1), CS 6146349 (SEQ ID NO: 2), CS_7650949 (SEQ ID NO: 3), CS_16184248 (SEQ ID NO: 4), CS_22082302 (SEQ ID NO: 5), CS_22999568 (SEQ ID NO: 6), CS_27555112 (SEQ ID NO: 7), and CS_34238211 (SEQ ID NO: 8).
  • the presence of an ‘A’ at CS 34238211 in the homozygous state indicates a female plant.
  • the heterozygous state at CS 34238211 in the heterozygous state indicates a male plant.
  • Hermaphrodite plants have an ‘A’ at CS 34238211 in the homozygous state in combination with some plurality of CS_5987852, CS_6146349, CS_7650949, CS_16184248, CS_22082302, CS_22999568, and CS_27555112 in the heterozygous state.
  • Methods of identifying the sex of a Cannabis plant comprising analyzing a nucleic acid sample from the Cannabis plant for the presence of an allele of one or more of the aforementioned markers and identifying the sex of the Cannabis plant based on the presence of the allele are provided.
  • Methods of identifying a female Cannabis plant in a population comprising analyzing a nucleic acid sample from the Cannabis plant for the presence of an allele of the aforementioned markers are provided.
  • Methods of determining the propensity of a female Cannabis plant to produce male flower parts comprising analyzing a nucleic acid sample from the female Cannabis plant for the presence of an allele of CS_22082302, CS_22999568, CS_27555112, CS_5987852, and CS 6146349 are provided.
  • Methods of improving female flower or cannabinoid production of a population of Cannabis plants comprising obtaining a nucleic sample from one or more Cannabis plants; analyzing the nucleic acid sample for the presence of an allele of the aforementioned markers that identifies a female Cannabis plant; removing Cannabis plants from the population that are not identified as female; and harvesting female flower and/or cannabinoids from the female Cannabis plants are provided.
  • Methods of Cannabis plant breeding comprising analyzing a nucleic acid sample from a Cannabis plant for the presence of an allele of the aforementioned markers; identifying the sex of the Cannabis plant based on the presence of the allele; selecting a female Cannabis plant, a male Cannabis plant, or a hermaphrodite Cannabis plant; and crossing the selected Cannabis plant with a second Cannabis plant to produce Cannabis seed are provided.
  • Methods of detecting in at least one Cannabis plant a genotype associated with sex determination comprising detecting in at least one Cannabis plant an allele of at least one of the aforementioned markers are provided.
  • Methods of introgressing a sex determination allele into a Cannabis plant comprising crossing a first Cannabis plant with a second Cannabis plant in order to form a population; genotyping at least one Cannabis plant in the population with respect to an aforementioned marker; and selecting from the population at least one Cannabis plant comprising the desired sex determination allele are provided.
  • isolated or recombinant nucleic acids comprising (a) a polynucleotide sequence that is at least about 80% identical to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8; or (b) a polynucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8; or (c) a fragment comprising at least about 20 contiguous nucleotides of (a) or (b); or (d) a complement of (a), (b) or (c).
  • Isolated or recombinant nucleic acid comprising (a) a polynucleotide sequence that is at least about 80% identical to the sequence set forth in one of more of SEQ ID NOs: 9-26; or (b) a polynucleotide sequence set forth in one of more of SEQ ID NOs: 9-26; or (c) a complement of (a) or (b) are provided.
  • Uses of the aforementioned isolated or recombinant nucleic acids for determining the sex of a Cannabis plant are also provided.
  • the present disclosure relates to Cannabis markers to accurately identify male, female, and hermaphrodite plants.
  • the ability to not only differentiate between male and female plants but additionally potential hermaphrodites provide the industry a unique and powerful capability.
  • the disclosure provides the information necessary to develop novel Cannabis lines and a mechanism to eliminate male plants and hermaphroditic pollen from production systems.
  • the term “about,” as used herein, refers to variation in the numerical quantity that can occur, for example, through typical measuring techniques and equipment, with respect to any quantifiable variable, including, but not limited to, mass, volume, time, and temperature. Further, given solid and liquid handling procedures used in the real world, there is certain inadvertent error and variation that is likely through differences in the manufacture, source, or purity of the ingredients used to make the compositions or carry out the methods and the like. The term “about” also encompasses these variations. Whether or not modified by the term “about,” the claims include equivalents to the quantities.
  • compositions of the present invention may comprise, consist essentially of, or consist of the components and ingredients of the present invention as well as other ingredients described herein.
  • consisting essentially of means that the methods, systems, apparatuses and compositions may include additional steps, components or ingredients, but only if the additional steps, components or ingredients do not materially alter the basic and novel characteristics of the claimed methods, systems, apparatuses, and compositions.
  • allele refers to one of two or more different nucleotide sequences that occur at a specific locus.
  • amplifying in the context of nucleic acid amplification is any process whereby additional copies of a selected nucleic acid (or a transcribed form thereof) are produced.
  • Typical amplification methods include various polymerase based replication methods, including the polymerase chain reaction (PCR), ligase mediated methods such as the ligase chain reaction (LCR) and RNA polymerase based amplification (e.g., by transcription) methods.
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • RNA polymerase based amplification e.g., by transcription
  • association with refers to, for example, a nucleic acid and a phenotypic trait, that are in linkage disequilibrium, i.e., the nucleic acid and the trait are found together in progeny plants more often than if the nucleic acid and phenotype segregated independently.
  • “Backcrossing” refers to the process whereby hybrid progeny are repeatedly crossed back to one of the parents.
  • the “donor” parent refers to the parental plant with the desired gene/genes, locus/loci, or specific phenotype to be introgressed.
  • the “recipient” parent (used one or more times) or “recurrent” parent (used two or more times) refers to the parental plant into which the gene or locus is being introgressed. For example, see Ragot, M. et al. (1995) Marker-assisted backcrossing: a practical example, in Techniques et Utilisations des Marqueurs Mole Les Colloques, Vol. 72, pp.
  • Croannabinoids refers to a class of chemical compounds that act on the cannabinoid receptors. “Endocannabinoids” are produced naturally in animals, including humans. “Phytocannabinoids” are naturally-occurring cannabinoids produced in plants. Cannabis species express at least 85 different phytocannabinoids, which are concentrated in resin produced in glandular trichomes. The phytocannabinoids are divided into subclasses based on, including cannabigerols, cannabichromenes, cannabidiols, tetrahydrocannabinol s, cannabinols and cannabinodiols, and other cannabinoids.
  • Cannabinoids found in Cannabis include, without limitation: cannabigerol (CBG), cannabichromene (CBC), cannabidiol (CBD), tetrahydrocannabinol (THC), cannabinol (CBN) and cannabinodiol (CBDL), cannabicyclol (CBL), cannabivarin (CBV), tetrahydrocannabivarin (THCV), cannabidivarin (CBDV), cannabichromevarin (CBCV), cannabigerovarin (CBGV), cannabigerol monomethyl ether (CBGM), cannabinerolic acid, cannabidiolic acid (CBDA), Cannabinol propyl variant (CBNV), cannabitriol (CBO), tetrahydrocannabinolic acid (THCA), and tetrahydrocannabivarinic acid (THCVA).
  • CBG cannabige
  • Cannabis ,” “ Cannabis species,” “hemp,” or “marijuana” refers to a flowering plant in the genus Cannabis including the species (or sub-species) Cannabis sativa, Cannabis ruderalis , and Cannabis indica.
  • Cannabis cultivar and “ Cannabis strain” are used interchangeably herein, and refer to Cannabis plants that have been selected for one or more desirable characteristics and propagated. Where the term cultivar or strain is used, it is to be understood that the cultivar or strain may be naturally-occurring, a result of breeding, and/or the result of genetic manipulation. Propagation may occur in any manner, including, without limitation, sexual reproduction (e.g., seed), cloning (e.g., cuttings, vegetative propagation), self- pollinization, and the like.
  • centiMorgan means a unit of measure of recombination frequency.
  • centimorgan is equal to a 1% chance that a marker at one genetic locus will be separated from a marker at a second locus due to crossing over in a single generation.
  • 1 centiMorgan is equivalent, on average, to 1 million base pairs. It is a unit of crossover frequency in linkage maps of chromosomes equal to one hundredth of a morgan.
  • crossing means the fusion of gametes via pollination to produce progeny (i.e., cells, seeds or plants).
  • progeny i.e., cells, seeds or plants.
  • the term encompasses both sexual crosses (the pollination of one plant by another) and selling (self- pollination, i.e., when the pollen and ovule are from the same plant or from genetically identical plants).
  • genomic is used to refer to, e.g., a cDNA and an mRNA encoded by the genomic sequence, as well as to that genomic sequence.
  • genetically linked refers to genetic loci that are in linkage disequilibrium and statistically determined not to assort independently. Genetically linked loci assort dependently from 51% to 99% of the time or any value there between, such as at least 60%, 70%, 80%, 90%, 95% or 99%.
  • genotype is the genetic constitution of an individual (or group of individuals) at one or more genetic loci, as contrasted with the observable trait (phenotype). Genotype is defined by the allele(s) of one or more known loci that the individual has inherited from its parents.
  • genotype can be used to refer to an individual's genetic constitution at a single locus, at multiple loci, or, more generally, the term genotype can be used to refer to an individual's genetic make-up for all the genes in its genome.
  • Fragments is intended to mean a portion of a nucleotide sequence. Fragments can be used as hybridization probes or PCR primers using methods disclosed herein.
  • An individual is “heterozygous” if more than one allele type is present at a given locus (e.g., a diploid individual with one copy each of two different alleles).
  • An individual is “homozygous” if the individual has only one type of allele at a given locus (e.g., a diploid individual has a copy of the same allele at a locus for each of two homologous chromosomes).
  • homologous refers to nucleic acid sequences that are derived from a common ancestral gene through natural or artificial processes (e.g., are members of the same gene family) and thus, typically, share sequence similarity. Typically, homologous nucleic acids have sufficient sequence identity that one of the sequences or its complement is able to selectively hybridize to the other under selective hybridization conditions.
  • selectively hybridizes includes reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non target nucleic acids.
  • Selectively hybridizing sequences have about at least 80% sequence identity, often at least 90% sequence identity and may have 95%, 97%, 99% or 100% sequence identity with each other.
  • a nucleic acid that exhibits at least some degree of homology to a reference nucleic acid can be unique or identical to the reference nucleic acid or its complementary sequence.
  • host cell means a cell which contains a heterologous nucleic acid, such as a vector and supports the replication and/or expression of the nucleic acid.
  • Host cells may be prokaryotic cells such as E. coli or eukaryotic cells such as plant, yeast, insect, amphibian or mammalian cells.
  • host cells are monocotyledonous or dicotyledonous plant cells.
  • one particularly preferred dicotyledonous host cell is a Cannabis host cell.
  • interval refers to a continuous linear span of chromosomal DNA with termini defined by and including molecular markers.
  • nucleic acid introduction when referring to a heterologous or isolated nucleic acid refers to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell where the nucleic acid can be incorporated into the genome of the cell (e.g., chromosome, plasmid, plastid or mitochondrial DNA), converted into an autonomous replicon or transiently expressed (e.g., transfected mRNA).
  • nucleic acid introduction means as “transfection,” “transformation” and “transduction.”
  • introgression refers to the transmission of a desired allele of a genetic locus from one genetic background to another.
  • introgression of a desired allele at a specified locus can be transmitted to at least one progeny plant via a sexual cross between two parent plants, where at least one of the parent plants has the desired allele within its genome.
  • transmission of an allele can occur by recombination between two donor genomes, e.g., in a fused protoplast, where at least one of the donor protoplasts has the desired allele in its genome.
  • the desired allele can be, e.g., a transgene or a selected allele of a marker or quantitative trait locus.
  • isolated refers to material, such as a nucleic acid or a protein, which is substantially free from components that normally accompany or interact with it in its naturally occurring environment.
  • the isolated material optionally comprises material not found with the material in its natural environment, e.g., a cell.
  • the material if the material is in its natural environment, such as a cell, the material has been placed at a location in the cell (e.g., genome or subcellular organelle) not native to a material found in that environment.
  • a naturally occurring nucleic acid e.g., a promoter
  • Nucleic acids which are “isolated” as defined herein, are also referred to as “heterologous” nucleic acids.
  • life cycle refers to the progression of a plant through various stages of growth. Cannabis plants go through a vegetative stage of growth, followed by a flowering cycle. The period of growth between germination or cutting rooting and flowering is known as the vegetative phase of plant development. Vegetation is the sporophytic state of the Cannabis plant. Plants do not produce resin or flowers during the vegetative stage and are bulking up to a desired production size for flowering. During the vegetative phase, plants are busy carrying out photosynthesis and accumulating resources that will be needed for flowering and reproduction.
  • “Vegetation cycle” or “vegetative phase” refers to the period of growth between germination or cutting rooting. Vegetation is the sporophytic state of the Cannabis plant. This is a form of asexual reproduction in plants during which plants do not produce resin or flowers. The plant is bulking up biomass to a desired production size for flowering. During the vegetative phase, plants are busy carrying out photosynthesis and accumulating resources that will be needed for flowering and reproduction.
  • “Flowering cycle” or “flowering phase” refers to the period during which the plant produces buds and flowers. This is the reproductive phase of plant growth. Cannabis is dioecious, having female and male reproduction parts on separate plants. Flowering is the gametophytic or reproductive state of Cannabis. For production, only female flowers are selected for cultivation.
  • linkage disequilibrium refers to a non-random segregation of genetic loci. This implies that such loci are in sufficient physical proximity along a length of a chromosome that they tend to segregate together with greater than random frequency.
  • marker refers to a genetic locus (a “marker locus”) used as a point of reference when identifying genetically linked loci.
  • the term may also refer to nucleic acid sequences complementary to the genomic sequences, such as nucleic acids used as probes or primers.
  • the primers may be complementary to sequences upstream or downstream of the marker sequences.
  • the term can also refer to amplification products associated with the marker.
  • the term can also refer to alleles associated with the markers. Allelic variation associated with a phenotype allows use of the marker to distinguish germplasm on the basis of the sequence.
  • a “marker allele”, alternatively an “allele of a marker locus”, can refer to one of a plurality of polymorphic nucleotide sequences found at a marker locus in a population.
  • “Marker assisted selection” is a process by which individual plants are selected based on marker genotypes.
  • the terms “nucleic acid,” “polynucleotide,” “polynucleotide sequence” and “nucleic acid sequence” refer to single-stranded or double-stranded deoxyribonucleotide or ribonucleotide polymers, or chimeras thereof.
  • the terms can additionally or alternatively include analogs of naturally occurring nucleotides having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids).
  • a particular nucleic acid sequence of this invention optionally encompasses complementary sequences, in addition to the sequence explicitly indicated.
  • a “plant” can be a whole plant, any part thereof, or a cell or tissue culture derived from a plant.
  • the term “plant” can refer to any of: whole plants, plant components or organs (e.g., leaves, stems, roots, etc.), plant tissues, seeds, plant cells, and/or progeny of the same.
  • a plant cell is a cell of a plant, taken from a plant, or derived through culture from a cell taken from a plant.
  • proximal or distal refer to a genetically linked marker being either closer (proximal) or further away (distal) to the marker region in reference.
  • recombinant indicates that the material (e.g., a nucleic acid or protein) has been synthetically (non-naturally) altered by human intervention.
  • the alteration to yield the synthetic material can be performed on the material within or removed from its natural environment or state.
  • a naturally occurring nucleic acid is considered a recombinant nucleic acid if it is altered, or if it is transcribed from DNA which has been altered, by means of human intervention performed within the cell from which it originates. See, e.g., “Compounds and Methods for Site Directed Mutagenesis in Eukaryotic Cells”, Kmiec, U.S. Pat. No. 5,565,350; “in Vivo Homologous Sequence Targeting in Eukaryotic Cells”. Zarling, et al., PCT/US93/03868.
  • single nucleotide polymorphism is a DNA sequence variation occurring when a single nucleotide — A, T, C or G — in the genome (or other shared sequence) differs between members of a species (or between paired chromosomes in an individual).
  • SNP single nucleotide polymorphism
  • the sex discrimination assay uses a single locus (CS 34238211) on Cannabis sativa chromosome 1 to determine if a plant will be male or female.
  • the sex discrimination assay also uses five additional loci (CS_22082302, CS_22999568, CS_27555112,
  • the present invention provides molecular markers, (i.e. including marker loci and nucleic acids corresponding to (or derived from) these marker loci, such as probes and amplification products) useful for genotyping plants, correlated with sex determination in Cannabis , for example CS_5987852, CS_6146349, CS_7650949, CS_16184248, CS_22082302, CS_22999568, CS_27555112, and CS_34238211 described below.
  • Such molecular markers are useful for selecting Cannabis plants that are female, male, or hermaphrodite. These markers are also useful for marker assisted selection (MAS) and breeding.
  • molecular markers can include restriction fragment length polymorphisms (RFLP), random amplified polymorphic DNA (RAPD), amplified fragment length polymorphisms (AFLP), single nucleotide polymorphisms (SNP) or simple sequence repeats (SSR).
  • RFLP restriction fragment length polymorphisms
  • RAPD random amplified polymorphic DNA
  • AFLP amplified fragment length polymorphisms
  • SNP single nucleotide polymorphisms
  • SSR simple sequence repeats
  • Simple sequence repeats or microsatellites are regions of DNA where one to a few bases are tandemly repeated for few to hundreds of times. For example, a dinucleotide repeat would resemble CACACACA and a trinucleotide repeat would resemble ATGATGATGATG. Simple sequence repeats are thought to be generated due to slippage mediated errors during DNA replication, repair and recombination. Over time, these repeated sequences vary in length between one cultivar and another.
  • An example of allelic variation in SSRs would be: Allele A being GAGAGAGA (4 repeats of the GA sequence) and allele B being GAGAGAGAGA (6 repeats of the GA sequence).
  • SSRs When SSRs occur in a coding region, their survival depends on their impact on structure and function of the encoded protein. Since repeat tracks are prone to DNA-slippage mediated expansions/deletions, their occurrences in coding regions are limited by non-perturbation of the reading frame and tolerance of expanding amino acid stretches in the encoded proteins. Among all possible SSRs, tri -nucleotide repeats or multiples thereof are more common in coding regions.
  • a single nucleotide polymorphism is a DNA sequence variation occurring when a single nucleotide — A, T, C or G — differs between members of a species (or between paired chromosomes in an individual). For example, two sequenced DNA fragments from two individuals, AAGCCTA to AAGCTTA, contain a difference in a single nucleotide. In this case, there are two alleles: C and T.
  • Markers corresponding to genetic polymorphisms between members of a population can be detected by numerous methods, well-established in the art (e.g., restriction fragment length polymorphisms, isozyme markers, allele specific hybridization (ASH), amplified variable sequences of the plant genome, self-sustained sequence replication, simple sequence repeat (SSR), single nucleotide polymorphism (SNP) or amplified fragment length polymorphisms (AFLP)).
  • restriction fragment length polymorphisms e.g., restriction fragment length polymorphisms, isozyme markers, allele specific hybridization (ASH), amplified variable sequences of the plant genome, self-sustained sequence replication, simple sequence repeat (SSR), single nucleotide polymorphism (SNP) or amplified fragment length polymorphisms (AFLP)
  • SSR simple sequence repeat
  • SNP single nucleotide polymorphism
  • AFLP amplified fragment length polymorphisms
  • hybridization formats include but are not limited to, solution phase, solid phase, mixed phase or in situ hybridization assays.
  • Markers which are restriction fragment length polymorphisms (RFLP) are detected by hybridizing a probe (which is typically a sub- fragment or a synthetic oligonucleotide corresponding to a sub-fragment of the nucleic acid to be detected) to restriction digested genomic DNA.
  • the restriction enzyme is selected to provide restriction fragments of at least two alternative (or polymorphic) lengths in different individuals and will often vary from line to line.
  • Determining a (one or more) restriction enzyme that produces informative fragments for each cross is a simple procedure, well known in the art. After separation by length in an appropriate matrix (e.g., agarose) and transfer to a membrane (e.g., nitrocellulose, nylon), the labeled probe is hybridized under conditions which result in equilibrium binding of the probe to the target followed by removal of excess probe by washing.
  • an appropriate matrix e.g., agarose
  • a membrane e.g., nitrocellulose, nylon
  • Nucleic acid probes to the marker loci can be cloned and/or synthesized.
  • Detectable labels suitable for use with nucleic acid probes include any composition detectable by spectroscopic, radioisotopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels include biotin for staining with labeled streptavidin conjugate, magnetic beads, fluorescent dyes, radiolabels, enzymes and colorimetric labels.
  • Other labels include ligands which bind to antibodies labeled with fluorophores, chemiluminescent agents and enzymes. Labeling markers is readily achieved such as by the use of labeled PCR primers to marker loci.
  • the hybridized probe is then detected using, most typically, autoradiography or other similar detection technique (e.g., fluorography, liquid scintillation counter, etc.). Examples of specific hybridization protocols are widely available in the art.
  • Amplified variable sequences refer to amplified sequences of the plant genome which exhibit high nucleic acid residue variability between members of the same species. All organisms have variable genomic sequences and each organism (with the exception of a clone) has a different set of variable sequences. Once identified, the presence of specific variable sequence can be used to predict phenotypic traits.
  • DNA from the plant serves as a template for amplification with primers that flank a variable sequence of DNA. The variable sequence is amplified and then sequenced.
  • RNA polymerase mediated techniques e.g., NASBA
  • RNA can be converted into a double stranded DNA suitable for restriction digestion, PCR expansion and sequencing using reverse transcriptase and a polymerase.
  • Oligonucleotides for use as primers are typically synthesized chemically according to the solid phase phosphoramidite triester method described by Beaucage and Caruthers, (1981) Tetrahedron Lett. 22:1859 or can simply be ordered commercially.
  • self-sustained sequence replication can be used to identify genetic markers.
  • Self-sustained sequence replication refers to a method of nucleic acid amplification using target nucleic acid sequences which are replicated exponentially in vitro under substantially isothermal conditions by using three enzymatic activities involved in retroviral replication: (1) reverse transcriptase, (2) Rnase H and (3) a DNA-dependent RNA polymerase (Guatelli, et al., (1990) Proc Natl Acad Sci USA 87: 1874). By mimicking the retroviral strategy of RNA replication by means of cDNA intermediates, this reaction accumulates cDNA and RNA copies of the original target.
  • markers of the present invention include single nucleotide polymorphisms.
  • sequence information from each locus was used to create a sex assay using molecular beacon technology.
  • Forward and reverse primers were designed to amplify the target region containing the single nucleotide polymorphism (SNP).
  • SNP single nucleotide polymorphism
  • two different competitive forward primers were designed which differ by the 3’ terminal base.
  • a unique 5’ tail sequence does not match the template DNA but rather is homologous to a secondary probe sequence (reporting cassette) that contains a specific fluorophore molecule.
  • Table 2 shows the primers and probe Set which determine male or female hemp ( Cannabis sativa ) plants.
  • the assay chemistry uses a common primer with two allele specific probes which target the single nucleotide polymorphism (SNP) conditioning the male or female trait.
  • SNP single nucleotide polymorphism
  • Table 3 shows the primers and probe sets which determine the propensity of a female hemp plant to produce male flowers.
  • the assay chemistry uses unique common primers with two allele specific probes for each SNP target at each of the five respective loci.
  • Marker loci and the alleles provided herein can be used in marker assisted selection (MAS) breeding.
  • MAS marker assisted selection
  • Markers containing the causal polymorphism for a trait, or that are within the coding sequence of a causative gene are ideal as no recombination is expected between them and the sequence of DNA responsible for the phenotype.
  • markers do not need to contain or correspond to casual polymorphisms in order to be effective in MAS. In fact, most MAS breeding only uses markers linked to a causal mutation.
  • Genetic markers are used to identify plants that contain a desired genotype at one or more loci, and that are expected to transfer the desired genotype, along with a desired phenotype to their progeny. Genetic markers can be used to identify plants containing a desired genotype at one locus, or at several unlinked or linked loci (e.g., a haplotype), and that would be expected to transfer the desired genotype, along with a desired phenotype to their progeny.
  • MAS uses polymorphic markers that have been identified as having a significant likelihood of co-segregation with a desired trait. Such markers are presumed to map near a gene or genes that give the plant its desired phenotype, and are considered indicators for the desired trait.
  • Identification of plants or germplasm that include a marker locus or marker loci linked to a desired trait or traits provides a basis for performing MAS. Plants that comprise favorable markers or favorable alleles are selected for, while plants that comprise markers or alleles that are negatively correlated with the desired trait can be selected against. Desired markers and/or alleles can be introgressed into plants having a desired (e.g., elite or exotic) genetic background to produce an introgressed plant or germplasm having the desired trait. In some aspects, it is contemplated that a plurality of markers for desired traits are sequentially or simultaneous selected and/or introgressed. The combinations of markers that are selected for in a single plant is not limited, and can include any combination of markers disclosed herein or any marker linked to the markers disclosed herein.
  • the present disclosure provides the means to identify Cannabis plants that comprise a sex determination locus by identifying plants having a specified allele, e.g., at one or more of markers CS_5987852, CS_6146349, CS_7650949, CS_16184248, CS_22082302, CS_22999568, CS_27555112, and CS_34238211. Similarly, by identifying plants lacking the desired allele, plants can be identified and, e.g., eliminated from subsequent crosses.
  • MAS is a powerful shortcut to select for desired phenotypes and for introgressing desired traits into cultivars (e.g., introgressing desired traits into elite lines).
  • MAS is easily adapted to high throughput molecular analysis methods that can quickly screen large numbers of plant or germplasm genetic material for the markers of interest and is much more cost effective than cultivating and observing plants for visible traits.
  • the efficiency of MAS compared to phenotypic screening becomes even greater, because all of the loci can be evaluated together from a single sample of DNA.
  • Backcross breeding is the process of crossing a progeny back to one of its parents. Backcrossing is usually done for the purpose of introgressing one or a few loci from a donor parent into an otherwise desirable genetic background from the recurrent parent. The more cycles of backcrossing that are done, the greater the genetic contribution of the recurrent parent to the resulting variety. This is often necessary, because donor parent plants may be otherwise undesirable, i.e., due to low yield, low fecundity or the like. In contrast, varieties which are the result of intensive breeding programs may have excellent yield, fecundity or the like, merely being deficient in one desired trait. As a skilled worker understands, backcrossing can be done to select for or against a trait.
  • the introgression of one or more desired loci from a donor line into another is achieved via repeated backcrossing to a recurrent parent accompanied by selection to retain one or more loci from the donor parent. Markers associated with sex determination are assayed in progeny and those progeny with one or more desired markers are selected for advancement. In another aspect, one or more markers can be assayed in the progeny to select for plants with the genotype of the parent. This invention anticipates that trait introgression activities will require more than one generation, wherein progeny are crossed to the recurrent parent or selfed.
  • Selections are made based on the presence of one or more sex determination markers and can also be made based on the recurrent parent genotype, wherein screening is performed on a genetic marker and/or phenotype basis.
  • the markers can be used in conjunction with other markers, ideally at least one on each chromosome of the Cannabis genome, to track the introgression of sex determination loci into elite germplasm.
  • the Cannabis plants of the disclosure may be used in a plant breeding program.
  • the goal of plant breeding is to combine, in a single variety or hybrid, various desirable traits.
  • these traits may include, for example, resistance to diseases and insects, tolerance to heat and drought, tolerance to chilling or freezing, reduced time to crop maturity, greater yield and better agronomic quality.
  • uniformity of plant characteristics such as germination and stand establishment, growth rate, maturity and plant and ear height is desirable.
  • Traditional plant breeding is an important tool in developing new and improved commercial crops.
  • This disclosure encompasses methods for producing a plant by crossing a first parent plant with a second parent plant wherein one or both of the parent plants is a plant displaying a phenotype as described herein.
  • Plant breeding techniques known in the art and used in a plant breeding program include, but are not limited to, recurrent selection, bulk selection, mass selection, backcrossing, pedigree breeding, open pollination breeding, restriction fragment length polymorphism enhanced selection, genetic marker enhanced selection, doubled haploids and transformation. Often combinations of these techniques are used.
  • a genetic trait which has been engineered into a particular plant using transformation techniques can be moved into another line using traditional breeding techniques that are well known in the plant breeding arts. For example, a backcrossing approach is commonly used to move a transgene from a transformed plant to an elite inbred line and the resulting progeny would then comprise the transgene(s). Also, if an inbred line was used for the transformation, then the transgenic plants could be crossed to a different inbred in order to produce a transgenic hybrid plant. As used herein, "crossing" can refer to a simple X by Y cross or the process of backcrossing, depending on the context.
  • the development of a hybrid in a plant breeding program involves three steps: (1) the selection of plants from various germplasm pools for initial breeding crosses; (2) the selfing of the selected plants from the breeding crosses for several generations to produce a series of inbred lines, which, while different from each other, breed true and are highly homozygous and (3) crossing the selected inbred lines with different inbred lines to produce the hybrids.
  • the vigor of the lines decreases. Vigor is restored when two different inbred lines are crossed to produce the hybrid.
  • An important consequence of the homozygosity and homogeneity of the inbred lines is that the hybrid created by crossing a defined pair of inbreds will always be the same.
  • Cannabis plants of the present disclosure may be used to produce, e.g., a single cross hybrid, a three-way hybrid or a double cross hybrid.
  • a single cross hybrid is produced when two inbred lines are crossed to produce the FI progeny.
  • a double cross hybrid is produced from four inbred lines crossed in pairs (Ax B and C x D) and then the two FI hybrids are crossed again (Ax B) times (C x D).
  • a three-way cross hybrid is produced from three inbred lines where two of the inbred lines are crossed (A x B) and then the resulting FI hybrid is crossed with the third inbred (Ax B) x C.
  • Much of the hybrid vigor and uniformity exhibited by FI hybrids is lost in the next generation (F2). Consequently, seed produced by hybrids is consumed rather than planted.
  • kits are also provided to facilitate the screening of germplasm for the markers of the present invention.
  • the kits comprise the polynucleotides of the present invention, fragments or complements thereof, for use as probes or primers to detect the markers for the sex determination markers.
  • the kits comprise the polynucleotides of SEQ ID NOs: 9-11, SEQ ID NOs: 12-14, SEQ ID NOs: 15-17, SEQ ID NOs: 18-20, SEQ ID NOs: 21-23, and/or SEQ ID NOs: 24-26. Instructions for using the polynucleotides, as well as buffers and/or other solutions may also be provided to facilitate the use of the polynucleotides.
  • a method of identifying the sex of a Cannabis plant comprising analyzing a nucleic acid sample from the Cannabis plant for the presence of an allele of a marker locus that is associated with sex determination, wherein the marker locus is selected from CS_5987852, CS_6146349, CS_7650949, CS_16184248, CS_22082302, CS_22999568, CS_27555112, and CS_34238211; and identifying the sex of the Cannabis plant based on the presence of the allele.
  • Cannabis plant to a location suitable for the identified sex.
  • a method of identifying a female Cannabis plant in a population comprising analyzing a nucleic acid sample from the Cannabis plant for the presence of an allele of CS 34238211; and identifying a Cannabis plant comprising an ‘A’ at
  • a method of determining the propensity of a female Cannabis plant to produce male flower parts comprising analyzing a nucleic acid sample from the female Cannabis plant for the presence of an allele of CS_22082302, CS_22999568,
  • a method of detecting in at least one Cannabis plant a genotype associated with sex determination comprising detecting in at least one Cannabis plant an allele of at least one marker that is associated with sex determination, wherein the marker is selected from CS_5987852, CS_6146349, CS_7650949, CS_16184248, CS_22082302, CS_22999568, CS_27555112, and CS_34238211.
  • a method of introgressing a sex determination allele into a Cannabis plant comprising crossing a first Cannabis plant with a second Cannabis plant in order to form a population; genotyping at least one Cannabis plant in the population with respect to at least one marker, wherein the marker is selected from CS_5987852, CS_6146349, CS_7650949, CS_16184248, CS_22082302, CS_22999568, CS_27555112, and CS_34238211; and selecting from the population at least one Cannabis plant comprising the desired sex determination allele.
  • a method of improving female flower or cannabinoid production of a population of Cannabis plants comprising: obtaining a nucleic sample from one or more Cannabis plants; analyzing the nucleic acid sample for the presence of an allele of CS_5987852, CS_6146349, CS_22082302, CS_22999568, CS_27555112, and CS 34238211; removing Cannabis plants from the population that are not identified as female; and harvesting female flower and/or cannabinoids from the female Cannabis plants.
  • a method of Cannabis plant breeding comprising analyzing a nucleic acid sample from a Cannabis plant for the presence of an allele of a marker locus that is associated with sex determination, wherein the marker locus is selected from CS_5987852, CS_6146349, CS_7650949, CS_16184248, CS_22082302, CS_22999568, CS_27555112, and CS_34238211; identifying the sex of the Cannabis plant based on the presence of the allele; selecting a female Cannabis plant, a male Cannabis plant, or a hermaphrodite Cannabis plant; and crossing the selected Cannabis plant with a second Cannabis plant to produce Cannabis seed.
  • any one of embodiments 1-16 comprising analyzing the nucleic acid sample of the Cannabis plant for the presence of an allele of two or more, three or more, four or more, five or more, six or more, or seven or more of the marker loci associated with sex determination.
  • any one of embodiments 1-22, wherein the analyzing or detecting comprises amplifying the nucleic acid sample using primers comprising the sequences set forth in SEQ ID NOs: 9-11, SEQ ID NOs: 12-14, SEQ ID NOs: 15-17, SEQ ID NOs: 18-20, SEQ ID NOs: 21-23, and/or SEQ ID NOs: 24-26, or a sequence that is at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, or at least about 99% identical to the sequences set forth in SEQ ID NOs: 9-26.
  • An isolated or recombinant nucleic acid comprising (a) a polynucleotide sequence that is at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, or at least about 99% identical to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8; or (b) a polynucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8; or (c) a fragment comprising at least about 20 contiguous nucleotides of (a) or (b); or (d) a complement of (a), (b) or (c).
  • An isolated or recombinant nucleic acid comprising (a) a polynucleotide sequence that is at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, or at least about 99% identical to the sequence set forth in one of more of SEQ ID NOs: 9-26; or (b) a polynucleotide sequence set forth in one of more of SEQ ID NOs: 9-26; or (c) a complement of (a) or (b).
  • nucleic acid of embodiment 24 or embodiment 25 further comprising a detectable label.
  • kits for determining the sex of a Cannabis plant comprising an isolated or recombinant nucleic acid of any one of embodiments 24-26.
  • the hemp sex algorithm was developed by carefully selecting a training set of lines which best represented global diversity in the species based on whole genome analyses. Individual plants from each line within the training set were evaluated to determine the sex state under controlled greenhouse conditions. The sex state (phenotypes) for each line in the training set were combined with the corresponding allele matrix (genotypes) and a neural net developed a preliminary prediction algorithm using its adaptive learning network. The prediction algorithm was then validated using cross validation with the appropriate hold-out strategy for the trait type, physical validation, and text mining of the global literature. The algorithm was then optimized using a selection network to identify the smallest subset of predictors or loci that accurately predict the trait.
  • the Hemp sex algorithm where predicted male, female, or hermaphrodite ( y ) is based on the corresponding allele state at the i th loci. All females (including hermaphrodites) have reference allele in the homozygous state (AA) at i 8 loci. The alternate allele in the homozygous state (aa) at i 1 , i 2 , i 3 , and i 6 loci and reference allele in the homozygous state (AA) at i 4 , i 5 , i 7 , and i 8 indicates a true female.
  • Hermaphrodites have the reference allele in the homozygous state (AA) at i 8 loci in combination with some plurality of loci in the heterozygous state (Aa).
  • the heterozygous state (Aa) at the i 8 loci in combination with some plurality of loci in the heterozygous state indicates a male.
  • the following allele states were observed in the evaluation of the individuals: where the allele state at the i locus can be homozygous for all loci (AA), some combination of reference homozygous and heterozygous (. AA-a ), or some combination of alternate homozygous and heterozygous (aa-A).
  • the results showed that 24 individuals representing 10 different varieties were female, while 19 individuals representing 10 varieties could enter a hermaphrodite state. Of the 19 individuals, 10 individuals from 5 varieties would more likely become a hermaphrodite and 9 individuals from 5 varieties would less likely become a hermaphrodite.
  • the sex discrimination assay was used to determine the sex of several hemp(C. sativa ) plants. Thirty-three of the female plants were then grown under varying levels of stress and observed for their propensity to develop male flower parts. The allele scores at each locus were used to define haplotypes, calculate a dosage effects, and develop categorical hermaphrodism propensities for each line. The data was then compared to herm type observations. Overall, only one female line which did not show signs of producing male flowers (Herm Type C & D) had a haplotype with high dosage effect and degree of hermaphrodism propensity.
  • Table 5 shows allele scores and dosage effect of the five loci controlling a female plants propensity to produce male flowers along with a categorical propensity score for 33 female lines showing varying levels of conversion to a hermaphrodite state after stress.

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Abstract

La présente divulgation se rapporte à des marqueurs moléculaires et à leur utilisation pour distinguer des plants femelles, mâles, et hermaphrodites de Cannabis. La présente divulgation se rapporte également à un procédé d'identification du sexe de plants de Cannabis à l'aide de ces marqueurs moléculaires. Sont également divulgués des kits de sélection de plants mâles, femelles, et hermaphrodites de Cannabis avant la floraison, des procédés de sélection d'un plant de Cannabis, et des procédés de production de Cannabis.
PCT/US2021/018993 2020-02-21 2021-02-22 Marqueurs de détermination du sexe chez le cannabis et leur utilisation en sélection Ceased WO2021168396A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023137336A1 (fr) * 2022-01-11 2023-07-20 Phylos Bioscience, Inc. Marqueurs de l'hermaphrodisme

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120311744A1 (en) * 2011-06-06 2012-12-06 Erich E. Sirkowski Marked Cannabis For Indicating Medical Marijuana
US20150191780A1 (en) * 2003-10-24 2015-07-09 Sue K. DeNise Methods and systems for inferring traits to breed and manage non-beef livestock
US20160177404A1 (en) * 2011-08-18 2016-06-23 Courtagen Life Sciences Inc. Cannabis genomes and uses thereof
WO2018184031A1 (fr) * 2017-03-31 2018-10-04 Wang Amanda Identificateur de sexe du cannabis
WO2019023751A1 (fr) * 2017-08-01 2019-02-07 Agriculture Victoria Services Pty Ltd Cannabis médicinal
US20190241904A1 (en) * 2015-12-15 2019-08-08 Bayer Cropscience Nv Brassicaceae plants resistant to plasmodiophora brassicae (clubroot)
WO2019222835A1 (fr) * 2018-05-22 2019-11-28 Anandia Laboratories Inc. Identification du sexe de plantes de cannabis
WO2020010102A1 (fr) * 2018-07-03 2020-01-09 New West Genetics Inc. Variété de cannabis permettant de produire plus de 50 % de plantes femelles

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20150191780A1 (en) * 2003-10-24 2015-07-09 Sue K. DeNise Methods and systems for inferring traits to breed and manage non-beef livestock
US20120311744A1 (en) * 2011-06-06 2012-12-06 Erich E. Sirkowski Marked Cannabis For Indicating Medical Marijuana
US20160177404A1 (en) * 2011-08-18 2016-06-23 Courtagen Life Sciences Inc. Cannabis genomes and uses thereof
US20190241904A1 (en) * 2015-12-15 2019-08-08 Bayer Cropscience Nv Brassicaceae plants resistant to plasmodiophora brassicae (clubroot)
WO2018184031A1 (fr) * 2017-03-31 2018-10-04 Wang Amanda Identificateur de sexe du cannabis
WO2019023751A1 (fr) * 2017-08-01 2019-02-07 Agriculture Victoria Services Pty Ltd Cannabis médicinal
WO2019222835A1 (fr) * 2018-05-22 2019-11-28 Anandia Laboratories Inc. Identification du sexe de plantes de cannabis
WO2020010102A1 (fr) * 2018-07-03 2020-01-09 New West Genetics Inc. Variété de cannabis permettant de produire plus de 50 % de plantes femelles

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HARM VAN BAKEL;JAKE M STOUT;ATINA G COTE;CARLING M TALLON;ANDREW G SHARPE;TIMOTHY R HUGHES;JONATHAN E PAGE: "The draft genome and transcriptome of Cannabis sativa", GENOME BIOLOGY, vol. 12, no. 10, R102, 20 October 2011 (2011-10-20), pages 1 - 18, XP021114902, ISSN: 1465-6906, DOI: 10.1186/gb-2011-12-10-r102 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023137336A1 (fr) * 2022-01-11 2023-07-20 Phylos Bioscience, Inc. Marqueurs de l'hermaphrodisme

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