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WO2019125188A1 - Marqueurs génétiques et leurs utilisations - Google Patents

Marqueurs génétiques et leurs utilisations Download PDF

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Publication number
WO2019125188A1
WO2019125188A1 PCT/NZ2018/050186 NZ2018050186W WO2019125188A1 WO 2019125188 A1 WO2019125188 A1 WO 2019125188A1 NZ 2018050186 W NZ2018050186 W NZ 2018050186W WO 2019125188 A1 WO2019125188 A1 WO 2019125188A1
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WO
WIPO (PCT)
Prior art keywords
animal
fgd4
cell
embryo
worth
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/NZ2018/050186
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English (en)
Inventor
Mathew Douglas LITTLEJOHN
Michael Dominic Keehan
Thomas John LOPDELL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
LIVESTOCK IMPROVEMENT Corp Ltd
Original Assignee
LIVESTOCK IMPROVEMENT Corp Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
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Publication of WO2019125188A1 publication Critical patent/WO2019125188A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • 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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/02—Breeding vertebrates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/101—Bovine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/02—Animal zootechnically ameliorated
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • 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
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
    • 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 generally relates to methods of identifying whether or not an animal carries a biological marker linked to animal productivity, more particularly, but not exclusively, to methods of identifying whether or not an animal carries a biological marker having a deleterious effect on animal productivity.
  • the invention also relates to methods for selecting or rejecting one or more animals, cells or embryos, animal evaluation, breeding animals, and herd formation.
  • the invention also relates to biological markers suitable for use in such methods.
  • Dairy animals are typically assessed to determine their suitability, or otherwise, for a particular purpose including breeding, inclusion in a herd, and milk production, on the basis of a number of different traits relating to productivity.
  • traits include production traits including milk protein yield, milk fat yield, milk volume, liveweight, and survival, and traits other than production (TOP) including milking speed, temperament and conformation, for example.
  • TOP production of these traits are used to determine or estimate an animal’s value or worth to a farmer.
  • assessment of and scoring an animal on the basis of a combination of such traits is used to determine or estimate Breeding Worth and/or Production Worth, for example. Similar evaluation systems may be used in other markets also.
  • Methods involving the analysis of such alterations or variations can be used for the purposes of selecting, screening and breeding animals, farm management, and for estimating an animal’s worth to a particular industry, for example.
  • the invention provides a method for identifying whether or not a non-human animal, cell or embryo carries a genetic marker linked to a deleterious effect on productivity and/or worth of an animal, the method comprising at least the step of analysing a nucleic acid of said animal, cell or embryo to identify whether or not it includes a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith, wherein where the nucleic acid includes a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith, it is identified to carry a genetic marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the method comprises as least the step of analysing the nucleic acid to identify whether or not it includes a genetic variation at a position corresponding to position 77632752 of chromosome 5 of Bos taurus.
  • the method comprises at least the step of analysing the nucleic acid to identify whether or not it includes the genetic marker 77632752T on chromosome 5 of Bos taurus and/or a genetic marker in linkage disequilibrium therewith. In one embodiment, where it includes the genetic marker 77632752T and/or a genetic marker in linkage disequilibrium therewith the animal, cell or embryo is identified to carry a genetic marker linked to a deleterious effect on productivity and/or worth of an animal.
  • cell or embryo is identified not to carry a genetic marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the method of the first aspect is used for selecting or rejecting a non-human animal, cell or embryo and/or estimating the worth of a non-human animal and/or its offspring.
  • the method of the first aspect is used to identify whether or not an animal is suitable for (or at least more or less suitable for) inclusion in a herd, production purposes and/or breeding purposes. In another embodiment, the method of the first aspect is used to identify whether or not a cell or embryo is suitable for (or at least more or less suitable for) producing an animal which is suitable for (or at least more suitable for) inclusion in a herd, production purposes and/or breeding purposes.
  • the method comprises identifying whether or not the animal, cell or embryo is heterozygous or homozygous for a genetic marker linked to a deleterious effect on productivity and/or worth of an animal and/or a genetic marker in linkage disequilibrium therewith.
  • the invention provides a method for selecting or rejecting a non-human animal, cell or embryo the method comprising selecting the non-human animal, cell or embryo where it has been identified not to carry a genetic marker linked to a deleterious effect on productivity and/or worth of an animal according to a method of the first aspect.
  • the invention provides a method for selecting or rejecting a non-human animal, cell or embryo, the method comprising at least the step of analysing a nucleic acid of said animal, cell or embryo to identify whether or not it includes a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith.
  • the method comprises at least the steps of: a) performing a method of the first aspect; and, b) selecting or rejecting the non-human animal, cell or embryo using the results of step a).
  • an animal, cell or embryo is selected if it does not have a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith.
  • an animal, cell or embryo is rejected if it has a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith.
  • the animal, cell or embryo has been identified to have the wild-type nucleotide sequence of FGD4 the animal, cell or embryo is selected.
  • the method comprises at least the step of analysing a nucleic acid of said animal, cell or embryo to identify whether or not it includes a variation at a position corresponding to position 77632752 on chromosome 5 of Bos taurus and/or a genetic marker in linkage disequilibrium therewith.
  • the variation is a nucleotide substitution.
  • the method comprises at least the step of analysing a nucleic acid of said animal, cell or embryo to identify whether or not it includes the genetic marker 77632752T on chromosome 5 of Bos taurus and/or a genetic marker in linkage disequilibrium therewith.
  • the method comprises at least the steps of:
  • nucleic acid includes the genetic marker 77632752T and/or a genetic marker in linkage disequilibrium therewith the animal
  • cell or embryo is identified to carry a genetic marker linked to a deleterious effect on productivity and/or worth of an animal and the animal.
  • the nucleic acid includes the genetic marker 77632752T and/or a genetic marker in linkage disequilibrium therewith the animal, cell or embryo is rejected.
  • the nucleic acid includes the genetic marker 77632752C the animal, cell or embryo is selected.
  • the method further comprises identifying whether or not the animal, cell or embryo is heterozygous or homozygous. In one embodiment, an animal, cell or embryo is rejected if it is heterozygous for a genetic marker linked to a deleterious effect on productivity and/or worth of an animal.
  • an animal, cell or embryo is selected if it is heterozygous for a genetic marker linked to a deleterious effect on productivity and/or worth of an animal.
  • an animal, cell or embryo is rejected if it is homozygous for a genetic marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the method is performed for the purpose of selecting or rejecting a non-human animal for production purposes. In one embodiment, the method is performed for the purpose of selecting or rejecting a non-human animal for milking purposes. In one embodiment, the method is performed for the purpose of selecting or rejecting a non-human animal for beef farming. In another embodiment, the method is performed for the purpose of selecting or rejecting a non-human animal for breeding purposes. In one embodiment, the method is performed for the purpose of selecting or rejecting a non-human animal for inclusion in a herd.
  • the method is performed for the purpose of selecting or rejecting non-human cell or embryo for use in cloning a non-human animal and/or breeding a non human animal.
  • breeding an animal may involve IVF.
  • the invention provides a method for estimating the worth of a non-human animal and/or its offspring, the method comprising at least the steps of: a) performing a method of the first aspect; and, b) estimating the worth of the animal and/or its offspring using the results of step a).
  • the method may comprise analysing a nucleic acid of said animal to identify whether or not it includes a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith.
  • the animal when a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith is present the animal is identified to carry a genetic marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the invention provides a method for estimating the worth of a non-human animal and/or its offspring, the method comprising at least the step of analysing a nucleic acid of the animal to identify whether or not it includes a variation at a position corresponding to position 77632752 on chromosome 5 of Bos taurus and/or a genetic marker in linkage disequilibrium thereof.
  • the variation is substitution of a nucleotide.
  • the invention provides a method for estimating the worth of a non-human animal and/or its offspring, the method comprising at least the step of analysing a nucleic acid of the animal to identify whether or not it includes the genetic marker 77632752T on chromosome 5 of Bos taurus and/or a genetic marker in linkage disequilibrium thereof.
  • the animal is identified to carry a genetic marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the genetic marker 77632752C and/or a genetic marker in linkage disequilibrium therewith is present the animal is identified not to carry a genetic marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the method further comprises identifying whether or not the animal is heterozygous or homozygous for a genetic marker linked to a deleterious effect on productivity and/or worth in an animal.
  • the invention provides a method for breeding a non-human animal which comprises selecting at least a first non-human animal that has been identified not to have a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith and mating said first animal with a second non-human animal.
  • the method further comprises selecting the second animal where it has been identified not to have a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith.
  • the invention provides a method for breeding a non-human animal which comprises selecting at least a first non-human animal that does not have a genetic variation at a position corresponding to position 77632752 on chromosome 5 of Bos taurus and/or a genetic marker in linkage disequilibrium therewith and mating said first animal with a second non human animal.
  • the method also comprises selecting a second non-human animal that does not have a genetic variation at a position corresponding to position 77632752 on chromosome 5 of Bos taurus and/or a genetic marker in linkage disequilibrium therewith.
  • the invention provides a method for breeding a non-human animal which comprises selecting the first and/or second non-human animal where it has been does not have the genetic marker 77632752T and/or a genetic marker in linkage disequilibrium therewith.
  • the first and/or second animal are selected where it has the genetic marker 77632752C and/or a genetic marker in linkage disequilibrium therewith.
  • the method comprises the steps of: a) performing a method of the first aspect of the invention for the first and/or second non-human animal; and, b) selecting the first and/or second non-human animal where it is identified not to have a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith.
  • the method further comprises identifying whether or not the animal is heterozygous or homozygous for a genetic marker linked to a deleterious effect on productivity and/or worth in an animal.
  • the invention provides a method for breeding a non-human animal the method comprising at least the step of selecting a first non-human gamete that has been identified not to have a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith and fusing said first gamete with a second non-human gamete to form a zygote.
  • the method further comprises selecting the second gamete where it has been identified not to have a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith.
  • the method comprises selecting the first and/or second gamete where it does not have a genetic variation at a position corresponding to position 77632752 on chromosome 5 of Bos taurus and/or a genetic marker in linkage disequilibrium therewith.
  • the method comprises selecting the first and/or second gamete where it has the genetic marker 77632752T and/or a genetic marker in linkage disequilibrium therewith.
  • the first and/or second gamete is selected where it has the genetic marker 77632752C and/or a genetic marker in linkage disequilibrium therewith.
  • the method further comprises the steps of: a) performing a method of the first aspect of the invention for the first non-human gamete; and, b) selecting the first and/or second non-human gamete it is identified not to have a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith.
  • the method further comprises identifying whether or not the gamete is heterozygous or homozygous for a genetic marker linked to a deleterious effect on productivity and/or worth in an animal.
  • the invention provides a method of breeding a non-human animal, the method comprising at least the step of selecting a non-human embryo that has been identified not to have a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith.
  • a selected embryo is used to breed an animal.
  • the method further comprises transferring an embryo to a gestational carrier.
  • the method comprises selecting the embryo where it does not have a genetic variation at a position corresponding to position 77632752 on chromosome 5 of Bos taurus and/or a genetic marker in linkage disequilibrium therewith.
  • the embryo is selected where it does not have the genetic marker 77632752T and/or a genetic marker in linkage disequilibrium therewith.
  • the embryo is selected where it has the genetic marker 77632752C and/or a genetic marker in linkage disequilibrium therewith.
  • the method further comprises the steps of: a) performing a method of the first aspect of the invention for the non-human embryo; and, b) selecting the embryo it is identified not to have a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith.
  • the method further comprises identifying whether or not the embryo is heterozygous or homozygous for a genetic marker linked to a deleterious effect on productivity and/or worth in an animal.
  • the invention provides a method of cloning a non-human animal, the method comprising at least the step of selecting a non-human cell that has been identified not to have a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith. One or more selected cells are used to clone an animal.
  • a cell is selected where it does not have a genetic alteration at a position corresponding to position 77632752 of chromosome 5 of Bos taurus and/or a genetic marker in linkage disequilibrium therewith.
  • a cell is selected where it has the genetic marker 77632752T and/or a genetic marker in linkage disequilibrium therewith.
  • a cell is selected where it has been identified to have the genetic marker 77632752C and/or a genetic marker in linkage disequilibrium therewith.
  • the method further comprises the steps of: a) performing a method of the first aspect of the invention for the non-human cell; and, b) selecting the cell where it is identified not to have a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith.
  • the method further comprises identifying whether or not the cell is heterozygous or homozygous for a genetic marker linked to a deleterious effect on productivity and/or worth in an animal.
  • the genetic variation which disrupts the FGD4 gene is located within the FGD4 gene at a position corresponding to position 77632752 or downstream of a position corresponding to position 77632752 of chromosome 5 of Bos Taurus. In one embodiment, the variation is a non-sense variant resulting in truncation of the FGD4 gene product.
  • the methods involve analysing a nucleic acid to identify whether or not it includes two or more genetic variations which disrupt the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith. In one embodiment, the methods involve analysing a nucleic acid to identify whether or not it includes two or more genetic variations which disrupt the FGD4 gene and/or two or more genetic markers in linkage disequilibrium therewith.
  • the methods of the invention involve analysing a nucleic acid to identify whether or not it includes a genetic variation which disrupts the FGD4 gene alone or in combination with a genetic marker in linkage disequilibrium therewith. In one embodiment, the methods of the invention involve analysing only a genetic marker in linkage disequilibrium with a genetic variation which disrupts the FGD4 gene.
  • the methods of the invention involve analysing a nucleic acid to determine whether or not it includes a genetic variation at a position corresponding to position 77632752 on chromosome 5 of Bos taurus alone or in combination with a genetic marker in linkage disequilibrium therewith. In one embodiment, the methods of the invention involve analysing only a genetic marker in linkage disequilibrium with the genetic variation 77632752 on chromosome 5 of Bos taurus.
  • the methods of the invention involve analysing a nucleic acid to determine whether or not it includes the genetic marker 77632752T on chromosome 5 of Bos taurus alone or in combination with a genetic marker in linkage disequilibrium therewith. In one embodiment, the methods of the invention involve analysing only a genetic marker in linkage disequilibrium with the genetic marker 77632752T on chromosome 5 of Bos taurus.
  • the various markers of use in the invention can be analysed in combination with another biological marker.
  • the invention provides a method for identifying whether or not a non-human animal, cell or embryo carries a biological marker linked to a deleterious effect on productivity and/or worth of an animal, the method comprising at least the step of analysing a FGD4, a precursor thereof, an isoform thereof and/or a fragment thereof of said animal, cell or embryo to identify whether or not it includes a variation which disrupts FGD4, wherein where it includes a variation which disrupts FGD4, the non-human animal, cell or embryo is determined to carry a biological marker linked to a deleterious effect on productivity and/or worth.
  • the method comprises at least the step of analysing a FGD4, a precursor thereof, an isoform thereof and/or a fragment thereof to identify whether or not it includes an amino acid variation resulting in a truncated protein of 624 amino acids in length.
  • the variation comprises insertion of an amino acid.
  • the variation comprises insertion of 2, 3, 4, 5 or more amino acids.
  • the variation comprises insertion of an amino acid at a position corresponding to position 620 of FGD4.
  • the variation results in insertion of 2, 3, 4, 5 or more amino acids at a position corresponding to position 620 of FGD4.
  • the amino acid or 2, 3, 4, 5 or more amino acids inserted at a position corresponding to position 620 of FGD4 are chosen from Val, Ser, Phe, Val, and Val, in any order or in the order in which they are listed.
  • the variation comprises insertion of an amino acid and truncation of the protein.
  • the variation is Leu6l9_Phe620insValSerPheValVal.
  • the animal where it includes one or more of the variations mentioned above the animal, cell or embryo is identified to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the method of the eighth aspect is used for selecting or rejecting a non-human animal, cell or embryo and/or estimating the worth of a non-human animal and/or its offspring.
  • the method of the eighth aspect is used to identify whether or not an animal is suitable for (or at least more or less suitable for) inclusion in a herd, production purposes and/or breeding purposes. In another embodiment, the method of the eighth aspect is used to identify whether or not a cell or embryo is suitable for (or at least more or less suitable for) producing an animal which is suitable for (or at least more suitable for) inclusion in a herd, production purposes and/or breeding purposes.
  • the invention provides a method for selecting or rejecting a non-human animal, cell or embryo the method comprising selecting the non-human animal, cell or embryo where it has been determined not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal according to a method of the eighth aspect.
  • the invention provides a method for selecting or rejecting a non-human animal, cell or embryo, the method comprising at least the step of analysing a FGD4, a precursor thereof, an isoform thereof and/or a fragment thereof of said animal, cell or embryo to identify whether or not it includes a variation in the amino acid sequence which disrupts FGD4.
  • the method comprises at least the steps of: a) performing a method of the eighth aspect; and, b) selecting or rejecting the non-human animal, cell or embryo using the results of step a).
  • an animal, cell or embryo is selected if it does not have a variation which disrupts FGD4. In one embodiment, an animal, cell or embryo is rejected if it has a variation which disrupts FGD4. In another embodiment, if the animal, cell or embryo has been identified to have the wild-type amino acid sequence of FGD4 the animal is selected. In one embodiment of the ninth aspect, where a FGD4, a precursor thereof, an isoform thereof and/or a fragment thereof comprises a truncation, the animal, cell or embryo is rejected. In one embodiment, the variation comprises a truncated protein of 624 amino acids. In one embodiment, the animal, cell or embryo is rejected if it has a variation comprising insertion of an amino acid into FDG4.
  • the variation comprises insertion of 2, 3, 4, 5 or more amino acids. In one embodiment, the variation comprises insertion of an amino acid at a position corresponding to position 620 of FGD4. In one embodiment, the variation results in insertion of 2, 3, 4, 5 or more amino acids at a position corresponding to position 620 of FGD4. In one embodiment, the amino acid or 2, 3, 4, 5 or more amino acids inserted at a position corresponding to position 620 of FGD4 are chosen from Val, Ser, Phe, Val, and Val, in any order or in the order in which they are listed. In one embodiment, the variation comprises insertion of an amino acid and truncation of the protein. In one embodiment, the animal, cell or embryo is rejected if it comprises Leu6l9_Phe620insValSerPheValVal.
  • the method comprises at least the step of analysing a FGD4, a precursor thereof, an isoform thereof and/or a fragment thereof of said animal, cell or embryo to identify whether or not it includes the biological marker Leu6l9_Phe620insValSerPheValVal.
  • the method comprises at least the steps of:
  • a FGD4 a precursor thereof, an isoform thereof and/or a fragment thereof comprises Leu6l9_Phe620insValSerPheValVal
  • the animal, cell or embryo is identified to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • a FGD4, a precursor thereof, an isoform thereof and/or a fragment thereof comprises Leu6l9_Phe620insValSerPheValVal
  • the animal, cell or embryo is rejected.
  • a FGD4, a precursor thereof, an isoform thereof and/or a fragment thereof comprises the wild type amino acid sequence of FGD4 (SEQ ID NO: 3)
  • the animal, cell or embryo is selected.
  • the method is performed for the purpose of selecting or rejecting a non-human animal for milking purposes.
  • the method is performed for the purpose of selecting or rejecting a non-human animal for beef farming.
  • the method is performed for the purpose of selecting or rejecting a non-human animal for breeding purposes.
  • the method is performed for the purpose of selecting or rejecting a non-human animal for inclusion in a herd.
  • the method is performed for the purpose of selecting or rejecting non-human cell or embryo for use in cloning a non-human animal and/or breeding a non human animal.
  • breeding an animal may involve IVF.
  • the invention provides a method for estimating the worth of an animal and/or its offspring, the method comprising at least the steps of: a) performing a method of the eighth aspect; and, b) estimating the worth of the animal and/or its offspring using the results of step a).
  • the method may comprise analysing a FGD4, a precursor thereof, an isoform thereof and/or a fragment thereof from said animal to identify whether or not it includes a variation which disrupts FGD4.
  • the animal is identified to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the variation comprises a truncation.
  • the variation comprises a truncated protein of 624 amino acids.
  • the variation comprises insertion of an amino acid into FDG4.
  • the variation comprises insertion of 2, 3, 4, 5 or more amino acids.
  • the variation comprises insertion of an amino acid at a position corresponding to position 620 of FGD4.
  • the variation results in insertion of 2, 3, 4, 5 or more amino acids at a position corresponding to position 620 of FGD4.
  • the amino acid or 2, 3, 4, 5 or more amino acids inserted at a position corresponding to position 620 of FGD4 are chosen from Val, Ser, Phe, Val, and Val, in any order or in the order in which they are listed.
  • the variation comprises insertion of an amino acid and truncation of the protein.
  • the variation comprises Leu6l9_Phe620insValSerPheValVal.
  • the invention provides a method for estimating the worth of a non-human animal and/or its offspring, the method comprising at least the step of analysing a FGD4, a precursor thereof, an isoform thereof and/or a fragment thereof from said animal to identify whether or not it includes the biological marker Leu6l9_Phe620insValSerPheValVal.
  • the biological marker Leu6l9_Phe620insValSerPheValVal is present the animal is identified to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the invention provides a method for breeding a non-human animal which comprises selecting at least a first animal that has been identified not to have an amino acid variation which disrupts FGD4 and mating said first animal with a second animal.
  • the method further comprises the step of selecting the second animal where it has been identified not to have an amino acid variation which disrupts FGD4.
  • the first and/or second animal is selected where it does not have an amino acid variation comprising a truncation.
  • the variation comprises a truncated protein of 624 amino acids.
  • the animal is selected if it does not have a variation comprising insertion of an amino acid into FDG4.
  • the variation comprises insertion of 2, 3, 4, 5 or more amino acids.
  • the variation comprises insertion of an amino acid at a position corresponding to position 620 of FGD4.
  • the variation results in insertion of 2, 3, 4, 5 or more amino acids at a position corresponding to position 620 of FGD4.
  • the amino acid or 2, 3, 4, 5 or more amino acids inserted at a position corresponding to position 620 of FGD4 are chosen from Val, Ser, Phe, Val, and Val, in any order or in the order in which they are listed.
  • the first and/or second animal is selected if it does not have a variation comprising insertion of an amino acid and truncation of the protein.
  • the first and/or second animal is selected if it does not have the biological marker Leu619_Phe620insValSerPheValVal.
  • the first and/or second animal is selected where it has the wild type amino acid sequence of FGD4 (SEQ ID No: 3).
  • the method further comprises the steps of: a) performing a method of the eighth aspect of the invention for the first and/or second animal; and, b) selecting the first and/or second animal where it is identified not to have an amino acid variation which disrupts FGD4.
  • the invention provides a method for breeding a non-human animal the method comprising at least the step of selecting a first non-human gamete that has been identified not to have a amino acid variation which disrupts FGD4 and fusing said first gamete with a second non-human gamete to form a zygote.
  • the method further comprises selecting the second gamete where it has been identified not to have an amino acid variation which disrupts FGD4.
  • the method comprises selecting the first and/or second gamete where it does not have an amino acid variation comprising a truncation.
  • the variation comprises a truncated protein of 624 amino acids.
  • the gamete is selected if it does not have a variation comprising insertion of an amino acid into FDG4.
  • the variation comprises insertion of 2, 3, 4, 5 or more amino acids.
  • the variation comprises insertion of an amino acid at a position corresponding to position 620 of FGD4.
  • the variation results in insertion of 2, 3, 4, 5 or more amino acids at a position corresponding to position 620 of FGD4.
  • the amino acid or 2, 3, 4, 5 or more amino acids inserted at a position corresponding to position 620 of FGD4 are chosen from Val, Ser, Phe, Val, and Val, in any order or in the order in which they are listed.
  • the first and/or second gamete is selected where it does not have an amino acid variation which comprises insertion of an amino acid and truncation of the protein.
  • the first and/or second gamete is selected if it does not have the biological marker Leu6l9_Phe620insValSerPheValVal.
  • the first and/or second gamete is selected where it has the wild type amino acid sequence of FGD4 (SEQ ID No: 3).
  • the method further comprises the steps of: a) performing a method of the eighth aspect of the invention for the first and/or second gamete; and, b) selecting the first and/or second gamete where it is identified not to have an amino acid variation which disrupts FGD4.
  • the invention provides a method of breeding a non-human animal, the method comprising at least the step of selecting a non-human embryo that has been identified not to have a amino acid variation which disrupts FGD4.
  • a selected embryo is used to breed an animal.
  • the method further comprises transferring one or more embryo to a gestational carrier.
  • the embryo is selected where it does not have an amino acid variation comprising a truncation.
  • the variation comprises a truncated protein of 624 amino acids.
  • the embryo is selected if it does not have a variation comprising insertion of an amino acid into FDG4.
  • the variation comprises insertion of 2, 3, 4, 5 or more amino acids.
  • the variation comprises insertion of an amino acid at a position corresponding to position 620 of FGD4.
  • the variation results in insertion of 2, 3, 4, 5 or more amino acids at a position corresponding to position 620 of FGD4.
  • the amino acid or 2, 3, 4, 5 or more amino acids inserted at a position corresponding to position 620 of FGD4 are chosen from Val, Ser, Phe, Val, and Val, in any order or in the order in which they are listed.
  • an embryo is selected if it does not have an amino acid variation comprising insertion of an amino acid and truncation of the protein.
  • the embryo is selected if it does not have the biological marker Leu6l9_Phe620insValSerPheValVal.
  • the embryo is selected where it has the wild type amino acid sequence of FGD4 (SEQ ID No: 3).
  • the method further comprises the steps of: a) performing a method of the eighth aspect of the invention for the embryo; and, b) selecting the embryo where it is identified not to have an amino acid variation which disrupts FGD4.
  • the invention provides a method of cloning a non-human animal, the method comprising at least the step of selecting a cell that has been identified not to have an amino acid variation which disrupts FGD4. One or more selected cells are used to clone an animal.
  • the cell is selected where it does not have an amino acid variation comprising a truncation.
  • the variation comprises a truncated protein of 624 amino acids.
  • the cell is selected if it does not have a variation comprising insertion of an amino acid into FDG4.
  • the variation comprises insertion of 2, 3, 4, 5 or more amino acids.
  • the variation comprises insertion of an amino acid at a position corresponding to position 620 of FGD4.
  • the variation results in insertion of 2, 3, 4, 5 or more amino acids at a position corresponding to position 620 of FGD4.
  • the amino acid or 2, 3, 4, 5 or more amino acids inserted at a position corresponding to position 620 of FGD4 are chosen from Val, Ser, Phe, Val, and Val, in any order or in the order in which they are listed.
  • the cell is not selected where it has a variation comprising insertion of an amino acid and truncation of the protein.
  • the cell is selected if it does not have the biological marker Leu6l9_Phe620insValSerPheValVal.
  • a cell is selected where it has the wild-type amino acid sequence of FGD4 (SEQ ID No: 3).
  • the method further comprises the steps of: a) performing a method of the eighth aspect of the invention for a cell; and, b) selecting a cell where it is identified not to have an amino acid variation which disrupts FGD4.
  • the methods involve analysing a FGD4, a precursor thereof, an isoform thereof and/or a fragment thereof to identify whether or not it includes two or more variations which disrupts FGD4.
  • the methods of the invention involve analysing a FGD4, a precursor thereof, an isoform thereof and/or a fragment thereof to identify whether or not it includes a variation which disrupts FGD4 alone or in combination with one or more other biological marker.
  • the variation which disrupts FGD4 is an amino acid variation resulting in a truncated protein of 624 amino acids in length.
  • the variation comprises insertion of an amino acid.
  • the variation comprises insertion of 2, 3, 4, 5 or more amino acids.
  • the variation comprises insertion of an amino acid at a position corresponding to position 620 of FGD4.
  • the variation results in insertion of 2, 3, 4, 5 or more amino acids at a position corresponding to position 620 of FGD4.
  • the amino acid or 2, 3, 4, 5 or more amino acids inserted at a position corresponding to position 620 of FGD4 are chosen from Val, Ser, Phe, Val, and Val, in any order or in the order in which they are listed.
  • the variation comprises insertion of an amino acid and truncation of the protein.
  • the variation is Leu6l9_Phe620insValSerPheValVal.
  • the methods of the invention involve analysing a FGD4, a precursor thereof, an isoform thereof and/or a fragment thereof to identify whether or not it includes the biological marker Leu6l9_Phe620insValSerPheValVal alone or in combination with one or more other biological marker.
  • the invention provides a method for identifying whether or not a non-human animal, cell or embryo carries a biological marker linked to a deleterious effect on productivity and/or worth of an animal, the method comprising at least the step of observing the level and/or activity of a FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof of an animal, cell or embryo.
  • the method comprises comparing the level and/or activity of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof against a standard.
  • the method of the fifteenth aspect is used for selecting or rejecting a non-human animal, cell or embryo and/or estimating the worth of a non-human animal and/or its offspring.
  • the method of the fifteenth aspect is used to identify whether or not an animal is suitable for (or at least more or less suitable for) inclusion in a herd, production purposes and/or breeding purposes. In another embodiment, the method is used to identify whether or not a cell or embryo is suitable for (or at least more or less suitable for) producing an animal which is suitable (or at least more suitable for) inclusion in a herd, production purposes and/or breeding purposes.
  • a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof the animal, cell or embryo is identified to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the invention provides a method for selecting or rejecting a non-human animal, cell or embryo, the method comprising selecting the non-human animal, cell or embryo where it has been identified not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal according to a method of the fifteenth aspect.
  • the invention provides a method for selecting or rejecting a non-human animal, cell or embryo the method comprising at least the step of observing the level and/or activity of a FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof.
  • the method comprises comparing the level and/or activity of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof against a standard. In one embodiment of the sixteenth aspect, the method comprises at least the steps of: a) performing a method of the fifteenth aspect; and, b) selecting or rejecting the non-human animal, cell or embryo using the results of step a).
  • an animal, cell or embryo is selected if it does not carry a biological marker linked to a deleterious effect on productivity and/or worth. In one embodiment, an animal, cell or embryo is rejected if it does carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the animal, cell or embryo is selected where the level and/or activity of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof is indicative of an animal(s) which is/are known not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • an animal is rejected if the level and/or activity of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof is indicative of an animal(s) which is/are known to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the animal, cell or embryo is rejected where it has substantially no level and/or activity of a FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof.
  • the method is performed for the purpose of selecting or rejecting a non-human animal for milking purposes. In one embodiment, the method is performed for the purpose of selecting or rejecting a non-human animal for beef farming. In another embodiment, the method is performed for the purpose of selecting or rejecting a non-human animal for breeding purposes. In one embodiment, the method is performed for the purpose of selecting or rejecting a non-human animal for inclusion in a herd.
  • the method is performed for the purpose of selecting or rejecting a non-human cell or embryo for use in cloning a non-human animal and/or breeding a non human animal.
  • breeding an animal may involve IVF.
  • the invention provides a method for estimating the worth of a non-human animal and/or its offspring, the method comprising at least the steps of: a) performing a method of the fifteenth aspect; and, b) estimating the worth of the animal and/or its offspring using the results of step a).
  • the method may comprise observing the level and/or activity of a FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof.
  • the method comprises comparing the level and/or activity of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof against a standard.
  • the animal where there is substantially no level and/or activity of a FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof the animal is identified to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the invention provides a method for breeding a non-human animal, the method comprising at least the step of selecting a first non-human animal that has been identified not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal and mating said first animal with a second non-human animal.
  • the method further comprises selecting a second non human animal that has been identified not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the invention provides a method for breeding a non human animal, the method comprising at least the step of selecting a first non-human animal where it has a level and/or activity of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof which is indicative of an animal(s) which is/are known not to carry a biological marker linked to a deleterious effect on productivity and/or worth and mating said first animal with a second non-human animal.
  • the method further comprises selecting a second non human animal where it has a level and/or activity of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof which is indicative of an animal(s) which is/are known not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the method further comprises the steps of: a) performing a method of the fifteenth aspect of the invention for the first and/or second non-human animal; and b) selecting the first and/or second non-human animal where it is identified not to have a level and/or activity of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof which is indicative of an animal(s) which is/are known not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • an animal where there is substantially no level and/or activity of a FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof an animal is identified to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the invention provides a method for breeding a non-human animal, the method comprising at least the step of selecting a first gamete that has been identified not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal and fusing said first gamete with a second gamete to form a zygote.
  • the method further comprises selecting the second gamete that has been identified not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the invention provides a method for breeding a non human animal, the method comprising at least the step of selecting a first gamete that has been identified to have a level and/or activity of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof is indicative of an animal(s) which is/are known not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal and fusing said first gamete with a second gamete to form a zygote.
  • the method further comprises selecting the second gamete where it has been identified to have a level and/or activity of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof is indicative of an animal(s) which is/are known not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the method further comprises the steps of: a) performing a method of the fifteenth aspect of the invention for the first and/or second gamete; and b) selecting the first and/or second gamete where it is identified not to have a level and/or activity of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof which is indicative of an animal(s) which is/are known not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof is identified to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the invention provides a method for breeding a non-human animal, the method comprising at least the step of selecting a non-human embryo that has been identified not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the invention provides a method of breeding a non-human animal, the method comprising at least the step of selecting a non-human embryo that has been identified to have a level and/or activity of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof is indicative of an animal(s) which is/are known not to carry a biological marker linked to a deleterious effect on productivity and/or worth.
  • a selected embryo is used to breed an animal.
  • the method further comprises transferring one or more embryo to a gestational carrier.
  • the method further comprises the steps of: a) performing a method of the fifteenth aspect of the invention for the non-human embryo; and, b) selecting the embryo where it is identified not to have a level and/or activity of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof which is indicative of an animal(s) which is/are known not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the invention provides a method of cloning a non-human animal, the method comprising at least the step of selecting a non-human cell that has been identified not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the invention provides a method of cloning a non human animal the method comprising at least the step of selecting a non-human cell that has been identified to have a level and/or activity of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof is indicative of an animal(s) which is/are known not to carry a biological marker linked to a deleterious effect on productivity and/or worth.
  • One or more selected cells are used to clone an animal.
  • the method further comprises the steps of: a) performing a method of the fifteenth aspect of the invention for the non-human cell; and, b) selecting the cell where it is identified not to have a level and/or activity of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof which is indicative of an animal(s) which is/are known not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • a cell where there is substantially no level and/or activity of a FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof a cell is identified to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the methods comprise the steps of:
  • the standard comprises a level of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof which is associated with an animal or animals which is/are known not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • an animal, cell or embryo has a lower level of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof compared to the standard it is identified to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • an animal, cell or embryo has substantially no level of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof compared to the standard it is identified to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • an animal, cell or embryo is rejected if it has a lower level of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof compared to the standard.
  • an animal, cell or embryo is rejected if it has substantially no level of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof.
  • a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof compared to the standard it is identified not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • an animal, cell or embryo is selected if it has substantially the same or a higher level of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof compared to the standard.
  • the standard comprises a level of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof which is associated with an animal or animals which is/are known to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the standard comprises substantially no level of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof.
  • an animal, cell or embryo has a higher level of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof compared to the standard it is identified not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the animal, cell or embryo has substantially the same or a lower level of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof compared to the standard it is identified to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • an animal, cell or embryo is selected if it has a higher level of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof compared to the standard.
  • an animal, cell or embryo is rejected if it has substantially the same or a lower level of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof compared to the standard.
  • the level of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof is the level of expression thereof.
  • the method involves observing the level of activity of FGD4, a precursor thereof, an isoform thereof, and/or a fragment thereof.
  • the methods comprise observing the level and/or activity of a combination of two or more of one or more FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof
  • the level and/or activity of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof is observed alone or in combination with one or more other biological markers.
  • the methods of the first to twenty first and thirty sixth to thirty ninth aspects may further involve analysing one or more additional biological markers.
  • the one or more biological markers is one or more genetic markers.
  • the methods of the first to twenty first and thirty sixth to thirty ninth aspects of the invention may further involve taking a sample from an animal.
  • the absence of a variation which disrupts FGD4, a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith identifies that the animal, cell, gamete or embryo is suitable for use (or at least more suitable for use than an animal, cell, gamete or embryo that has a variation which disrupts FGD4, a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith, or at least more suitable than the same animal, cell, gamete or embryo if it had a variation which disrupts FGD4, a genetic variation which disrupts the FGD4 gene and/or marker in linkage disequilibrium therewith) for breeding and cloning purposes or that the animal is suitable for (or at least more suitable for as beforementioned) production purposes, breeding purposes and/or inclusion in a herd.
  • an animal, cell, embryo or gamete is selected if it has the wild-type allele, wild-type nucleic acid sequence encoding FGD4 or wild-type amino acid sequence.
  • the presence of a variation which disrupts FGD4, a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith identifies that the animal, cell, gamete or embryo is not suitable for use (or at least less suitable for use than an animal, cell, gamete or embryo that has a variation which disrupts FGD4, a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith, or at least less suitable than the same animal, cell, gamete or embryo if it had a variation which disrupts FGD4, a genetic variation which disrupts the FGD4 gene and/or marker in linkage disequilibrium therewith) for breeding and cloning purposes
  • an animal is identified to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal it is identified not to be suitable (or at least to be less suitable than an animal or animals that do not have a biological marker linked to a deleterious effect on productivity and/or worth of an animal, or at least less suitable than the same animal if it did not carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal) for inclusion in a herd, for production and/or breeding purposes.
  • a cell or embryo is identified to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal it is identified not to be suitable (or at least less suitable than a cell or embryo that does not carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal, or at least to be less suitable than the same cell or embryo if it did not carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal) to produce an animal which is suitable for inclusion in a herd, for production and/or breeding purposes.
  • an animal is identified not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal it is identified to be suitable (or at least to be more suitable than an animal or animals that do carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal, or at least more suitable than the same animal if it did carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal) for inclusion in a herd, for production and/or breeding purposes.
  • a cell or embryo is identified not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal it is identified to be suitable (or at least to be more suitable than a cell or embryo that does carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal or to be more suitable than the same cell or embryo if it did carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal) to produce an animal which is suitable for production and/or breeding purposes.
  • an animal is identified to be homozygous for a genetic marker linked to a deleterious effect on productivity and/or worth of an animal it is identified not to be suitable (or at least less suitable than an animal or animals that are not homozygous for a genetic marker linked to a deleterious effect on productivity and/or worth, or at least to be less suitable than the same animal if it was not homozygous for a genetic marker linked to a deleterious effect on productivity and/or worth of an animal) for inclusion in a herd, for production and/or breeding purposes.
  • a cell or embryo is identified to be homozygous for a genetic marker linked to a deleterious effect on productivity and/or worth of an animal it is identified not to be suitable (or at least to be less suitable than a cell or embryo that is not homozygous for a genetic marker linked to a deleterious effect on productivity and/or worth of an animal, or at least less suitable than the same cell or embryo if it was not homozygous for a genetic marker linked to a deleterious effect on productivity and/or worth of an animal) to produce an animal which is suitable for inclusion in a herd, for production and/or breeding purposes.
  • an animal is identified to be heterozygous for a genetic marker linked to a deleterious effect on productivity and/or worth of it is identified not to be suitable (or at least to be less suitable than an animal or animals that do not carry a genetic marker linked to a deleterious effect on productivity and/or worth of an animal, or at least to be less suitable than the same animal if it did not carry a genetic marker linked to a deleterious effect on productivity and/or worth of an animal) for inclusion in a herd, for production and/or breeding purposes.
  • a cell or embryo is identified to be heterozygous for a genetic marker linked to a deleterious effect on productivity and/or worth of an animal it is identified not to be suitable (or at least to be less suitable than a cell or embryo that does not carry a genetic marker linked to a deleterious effect on productivity and/or worth of an animal, or at least to be less suitable than the same cell or embryo if it did not carry a genetic marker linked to a deleterious effect on productivity and/or worth of an animal) to produce an animal which is suitable for inclusion in a herd, for production and/or breeding purposes.
  • the invention provides a method of forming a herd of non-human animals, the method comprising selecting an animal where it has been identified not to carry a biological marker linked to a deleterious effect on productivity, worth and/or one or more related traits as described herein of an animal and forming a herd of selected animals.
  • the invention provides a method of forming a herd of non-human animals, the method comprising at least the steps of: a) selecting or rejecting a non-human animal based on the results of a method according to any one or more of the first, second, third, eighth, ninth, tenth, fifteenth, sixteenth, seventeenth, thirty sixth, thirty eighth or thirty ninth aspects of the invention; and, b) forming a herd of selected animals.
  • the invention provides a method of forming a herd of non-human animals, the method comprising at least the steps of:
  • step b selecting or rejecting an animal based on the results of step a.;
  • an animal is rejected if it has a biological marker linked to a deleterious effect on productivity and/or worth of an animal. In one embodiment, an animal is selected if it does not have a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • an animal is rejected if it includes one or more of:
  • a genetic variation which disrupts the FGD4 gene for example, a genetic variation at a position corresponding to position 77632752 of chromosome 5 of Bos Taurus, including, for example, 77632752T) and/or a genetic marker in linkage disequilibrium therewith;
  • an amino acid variation which disrupts FGD4 for example, a truncated FDG4 and/or, a level or activity of FGD4, a precursor thereof, an isoform thereof, a fragment thereof and/or a nucleic acid encoding any one or more thereof which is indicative of an animal or animals who is/are known to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • an animal is selected if it:
  • FGD4 has a nucleic acid encoding FGD4 that has the wild-type allele or sequence (for example 77632752C or SEQ ID No: 1 and/or a genetic marker in linkage disequilibrium therewith; does not have an amino acid variation which disrupts FGD4 (for example, truncation of FGD4);
  • FGD4 has a level or activity of FGD4, a precursor thereof, an isoform thereof, a fragment thereof and/or a nucleic acid encoding any one or more thereof which is indicative of an animal or animals who is/are known not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the invention provides a method for generating an animal, the method comprising at least the step of introducing a genetic alteration to the FGD4 gene of a cell used to generate the animal.
  • the genetic alteration introduced to the FGD4 gene corrects a variation in the FGD4 gene which is linked to a deleterious effect on productivity and/or worth of an animal.
  • the variation in the FGD4 gene which is linked to a deleterious effect on productivity and/or worth of an animal disrupts the FGD4 gene.
  • the method is conducted for the purpose of generating (or at least increasing the likelihood of generating) an animal which does not carry a biological marker which is linked to a deleterious effect on productivity and/or worth of an animal. In one embodiment, the method is conducted for the purpose of generating (or at least increasing the likelihood of generating) an animal which is suitable for production and/or breeding purposes. In one embodiment, the method is conducted for the purpose of generating (or at least increasing the likelihood of generating) an animal having desirable worth and/or productivity.
  • the invention provides a method for generating a cell or embryo which is of use in a method for generating an animal, the method comprising at least the step of introducing a genetic alteration to the FGD4 gene of the cell or embryo.
  • the genetic alteration introduced to the FGD4 gene corrects a variation in the FGD4 gene which is linked to a deleterious effect on productivity and/or worth of an animal.
  • the variation in the FGD4 gene which is linked to a deleterious effect on productivity and/or worth of an animal disrupts the FGD4 gene.
  • the method is conducted for the purpose of generating (or at least increasing the likelihood of generating) a cell or embryo of use in generating an animal which does not carry a biological marker which is linked to a deleterious effect on productivity and/or worth of an animal.
  • the method is conducted for the purpose of generating (or at least increasing the likelihood of generating) a cell or embryo of use in generating an animal which is suitable for production and/or breeding purposes.
  • the method is conducted for the purpose of generating (or at least increasing the likelihood of generating) a cell or embryo which may be used to generate an animal having desirable worth and/or productivity
  • the method for generating the animal involves IVF. In one embodiment, the method for generating the animal is a cloning method.
  • a genetic alteration which is introduced includes a genetic alteration located within the FGD4 gene at a position corresponding to position 77632752 or downstream of a position corresponding to position 77632752 of chromosome 5 of Bos Taurus.
  • the genetic alteration which is introduced includes a genetic alteration at a position corresponding to position 77632752 on chromosome 5 of Bos taurus.
  • the genetic alteration which is introduced includes a substitution of a T at the position corresponding to position 77632752 on chromosome 5 of Bos taurus.
  • the T is substituted with a C.
  • the genetic alteration which is introduced results in the FGD4 gene having the sequence of SEQ ID No: 1.
  • the cell is chosen from a gamete or a zygote. In one embodiment, the cell is a somatic cell or a cell from a cell line.
  • a method of the twenty third or twenty fourth aspects may further comprise the step of selecting or rejecting an animal, cell or embryo using a method of any one or more of the methods of the first, second, third, eighth, ninth, tenth, fifteenth, sixteenth or seventeenth aspects of the invention. This step may be conducted prior to and/or after the step of introducing a genetic alteration to the FGD4 gene to select a starting cell and/or to confirm the alteration is present in a resulting cell, embryo and/or animal.
  • a method of the twenty third or twenty fourth aspects uses IVF, it may further comprise the step of selecting or rejecting an animal, cell or embryo using a method of any one or more of the methods of the first, second, third, eighth, ninth, tenth, fifteenth, sixteenth or seventeenth aspects of the invention. This step may be conducted prior to and/or after the step of introducing a genetic alteration to the FGD4 gene to select a starting cell and/or to confirm the alteration is present in a resulting cell, embryo and/or animal.
  • the invention provides a cell capable of use in a method of generating a non human animal, wherein a genetic alteration has been introduced to a FGD4 gene of the cell, and the genetic alteration corrects a variation in the FGD4 gene which is linked to a deleterious effect on productivity and/or worth of an animal.
  • the invention provides an embryo capable of use in a method of generating a non-human animal, wherein a genetic alteration has been introduced to a FGD4 gene of the embryo, and the genetic alteration corrects a variation in the FGD4 gene which is linked to a deleterious effect on productivity and/or worth of an animal.
  • the invention provides a method for generating a non-human animal, the method comprising using a cell or embryo to which a genetic alteration to a FGD4 gene has been introduced to generate the non-human animal, wherein the genetic alteration corrects a variation in the FGD4 gene which is linked to a deleterious effect on productivity and/or worth of an animal.
  • the invention provides a method for increasing the likelihood that a non human animal will have a desirable worth and/or productivity, the method comprising using a cell or embryo to which a genetic alteration to a FGD4 gene has been introduced to generate the non-human animal, wherein the genetic alteration corrects a variation in the FGD4 gene which is linked to a deleterious effect on productivity and/or worth of an animal.
  • the invention provides a method for increasing the likelihood that a non human animal is suitable for inclusion in a herd, for production and/or breeding purposes, the method comprising using a cell or embryo to which a genetic alteration to a FGD4 gene has been introduced to generate the non-human animal, wherein the genetic alteration corrects a variation in the FGD4 gene which is linked to a deleterious effect on productivity and/or worth of an animal.
  • the invention provides a method for generating a non-human animal, the method comprising at least the step of introducing a genetic alteration to a FGD4 gene of a cell or embryo and using the cell or embryo to generate a non-human animal, wherein the genetic alteration corrects a variation in the FGD4 gene which is linked to a deleterious effect on productivity and/or worth of an animal.
  • the invention provides a method for increasing the likelihood that a non-human animal to be generated will have a desirable worth and/or productivity, the method comprising at least the step of introducing a genetic alteration to a FGD4 gene of a cell or embryo and using the cell or embryo to generate a non-human animal, wherein the genetic alteration corrects a variation in the FGD4 gene which is linked to a deleterious effect on productivity and/or worth of an animal.
  • the invention provides a method for increasing the likelihood that a non human animal to be generated will be suitable for inclusion in a herd, for production and/or breeding purposes, the method comprising at least the step of introducing a genetic alteration to a FGD4 gene of a cell or embryo and using the cell or embryo to generate a non-human animal, wherein the genetic alteration corrects a variation in the FGD4 gene which is linked to a deleterious effect on productivity and/or worth of an animal.
  • the invention provides a method for generating an animal that will not carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal, the method comprising using a cell or embryo to which a genetic alteration to a FGD4 gene has been introduced to generate the non-human animal, wherein the genetic alteration corrects a variation in the FGD4 gene which is linked to a deleterious effect on productivity and/or worth of an animal.
  • the invention provides a method for increasing the likelihood that a non-human animal will not carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal, the method comprising using a cell or embryo to which a genetic alteration to a FGD4 gene has been introduced to generate the non-human animal, wherein the genetic alteration corrects a variation in the FGD4 gene which is linked to a deleterious effect on productivity and/or worth of an animal.
  • the invention provides a method for increasing the likelihood that a non-human animal to be generated will not carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal, the method comprising at least the step of introducing a genetic alteration to a FGD4 gene of a cell or embryo and using the cell or embryo to generate a non-human animal, wherein the genetic alteration corrects a variation in the FGD4 gene which is linked to a deleterious effect on productivity and/or worth of an animal.
  • the variation in the FGD4 gene which is linked to a deleterious effect on productivity and/or worth of an animal disrupts the FGD4 gene.
  • more than one genetic alteration is introduced into a cell or embryo.
  • one or more genetic alteration introduced to a cell or embryo is introduced to correct one or more variation in the FGD4 gene which is linked to a deleterious effect on productivity and/or worth of an animal.
  • the invention provides a method to identify whether or not an animal is suitable for (or at least more or less suitable for) inclusion in a herd, production purposes and/or breeding purposes.
  • the method comprises analysing a nucleic acid of said animal to identify whether or not it includes a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith.
  • the method comprises analysing a FGD4, a precursor thereof, an isoform thereof and/or a fragment thereof of said animal to identify whether or not it includes a variation which disrupts FGD4.
  • the method comprises observing the level and/or activity of a FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof of the animal.
  • the animal is identified to have a variation which disrupts the FGD4 gene or FGD4 or to have a level or level of activity of FGD4, a precursor thereof, an isoform thereof and/or a fragment thereof which is indicative of an animal(s) which is/are known to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal, the animal is identified not to be suitable for (or at least less suitable for) inclusion in a herd, production purposes and/or breeding purposes.
  • the animal is identified not to have a variation which disrupts the FGD4 gene or FGD4 or to have a level or level of activity of FGD4, a precursor thereof, an isoform thereof and/or a fragment thereof which is indicative of an animal(s) which is/are known not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal, the animal is identified to be suitable for (or at least more suitable for) inclusion in a herd, production purposes and/or breeding purposes.
  • the invention provides a method to identify whether or not an embryo or cell is suitable for (or at least more or less suitable for) producing an animal which is suitable for inclusion in a herd, production purposes and/or breeding purposes.
  • the method comprises analysing a nucleic acid of said cell or embryo to identify whether or not it includes a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith.
  • the method comprises analysing a FGD4, a precursor thereof, an isoform thereof and/or a fragment thereof of said cell ore embryo to identify whether or not it includes a variation which disrupts FGD4.
  • the method comprises observing the level and/or activity of a FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof of the cell or embryo.
  • a FGD4 a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof of the cell or embryo.
  • the cell or embryo is identified to have a variation which disrupts the FGD4 gene or FGD4 or to have a level or level of activity of FGD4, a precursor thereof, an isoform thereof and/or a fragment thereof which is indicative of an animal(s) which is/are known to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal, the cell or embryo is identified not to be suitable for (or at least less suitable for) producing an animal which is suitable for inclusion in a herd, production purposes and/or breeding purposes.
  • the cell or embryo is identified not to have a variation which disrupts the FGD4 gene or FGD4 or to have a level or level of activity of FGD4, a precursor thereof, an isoform thereof and/or a fragment thereof which is indicative of an animal(s) which is/are not known to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal
  • the cell or embryo is identified to be suitable for (or at least more suitable for) producing an animal which is suitable for inclusion in a herd, production purposes and/or breeding purposes.
  • the methods comprise: a) performing method of the first, eighth or fifteenth aspects; and b) identifying whether or not an animal is suitable (or at least more or less suitable) for inclusion in a herd, production purposes and/or breeding purposes or whether or not a cell or embryo is suitable (or at least more or less suitable) for producing an animal which is suitable (or at least more or less suitable) for inclusion in a herd, production purposes and/or breeding purposes.
  • the invention provides a method for identifying whether or not a non-human animal, cell or embryo carries a biological marker linked to a deleterious effect on protein yield, milk yield, fat yield, liveweight, stature, body condition score, dairy conformation, and/or overall opinion.
  • the method comprises at least the step of analysing a nucleic acid of said animal, cell or embryo to identify whether or not it includes a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith.
  • the method comprises analysing a FGD4, a precursor thereof, an isoform thereof and/or a fragment thereof of said cell or embryo to identify whether or not it includes a variation which disrupts FGD4.
  • the method comprises observing the level and/or activity of a FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof of the animal, cell or embryo.
  • a FGD4 a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof of the animal, cell or embryo.
  • the animal, cell or embryo is identified to have a variation which disrupts the FGD4 gene or FGD4 or to have a level or level of activity of FGD4, a precursor thereof, an isoform thereof and/or a fragment thereof which is indicative of an animal(s) which is/are known to carry a biological marker linked to a deleterious effect on protein yield, milk yield, fat yield, liveweight, stature, body condition score, dairy conformation, and/or overall opinion it is identified to carry a biological marker linked to a deleterious effect on protein yield, milk yield, fat yield, liveweight, stature,
  • the method is for identifying whether or not a non human animal, cell or embryo carries a biological marker linked to a deleterious effect on liveweight, protein yield, milk yield, and fat yield. In one embodiment, the method is for identifying whether or not a non-human animal, cell or embryo carries a biological marker linked to a deleterious effect on protein yield, milk yield, and fat yield. In one embodiment, the method is for identifying whether or not a non-human animal, cell or embryo carries a biological marker linked to a deleterious effect on liveweight.
  • the method is for identifying whether or not a non-human animal, cell or embryo carries a biological marker linked to a deleterious effect on liveweight, stature, body condition score, dairy conformation, and overall opinion. In one embodiment, the method is for identifying whether or not a non-human animal, cell or embryo carries a biological marker linked to a deleterious effect on stature, body condition score, dairy conformation, and/or overall opinion.
  • the variation which disrupts FGD4 is an amino acid variation resulting in a truncated protein of 624 amino acids in length.
  • the variation comprises insertion of an amino acid.
  • the variation comprises insertion of 2, 3, 4, 5 or more amino acids.
  • the variation comprises insertion of an amino acid at a position corresponding to position 620 of FGD4.
  • the variation results in insertion of 2, 3, 4, 5 or more amino acids at a position corresponding to position 620 of FGD4.
  • the amino acid or 2, 3, 4, 5 or more amino acids inserted at a position corresponding to position 620 of FGD4 are chosen from Val, Ser, Phe, Val, and Val, in any order or in the order in which they are listed.
  • the variation comprises insertion of an amino acid and truncation of the protein.
  • the variation is Leu6l9_Phe620insValSerPheValVal.
  • the invention provides a method for selecting or rejecting a non-human animal, cell or embryo, the method comprising selecting the non-human animal, cell or embryo where it has been determined not to carry a biological marker linked to a deleterious effect on protein yield, milk yield, fat yield, liveweight, stature, body condition score, dairy conformation, and/or overall opinion.
  • the method comprises at least the steps of: a) performing a method of the thirty eighth aspect; and b) selecting or rejecting the non -human animal, cell or embryo using the results of step a).
  • the invention provides:
  • a cell generated by a method of the twenty fourth aspect and/or,
  • the invention provides:
  • a method for selecting or rejecting an animal comprising selecting and/or rejecting an animal based on the results of a method of the third, tenth and/or seventeenth aspects;
  • a method for selecting or rejecting an animal comprising: a) performing a method of the third, tenth and/or seventeenth aspects; and b) selecting or rejecting an animal based on the results of a).
  • a method to identify whether or not an animal is suitable for (or at least more or less suitable for) inclusion in a herd, for production purposes and/or breeding purposes comprising predicting the animal’s suitability based on the results of a method of the first, third, eighth, tenth, fifteenth and/or seventeenth aspects;
  • a method to identify whether or not an animal is suitable for (or at least more or less suitable for) inclusion in a herd, for production purposes and/or breeding purposes comprising: a) performing a method of the first, third, eighth, tenth, fifteenth and/or seventeenth aspects; and, b) predicting the animal’s suitability based on the results of a);
  • a method to identify whether or not a cell or embryo is suitable for (or at least more or less suitable for) generating an animal which is suitable for inclusion in a herd, for production purposes and/or breeding purposes comprising: a) performing a method of the first, third, eighth, tenth, fifteenth and/or seventeenth aspects; and, b) predicting the animal’s suitability based on the results of a);
  • a method for estimating the worth of an animal comprising the step of incorporating information on a biological marker of the invention of the animal in the worth calculation;
  • a method for selecting or rejecting an animal cell or embryo comprising the step of selecting or rejecting an animal, cell or embryo using information on a biological marker of the animal, cell or embryo.
  • the invention provides a method for identifying a genetic variation in the FGD4 gene which is linked to a deleterious effect on productivity and/or worth of an animal.
  • the method comprises identifying a genetic variation in the FGD4 gene which disrupts the gene.
  • the genetic variation disrupts the FGD4 gene
  • the variation is identified to be linked to a deleterious effect on productivity and/or worth of an animal.
  • the method further comprises identifying whether or not the alteration results in a decrease in the level and/or activity of FGD4.
  • the alteration results in a decrease in the level and/or activity of FGD4 the genetic alteration is identified as being linked to a deleterious effect on productivity and/or worth of an animal.
  • the method further comprises determining whether or not the identified genetic variation in the FGD4 gene is associated with an animal or animals known to have a variation which disrupts FGD4 and/or less than desirable productivity and/or worth.
  • the invention provides a method for identifying an amino acid variation in FGD4 which is linked to a deleterious effect on productivity and/or worth of an animal.
  • the method comprises identifying a variation in FGD4 which disrupts FGD4.
  • the variation is identified to be linked to a deleterious effect on productivity and/or worth of an animal.
  • the method further comprises identifying whether or not the variation results in a decrease in the level and/or activity of FGD4.
  • the alteration results in a decrease in the level and/or activity of FGD4 the variation is identified as being linked to a deleterious effect on productivity and/or worth of an animal.
  • the method further comprises determining whether or not the identified variation in FGD4 is associated with an animal or animals known to have a variation which disrupts FGD4 and/or less than desirable productivity and/or worth.
  • the non-human animal is, or the non-human cell or embryo is from, a production animal.
  • the production animal is a mammal.
  • the animal is, or the cell or embryo is from a ruminant.
  • the animal, cell or embryo is from the Bovidae family.
  • the animal, cell or embryo is bovine.
  • the bovine is Bos taurus or Bos indicus.
  • the bovine is chosen from the group consisting Jersey, Holstein- Friesian or crossbred dairy cattle. In one particular embodiment of any one or more of the aspects of the invention, the bovine is Holstein-Friesian.
  • the deleterious effect on productivity and/or worth includes a deleterious effect on one or more of the following animal assessment parameters: milk yield; protein yield; and fat yield. In one embodiment of any one or more aspects of the invention, the deleterious effect on productivity and/or worth includes a deleterious effect on one or more of the following animal assessment parameters: liveweight; milk yield; protein yield; and fat yield. In one embodiment of any one or more aspects of the invention, the deleterious effect on productivity and/or worth includes a deleterious effect on one or more of the following animal assessment parameters: liveweight; stature; body condition score; dairy conformation; and overall opinion.
  • the deleterious effect on productivity and/or worth includes a deleterious effect on one or more of the following animal assessment parameters: stature; body condition score; dairy conformation; and overall opinion.
  • the deleterious effect on productivity and/or worth includes a deleterious effect on one or more of the following animal assessment parameters: liveweight; stature; body condition score; protein yield; milk yield; fat yield; dairy conformation; and, overall opinion.
  • the deleterious effect on productivity and/or worth includes a deleterious effect on all of the following animal assessment parameters: protein yield; milk yield; and, fat yield.
  • the deleterious effect on productivity and/or worth includes a deleterious effect on all of the following animal assessment parameters: liveweight; protein yield; milk yield; and, fat yield.
  • the deleterious effect on productivity and/or worth includes a deleterious effect on all of the following animal assessment parameters: liveweight; stature; body condition score; dairy conformation; and, overall opinion.
  • the deleterious effect on productivity and/or worth includes a deleterious effect on all of the following animal assessment parameters: stature; body condition score; dairy conformation; and overall opinion.
  • the deleterious effect on productivity and/or worth includes a deleterious effect on all of the following animal assessment parameters: liveweight; stature; body condition score; protein yield; milk yield; fat yield; dairy conformation; and, overall opinion.
  • the invention provides an isolated nucleic acid comprising the genetic marker 77632752T on chromosome 5 of Bos taurus and/or a genetic marker in linkage disequilibrium therewith.
  • the invention provides a nucleic acid comprising or consisting the sequence of SEQ ID No. 2 or a functionally equivalent variant thereof.
  • the invention provides an oligonucleotide or part of the nucleic acid comprising or consisting of the sequence of SEQ ID No. 2.
  • the invention provides a nucleic acid which is complementary to or can hybridise to said nucleic acids, in one embodiment under stringent conditions.
  • the nucleic acid is chosen from the group comprising DNA, cDNA and mRNA, including single- and double-stranded nucleic acids, for example.
  • the invention provides an isolated peptide encompassing a variation which disrupts FGD4.
  • the peptide comprises Leu6l9_Phe620insValSerPheValVal.
  • the peptide comprises or consists the sequence of SEQ ID No. 4.
  • the invention provides a fragment, isoform or precursor of the peptide comprising or consisting the sequence of SEQ ID No. 4 or a part of any one or more thereof or a functionally equivalent variant thereof.
  • the invention provides other isolated nucleic acids and/or peptides and/or proteins as may be described herein.
  • any of the aspects includes analysing a nucleic acid or analysing FGD4, gene product or FGD4 protein, a precursor thereof, an isoform thereof and/or a fragment thereof, or observing the level and/or activity of a FGD4, this may be done in vitro.
  • the invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.
  • Figure 1 shows a version of SEQ ID NO: 1 showing lOOObp of non-coding sequences both 5’ and 3’ of the first and last exons, though for brevity, all introns have been removed aside from the intron of interest (i.e. intron 10 that encompasses the chr5:77632752 substitution).
  • Intron-Exon boundaries for introns that have been removed are demarcated by‘pipe’ symbols, and where relevant, exons are provided in uppercase, and intronic and intergenic sequences are provided in lowercase.
  • Site of the chr5:77632752C>T substitution is bracketed, though note a‘G’ allele is indicated due to the gene being transcribed from the reverse direction (i.e.
  • Figure 2 shows a version of SEQ ID NO: 2 showing lOOObp of non-coding sequences both 5’ and 3’ of the first and last exons, though for brevity, all introns have been removed aside from the intron of interest (i.e. intron 10 that encompasses the chr5:77632752 substitution).
  • Intron-Exon boundaries for introns that have been removed are demarcated by‘pipe’ symbols, and where relevant, exons are provided in uppercase, and intronic and intergenic sequences are provided in lowercase.
  • Site of the chr5:77632752C>T substitution is bracketed, though note an‘A’ allele is indicated due to the gene being transcribed from the reverse direction (i.e. base shown on minus strand).
  • Figure 3 shows SEQ ID No. 3 protein sequence indicating the reference (wild type) form of FGD4.
  • Figure 4 shows SEQ ID No. 4 - protein sequence indicating the alternate (mutant) form of FGD4.
  • NCBI Reference Sequence XP 005206940.1 FYVE, RhoGEF and PH domain-containing protein 4 isoform XI [Bos taurus].
  • Consequence of the chr5:77632752C>T splice donor variant is shown, where loss of the splice donor site leads to exon 10 frame extension, with the addition of five new amino acids and a premature termination codon. The accompanying amino acid changes are bolded and underlined.
  • Figure 5 shows SEQ ID No. 5 sequence representing the wild type gene mRNA transcript of FGD4 (Refseq ID XM_005206883.3). Demonstrates that the position of the nucleotide substitution is not shown in this transcript, since the chr5:77632752 site occurs in intron 10, and only spliced mRNA is indicated.
  • Figure 6 shows SEQ ID No. 6 sequence representing the predicted mutant transcript of FDG4 (Refseq ID XM_005206883.3).
  • the site of the nucleotide substitution in the mutant transcript is bracketed, forming the first base of the intron 10 inclusion event resulting from loss of splicing fidelity.
  • the position of the variant is nucleotide 2176.
  • Figure 7 shows RNA sequence alignments for the FGD4 gene in six animals of contrasting chr5:77632752C>T genotype.
  • the three top-most animals are heterozygous for the FGD4 variant (chr5:77632752CT), with the bottom three animals homozygous wild-type (chr5:77632752CC).
  • the exon-intron gene structure is indicated by the FGD4 gene model at bottom of field (only exons 8-15 are shown), with the exon 10 splice boundary impacted by the chr5:77632752C>T mutation indicated by the vertical bar.
  • Substantially increased intron 10 sequence read depth is evident in the three heterozygous animals, suggesting intron 10 retention as a consequence of the chr5:77632752C>T splice donor mutation.
  • the inventors have identified a genetic variation in the FGD4 (FYVE, RhoGEF and PH domain containing 4) gene which has a deleterious effect on a number of traits representative of productivity and used in worth calculations for an animal, when in a homozygous state.
  • FGD4 FYVE, RhoGEF and PH domain containing 4
  • the inventors contemplate that methods involving the analysis of nucleic acid to determine whether or not it includes such an alternation in FGD4 can be used, inter alia, to identify whether or not an animal, cell or embryo carries a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the inventors also contemplate that the analysis of one or more genetic marker which is in linkage disequilibrium with the specific marker they have identified may also be used for the same purpose.
  • haplotypes including the genetic marker of the invention and/or one or more marker in linkage disequilibrium therewith may also be used for this purpose.
  • methods of the invention may comprise the analysis of a FGD4, fragments, isoforms, and/or precursors thereof, and/or of the levels (including size, sequence, amount, expression and activity levels) of FGD4.
  • the invention may also comprise the analysis of the nucleotide sequence of a FGD4 transcript or a fragment thereof.
  • the invention should be taken to encompass methods involving the analysis of nucleic acid from an animal to determine whether or not it includes any such alteration or variation in the gene (including reference to the transcript), analysis of the amino acid sequence of FGD4 (including reference to fragments, isoforms, and/or precursors thereof) to determine whether or not it includes any alteration that disrupts FGD4, and/or the level (including the amount and level of expression, for example) and/or activity of FGD4 to identify whether or not it has a marker linked to a deleterious effect on productivity and/or whether or not an animal is suitable for (or at least more or less suitable for) production purposes, breeding purposes, and/or inclusion in a herd, or whether or not a cell or embryo is suitable for (or at least more or less suitable for) producing an animal that is suitable for production purposes, breeding purposes and/or inclusion in a herd, for example. While the description which follows may focus on the analysis of the nucleotide sequence or amino acid sequence at a particular position, it should be understood to extend to the analysis
  • the specific genetic variation identified by the inventors is annotated as being a splice donor variant.
  • methods of the invention may comprise the analysis of nucleic acids/proteins to identify splice variants which disrupt FGD4.
  • Analysis of a biological marker of FGD4 in accordance with the invention may also be used for selecting or rejecting a cell or embryo. Such analysis may aid in methods for breeding or cloning animals, for example.
  • Analysis of a biological marker in accordance with the invention may assist in: predicting phenotypic performance, including use in production management systems known as Marker Assisted Selection; the selection or rejection of animals for breeding and/or other purposes; managing animals in order to maximise their individual potential performance and value; estimating the worth or economic value of an animal; improving profits related to selling animals and/or products produced from the animals; improving the genetics of a population of animals by selecting and breeding desirable animals;
  • animals may be tested or screened any time during their life, including early at birth or as embryos or foetuses, to predict life time performance and segregated or managed to suit their genotype and therefore predicted phenotype. Any time during their life includes, but is not limited to, early at birth, as gametes, zygotes, embryos, foetuses.
  • “Productivity” refers to the level and/or efficiency of production of a particular product by an animal. In one embodiment, it includes efficiency of production per unit liveweight of an animal.
  • the product may be meat, or milk. Such raw products may be further processed into consumer products such as milk powder, cheeses, whey protein, specialty beverages and meat products.
  • Productivity can be measured based on an assessment of one or more parameters, having regard to the product to be produced, as will be understood by persons skilled in the art. However, by way of example, where the product is milk (or a derivative thereof), milk yield, protein yield, and/or fat yield can be assessed, alone or in combination with one or more other animal assessment parameters as described herein.
  • liveweight can be assessed, alone or in combination with one or more other animal assessment parameters as described herein.
  • the“worth” of an animal refers to an index used to evaluate the value of an animal, for breeding purposes, production purposes, inclusion in a herd, herd management, for example.
  • the “worth” is the sum of the estimated value of one or more characteristics (or animal assessment parameters) which may be associated with the animal, typically weighted by an economic value.
  • Exemplary assessment parameters may include those which may be referred to as“production traits” and those which may be referred to as“traits other than production” or“TOP”; as described in “Evaluation System for Traits Other Than Production (TOP) for Dairy Cattle in New Zealand” published in August 2014 by Advisory Committee on Traits Other than Production at dairynz.co.nz/media/l205535/TOP_Booklet_A4.pdf and “Your Index Your Animal Evaluation System (Second Edition)” published by NZ Animal Evaluation Unit at dairynz.co.nz/media/58l307/Your_Index_Your_AE_System.pdf.
  • TOP Evaluation System for Traits Other Than Production
  • breeding worth should be taken to encompass“breeding worth”,“lactation worth”,“production worth” and other known indexes used to assess the value of an animal. Breeding worth, lactation worth and production worth, are described in“Your Index Your Animal Evaluation System (Second Edition)” as detailed above, for example. Other exemplary indexes include the Balanced Performance Index (BPI) in Australia, and the Economic Breeding Index (EBI) applied in Ireland. These are described, for example in“Your Herd. Your Test. Your Future.
  • BPI Balanced Performance Index
  • EBI Economic Breeding Index
  • results, data and/or information generated by one or more method of the invention may be used in calculations for estimating“worth”.
  • an animal may be selected or rejected for a particular purpose (for example, inclusion in a herd, for production purposes and/or for breeding purposes) based on its worth.
  • the methods of the invention are described in terms of estimating the worth of an animal“and/or its offspring”. This should be taken to mean that the worth of the offspring of an animal or pair of animals can be estimated indirectly based on evaluation of one or both parents without the need to directly assess the offspring. Such evaluation would at least involve identifying whether or not one or both parent has a biological marker in accordance with the invention.
  • the method may comprise identifying whether or not a parent animal has a biological marker linked to a deleterious effect on productivity and/or worth of an animal and using this information to in a calculation to estimate the worth of the offspring. Such methods may be used to estimate the potential worth of an offspring if a pair of chosen animals was mated, for example.
  • worth is calculated taking into consideration one or more of the animal assessment parameters (or traits) of milk yield, milk fat yield and milk protein yield (production traits), liveweight, stature, body condition score, dairy conformation, and overall opinion (TOP), alone or in combination with one or more other animal assessment parameters.
  • worth is calculated taking into consideration one or more of protein yield, milk yield, and fat yield, alone or in combination with one or more other animal assessment parameters.
  • worth is calculated taking into consideration one or more of protein yield, milk yield, fat yield, and liveweight, alone or in combination with one or more other animal assessment parameters.
  • worth is calculated taking into consideration one or more of liveweight, stature, body condition score, dairy conformation, and overall opinion, alone or in combination with one or more other animal assessment parameters. In one embodiment, worth is calculated taking into consideration one or more of stature, body condition score, dairy conformation, and overall opinion, alone or in combination with one or more other animal assessment parameters. In one embodiment of the invention, worth is calculated taking into consideration all of the production traits of milk yield, milk fat yield and milk protein yield, alone or in combination with one or more other animal assessment parameters. In one embodiment, worth is calculated taking into consideration all of protein yield, milk yield, fat yield, and liveweight, alone or in combination with one or more other animal assessment parameters.
  • worth is calculated taking into consideration all of the TOP of liveweight, stature, body condition score, dairy conformation, and overall opinion, alone or in combination with one or more other animal assessment parameters. In another embodiment, worth is calculated taking into consideration all of the TOP of stature, body condition score, dairy conformation, and overall opinion, alone or in combination with one or more other animal assessment parameters. In another embodiment of the invention, worth is calculated taking into consideration all of milk yield, milk fat yield and milk protein yield, liveweight, stature, body condition score, dairy conformation, and overall opinion, alone or in combination with one or more other animal assessment parameters.
  • A“deleterious effect on productivity” should be understood to be mean any level of detrimental effect on the level and/or efficiency of production of one or more product by an animal. In one embodiment, it means the animal has or is predicted to have a reduced level of production compared to an animal or animals having at least a desirable level of productivity or the same animal if it did not have a genetic variation which disrupts the FGD4 gene.
  • the product is milk and the animal has or is predicted to have a reduced level of milk yield, protein yield and/or fat yield.
  • the product is meat and the animal has or is predicted to have a reduced liveweight.
  • a“desirable level of productivity” may differ from one animal or breed or farm or herd or country to another, for example. It may also differ depending on the product which an animal is being used to produce. Skilled persons will readily appreciate a benchmark desirable level of productivity having regard to the product to be produced, information on breed, the herd or population of animals used and one or more assessment criteria known in the art.
  • a desirable level of productivity is the mean of a population of animals. In another embodiment, a desirable level of productivity is the mean of a population of animals that is homozygous for the wild- type allele of FGD4. In another embodiment, a desirable level of productivity is the mean of a population of animals that is heterozygous for a genetic marker of the invention. In another embodiment, a desirable level of productivity is the level of a chosen highly productive animal.
  • A“deleterious effect on worth” or like phrases is intended to mean any level of detrimental effect on the estimated worth of an animal. In one embodiment, it means the animal has or is predicted to have a lower worth compared to an animal or animals having a desirable worth or the worth of same animal if it did not have a genetic variation which disrupts the FGD4 gene. In some embodiments, there is at least a 5% level of reduction, at least a 10% level of reduction, or at least a 15% level of reduction in the worth of an animal compared to an animal or animals having a desirable worth or the worth of same animal if it did not have a genetic variation which disrupts the PLCD4 gene, for example.
  • a“desirable worth” may differ from one animal or breed or farm or herd or country to another, for example. It may also differ depending on the purpose for which an animal is to be used, the industry in which an animal is to be used, and/or the product which an animal is being used to produce. Persons skilled in the art will readily be able to appreciate or calculate a benchmark desirable worth having regard to such factors and using one or more known methods to calculate worth of an animal (for example, those herein before described).
  • a desirable worth is the mean of a population of animals.
  • a desirable worth is the mean of a population of animals that is homozygous for the wild-type allele of FGD4.
  • a desirable worth is the mean of a population of animals that is heterozygous for a genetic marker of the invention.
  • a desirable worth is the worth of a chosen animal.
  • “a deleterious effect on productivity and/or worth” includes reference to a deleterious effect on one or more animal assessment parameters (or traits) representative of productivity and used in worth estimations. These traits include“production traits” and“traits other than production (TOP)”, as described herein before.
  • the one or more animal assessment parameters representative of productivity include the“production traits” protein yield, milk yield, fat yield, and the“TOP” liveweight, stature, body condition score, dairy conformation, and overall opinion.
  • the invention provides methods to identify whether or not an animal, cell or embryo carries a biological marker linked to a deleterious effect on one or more of protein yield, milk yield, fat yield, liveweight, stature, body condition score, dairy conformation, and overall opinion, and the various aspects of the invention described herein should be taken to include this.
  • the invention provides methods to identify whether or not an animal, cell or embryo carries a biological marker linked to a deleterious effect on one or more of protein yield, milk yield, and fat yield.
  • the invention provides methods to identify whether or not an animal, cell or embryo carries a biological marker linked to a deleterious effect on one or more of protein yield, milk yield, fat yield, and liveweight.
  • the invention provides methods to identify whether or not an animal, cell or embryo carries a biological marker linked to a deleterious effect on one or more of liveweight, stature, body condition score, dairy conformation, and overall opinion. In one embodiment, the invention provides methods to identify whether or not an animal, cell or embryo carries a biological marker linked to a deleterious effect on one or more of stature, body condition score, dairy conformation, and overall. In another embodiment, the invention provides methods to identify whether or not an animal, cell or embryo carries a biological marker linked to a deleterious effect on all of the following: protein yield, milk yield, and fat yield.
  • the invention provides methods to identify whether or not an animal, cell or embryo carries a biological marker linked to a deleterious effect on all of the following: liveweight; protein yield; milk yield; and fat yield. In another embodiment, the invention provides methods to identify whether or not an animal, cell or embryo carries a biological marker linked to a deleterious effect on all of the following: stature; body condition score; dairy conformation; and overall opinion. In another embodiment, the invention provides methods to identify whether or not an animal, cell or embryo carries a biological marker linked to a deleterious effect on all of the following: liveweight, stature, body condition score, dairy conformation, and overall opinion.
  • the invention provides methods to identify whether or not an animal, cell or embryo carries a biological marker linked to a deleterious effect on all of the following: protein yield, milk yield, fat yield, liveweight, stature, body condition score, dairy conformation and overall opinion.
  • the animal assessment parameters may be assigned a measure or a score.
  • the animal assessment parameters may be assigned a score on a scale.
  • liveweight may be assigned a measure in Kilograms (or other appropriate units of measure)
  • stature may be assigned a measure in centimeters (or other appropriate units of measure)
  • protein yield, milk yield, and/or fat yield may be assigned a measure in Kg/day (or other appropriate units of measure), for example.
  • A“deleterious effect on one or more animal assessment parameters” or like phrases is intended to mean any level of detrimental effect on an assessment parameter.
  • a“deleterious effect on one or more animal assessment parameters” means the animal has or is predicted to have a lower measure or lower score for stature, body condition, dairy conformation, overall opinion, liveweight, protein yield, milk yield, and/or fat yield compared to an animal or animals having a desirable assessment measure or score or the same animal if it did not have a genetic variation which disrupts the FGD4 gene.
  • a“desirable assessment measure” or“desirable assessment score” may differ from one animal or breed or farm or herd or country to another, for example. It may also differ depending on the product which an animal is being used to produce. Persons skilled in the art will readily be able to appreciate or calculate a benchmark desirable assessment measure or score using one or more known methods (for example, those described in “Evaluation System for Traits Other Than Production (TOP) for Dairy Cattle in New Zealand” and“Your Index Your Animal Evaluation System (Second Edition)” as referred to herein before).
  • a desirable assessment measure or score is the mean value in a population.
  • a desirable assessment measure or score is the mean of a population of animals that is homozygous for the wild-type allele of FGD4. In another embodiment, a desirable assessment measure or score is the mean of a population of animals that is heterozygous for a genetic marker of the invention. In another embodiment, a desirable assessment measure or score is the value of a specific individual.
  • biological marker(s) should be taken broadly and includes, for example, genetic markers, the level of a protein (including reference to a fragment thereof, a precursor thereof, an isoform thereof) or a nucleic acid encoding a protein (including reference to a fragment thereof, a precursor thereof, an isoform thereof), the level of expression of a gene or protein (including reference to a fragment thereof, a precursor thereof, an isoform thereof), the level of activity of a protein (including reference to a fragment thereof, a precursor thereof, an isoform thereof) and/or variation in the amino acid sequence of a protein (including reference to a fragment thereof, a precursor thereof, an isoform thereof) which may include observation of the size of a protein (including reference to a fragment thereof, a precursor thereof, an isoform thereof) or nucleic acid (including for example a transcript).
  • a biological marker comprises a splice variant.
  • genetic marker refers to nucleic acids or specific genetic loci (including specific nucleotide positions) that are polymorphic or contain sequence alterations or variations within a population, the alleles of which can be detected and distinguished by one or more analytic methods.
  • genetic marker further includes within its scope a plurality of genetic markers co segregating, in the form of a "haplotype".
  • haplotype refers to a plurality of genetic markers that are generally inherited together. Typically, genetic markers within a haplotype are in linkage disequilibrium.
  • references herein to“analysing” a nucleic acid or nucleic acid sequence of an animal, cell or embryo to identify whether or not it includes a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith should be taken to include the physical, biological and/or chemical analysis of a nucleic acid from an animal, cell or embryo, as well as the analysis of data already available for the nucleic acid of a particular animal, cell or embryo.
  • References to “analysing” a nucleic acid should be taken to include analysis of DNA or RNA (for example, a FGD4 transcript) and either or both strands of a double -stranded nucleic acid.
  • nucleotide or nucleic acid“sequence” should be taken broadly to include both a single nucleotide as well as two or more nucleotides.
  • reference to determining or identifying whether or not a nucleic acid includes a specific genetic marker should be read to encompass determining or identifying the genetic marker by observing the nucleotide or sequence on either strand of a DNA molecule.
  • references to“analysing” an amino acid sequence or a FGD4 protein, a precursor thereof, an isoform thereof, and/or a fragment thereof of an animal, cell or embryo to identify whether or not it includes a variation which disrupts FGD4 should be taken to include the physical, biological and/or chemical analysis of a FGD4 protein, a precursor thereof, an isoform thereof, and/or a fragment thereof from an animal, cell or embryo, as well as the analysis of data already available for a FGD4, a precursor, an isoform, a fragment of a particular animal, cell or embryo.
  • references to“analysing a nucleic acid of a cell or embryo” or“analysing a FGD4, a fragment thereof, an isoform thereof, and/or a precursor thereof of a cell or embryo” and like phrases should be taken to include reference to analysing a nucleic acid or a FGD4, a fragment thereof, an isoform thereof, and/or a precursor thereof of an animal from which the cell or embryo is derived in lieu of directly analysing a nucleic acid or a FGD4, a fragment thereof, an isoform thereof, and/or a precursor thereof of said cell or embryo.
  • the step of“analysing” a nucleic acid of an animal, cell or embryo to identify whether or not it includes a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith may comprise imputing a genotype of said animal (including the genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith).
  • the step of analysing may comprise directly genotyping (for example, involving physical, biological, or chemical analysis of a nucleic acid) the animal, cell or embryo for a marker and comparing the genotype of the animal, cell or embryo to the genotype of a family member for which an additional marker has been genotyped.
  • the step may comprise identifying a genotype of the animal, cell or embryo for a marker (for example, involving analysis of genetic data already available for a particular animal, cell or embryo) and comparing the genotype of the animal, cell or embryo to the genotype of a family member for which an additional marker has been genotyped.
  • a genetic marker of the invention and/or a marker in linkage disequilibrium is imputed to be present or absent from the animal, cell or embryo based on the comparison.
  • the step of“analysing” may comprise directly genotyping the animal, cell or embryo for a marker and comparing the genotype of the animal, cell or embryo to the genotype(s) of a reference population comprising apparently unrelated individuals for which an additional marker has been genotyped.
  • the step may comprise identifying a genotype of the animal, cell or embryo for a marker (for example, involving analysis of genetic data already available for a particular animal, cell or embryo) and comparing the genotype of the animal, cell or embryo to the genotype(s) of a reference population comprising apparently unrelated individuals for which an additional marker has been genotyped.
  • a genetic marker of the invention and/or a marker in linkage disequilibrium is imputed to be present or absent from the animal, cell or embryo based on the comparison.
  • a reference population may be assembled from animals of a similar breed or crossbreed composition, or animals otherwise expected to share a similar haplotype structure, for example. Skilled persons will readily appreciate other appropriate reference populations.
  • identifying whether or not an animal, cell or embryo carries a genetic marker linked to a deleterious effect on productivity and/or worth of an animal and/or a genetic marked in linkage disequilibrium, and/or whether or not a nucleic acid has a genetic variation which disrupts the FGD4 gene and/or a genetic marked in linkage disequilibrium can include identifying by imputation.
  • reference to an animal, cell or embryo having been identified to carry or not carry a genetic marker should also be taken to include it having been identified by imputation. Similar references to“identify”,“identifying” and the like should be construed in the same way, unless the context requires otherwise.
  • Reference to“observing the level and/or activity of a FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof’ of an animal, cell or embryo should be taken to include the physical or chemical analysis of the level and/or activity of a FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof from an animal, cell or embryo, as well as the analysis of data already available for the level and/or activity of a FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof of a particular animal, cell or embryo.
  • references to“observing the level and/or activity of a FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof’ of a cell or embryo should be taken to include reference to observing the level and/or activity of a FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof of an animal from which the cell or embryo is derived in lieu of directly observing the level and/or activity of a FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof of said cell or embryo.
  • single nucleotide polymorphism refers to nucleic acid sequence variations that occur when a single nucleotide in the genome sequence is altered.
  • a single nucleotide polymorphism may also be a single nucleotide insertion or deletion.
  • the different nucleotides within a SNP are referred to as an allele.
  • genetictype means the genetic constitution or nucleotide sequence at one or more genetic locus, in particular the nucleotide sequence of an allele of a genetic locus.
  • Linkage disequilibrium should be taken broadly to refer to the tendency of the presence of an allele at one genetic locus to predict the presence of an allele at one or more other genetic loci (for example a distinct genetic marker).
  • the genetic loci need not necessarily be on the same chromosome. However, in a preferred embodiment, the genetic loci are located on the same chromosome.
  • DELTA 2 is calculated using the formula described by Devlin et al (Genomics 29 (2): 311-22 (1995)), and is a measure of how well an allele X at a first genetic loci predicts the occurrence of an allele Y at a second genetic loci.
  • a DELTA 2 value of 1.0 indicates the prediction is perfect (for example, if Y is present then X is present). It should be appreciated that reference to linkage disequilibrium herein should not be taken to imply a DELTA 2 value of 1.0.
  • the linkage disequilibrium between an allele at one genetic locus and an allele at a second genetic locus has a DELTA 2 value of at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.95, and most preferably 1.0.
  • Other measures of linkage disequilibrium are described, for example, in the Encyclopedia of Cancer at: link. springer.com referenceworkentry/10.1007%2F978-3-642- 16483 -5 3368
  • A“variation in the amino acid sequence” of FGD4, isoform, fragment and/or precursor thereof should be considered broadly to include any change in the amino acid sequence. By way of example only, it should be taken to include substitution of any one or more amino acid, addition of one or more amino acid and/or deletion of one or more amino acid. In one embodiment, a variation may be a truncation of a protein.
  • A“genetic variation” in the FGD4 gene should be considered broadly to include any change in the nucleotide sequence. By way of example only, it should be taken to include substitution of any one or more nucleotide, addition of one or more nucleotide and/or deletion of one or more nucleotide. In certain embodiments, a variation may result in frameshift and/or introduction of a premature stop codon.
  • nucleic acid includes a genetic variation or a peptide or protein includes a variation in the amino acid sequence
  • variation is a difference in the nucleic acid or amino acid sequence compared to the nucleic acid sequence or amino acid sequence associated with one or more animal which is known not to carry a biological marker which disrupts FGD4 and is linked to a deleterious effect on productivity and/or worth.
  • the variation will be determined in relation to a “reference sequence”.
  • the reference sequence is a nucleic acid encoding part or all of a FGD4 gene (or transcript) and/or a genetic marker in linkage disequilibrium therewith or the amino acid sequence of FGD4, an isoform, a precursor or a fragment thereof, which is associated with an animal that is known not to carry such a marker.
  • the nucleic acid reference sequence is SEQ ID No. 1, SEQ ID No. 5 or a part of these.
  • the amino acid reference sequence is SEQ ID No. 3 or a part thereof.
  • the reference sequence could be a nucleic acid or peptide or protein having a sequence which is known to be associated with an animal or animals who is/are known to carry a biological marker which disrupts FGD4 and is linked to a deleterious effect on productivity and/or worth. Accordingly, reference to “variation” should not be restricted to mean that the nucleic acid, peptide or protein being analysed is different from the reference sequence.
  • such a“part” comprises or consists at least 4, 10, 20, 30, 40, 50, 60, 70, 80 or more consecutive amino acids of a FGD4 which includes a variation which disrupts FGD4 (in one embodiment, it comprises the variation Leu6l9_Phe620insValSerPheValVal). In one embodiment, it comprises or consists at least 4 consecutive amino acids of SEQ ID No. 4, or at least 10, 20, 30, 40, 50, 60, 70, 80 or more consecutive amino acids of SEQ ID No. 4. In one embodiment, such a“part” comprises or consists at least 4 consecutive amino acids of SEQ ID No.
  • a region of the FGD4 gene or transcript.
  • the region will encompass a part of a FGD4 gene or transcript in which a genetic alteration which disrupts the FGD4 gene resides.
  • such a region comprises or consists at least 4 consecutive nucleotides, or at least 10, 20, 30, 40, 50, 60, 70 80 or more consecutive nucleotides of a FGD4 gene or transcript.
  • the region will encompass a part of a FGD4 gene or transcript in which the genetic marker 77632752T and/or a marker in linkage disequilibrium therewith resides.
  • such a region comprises or consists at least 4 consecutive nucleotides of SEQ ID No. 2 or SEQ ID No. 6, or at least 10, 20, 30, 40, 50, 60, 70 80 or more consecutive nucleotides of SEQ ID No. 2 or 6.
  • protein isoforms are different forms of a protein coded from the same gene.
  • different forms of a protein can be produced by alternative splicing of RNA transcripts to form different mRNA sequences or by different glycosylation or other posttranslational modification.
  • protein precursors including pre-pro-proteins or pre-pro-peptides and pro-proteins or pro-peptides
  • protein precursors are inactive forms of a protein or peptide that can be activated by post- and/or co-translation modification.
  • A“fragments” of FGD4 may include peptides of any length suitable to identify an amino acid variation in accordance with the invention.
  • A“fragment” will include or encompass the amino acid variation to be detected (for example, one or more amino acid substitution, addition or deletion at one or more particular position(s) in FGD4).
  • a “fragment” may consist of the truncated protein.
  • a genetic alteration or variation which“disrupts” the FGD4 gene may be any genetic change which has an affect on the level, expression or activity of the FGD4 gene product (including reference to isoforms, fragments and/or precursors thereof). By way of example, it may decrease the level of expression or alter the structure or function of the gene product.
  • a variation or alteration in the amino acid sequence which disrupts FGD4 may be any change in the amino acid sequence of FGD4 which has an effect on the level or level of activity of FGD4 (including reference to isoforms, fragments and/or precursors thereof).
  • a variation which“disrupts” FGD4 or its gene is one which is associated with a deleterious effect on productivity and/or worth in an animal, as herein before described.
  • an“increase” or“decrease” in the level or activity of FGD4 an isoform thereof, a fragment thereof, a precursor thereof, and/or a nucleic acid encoding any one or more thereof it should be taken broadly to include any increase or decrease in said level or activity compared to a reference animal or animals or a standard.
  • the reference animal or animals are those having wild-type FGD4.
  • the reference animal or animals are those having a genetic or amino acid variation which is linked to or associated with a deleterious effect on productivity and/or worth (ie a variation which disrupts FGD4).
  • Reference to the“FGD4 gene” should be taken to include reference to the coding and non-coding regions of the gene, including upstream and downstream regulatory elements.
  • the term“animal” is used herein primarily in reference to mammals.
  • the mammal is a ruminant.
  • the mammal is one within within the Bovidae family.
  • the animal is a bovine animal. More particularly the animal is Bos taurus or Bos indicus.
  • the animal is a beef or dairy breed.
  • the animal may be chosen from the group of animals including, but not limited to, Jersey, Holstein-Friesian, Ayrshire, crossbred dairy cattle, Angus, Hereford, Simmental and crossbred beef cattle.
  • the animal is Holstein-Friesian.
  • A“functionally equivalent variant” of any particular nucleic acid, protein or peptide referred to herein should be taken broadly to encompass any nucleic acid, peptide or protein whose sequence may vary from the specific sequence provided but which nucleic acid, peptide or protein retains substantially the same function; for example, in the case of an oligonucleotide used to detect a genetic marker of the invention, the ability to bind to a particular target nucleic acid or prime a particular reaction with the desired specificity).
  • the phrase“functionally equivalent” should not be taken to imply that the variant has the same level of activity as the nucleic acid, peptide or protein of which it is a variant, although this may be desired.
  • “functionally equivalent variants” of any particular nucleic acid, peptide or protein will have at least approximately 80%, approximately 90%, approximately 95%, or approximately 99% sequence homology or similarity to the sequence of which they are a variant. In one particular embodiment, the“functionally equivalent variants” of any particular nucleic acid, peptide or protein will have at least approximately 80%, approximately 90%, approximately 95%, or approximately 99% sequence identity to the sequence of which they are a variant.
  • any appropriate breeding methods may be utilised including for example natural insemination, artificial insemination and in vitro fertilisation.
  • the word“mating” should be construed broadly and not limited to the physical pairing of two animals.
  • the methods of the invention may be used to identify animals suitable for cloning. They may also be used during cloning processes, to determine for example whether or not a cell, embryo or cloned animal has or doesn’t have a genetic variation in the FGD4 gene. Any appropriate cloning method could be used. However, by way of example, such cloning techniques include somatic cell nuclear transfer, chromatin transfer, and embryo splitting.
  • IVF IVF
  • any appropriate IVF methodology may be used, as will be apparent to persons of general skill in the art to which the invention relates.
  • appropriate methods are described, for example, in: Imai K, Tagawa M, Yoshioka H, Matoba S, Narita M, et al. (2006) The efficiency of embryo production by ovum pick-up and in vitro fertilization in cattle. J Reprod Dev 52: 19-29.
  • methods of the invention may involve taking a sample from an animal to be tested.
  • the sample may be any appropriate tissue or body fluid sample.
  • the sample is one or more of a cell, blood, muscle, bone, somatic cell(s), saliva, or semen.
  • Such samples can be taken from the animal using standard techniques known in the art. It should be appreciated that a sample may be taken from an animal at any stage of life, including prior to birth; by way of non-limiting example, a zygote, an embryo, a feotus. Individual gametes could also be tested using the methods of the invention. This may assist in breeding and/or cloning programmes.
  • sample should be taken to include a zygote, embryonic tissue, foetal tissue and gametes.
  • a sample may also be taken after the death of an animal.
  • the samples are analysed using techniques which allow for the observation or analysis of a biological marker, as will be described further herein after.
  • analysis or observation of a biolocial marker in an animal could be conducted by analysing protein, peptide, nucleic acid or a cell of that animal that may be present in the maternal blood supply, placenta, amniotic fluid or any other maternal tissue or fluid prior to birth of the animal.
  • reference to analysing a nucleic acid of an animal analysing FGD4, a precursor, an isoform and/or fragment thereof of an animal, observing the level or activity of a FGD4, a precursor, an isoform and/or fragment thereof of an animal, and the like, should be taken to include reference to analysing and/or observing one or more of these from that animal that may be present in a maternal tissue or fluid.
  • Embryo should be taken broadly to include an organism from the first division of the zygote. In certain embodiments, an embryo is an organism between the first division of the zygote until the time it becomes a foetus. Reference to an“embryo” should be taken to include reference to an organism at different developmental stages, including a blastula, blastocyst, gastrula, and morula for example.
  • the invention provides methods for the selection or rejection of one or more cells.
  • such“cells” may include a gamete (for example, sperm or ovum) or zygote. Selection of such cells may be of use in an IVF program, for example.
  • such “cells” may be somatic cells, embryonic cells, embryonic stem cells, cells in a cell line, cells of use in cloning, for example. Selection of these cells may be of use in cloning procedures, or preparing cell lines for use in cloning and other procedures, for example.
  • the methods of the invention may be described herein after in terms of analysing a biological marker (such as a nucleic acid sequence, amino acid sequence, level of a protein and peptide or activity level of a protein or peptide) in, of or from an“animal” or to determine whether or not an“animal” has a particular marker linked to a deleterious effect on productivity and/or worth or determining the genotype of an“animal”, and the like.
  • a biological marker such as a nucleic acid sequence, amino acid sequence, level of a protein and peptide or activity level of a protein or peptide
  • the invention may be described herein with reference to a level or level of activity of FGD4 (including reference to an isoform, precursor or fragment thereof and/or a nucleic acid encoding any one or more thereof) being“indicative” of an animal or animals which is/are known not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal, or being “indicative” of an animal or animals which is/are known to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal. “Indicative” should be taken to mean a level which is associated with an animal or animals known to have or not to have a variation in an amino acid or nucleic acid sequence which disrupts FGD4/the FGD4 gene.
  • the level is exactly the same as a level associated with such animal or animals. However, in one embodiment the level or level of activity is substantially similar or substantially the same as that associated with such animal or animals.
  • Reference to“animal or animals” should be taken to mean that a particular reference or standard is based on a value from a single animal or pooled or averaged from a group of animals.
  • Reference to an“animal or animals” should be taken to mean that the relevant benchmark is based on a value from a single animal or pooled or averaged from a group of animals. The same is the case of a cell or cells or an embryo or embryos.
  • the various embodiments of the invention may be described herein with reference to a reference animal (or animals) being one which is known to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal or being one which is known not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • a reference animal or animals
  • the various embodiments of the invention may be described herein which reference to a standard or control being one which is associated with an animal (or animals) known to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal or being one which is associated with an animal (or animals) known not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • a combination of both types of reference animal(s), standard(s) and/or control(s) could be used.
  • various embodiments of the invention may be described herein with reference to making a decision (such as a selection decision) based on a parameter being tested being“indicative” of an animal (or animals) known to carry a relevant biological marker, it should be appreciated that in some embodiments decisions could alternatively be made on the basis of the parameter being“indicative” of an animal (or animals) known not to carry a relevant biological marker.
  • a combination of both could be used. The description should be read accordingly, unless the context requires otherwise.
  • the production purpose is milking.
  • the production purpose is meat production.
  • a cell, animal or embryo is identified as unsuitable or less suitable where it includes a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • a cell, animal or embryo is identified as suitable or more suitable where it does not include a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • “More or less” suitable refers to a comparison between the animal, cell or embryo and an animal, cell or embryo that has (or in some embodiments does not have) a biological marker linked to a deleterious effect on productivity and/or worth of an animal, or a comparison between the same animal, cell or embryo if it had (or in some embodiments did not have) a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • Leu6l9_Phe620insValSerPheValVal* is used herein to describe a variation in FGD4 that has been identified by the inventors. This should be read to mean that Leu is present at a position corresponding to position 619 of FGD4, Phe which is normally present in wild type FGD4 at a position corresponding to position 620 is absent, and the 5 amino acids, being Val, Ser, Phe, Val and Val have been inserted into FDG4 starting at a position corresponding to position 620. The inserted amino acids therefore take positions 620, 621, 622, 623, and 624 of the truncated protein.
  • a method of the invention involves the analysis of a nucleic acid from an animal to determine whether or not it includes a genetic marker as herein before described (for example, a genetic alteration which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith).
  • a genetic marker as herein before described (for example, a genetic alteration which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith).
  • the invention may employ analysis of the gene encoding FGD4 and/or analysis of a transcript encoding FGD4, or a part thereof.
  • the specific marker identified by the inventors is a substitution of a C with an A at position 77632752 within the FGD4 gene on chromosome 5 of Bos taurus.
  • This alteration is annotated by the inventors as a splice donor variant, resulting in an extension of the coding frame by 5 amino acids and then the introduction of a new stop codon; Leu6l9_Phe620insValSerPheValVal*. This results in a truncated protein of 624 amino acids in length.
  • the two alleles of this marker may be referred to herein as 77632752C (wild type) and 77632752T (mutant).
  • the Entrez Gene ID for the FGD4 gene is (http://www.ncbi.nlm.nih.gov/gene/505234).
  • sequence and position is based on the genomic sequence of chromosome 5 in bovine build UMD3.1.1 (AC_000162.1 in the GenBank database http://www.ncbi.nlm.nih.gOv/T Further sequence information is provided in SEQ ID No. 1 (wild- type) and SEQ ID No. 2 (mutant) herein after.
  • the inventors predict the genetic alteration is likely to result in an mRNA transcript having the sequence shown in Figure 6 (SEQ ID No: 6).
  • a particular genetic marker of the invention may vary slightly from genome to genome; for example, in a different species of animal, or different breed of animal, the location of the marker may vary.
  • persons of skill in the art to which the invention relate will be able to readily identify a particular marker in different genomes through routine sequence alignment and with knowledge that it resides in the FGD4 gene.
  • reference to a position of a particular marker herein should be taken to mean a position“corresponding to” a particular position of chromosome 5 of Bos taurus” in the UMD3.1.1 genome build.
  • reference to the genetic marker 77632752T on chromosome 5 of Bos Taurus should be taken to mean that the genetic marker is at a position corresponding to position 77632752 on chromosome 5.
  • a particular biological marker of the invention may vary slightly from one transcript or protein to another; for example, in a different species of animal, or different breed of animal, the location of the marker may vary.
  • persons of skill in the art to which the invention relates will be able to readily identify a particular marker in different transcripts or proteins through routine sequence alignment. To account for this variation in the location of a marker across transcripts or proteins reference to a position of a particular marker herein should be taken to mean a position“corresponding to” a particular position of a transcript or protein reference sequence.
  • reference to a marker of the invention being located at a position or in a particular region of a transcript should be taken to mean that the marker on any given transcript is at a position or in a region“corresponding” to the position(s) (ie position or the region may be defined by different positions in a different transcript).
  • reference to a marker of the invention being located at a particular position or in a particular region of the protein should be taken to mean that the marker of any given protein is at a position or in a region “corresponding” to those positions (ie the position or region may be defined by different positions in a different protein).
  • the genetic marker 77632752T is linked to a deleterious effect on productivity and/or worth of an animal, they contemplate that any variation in the nucleotide sequence at this genetic position may be linked to a deleterious effect on productivity and/or worth of an animal. This includes deletion, addition or substitution at this position. The invention should be interpreted accordingly.
  • the methods of the invention involve the analysis of a nucleic acid to determine whether or not it includes a variation in the nucleotide sequence which results in a splice variant.
  • the splice variant is at the exon lOdntron 10 junction.
  • the methods of the invention involve the analysis of a nucleic acid to determine whether or not it includes a variation in the nucleotide sequence predicted to result in truncation of the protein.
  • the variation results in a truncated protein of 624 amino acids in length.
  • the variation comprises insertion of an amino acid.
  • the variation comprises insertion of an amino acid and truncation of the protein.
  • the methods of the invention involve analysis of a nucleic acid to determine whether or not it includes a variation in the nucleotide sequence in the region corresponding to positions 619 to 624 of the FGD4 protein.
  • the nucleic acid is a mRNA transcript.
  • the methods of the invention involve the analysis of a nucleic acid to determine whether or not it includes a variation in the nucleotide sequence (or codon) coding for an amino acid at a position corresponding to position 619, 620, 621, 622, 623, and/or 624.
  • the methods comprise analysing a nucleic acid to identify whether or not it includes a variation in the nucleotide sequence coding for the amino acids at two, three, four or five of the positions corresponding to positions 619, 620, 621, 622, 623 and 624 of FGD4.
  • the nucleic acid is a mRNA transcript.
  • the methods of the invention involve analysis of a nucleic acid to determine whether or not it includes a variation which results in insertion of one, two, three, four, five or more amino acids at a position corresponding to position 620 of FGD4.
  • the variation results in the protein sequence, Leu6l9_Phe620insValSerPheValVal.
  • the nucleic acid is a mR A transcript.
  • the methods of the invention involve analysis of a nucleic acid to determine whether or not it includes a variation in the FGD4 gene at a position corresponding to position 77632752 or downstream of a position corresponding to position 77632752.
  • the variation is a non-sense variant resulting in truncation of the FGD4 gene product.
  • the methods involve the analysis of a nucleic acid to determine whether or not it includes the genetic marker 77632752T on chromosome 5 of Bos taurus.
  • the methods may involve analysing the nucleotide sequence of a nucleic acid to determine the nucleotide sequence of a genetic marker in linkage disequilibrium with this genetic marker.
  • the methods of the invention may involve analysing the nucleotide sequence of a nucleic acid to determine the haplotype.
  • the haplotype includes a genetic marker at a position coresponding to position 77632752 on chromosome 5 of Bos taurus.
  • a variation as above mentioned and/or a genetic marker in linkage disequilibrium therewith is present it is identified as carrying a biological marker linked to a deleterious effect in productivity and/or worth of an animal and it is inferred that an animal is not suitable for production purposes, breeding purposes or inclusion in a herd, or that a cell or embryo is not suitable for generating an animal for such purposes, for example. In such cases, the animal, cell or embryo may be rejected.
  • the animal, cell or embryo may be selected for use in a herd or breeding programme or for production purposes, and the cell or embryo may be selected for use in generating an animal for such purposes, for example.
  • the genetic marker 77632752T and/or a genetic marker in linkage disequilibrium therewith is present it is identified as carrying a biological marker linked to a deleterious effect in productivity and/or worth of an animal and it is inferred that an animal is not suitable for production purposes, breeding purposes or inclusion in a herd, or that a cell or embryo is not suitable for generating an animal for such purposes, for example. In such cases, the animal, cell or embryo may be rejected.
  • the animal, cell or embryo may be selected for use in a herd or breeding programme or for production purposes, and the cell or embryo may be selected for use in generating an animal for such purposes, for example.
  • a genetic alteration which disrupts the FGD4 gene involves insertion and/or deletion of one or more nucleotide
  • the methods of the invention may involve analysis of the size of the FGD4 gene (or transcript) or a part or region thereof.
  • nucleic acid sequence of either strand of a double-stranded nucleic acid could be analysed.
  • nucleic acid sequence variations on such opposite strand which correlate with the genotypes mentioned above, having regard to the information contained herein and nucleic acid base pairing principles (ie, A pairs with T and C pairs with G).
  • SEQ ID No. 1 and 2 herein after reflect the minus (or reverse) strand relative to the UMD3.1.1 reference.
  • nucleic includes a specific genetic marker (such as the 77632752T allele) it should be read to encompass determining the genetic marker by observing the nucleotide or sequence on either strand.
  • the invention also encompasses use of a genetic marker which is in linkage disequilibrium with a marker of the invention. Such markers may be analysed instead of or in addition to a genetic marker of the invention.
  • Nucleic acids can be analysed to determine the genotype/sequence of the genetic markers described herein according to any appropriate technique.
  • Such techniques include for example polymerase chain reaction (PCR), including allele-specific PCR, gel electrophoresis, the use of oligonucleotide probe hybridisation, Southern blotting, direct sequencing, restriction digestion, restriction fragment length polymorphism (RFLP), single-strand confirmation polymorphism (SSCP), LCR (ligase chain reaction), denaturing gradient gel electrophoresis (DGGE), the use of allele-specific oligonucleotides (ASOs), the use of proteins which recognize nucleic acid mismatches, such as E.coli mutS protein, RNAse protection assays, oligonucleotide array hybridisation (for example genotyping microarrays or “SNP chips”), denaturing HPLC (dHPLC), fluorescence quenching PCR (TaqManTM Applied Biosystems, CA 94404, USA), High Resolution Mel
  • SNPs single nucleotide polymorphisms
  • ASOs allele-specific oligonucleotides
  • Any one or more of the techniques mentioned hereinbefore may be used to analyse genetic markers which may include insertion or deletion of one or more nucleotide.
  • oligonucleotides which hybridise to a genetic region encompassing the marker, adjacent to the marker, or flanking the marker.
  • oligonucleotides may be DNA, RNA or derivatised forms thereof and include nucleic acid primers, such as PCR and LCR primers, and nucleic acid probes.
  • nucleic acid sequence of chromosome 5 particularly in the genetic regions proximal to the genetic marker, the nature of the genetic marker to be analysed, and the general principles of nucleic acid hybridisation.
  • the nucleic acids will be capable of hybridising in a specific manner to a target nucleic acid and in the case of primers they will be capable of priming a PCR or like reaction.
  • nucleic acids While such nucleic acids will preferably have 100% complementarity to their target region of the mRNA or cDNA of the protein of interest, they may contain one or more non-complementary nucleotides at a particular position while still substantially retaining specificity for the target nucleic acid to which they are designed to bind.
  • the nucleic acids may have approximately 80%, approximately 90%, approximately 95%, or approximately 99% complementarity or homology to its target.
  • the oligonucleotides may be designed such that a mismatch at a particular nucleotide position is indicative of the nature of the genetic marker being analysed (for example, a SNP).
  • a mismatch in the nucleotide present at the 3’ end of an LCR primer will inhibit the reaction providing an indication of the nature of the nucleotide at that position.
  • Mismatches may similarly be utilised in techniques including RNAse protection assays and allele- specific PCR, as well as in fluorescence quenching PCR, for example.
  • the nucleic acids will hybridise to their target nucleic acid under stringent hybridisation conditions (see for example, Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).
  • oligonucleotide probes or primers may be of any length as is appropriate for a particular application, having regard to the sequence of the genetic region to which they are designed to bind.
  • a probe or primer will typically be capable of forming a stable hybrid with the complementary sequence to which it is designed to hybridise. Accordingly, the length is dependent on the nucleic acid composition and percent homology between the oligonucleotide and its complementary sequence, as well as the hybridisation conditions which are utilised (for example, temperature and salt concentrations). Such hybridisation factors are well known in the art to which the invention relates.
  • oligonucleotides of use in the present invention may be from 2 to 500 nucleotides in length. In one embodiment, particularly where they are used as primers, the oligonucleotides may be of approximately 15 nucleotides to 30 nucleotides in length.
  • Oligonucleotide probes and primers of use in the invention may be prepared by any number of conventional DNA synthesis methods including recombinant techniques and chemical synthesis, or they may be purchased commercially. It will be appreciated that the usefulness of any probe or primer may be evaluated, at least notionally, using appropriate software and sequence information for the nucleic acid encoding the protein of interest. For example, software packages such as Primer3 (http://primer3.sourceforge.net/), PC Oligo5 (National Bioscience Inc), Amplify (University of Wisconsin), and the PrimerSelect program (DNAStar Inc) may be used to design and evaluate primers.
  • Primer3 http://primer3.sourceforge.net/
  • PC Oligo5 National Bioscience Inc
  • Amplify Universality of Wisconsin
  • DNAStar Inc the PrimerSelect program
  • amplification may be conducted according to conventional procedures in the art to which this invention relates, such as described in US Patent No 4,683,202.
  • PCR reactions will generally include O.ImM-ImM of each primer, 200mM each dNTP, 3-7mM MgCL, and 1U Taq DNA polymerase.
  • exemplary PCR cycling conditions include: denaturation at a temperature of approximately 94°C for 30 to 60 seconds, annealing at a temperature calculated on the basis of the sequence and length of the primer (as herein after discussed) for 30 to 60 seconds, and extension at a temperature of approximately 70°C to 72°C for 30 to 60 seconds. By way of example, between 25 and 45 cycles are run.
  • any amplification conditions provided herein are merely exemplary and may be varied so as to optimise conditions where, for example, alternative PCR cyclers or DNA polymerases are used, where the quality of the template DNA differs, or where variations of the primers not specifically exemplified herein are used, without departing from the scope of the present invention.
  • the PCR conditions may be altered or optimised by changing the concentration of the various constituents within the reaction and/or changing the constituents of the reaction, altering the number of amplification cycles, the denaturation, annealing or extension times or temperatures, or the quantity of template DNA, for example.
  • PCR conditions may be optimised to overcome variability between reactions.
  • annealing temperatures for any primer within the scope of the present invention may be derived from the calculated melting temperature of that primer. Such melting temperatures may be calculated using standard formulas, such as that described in Sambrook and Russell, 2001. As will be understood by those of ordinary skill in the art to which this invention relates annealing temperatures may be above or below the melting temperature but generally an annealing temperature of approximately 5°C below the calculated melting temperature of the primer is suitable.
  • Oligonucleotides used for detection and/or analysis of genetic markers in accordance with the invention may be modified to facilitate such detection.
  • nucleic acid products obtained using techniques such as PCR may be modified to facilitate detection and/or analysis.
  • the nucleic acid molecules may be labelled to facilitate visual identification using techniques standard in the art.
  • nucleic acids may be radio-labelled using P 32 as may be described in Sambrook and Russell, 2001. Further, nucleic acids may be appropriately labelled for use in colorigenic, fluorogenic or chemiluminescence procedures.
  • control samples may be positive or negative controls for a particular genetic marker.
  • the type of control samples used may vary depending on such factors as the nature of the genetic marker being analysed and the specific technique being used for such detection and analysis.
  • Positive controls may include samples having known nucleic acid sequences (including nucleic acids of a known size), for example.
  • Negative controls may include samples having no nucleic acid present.
  • positive control samples could include one or more nucleic acids known to have a particular nucleotide sequence at a relevant position.
  • the method may utilise a control sample having a sequence which is associated with an animal known to carry a genetic marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the method may utilise a control sample having a sequence which is associated with an animal known not to carry a genetic marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the methods of this embodiment of the invention may involve comparing the sequence of a nucleic acid being tested to one or more reference sequences, as herein before described.
  • the methods of the invention will involve taking a sample from an animal to be tested, as noted herein before.
  • a sample may be processed prior to analysis.
  • the sample may be processed to isolate nucleic acid from the sample to be analysed or to amplify a specific genetic region to be analysed.
  • nucleic acid is isolated or extracted from the sample prior to analysis.
  • genomic DNA is isolated or extracted from the sample.
  • mRNA may be isolated or extracted from the sample. In such a case, the mRNA may be converted to cDNA using reverse transcription techniques known in the art. Techniques for isolating nucleic acids from samples will be readily appreciated by skilled persons. By way of Example, methods of use in isolating nucleic acids are described in Sambrook and Russell, 2001.
  • analysis of the nucleic acid may occur in situ obviating the need to extract nucleic acid from the sample. This may be done using PCR for example. Skilled persons will readily appreciate appropriate techniques and methodology to this end (see for example, Sambrook and Russell, 2001).
  • the step of analysing a nucleic acid of an animal, cell or embryo to determine whether or not it includes a genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith may comprise imputing a genotype of said animal (including the genetic variation which disrupts the FGD4 gene and/or a genetic marker in linkage disequilibrium therewith), as herein before described.
  • imputing genotypes are described, for example, in Li, Y., Willer, C., Sanna, S., & Abecasis, G. (2009). Genotype imputation. Annual review of genomics and human genetics, 10, 387.
  • Computational, statistical and/or in silico methods for genotype imputation will be readily known to those skilled in the art to which the invention relates. In some embodiments, these methods may be practised using readily available algorithms, software packages and tools such as, by way of non limiting example, IMPUTE, MACH, fastPHASE, BIMBAM, PLINK, TUNA, WHAP, FImpute and/or BEAGLE.
  • the methods of the invention may be combined with other methods of use in assessing genotype, predicting phenotype, selecting an animal based on certain characteristics, estimating breeding values or estimating worth and the like. Accordingly, the methods of the invention may include, in addition to analysis of a genetic marker identified herein, analysis of additional genetic markers, and/or the level of expression of certain genes/proteins, and/or one or more phenotypic traits, for example.
  • the methods of the invention comprise analysing a FGD4, a precursor thereof, an isoform thereof, and/or a fragment thereof to determine whether or not it includes a variation in the amino acid sequence which disrupts FGD4, in one particular embodiment, one which results in a decrease in the activity of FGD4.
  • the variation could comprise a deletion, insertion and/or substitution of one or more amino acid, for example.
  • the variation is located in a region of the protein encoded by the genetic region of the FGD4 gene at a position corresponding to position 77632752 or downstream of a position corresponding to position 77632752 of chromosome 5 of Bos Taurus. In one embodiment, the variation is a truncation.
  • the variation comprises a variation in the region defined by positions corresponding to positions 619 to 624 of the FGD4 protein.
  • the variation in the amino acid sequence comprises a truncation of the FGD4 protein. In one embodiment, the variation comprises a truncated protein of 624 amino acids. In one embodiment, the variation comprises insertion of an amino acid. In one embodiment, the variation comprises insertion of 2, 3, 4, 5 or more amino acids. In one embodiment, the variation comprises insertion of an amino acid at a position corresponding to position 620 of FGD4. In one embodiment, the variation results in insertion of 2, 3, 4, 5 or more amino acids at a position corresponding to position 620 of FGD4.
  • the amino acid or 2, 3, 4, 5 or more amino acids inserted at a position corresponding to position 620 of FGD4 are chosen from Val, Ser, Phe, Val, and Val, in any order or in the order in which they are listed.
  • the variation comprises insertion of one or more amino acid and truncation of the protein.
  • the variation is Leu6l9_Phe620insValSerPheValVal.
  • the variation in the PGD4 protein is a truncation downstream of the amino acid corresponding to position 619.
  • an animal, cell or embryo is identified to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal and it is inferred that an animal is not suitable for production purposes, breeding purposes or inclusion in a herd, or that a cell or embryo is not suitable for generating an animal for such purposes, for example. In such cases, the animal, cell or embryo may be rejected.
  • the animal, cell or embryo has the wild-type amino acid sequence the animal may be selected for use in a herd or breeding programme or for production purposes, and the cell or embryo may be selected for use in generating an animal for such purposes, for example.
  • the FGD4, precursor thereof, isoform thereof and/or fragment thereof comprises the variation Leu6l9_Phe620insValSerPheValVal or comprises or consists the amino acid sequence SEQ ID No. 4 or a part thereof an animal, cell or embryo is identified to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal and it is inferred that an animal is not suitable for production purposes, breeding purposes or inclusion in a herd, or that a cell or embryo is not suitable for generating an animal for such purposes, for example. In such cases, the animal, cell or embryo may be rejected.
  • the animal, cell or embryo has the wild-type amino acid sequence the animal may be selected for use in a herd or breeding programme or for production purposes, and the cell or embryo may be selected for use in generating an animal for such purposes, for example.
  • amino acid sequences and positions described herein are based on the sequence of FGD4 in GeneBank XP_005206940.l . Further sequence information is provided in SEQ ID No. 3 (wild-type) and SEQ ID No. 4 (mutant) herein after, as well as the description of the Leu6l9_Phe620insValSerPheValVal variant described herein.
  • FGD4, isoforms thereof, precursors thereof, and/or fragments thereof may be analysed using standard techniques known in the art. However, by way of example, peptide sequencing methods, mass spectrometry, Western blotting and ELISA could be used. It should be appreciated that the analysis of FGD4, isoforms thereof, precursors thereof, and/or fragments thereof to identify a variation may comprise analysis of the size of FGD4, a precursor thereof, an isoform thereof, and/or a fragment thereof.
  • the methods may employ one or more control samples, such as positive and/or negative controls for a particular amino acid sequence variation.
  • the type of control samples used may vary depending on such factors as the type of variation being analysed and the specific technique being used for detection and analysis.
  • Positive controls may include samples having known amino acid sequences (including proteins and peptides of a known size), for example.
  • Negative controls may include samples having no peptide present.
  • the method may utilise a control sample having a sequence which is associated with an animal known to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the method may utilise a control sample having a sequence which is associated with an animal known not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the methods of this embodiment of the invention may involve comparing the sequence of a peptide or protein being tested to one or more reference sequences, as herein before described.
  • the methods of the invention may involve taking a sample for an animal to be tested.
  • a sample may be processed prior to analysis according to any of a number of known methods.
  • the sample may be processed to remove one or more one or more high abundance proteins that might make it difficult to analyse FGD4, an isoform, fragment or precursor thereof.
  • Exemplary techniques which may be employed to process a sample prior to analysis of FGD4, an isoform, fragment or precursor thereof are described elsewhere herein.
  • the methods of the invention involve observing the level of one or more of FGD4 (including reference to any isoform of FGD4, any precursor of FGD4, any fragment of FGD4), and/or any one or more nucleic acid encoding one or more of the foregoing.
  • An animal, cell or embryo is identified as carrying a biological marker linked to a deleterious effect in productivity and/or worth of an animal where there is a decrease in the expected level of FGD4 (including reference to an isoform, a precursor or a fragment thereof) and/or a nucleic acid encoding same.
  • any decrease in the level indicates the animal, cell or embryo carries a biological marker linked to a deleterious effect in productivity and/or worth of an animal.
  • the decrease is at least approximately 20%, at least approximately 30%, at least approximately 40% or at least approximately 50% compared to an animal or animals that do not carry a biological marker linked to a deleterious effect in productivity and/or worth of an animal (including reference to the same animal if it did not have such biological marker).
  • there is substantially no FGD4 including reference to any isoform of FGD4, any precursor of FGD4, any fragment of FGD4 and/or a nucleic acid encoding same in an animal, cell or embryo.
  • the methods of this aspect of the invention will involve taking a sample from an animal, observing the level (in one embodiment the level of expression) of FGD4 (including reference to an isoform, a precursor and/or a fragment thereof) or a nucleic acid encoding same, and comparing the level against one or more standard.
  • a difference in the level between the sample and the one or more standard can be used to identify whether the animal, cell or embryo carries a biological marker linked to a deleterious effect in productivity and/or worth of an animal.
  • a standard comprises a level of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof which is associated with an animal or animals which is/are known not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • an animal, cell or embryo has a lower level of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof compared to the standard it is identified to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • an animal, cell or embryo has substantially no level of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof compared to the standard it is identified to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • an animal, cell or embryo is rejected if it has a lower level of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof compared to the standard.
  • an animal, cell or embryo is rejected if it has substantially no level of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof.
  • a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof compared to the standard it is identified not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • an animal, cell or embryo is selected if it has substantially the same or a higher level of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof compared to the standard.
  • the standard comprises a level of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof which is associated with an animal or animals which is/are known to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the standard comprises substantially no level of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof.
  • a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof compared to the standard it is identified not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • an animal, cell or embryo has substantially the same or a lower level of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof compared to the standard it is identified to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • an animal, cell or embryo is selected if it has a higher level of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof compared to the standard.
  • an animal, cell or embryo is rejected if it has substantially the same or a lower level of FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof compared to the standard.
  • the method of this aspect of the invention may comprise using two or more standards.
  • it may comprise measuring the level of a FGD4, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding any one or more thereof across both one or a pool of wild-type animals and one or a pool of animals known to carry an alteration which disrupts FGD4.
  • the samples from the animals to be tested would then be compared with the pooled values to determine whether they are indicative of an animal carrying an alteration or indicative of a wild-type animal.
  • the animal may be selected for production purposes, inclusion in a herd or breeding programme, for example. Where it is identified that an animal does carry a biological marker in accordance with the invention, the animal may be rejected for production purposes, inclusion in a herd or breeding programme, for example. Where it is identified that a cell or embryo does not carry a biological marker in accordance with the invention, the cell or embryo may be selected for use in breeding or cloning processes, for example. Where it is identified that a cell or embryo does carry a biological marker in accordance with the invention, the cell or embryo may be rejected for use in breeding or cloning processes, for example.
  • FGD4 (including reference to precursors, fragments and/or isoforms thereof) and nucleic acids encoding same may be detected and the levels thereof compared to a standard using any one or a combination of techniques which are of use in identifying, quantifying and/or highlighting differential levels or expression of one or more proteins. Such techniques will be readily appreciated by persons of ordinary skill in the art to which the invention relates.
  • the levels of FGD4 may be measured using protein purification methods, immunological techniques, separation of proteins based on characteristics such as molecular weight and isoelectric point including gel electrophoresis (for example, PAGE, including 2D PAGE) and microfluidics-based technologies as for example in gel- free protein separation techniques, and mass spectrometry (MS) utilizing isobaric label based MS such as iTRAQ or label-free approaches such as multiple reaction monitoring (MRM).
  • protein purification methods immunological techniques, separation of proteins based on characteristics such as molecular weight and isoelectric point including gel electrophoresis (for example, PAGE, including 2D PAGE) and microfluidics-based technologies as for example in gel- free protein separation techniques, and mass spectrometry (MS) utilizing isobaric label based MS such as iTRAQ or label-free approaches such as multiple reaction monitoring (MRM).
  • MS mass spectrometry
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • Western blotting immunohistochemical staining
  • antibody arrays antibody arrays
  • agglutination assays Protocols for carrying out such techniques are readily available; for example, see“Antibodies a Laboratory Manual”, Cold Spring Harbor Laboratory Press (1988).
  • Antibodies of use in such immunological techniques may be purchased commercially or produced according to standard methodology in the art having regard to the nature of the proteins to be tested.
  • polyclonal antibodies and monoclonal antibodies may be produced in accordance with the procedures described in the text “Antibodies a Laboratory Manual” (Cold Spring Harbor Laboratory Press, 1988) using one or more of the proteins or a fragment thereof as antigen.
  • monoclonal antibodies are used.
  • Nucleic acid-based techniques of use in determining the level of a nucleic acid may include differential display procedures, Northern Blotting, competitive PCR, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), microarray analysis, and RNA sequencing. Persons skilled in the art to which the invention relates will readily appreciate methodology for performing these techniques.
  • Nucleic acids such as oligonucleotide probes and primers, of use in detecting expression levels of proteins in accordance with the invention (for example using Northern blotting or competitive PCR) will be readily appreciated by skilled persons having regard to the information contained herein and any published amino acid and/or nucleic acid sequence information for FGD4.
  • the nucleic acids will be capable of hybridising in a specific manner to an mRNA or cDNA associated with FGD4 and in the case of primers they will be capable of priming a PCR or like reaction.
  • Mass spectroscopy techniques of use in the invention are described for example in“Proteins and proteomics-A laboratory manual” (RJ Simpson, Cold Spring Harbour Laboratory Press (2002).
  • the difference in the levels of FGD4 (including reference to a fragment, precursor and/or isoform) or a nucleic acid encoding same in a sample versus a standard may be compared using standard technology having regard to the method employed to detect the protein or nucleic acid.
  • colorimetric and fluorometric techniques may be used in which a detection molecule (such as an antibody or nucleic acid probe or primer) is labelled with a molecule which can be visualised by the naked eye or otherwise detected using a spectrophotometer, or fluorometer for example.
  • detection molecules could be labelled with radio-isotopes.
  • Incorporating labels into nucleic acids during PCR amplification where it is employed (as opposed to labelling a detection molecule such as a probe or primer), is also contemplated.
  • the methods of the invention may include the testing of one or more positive or negative control samples to ensure the integrity of the results.
  • the sample may be processed prior to analysing FGD4 (including reference to an isoform, precursor and/or fragment) and/or a nucleic acid encoding same to facilitate analysis of the proteins or nucleic acids. Skilled persons will readily appreciate appropriate processing steps and techniques suitable for performing them.
  • high abundance proteins which have the potential to make it difficult to analyse such as detect and/or measure the level of FGD4 (including reference to one or more isoform, precursor and/or fragment) may be removed from the sample.
  • detect and/or measure the level of FGD4 including reference to one or more isoform, precursor and/or fragment
  • Top6 or Top7 depletion may be used.
  • the sample may also be subject to proteolytic digestion.
  • detection of a protein or isoform in accordance with the invention should be taken to include detection of any one or more fragments thereof. Fragments should be of a length sufficient to ensure specificity to FGD4. Such fragments will for example be at least 8 amino acids in length, more preferably at least 10, 15 or 20 amino acids in length.
  • Processing steps for preparing the sample for analysis of nucleic acids encoding FGD4 may include lysing cells, isolating mRNA, and generating cDNA using standard procedures such as reverse transcription-PCR as will be known in the art to which the invention relates.
  • mRNA may be observed in situ.
  • methods of the invention may involve observing the level of activity of FGD4 (including reference to a precursor, isoform and/or fragment thereof).
  • An animal, cell or embryo is identified as carrying a biological marker linked to a deleterious effect in productivity and/or worth of an animal where there is a decrease in the expected level of activity of FGD4 (including reference to an isoform, a precursor or a fragment thereof).
  • any decrease in the level indicates the animal, cell or embryo carries a biological marker linked to a deleterious effect in productivity and/or worth of an animal.
  • the decrease is at least approximately 20%, at least approximately 30%, at least approximately 40% or at least approximately 50% compared to an animal or animals that do not carry a biological marker linked to a deleterious effect in productivity and/or worth of an animal (including reference to the same animal if it did not have such biological marker).
  • there is substantially no level of activity of FGD4 (including reference to any isoform of FGD4, any precursor of FGD4, any fragment of FGD4) in an animal, cell or embryo.
  • the methods of this aspect of the invention will involve taking a sample from an animal, observing the level of activity of FGD4 (including reference to an isoform, a precursor and/or a fragment thereof), and comparing the level against one or more standard.
  • a difference in the level between the sample and the one or more standard identifies whether the animal, cell or embryo carries a biological marker linked to a deleterious effect in productivity and/or worth of an animal.
  • a standard comprises a level of activity of FGD4, a precursor thereof, an isoform thereof, and/or a fragment thereof which is associated with an animal or animals which is/are known not to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • a lower level of activity of FGD4, a precursor thereof, an isoform thereof, and/or a fragment thereof compared to the standard identifies that an animal, cell or embryo carries a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • substantially no level of activity of FGD4, a precursor thereof, an isoform thereof, and/or a fragment thereof identifies that an animal, cell or embryo carries a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • an animal, cell or embryo is rejected if it has a lower level of activity of FGD4, a precursor thereof, an isoform thereof, and/or a fragment thereof compared to the standard.
  • an animal, cell or embryo is rejected if it has substantially no level of activity of FGD4, a precursor thereof, an isoform thereof, and/or a fragment thereof compared to the standard.
  • substantially the same and/or a higher level of activity of FGD4, a precursor thereof, an isoform thereof, and/or a fragment thereof compared to the standard identifies that an animal, cell or embryo does not carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • an animal, cell or embryo is selected if it has substantially the same or a higher level of activity of FGD4, a precursor thereof, an isoform thereof, and/or a fragment thereof compared to the standard.
  • the standard comprises a level of activity of FGD4, a precursor thereof, an isoform thereof, and/or a fragment thereof which is associated with an animal or animals which is/are known to carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • the standard comprises substantially no level of activity FGD4, a precursor thereof, an isoform thereof, and/or a fragment thereof.
  • a higher level of activity of FGD4, a precursor thereof, an isoform thereof, and/or a fragment thereof compared to the standard identifies that an animal, cell or embryo does not carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • substantially the same or a lower level of activity of FGD4, a precursor thereof, an isoform thereof, and/or a fragment thereof compared to the standard identifies that an animal, cell or embryo does carry a biological marker linked to a deleterious effect on productivity and/or worth of an animal.
  • an animal, cell or embryo is selected if it has a higher level of activity of FGD4, a precursor thereof, an isoform thereof, and/or a fragment thereof compared to the standard.
  • an animal, cell or embryo is rejected if it has substantially the same or a lower level of activity of FGD4, a precursor thereof, an isoform thereof, and/or a fragment thereof compared to the standard.
  • the method of this aspect of the invention may comprise using two or more standards.
  • it may comprise measuring the level of activity of a FGD4, a precursor thereof, an isoform thereof, a fragment thereof across both one or a pool of wild-type animals and one or a pool of animals known to carry an alteration which disrupts FGD4.
  • the samples from the animals to be tested would then be compared with the pooled values to determine whether they are indicative of an animal carrying an alteration or indicative of a wild-type animal.
  • the animal may be selected for production purposes, inclusion in a herd or breeding programme, for example. Where it is identified that an animal does carry a biological marker in accordance with the invention, the animal may be rejected for production purposes, inclusion in a herd or breeding programme, for example. Where it is identified that a cell or embryo does not carry a biological marker in accordance with the invention, the cell or embryo may be selected for use in breeding or cloning processes, for example. Where it is identified that a cell or embryo does carry a biological marker in accordance with the invention, the cell or embryo may be rejected for use in breeding or cloning processes, for example.
  • the level of activity of FGD4 may be measured using standard methodology as known in the art, having regard to the function of FGD4.
  • the methods used may involve one or more of the following techniques: Immunoprecipitation, Western blotting, ELISA, mass spectrometry, surface plasmon resonance, isothermal titration calorimetry, luciferase assays and reporter gene assays.
  • the difference in the level of FGD4 (including reference to one or more fragment, precursor and/or isoform) activity in a sample versus a standard may be compared using standard technology having regard to the method employed to detect the activity.
  • colorimetric and fluorometric techniques may be used in which a detection molecule (such as an antibody or nucleic acid probe or primer) is labelled with a molecule which can be visualised by the naked eye or otherwise detected using a spectrophotometer, or fluorometer for example.
  • detection molecules could be labelled with radio-isotopes.
  • the methodology described in Perrot- Applanat et al, 1997 Mol Endo 11(8) may be used.
  • the methods of the invention may include the testing of one or more positive or negative control samples to ensure the integrity of the results.
  • the sample may be processed prior to analysing FGD4 (including reference to one or more isoform, precursor and/or fragment) activity. Skilled persons will readily appreciate appropriate processing steps and techniques suitable for performing them.
  • high abundance proteins which have the potential to make it difficult to analyse such as detect and/or measure the level of activity of FGD4 (including reference to one or more isoform, precursor and/or fragment) may be removed from the sample.
  • detect and/or measure the level of activity of FGD4 including reference to one or more isoform, precursor and/or fragment
  • Top6 or Top7 depletion may be used.
  • immunoprecipitation of the protein of interest could also be used.
  • Processing steps for preparing the sample for analysis of FGD4 (including reference to one or more isoform, precursor and/or fragment) activity may include cell lysis, immunoprecipitation and preparation of cell membranes, for example. Persons skilled in the art will readily appreciate other useful techniques that may be used.
  • the invention also provides methods for breeding and cloning animals. Methods of embodiments of the invention described herein before may be used for selecting animals (including selecting their gametes, for example) for such purposes.
  • Such methods may comprise identifying at least one first animal that does not carry a biological marker linked to a deleterious effect in productivity and/or worth of an animal (in one embodiment, using one or more method as described herein) and mating said animal with a second animal.
  • the method may comprise further identifying at least one second animal that is identified not to carry a biological marker linked to a deleterious effect in productivity and/or worth of an animal (in one embodiment, using one or more method as described herein).
  • the mating will produce one or more offspring.
  • the invention also encompasses breeding methods which comprise selecting a first and/or a second gamete where it/they have been identified not to carry a biological marker linked to a deleterious effect in productivity and/or worth of an animal and fusing the first gamete and second gamete to form a zygote.
  • the method may comprise: 1) selecting a first gamete and/or a second gamete using a method of the invention and fusing said first gamete with said second gamete to form a zygote.
  • the method may further comprise selecting any embryo that results from this process using one or more method of the invention.
  • the invention also encompasses breeding methods which comprise selecting an embryo that has been identified not to carry a biological marker linked to a deleterious effect in productivity and/or worth of an animal. A selected embryo is used to breed an animal.
  • the invention also provides a method of cloning an animal comprising selecting a cell for cloning where it has been identified not to carry a biological marker linked to a deleterious effect in productivity and/or worth of an animal. A selected cell is used to clone an animal.
  • one or more methods of the invention may be used to identify and select appropriate gametes and embryos for these breeding methods.
  • a method of the invention may comprise selecting a gamete, embryo or cell identified not to have one or more biological marker linked to a deleterious effect in productivity and/or worth of an animal.
  • the invention also provides methods for determining whether or not one or more cell or embryo is suitable for being used to produce an animal (for example, using breeding or cloning methods) which does not carry a biological marker linked to a deleterious effect in productivity and/or worth of an animal.
  • the breeding and/or cloning methods of the invention further comprise transferring one or more embryo to a gestational carrier, in accordance with any number of techniques known in the art.
  • animals may be mated using any appropriate methods including naturally, artificial insemination or IVF.
  • individual gametes may be selected for use in the process.
  • gametes may be selected using a method of the invention; for example, a method of the invention may be used to identify animals that do not carry a biological marker linked to a deleterious effect in productivity and/or worth and gametes from those animals selected for use in a breeding program or process or gametes may be tested in accordance with the invention and then selected for use in a breeding program or process.
  • a method of selecting or rejecting one or more animal could be used to select the first and/or second animal and their gametes used in IVF.
  • a method of selecting or rejecting one or more cells could be used to select a first and/or second gamete and selected gametes used in IVF. Following selection of male and female gametes, the female gamete is fertilised in vitro. At the relevant time, one or more embryo is transferred to a gestational carrier.
  • in vitro fertilisation of a female gamete may occur and then a method of the invention used to determine whether or not an embryo has a desired genotype/phenotype and should be selected or rejected for further use in a breeding programme.
  • a method of the invention used to determine whether or not an embryo has a desired genotype/phenotype and should be selected or rejected for further use in a breeding programme. This might occur where individual gametes, or animals from which they have been obtained or derived, have not been tested to identify whether or not they carry a biological marker linked to a deleterious effect in productivity and/or worth prior to fertilisation (accordingly, the invention should be taken to include methods of breeding where the first and/or second animal and/or gametes are not selected on the basis of such a test, but a resulting embryo or offspring is tested and selected).
  • a method of the invention could be used where the individual gametes or animals from which they have been obtained or derived have been tested and selected on the basis of having a desirable genotype/phenotype, for quality control purposes or to double check that the resulting embryos have the same desirable genotype/phenotype.
  • one or more method of the invention may be used to determine whether or not any offspring has or may be inferred to have the desired characteristics.
  • Such testing may occur at any time during the life of the offspring, including before birth; by way of example only, testing an embryo, a foetus, amniotic fluid, placenta, maternal blood, at birth.
  • gametes are fused to form a zygote.
  • Methods for fusing gametes are known in the art.
  • cloning may be used to generate an animal.
  • the method may comprise selecting at least one first animal that has been identified not to carry a biological marker linked to a deleterious effect in productivity and/or worth (in one embodiment, using one or more method as described herein) and using the nucleus or chromatin from one or more cell of that animal in a cloning procedure (such as somatic cell nuclear transfer, chromatin transfer techniques, and embryo splitting).
  • a cloning procedure such as somatic cell nuclear transfer, chromatin transfer techniques, and embryo splitting.
  • a cloning procedure may utilise a cell derived from a cell line.
  • a method of the invention may be used to select such a cell which is, or cell line whose cells are, capable of being used to generate an animal who does not carry a biological marker linked to a deleterious effect in productivity and/or worth.
  • the cell line may be an embryonic cell line.
  • One or more cell of use in cloning may be selected using a method of the invention. Following selection of one or more cells a cloning procedure can be conducted.
  • a method of the invention may be used to identify animals that do not carry a biological marker linked to a deleterious effect in productivity and/or worth and cells from those animals selected for use in a cloning process.
  • a method of the invention may be used to identify cells from a cell line which do not carry a biological marker linked to a deleterious effect in productivity and/or worth and may be of use in generating an animal that does not carry a biological marker linked to a deleterious effect in productivity and/or worth.
  • methods of the first, eighth, fifteenth and/or other aspects of the invention may be used for such purposes. Methods of the invention could also be used to identify animals whose cells could be used to generate cell lines for cloning purposes.
  • a method of selecting or rejecting one or more animal could be used to select an animal for cloning.
  • a method of selecting or rejecting one or more cells could be used to select one or more cells of use in cloning.
  • one or more method of the invention may be used to identify whether or not any cloned animal carries a biological marker linked to a deleterious effect in productivity and/or worth.
  • Such testing may occur at any time during the life of the cloned animal.
  • testing of a blastocyst, an embryo, a foetus, amniotic fluid, placenta, maternal blood, at birth may occur at any time during the life of the cloned animal.
  • a cloning method of the invention may involve selecting desirable cells without testing those cells or the animals or cell line from which they came for the presence or absence of a biological marker linked to a deleterious effect in productivity and/or worth.
  • the cloning procedure can be initiated and then a method of the invention used to determine whether an embryo, foetus or animal resulting from the cloning procedure has a relevant biological marker in accordance with the invention and an embryo, foetus or animal selected where it has a desirable genotype/phenotype.
  • the breeding and cloning methods of the invention may involve subjecting one or more cell, zygote, embryo and/or feotus, for example, to any one of a number of standard growth and/or gestation methods.
  • an animal, cell or embryo is identified to be heterozygous for a biological marker linked to a deleterious effect on productivity and/or worth of an animal, it is not selected for the purposes of breeding or cloning. This will help avoid the possible mating of carriers of the deleterious effect on productivity and/or worth during current or future breeding programme, for example.
  • the invention also provides methods for forming a herd of animals. Such methods comrpise selecting or rejecting an animal based on whether or not it carries a biological marker linked to a deleterious effect on productivity and/or worth.
  • the method comprises selecting or rejecting an animal on the basis of the results of a method of the first, second third, eighth, ninth, tenth, fifteenth, sixteenth and/or seventeenth aspect of the invention described herein, and forming a herd of selected animals.
  • methods of the invention which involve selecting or rejecting one or more cells may also be used to select one or more animals for inclusion in a herd.
  • an animal where an animal is identified to have a biological marker linked to a deleterious effect on productivity and/or worth it may be rejected and not selected for inclusion in the herd.
  • an animal where an animal is identified not to have a biological marker linked to a deleterious effect on productivity and/or worth, it may be selected for inclusion in the herd.
  • an animal that is or has been identified as being heterozygous for a biological marker linked to a deleterious effect on productivity and/or worth of an animal is chosen for inclusion in a herd.
  • a heterozygous animal is not chosen for inclusion in a herd.
  • the invention should also be taken to include a herd formed by the methods described herein.
  • the herd of animals may be formed for any desirable reason. However, by way of example only, it may desirable to form a herd for: beef farming; milk production. Nucleic Acids, Peptides, Antibodies
  • the invention also provides isolated nucleic acids carrying one or more genetic marker of the invention.
  • the isolated nucleic acid emcompasses a genetic variation at a position corresponding to position 77632752 on chromosome 5 of Bos taurus.
  • the isolated nucleic acid includes the genetic marker 77632752T and/or a marker in linkage disequilibrium therewith reside are encompassed by the invention.
  • the nucleic acid comprises or consists of the sequence of SEQ ID No. 2, or is a functionally equivalent variant thereof.
  • the nucleic acid comprises or consists of the sequence of SEQ ID No. 6 or is a functionally equivalent variant thereof.
  • the invention also encompasses nucleic acids which can hydridise, preferably under stringent conditions (as herein before described), to a region of a FGD4 gene in which the genetic marker 77632752T resides.
  • nucleic acids may be used as probe or primers or otherwise in analysis of genetic markers of the invention, as herein before described.
  • the invention provides nucleic acids with are completmentary to or can hydridise (preferably under stringent conditions) to a nucleic acid of SEQ ID No. 2 or SEQ ID No. 6.
  • Nucleic acids of the invention may have 100% sequence identity, homology or complementarity to the relevant region of a FGD4 gene, but may also have some sequence variation.
  • nucleic acids of the invention may have approximately 80%, approximately 90%, approximately 95% or approximately 99% sequence identity, homology or complementarity.
  • the nucleic acids may be of any appropriate length. In one embodiment, they are at least 4 nucleotides in length, or at least 10, 20, 30, 40, 50, 60, 70, 80 or more nucleotides in length.
  • nucleic acids of the invention include DNA, mRNA and cDNA.
  • the invention also provides peptides or proteins encompassing an amino acid variation associated with one or more genetic marker of the invention. These may be of any appropriate length. In one embodiment, they are at least 4 amino acids in length, or at least 10, 20, 30, 40, 50, 60, 70, 80 or more amino acids in length. In one embodiment, the peptide comprises an amino acid variation in the region defined by positions corresponding to positions 619 to 624 of FGD4. In one embodiment, the peptide comprises or consists Leu6l9_Phe620insValSerPheValVal as defined herein. In one embodiment, a peptide of the invention comprises or consists the amino acid sequence of SEQ ID No. 4 or is a functionally equivalent variant thereof. In another embodiment, the invention provides an isoform, precursor or fragment of a peptide comprising or consisting the amino acid sequence of SEQ ID No. 4 or a part thereof.
  • the invention provides one or more antibodies which bind to a peptide or protein encompassing the amino acid variation associated with one or more genetic marker of the invention.
  • the one or more antibodies are monoclonal antibodies.
  • the one or more antibodies are polyclonal antibodies.
  • an alteration in the FGD4 gene is associated with a deleterious effect on productivity and/or worth of an animal allows for the targeted correction of an alteration in the gene having this effect using cloning and/or gene editing processes in which one or more genetic alteration is introduced into the FGD4 gene.
  • a specific alteration may be introduced into a cell or embryo that may be used to generate an animal.
  • the invention provides any cell or embryo into which an alteration has been introduced in accordance with the invention.
  • the correction of an alteration in FGD4 linked to a deleterious effect on productivity and/or worth of an animal may allow for the production of animals and more broadly the formation of herds which have desirable productivity and/or worth or an increased level of productivity and/or worth compared to an animal/animals which have an alteration linked to a deleterious effect on productivity and/or worth of an animal.
  • the genetic alteration introduced into the FGD4 gene may be of any nature, including insertion of one or more nucleotide, deletion of one or more nucleotide, and/or substitution of one or more nucleotide.
  • the genetic alteration corrects a variation in the FGD4 gene which is linked to a deleterious effect on productivity and/or worth of an animal.
  • the genetic alteration is one which corrects a variation in the FGD4 gene which disrupts the FGD4 gene.
  • the genetic alteration is one which corrects a splice variant of FGD4.
  • the genetic alteration is one which increases the level and/or activity of FGD4 compared to the level and/or activity of FGD4 if it did not include the alteration.
  • the genetic alteration includes a genetic alteration located within the FGD4 gene at a position corresponding to position 77632752 or downstream of the nucleotide corresponding to position 77632752 of chromosome 5 of Bos taurus.
  • the genetic alteration includes a genetic alteration located at a position corresponding to position 77632752 on chromosome 5 of Bos taurus.
  • the one or more genetic alteration is a substitution of the nucleotide at the position corresponding to position 77632752 on chromosome 5 of Bos Taurus with a C.
  • the one or more genetic alteration includes substitution of a T at the position corresponding to position 77632752 on chromosome 5 of Bos Taurus with another nucleotide.
  • the one or more genetic alteration incudes substitution of a T with a C at the position corresponding to position 77632752 of chromosome 5 of Bos Taurus.
  • the one or more genetic alteration is as described herein before.
  • one or more cell used to generate an animal includes individual gametes, zygotes, embryos, somatic cells, cells from a cell line, for example.
  • a genetic alteration may be introduced into a gamete or zygote, for example.
  • a genetic alteration may be introduced into a somatic cell or cell from a cell line, for example.
  • Such methods may further comprise testing or screening one or more cell, embryo, or animal after a gene editing step to ensure they include the desired genetic alteration.
  • any one of a number of standard methods may be used to introduce one or more genetic alteration to the FGD4 gene in accordance with the invention.
  • TALEN or CRISPR methods may be used.
  • CRISPR embryo genomes can be directly modified by injection of Cas9 mRNA and sgRNA into the fertilised egg resulting in the efficient production of animals carrying biallelic mutations in a given gene, for example.
  • Such techniques are described for example in: Precision Editing of Large Animal Genomes, Wenfang (Spring) Tan, Daniel F. Carlson, Mark W. Walton, Scott C. Fahrenkrug and Perry B. hackett, Adv Genet. 2012 ; 80: 37-97.
  • the breeding method may involve IVF.
  • IVF IVF
  • individual male and female gametes may be chosen, the female gamete fertilised in vitro, and then a gene editing method conducted on the fertilised zygote to introduce at least one desired alteration into the FGD4 gene.
  • one or more other alteration could also be introduced into the genome of a gamete or zygote.
  • one or more embryo may be transferred to a gestational carrier according to methods known in the art.
  • the methods of this embodiment may optionally comprise conducting a method of the invention described herein before to determine whether or not any cell or animal has a desired alteration.
  • Such testing may occur at any time during the process and life of any animal.
  • testing of a blastocyst, an embryo, a foetus, amniotic fluid, placenta, maternal blood, at birth By way of example testing of a blastocyst, an embryo, a foetus, amniotic fluid, placenta, maternal blood, at birth.
  • an embyo is tested prior to transferring to the gestational carrier.
  • the animal is tested at birth.
  • gene editing is combined with cloning.
  • one may first chose an animal for use in cloning based on its genetic merit or otherwise.
  • a cell from the animal may be subject to gene editing methods to introduce at least one desired alteration into the FGD4 gene.
  • the nucleus from such cell may then be used in known cloning processes, such as chromatin transfer, somatic cell nuclear transfer and embryo splitting. It should be appreciated that one or more other alteration could also be introduced into the genome if desired.
  • one or more embryo may be implanted into a carrier female animal for gestation according to methods known in the art.
  • the methods of this embodiment one may optionally comprise conducting a method of the invention described herein before to determine whether or not any cloned cell or animal has a desired alteration.
  • Such testing may occur at any time during the process and life of any animal.
  • testing of an embryo, a foetus, amniotic fluid, placenta, maternal blood, at birth By way of example testing of an embryo, a foetus, amniotic fluid, placenta, maternal blood, at birth.
  • an embyo is tested prior to transferring to the gestational carrier.
  • the animal is tested at birth.
  • the gene editing methods of this aspect of the invention are conducted for the purpose of generating (or at least increasing the likelihood of generating) an animal which does not have a biological marker linked to a deleterious effect on productivity and/or worth in accordance with the invention. In one embodiment, the gene editing methods are conducted for generating (or at least increasing the likelihood of generating) an animal having desirable worth and/or productivity. In one embodiment, the gene editing methods are conducted for the purpose of generating (or at least increasing the likelihood of generating) an animal which is suitable for production and/or breeding purposes.
  • the methods are conducted for the purpose of generating (or at least increasing the likelihood of generating) a cell or embryo which may be used to generate an animal which does not have a biological marker linked to a deleterious effect on productivity and/or worth or an animal having desirable worth and/or productivity.
  • the method is conducted for the purpose of generating (or at least increasing the likelihood of generating) a cell or embryo of use in generating an animal which is suitable for production and/or breeding purposes.
  • the methods of this aspect of the invention provide means for generating a cell or embryo which is of use in a method for generating an animal, the method comprising at least the step of introducing a genetic alteration to the FGD4 gene of the cell or embryo.
  • the invention also provides cells or embryos generated using a gene editing method of the invention and which are capable of use in a method of generating a non-human animal.
  • the invention also relates to kits which are of use in a method of the invention.
  • the kit comprises at least one or more reagents suitable for analysis of one or more biological marker in accordance with the invention.
  • Reagents suitable for analysis of one or more of the markers include one or more nucleic acid probes and/or primers and one or more antibodies, as herein before described. Skilled persons will readily appreciate other appropriate reagents suitable for detecting or observing the biological markers and having regard to the various techniques mentioned herein before.
  • kits of the invention may also comprise one or more standard and/or other controls including nucleic acids, peptides or proteins whose sequence at a particular position is known. Further, kits of the invention can also comprise instructions for the use the components of the kit as well as printed charts or the like that could be used as standards against which results obtained from test samples could be compared. Reagents may be held in any suitable container.
  • the inventors have identified a genetic variation on chromosome 5.
  • the name of the gene that this variation is in is called FGD4 (FYVE, RhoGEF and PH domain containing 4) and denoted with locus tag of XM_005206883.3.
  • the variation is a nucleotide substitution at position 77632752 (77632752 C ⁇ T) on chromosome 5 of Bos taurus. This results in a splice donor substitution (exon 10).
  • the variation results in an extension of the coding frame by 5 amino acids and then the introduction of a new stop codon (Leu6l9_Phe620insValSerPheValVal*).
  • the data generated and observations made by the inventors indicates that the presence of the variation (77632752T) results in a decrease in milk production, liveweight, and TOP (traits other than production) traits in an animal.
  • the inventors note a significant decrease in liveweight, stature, body condition score, protein yield, milk yield, fat yield, dairy conformation and overall opinion.
  • Genomic sequence (minus strand) indicating the reference form of the FGD4 gene (XM_005206883.3 annotation).
  • Genomic sequence (minus strand) indicating the mutant form of the FGD4 gene (XM_005206883.3 annotation).
  • SEQ ID No. 3 Protein sequence indicating the reference (wild type) form of FGD4.
  • SEQ ID No. 4 Protein sequence indicating the alternate (mutant) form of FGD4.
  • SEQ ID No.5 Sequence representing the wild type gene mRNA transcript of FGD4 (Refseq
  • SEQ ID No. 6 Sequence representing the predicted mutant transcript of FDG4 (Refseq ID
  • the position of the variant is nucleotide 2176.
  • the variant described was identified by applying recessive inheritance models in genome wide association mapping studies of various phenotypic traits. Association analyses were conducted using imputed whole genome sequence data, comprising a‘full-resolution’ analysis that has the potential to allow mapping of causal mutations directly.
  • Genome wide association mapping was conducted using PLINK software (vsl.9), using 65,500 animals measured for the traits referenced in Table 1. Analyses were conducted assuming a recessive mode of inheritance, testing >16.2M imputed whole genome sequence variants per trait. Prior to association analysis, phenotypes were adjusted using covariates calculated to address population structure in the mixed breed cattle population, applying the identity by state and multidimensional scaling procedure implemented in PLINK to produce 1000 covariates based on markers from the Illumina SNP50k SNP chip platform. Covariates were fitted as fixed effects using JMP software, with phenotypic residuals subsequently used as input into the recessive association models.
  • genotypes for the FGD4 77632752 variant were also recoded to merge the heterozygote and major allele homozygous classes prior to analysis.
  • results of the genome scan implicating the FGD4 77632752T allele as having a deleterious impact was validated in a population of 635 calves.
  • animals potentially homozygous for this variant (77632752TT) were identified from pedigree records, where young animals that had a carrier sire, and carrier maternal grandsire from a national animal information database were considered as candidates.
  • a subset of these animals were then tissue sampled and genotyped for the FGD4 variant using a custom Taqman assay (Thermo Fisher Scientific). Animals were also weighed on an electronic scale during tissue sampling, with both weighing and sampling activities performed by AsureQuality.
  • These 635 calves had a mean age of 270 days and mean weight of 197 kgs.
  • RNA-sequence data As the loss of splicing fidelity should result in generation of mutant transcripts with effects visualisable using RNA-sequence data, the inventors made use of a large mammary RNA sequence resource representing 376 lactating cows to investigate the functional effects of the chr5:77632752C>T variant. Although no animals were homozygous for the chr5:77632752T SNP, eight individuals carried the variant. Examining the representation of FGD4 transcript structures in these heterozygous animals, clear differences in splicing were apparent, where the sequence read depth of FGD4 intron 10 was substantially higher than other FGD4 introns (Figure 7; three mutant and three wildtype individuals shown).

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Abstract

La présente invention concerne de manière générale des méthodes permettant d'identifier si un animal porte ou non un marqueur biologique lié à la productivité animale, et plus particulièrement, mais pas exclusivement, des méthodes pour identifier si un animal porte ou non un marqueur biologique ayant un effet délétère sur la productivité animale. L'invention concerne également des méthodes pour sélectionner ou rejeter un ou plusieurs animaux, cellules ou embryons, l'évaluation animale, l'élevage d'animaux et la formation de troupeau. L'invention concerne également des marqueurs biologiques appropriés pour une utilisation dans de telles méthodes. En particulier, la présente invention concerne des variations génétiques qui perturbent le gène FGD4.
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CN118553304A (zh) * 2024-05-20 2024-08-27 蘑米(广州)生物科技有限公司 筛选与菌株的蛋白质产量相关的标志蛋白质或标志基因的方法以及评价菌株的蛋白质产量的方法和相关设备

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CN118553304A (zh) * 2024-05-20 2024-08-27 蘑米(广州)生物科技有限公司 筛选与菌株的蛋白质产量相关的标志蛋白质或标志基因的方法以及评价菌株的蛋白质产量的方法和相关设备

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