WO2011028134A9 - Marqueurs biologiques et leurs utilisations - Google Patents

Marqueurs biologiques et leurs utilisations Download PDF

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Publication number
WO2011028134A9
WO2011028134A9 PCT/NZ2010/000172 NZ2010000172W WO2011028134A9 WO 2011028134 A9 WO2011028134 A9 WO 2011028134A9 NZ 2010000172 W NZ2010000172 W NZ 2010000172W WO 2011028134 A9 WO2011028134 A9 WO 2011028134A9
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Prior art keywords
animal
proteins
potential
offspring
level
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PCT/NZ2010/000172
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English (en)
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WO2011028134A1 (fr
Inventor
Richard John Spelman
Stephen Richard Davis
Michel Alphonse Jumien Georges
Wouter Robert Herman Coppieters
Latifa Karim
Li Lin
Tom Jean-Marc Druet
Bevin Lyal Harris
Michael Dominic Keehan
Mathew Douglas Littlejohn
Juan Antonio Castro Arias
Haruko Takeda
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Livestock Improvement Corporation Limited
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Publication of WO2011028134A1 publication Critical patent/WO2011028134A1/fr
Publication of WO2011028134A9 publication Critical patent/WO2011028134A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • 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 relates generally to methods for inferring the size potential of an animal and/or its offspring, particularly but not exclusively, methods for identifying and selecting animals on the basis of their liveweight and/or growth rate potential.
  • the invention also relates to biological markers suitable for use in such methods.
  • the size of an animal may have an effect on its value for a particular purpose. For example, for beef farming it is desirable for animals to have a relatively larger weight or growth rate, whereas for dairy farming efficiencies can be gained with smaller animals, as less feed is required to support milk production.
  • the inventors have identified a number of genetic markers whose sequence or genotype can be used to predict or infer the potential size of an animal and/or its offspring. Such information may be used in methods for selecting, screening and breeding animals, farm management, and for estimating an animals worth to a particular industry, for example.
  • the invention provides a method for inferring the size potential of an animal and/or its offspring, the method comprising at least the step of analysing a nucleic acid from said animal to determine the nucleotide present at one or more of the following positions on chromosome 14 of Bos taurus 22986260, 23186380, 23186648, 23187238, 23205280, 23211989, 23215765, 23218552, 23228064, 23232324, 23235239, 23243588, 23243734, 23264810, 23264854, 23272034, and 23273471, or to determine the nucleotide sequence of one or more genetic marker in linkage disequilibrium with one or more thereof, wherein the nucleotide present at the one or more positions infers the size potential of the animal and/or its offspring.
  • the invention provides a method for inferring the growth rate potential of an animal and/or its offspring, the method comprising at least the step of analysing a nucleic acid from said animal to determine the nucleotide present at one or more of the following positions on chromosome 14 of Bos taurus 22986260, 23186380, 23186648, 23187238, 23205280, 23211989, 23215765, 23218552, 23228064, 23232324, 23235239, 23243588, 23243734, 23264810, 23264854, 23272034, and 23273471, or to determine the nucleotide sequence of one or more genetic marker in linkage disequilibrium with one or more thereof, wherein the nucleotide present at the one or more positions infers the growth rate potential of the animal and/or its offspring.
  • the invention provides a method for inferring the liveweight potential of an animal and/or its offspring, the method comprising at least the step of analysing a nucleic acid from said animal to determine the nucleotide present at one or more of the following positions on chromosome 14 of Bos taurus 22986260, 23186380, 23186648, 23187238, 23205280, 23211989, 23215765, 23218552, 23228064, 23232324, 23235239, 23243588, 23243734, 23264810, 23264854, 23272034, and 23273471, or to determine the nucleotide sequence of one or, more genetic marker in linkage disequilibrium . with one or more thereof, wherein the nucleotide present at the one or more positions infers the liveweight potential of the animal and/or its offspring.
  • 23264854G, 23272034A, and 2327347 IT infers a larger size for the animal and/or its offspring.
  • the presence of one or more of these markers infers a higher growth rate and/or higher liveweight for the animal and/or its offspring.
  • 23232324G, 23235239G, 23243588TT, 23243734G, 23264810(CCG)9, 23264854A, 23272034G, and 23273471C infers a smaller size for the animal and/or its offspring.
  • the presence of one or more of these markers infers a lower growth rate and/or lower liveweight for the animal and/or its offspring.
  • the invention provides a method for selecting or rejecting an animal, the method comprising at least the step of analysing a nucleic acid from said animal to determine the nucleotide present at one or more of the following positions on chromosome 14 of Bos taurus 22986260, 23186380, 23186648, 23187238, 23205280, 23211989, 23215765, 23218552, 23228064, 23232324, 23235239, 23243588, 23243734, 23264810, 23264854, 23272034, and 23273471, or to determine the nucleotide sequence of one or more genetic marker in linkage disequilibrium with one or more thereof, wherein the nucleotide present at the one or more positions infers the size potential of the animal and/or its offspring.
  • the method comprises at least the steps of: a) analysing a nucleic acid from the animal to determine the nucleotide present at one or more of the following positions on chromosome 14 of Bos taurus 22986260, 23186380, 23186648, 23187238, 23205280, 23211989, 23215765, 23218552, 23228064, 23232324, 23235239, 23243588, 23243734, 23264810, 23264854, 23272034, and 23273471 or to determine the nucleotide sequence of one or more genetic marker in linkage disequilibrium with one or more thereof; and,
  • 23264854G, 23272034A, and 2327347 IT infers a larger size for the animal and/or its offspring.
  • the presence of one or more of these markers infers a higher growth rate and/or higher liveweight for the animal and/or its offspring.
  • 23232324G, 23235239G, 23243588TT, 23243734G, 23264810(CCG)9, 23264854A, 23272034G, and 23273471C infers a smaller size for the animal and/or its offspring.
  • the presence of one or more of these markers infers a lower growth rate and/or lower liveweight for the animal and/or its offspring.
  • the invention provides a method of identifying animals which are more likely or less likely to produce one or more desirable trait the method comprising at least the step of analysing a nucleic acid from the animal to determine the nucleotide present at one or more of the following positions on chromosome 14 of Bos taurus 22986260, 23186380, 23186648, 23187238, 23205280, 23211989, 23215765, 23218552, 23228064, 23232324, 23235239, 23243588, 23243734, 23264810, 23264854, 23272034, and 23273471 or to determine the nucleotide sequence of one or more genetic marker in linkage disequilibrium with one or more thereof.
  • the method comprises at least the steps of: a) analysing a nucleic acid from the animal to determine the nucleotide present at one or more of the following positions on chromosome 14 of Bos taurus 22986260, 23186380, 23186648, 23187238, 23205280, 23211989, 23215765, 23218552, 23228064, 23232324, 23235239, 23243588, 23243734, 23264810, 23264854, 23272034, and 23273471 or to determine the nucleotide sequence of one or more genetic marker in linkage disequilibrium with one or more thereof; and,
  • the one or more desirable trait is size. In one embodiment, the trait is growth rate. In one embodiment, the trait is liveweight. In one embodiment, animals which are more likely to have a smaller size are identified on the basis of the presence of one or more of 22986260C, 23186380C, 23186648G,
  • animals which are more likely to have a lower growth rate and/or lower liveweight are identified on the basis of the presence of one or more of these markers.
  • animals which are more likely to have a larger size are identified on the basis of the presence of one or more 22986260T, 23186380T, 23186648A,
  • animals more likely to have a higher growth rate and/or higher liveweight are identified on the basis of the presence of one or more of these markers.
  • the invention provides a method for identifying an animal having a higher growth rate potential and/or higher liveweight potential, the method comprising at least the step of analysing a nucleic acid from said animal to determine the nucleotide sequence at one or more of the following positions on chromosome 14 of Bos taurus 22986260, 23186380, 23186648, 23187238, 23205280, 23211989, 23215765, 23218552, 23228064, 23232324, 23235239, 23243588, 23243734, 23264810, 23264854, 23272034, and 23273471, or to determine the nucleotide sequence of one or more genetic marker in linkage disequilibrium with one or more thereof, wherein the presence of one or more of 22986260T, 23186380T, 23186648 A, 23187238A, 23205280A,
  • the invention provides a method for identifying an animal having a lower liveweight potential and/or a lower growth rate potential, the method comprising at least the step of analysing a nucleic acid from said animal to determine the nucleotide sequence at one or more of the following positions on
  • chromosome 14 of Bos taurus 22986260, 23186380, 23186648, 23187238, 23205280, 23211989, 23215765, 23218552, 23228064, 23232324, 23235239, 23243588, 23243734, 23264810, 23264854, 23272034, and 23273471, or to determine the nucleotide sequence of one or more genetic marker in linkage disequilibrium with one or more thereof, wherein the presence of one or more of 22986260C, 23186380C, 23186648G, 23187238G, 23205280G, 23211989G, 23215765G, 23218552C, 23228064T, 23232324G,
  • 23235239G, 23243588TT, 23243734G, 23264810(CCG)9, 23264854A, 23272034G, and 23273471C infers a lower liveweigh and/or lower growth rate potential.
  • the invention provides a method for estimating the worth of an animal and/or its offspring, the method comprising at least the step of analysing a nucleic acid from the animal to determine the nucleotide present at one or more of the following positions on chromosome 14 of Bos taurus 22986260, 23186380, 23186648, 23187238, 23205280, 23211989, 23215765, 23218552, 23228064, 23232324, 23235239, 23243588, 23243734, 23264810, 23264854, 23272034, and 23273471 or to determine the nucleotide sequence of one or more genetic marker in linkage disequilibrium with one or more thereof, wherein the nucleotide present infers of the size potential of the animal and/or its offspring.
  • the invention provides a method for estimating the worth of an animal and/or its offspring, the method comprising at least the step of analysing a nucleic acid from the animal to determine the nucleotide present at one or more of the following positions on chromosome 14 of Bos taurus 22986260, 23186380, 23186648, 23187238, 23205280, 23211989, 23215765, 23218552, 23228064, 23232324, 23235239, 23243588, 23243734, 23264810, 23264854, 23272034, and 23273471 or to determine the nucleotide sequence of one or more genetic marker in linkage disequilibrium with one or more thereof, wherein the nucleotide present infers of the growth rate potential of the animal and/or its offspring.
  • the invention provides a method for estimating the worth of an animal and/or its offspring, the method comprising at least the step of analysing a nucleic acid from the animal to determine the nucleotide present at one or more of the following positions on chromosome 14 of Bos taurus 22986260, 23186380, 23186648, 23187238, 23205280, 23211989, 23215765, 23218552, 23228064, 23232324, 23235239, 23243588, 23243734, 23264810, 23264854, 23272034, and 23273471 or to determine the nucleotide sequence of one or more genetic marker in linkage disequilibrium with one or more thereof, wherein the nucleotide present infers of the liveweight potential of the animal and/or its offspring.
  • 23264854G, 23272034A, and 2327347 IT infers a larger size potential of the animal and/or its offspring.
  • the presence of one or more of these markers infers a higher growth rate and/or higher liveweight for the animal and/or its offspring.
  • 23232324G, 23235239G, 23243588TT, 23243734G, 23264810(CCG)9, 23264854A, 23272034G, and 23273471C infers a smaller size potential of the animal and/or its offspring.
  • the presence of one or more of these markers infers a lower growth rate and/or lower liveweight for the animal and/or its offspring.
  • the invention provides a method for breeding animals to produce offspring, which comprises selecting at least a first animal that has one or more of the following genotypes 22986260T, 23186380T, 23186648 A, 23187238 A, 23205280A, 23211989(deletion), 23215765C, 23218552T, 23228064G, 23232324A, 23235239C, 23243588 (deletion), 23243734A, 23264810(CCG)11, 23264854G, 23272034A, and 2327347 IT (read in relation to chromosome 14 of Bos Taurus) and mating said first animal with a second animal to produce offspring.
  • the second animal is selected on the basis it has one or more of the following genotypes 22986260T, 23186380T, 23186648A, 23187238A, 23205280A, 23211989(deletion), 23215765C, 23218552T, 23228064G, 23232324A, 23235239C, 23243588 (deletion), 23243734A, 23264810(CCG)11, 23264854G, 23272034A, and 23273471T.
  • the invention provides a method for breeding animals to produce offspring, which comprises selecting at least a first animal that has one or more of the following genotypes 22986260C, 23186380C, 23186648G, 23187238G, 23205280G, 23211989G, 23215765G, 23218552C, 23228064T, 23232324G, 23235239G,
  • the second animal is selected on the basis it has one or more of the following genotypes 22986260C, 23186380C, 23186648G, 23187238G, 23205280G, - 23211989G, 23215765G, 23218552C, 23228064T, 23232324G, 23235239G,
  • the methods of the invention further involve taking a sample from the animal.
  • the methods further involve analysing one or more additional biological markers.
  • analysis of the nucleotide sequence of the one or more genetic markers occurs using one or more of: polymerase chain reaction (PCR); gel electrophoresis; Southern blotting; nucleic acid sequencing; restriction fragment length polymorphism (RFLP); single-strand confirmation polymphism (SSCP); LCR (ligase chain reaction); denaturing gradient gel electrophoresis (DGGE); allele-specific oligonucleotides (ASOs); proteins which recognize nucleic acid mismatches; RNAse protection; oligonucleotide array hybridisation; denaturing HPLC (dHPLC); high resolution melting (HRM); and, matrix- assisted laser desorption/ionisation time-of-flight mass spectroscopy (MALDI-TOF MS).
  • PCR polymerase chain reaction
  • RFLP restriction fragment length polymorphism
  • SSCP single-strand confirmation polymphism
  • LCR ligase chain reaction
  • DGGE denaturing gradient gel electrophoresis
  • the invention provides a method for inferring the size potential of an animal and/or its offspring, the method comprising at least the step of observing the level of one or more of the following proteins, precursors thereof, isoforms thereof, fragments thereof, and/or nucleic acids encoding the proteins: RPS20; MOS; PLAG1; CHCHD7; RDHE2; SDR16C6; and, PENK; wherein the level infers the size potential of the animal and/or its offspring.
  • the invention provides a method for inferring the growth rate potential of an animal and/or its offspring, the method comprising at least the step of observing the level of one or more of the following proteins, precursors thereof, isoforms thereof, fragments thereof, and/or nucleic acids encoding the proteins: RPS20; MOS; PLAG1; CHCHD7; RDHE2; SDR16C6; and, PENK; wherein the level infers the growth rate potential of the animal and/or its offspring.
  • the invention provides a method for inferring the liveweight potential of an animal and/or its offspring, the method comprising at least the step of observing the level of one or more of the following proteins, precursors thereof, isoforms thereof, fragments thereof, and/or nucleic acids encoding the proteins: RPS20; MOS; PLAG1; CHCHD7; RDHE2; SDR16C6; and, PENK; wherein the level infers the liveweight potential of the animal and/or its offspring.
  • the invention provides a method of identifying animals which are more or less likely to produce one or more desirable trait the method comprising at least the step of observing the level of one or more of the following proteins, precursors thereof, isoforms thereof, fragments thereof, and/or nucleic acids encoding the proteins: RPS20; MOS; PLAG1; CHCHD7; RDHE2; SDR16C6; and, PENK.
  • the one or more desirable trait is size.
  • the trait is growth rate.
  • the trait is liveweight.
  • the methods involve comparing the level of expression of one or more of said proteins, precursor thereof, isoforms thereof, fragments thereof, and/or nucleic acids encoding said proteins against a standard, wherein a difference in the level of expression in the sample compared to the standard infers the size potential, growth rate potential and/or liveweight potential of the animal and/or its offspring. In one embodiment, an increase in the level of expression infers a higher growth rate and/or higher liveweight potential for the animal and/or its offspring.
  • the invention provides a method for identifying an animal having a higher growth rate potential and/or a higher liveweight potential, the method comprising at least the step of observing the level of expression of one or more of the following proteins, precursors thereof, isoforms thereof, fragments thereof, and/or nucleic acids encoding the proteins: RPS20; MOS; PLAG1; CHCHD7; RDHE2; SDR16C6; and, PENK, wherein an increase in the level of expression compared to a standard infers a higher growth rate potential and/or higher liveweight potential.
  • the invention provides a method for identifying an animal having a lower growth rate potential and/or a lower liveweight potential, the method comprising at least the step of observing the level of expression of one or more of the following proteins, precursors thereof, isoforms thereof, fragments thereof, and/or nucleic acids encoding the proteins: RPS20; MOS; PLAG1; CHCHD7; RDHE2; SDR16C6; and, PENK, wherein a decrease or substantially no increase in the level of expression compared to a standard infers a lower growth rate potential and/or lower liveweight potential.
  • the invention provides a method for selecting or rejecting an animal the method comprising at least the step of observing the level of one or more of the following proteins, precursors thereof, isoforms thereof, fragments thereof, and/or nucleic acids encoding the proteins: RPS20; MOS; PLAG1; CHCHD7; RDHE2; SDR16C6; and, PENK; and selecting or rejecting an animal based on the level of one or more of said proteins, precursors, isoforms or fragments thereof, and/or nucleic acids encoding same.
  • the method involves comparing the level of expression of one or more of said proteins, precursor thereof, isoforms thereof, fragments thereof, and/or nucleic acids encoding said proteins against a standard, wherein a difference in the level of expression in the sample compared to the standard infers the size potential, growth rate potential and/or liveweight potential of the animal and/or its offspring.
  • a difference in the level of expression in the sample compared to the standard infers the size potential, growth rate potential and/or liveweight potential of the animal and/or its offspring.
  • an increase in the level of expression infers a higher growth rate and/or higher liveweight potential for the animal and/or its offspring.
  • a decrease or substantially no increase in the level of expression compared to the standard infers a lower growth rate potential and/or lower liveweight potential for the animal and/or its offspring.
  • the method is performed for the purpose of selecting or rejecting an animal for milking purposes. In one embodiment, the method is performed for the purpose of selecting or rejecting an animal for beef farming. In another embodiment, the method is performed for the purpose of selecting or rejecting an animal for breeding purposes.
  • the animal is selected if there is a decrease or no substantial increase in the level of expression compared to the standard or the animal is rejected where there is an increase in the level of expression compared to the standard.
  • the animal is selected if there is an increase in the level of expression compared to the standard or the animal is rejected where there is a decrease or no substantial increase in the level of expression compared to the standard.
  • the invention provides a method for estimating the worth of an animal and/or its offspring, the method comprising at least the step of observing the level of one or more of the following proteins, precursors thereof, isoforms thereof, fragments thereof, and/or nucleic acids encoding the proteins: RPS20; MOS; PLAG1; CHCHD7; RDHE2; SDR16C6; and, PENK; wherein the level infers the size potential of the animal and/or its offspring. In one embodiment, the level infers the growth rate potential and/or the liveweight potential of the animal and/or its offspring.
  • the method involves comparing the level of expression of one or more of said proteins, precursor thereof, isoforms thereof, fragments thereof, and/or nucleic acids encoding said proteins against a standard, wherein a difference in the level of expression in the sample compared to the standard infers the size potential, growth rate potential and/or liveweight potential of the animal and/or its offspring.
  • a difference in the level of expression in the sample compared to the standard infers the size potential, growth rate potential and/or liveweight potential of the animal and/or its offspring.
  • an increase in the level of expression infers a higher growth rate potential and/or higher liveweight potential for the animal and/or its offspring.
  • a decrease or substantially no increase in the level of expression compared to the standard infers a lower growth rate potential and/or lower liveweight potential for the animal and/or its offspring.
  • the invention provides a method for breeding animals to produce offspring, the method comprising observing the level of one or more of the following proteins, precursors thereof, isoforms thereof, fragments thereof, and/or nucleic acids encoding the proteins: RPS20; MOS; PLAG1 ; CHCHD7; RDHE2; SDR16C6; and, PENK; in at least a first animal, selecting the first animal where there is an increase in the expression of one or more of said proteins, precursors, isoforms, fragments and/or nucleic acids encoding same compared to a standard, and mating said first animal with a second animal to produce offspring.
  • the method further comprises observing the level of one or more of the following proteins, precursors thereof, isoforms thereof, fragments thereof, and/or nucleic acids encoding the proteins: RPS20; MOS; PLAG1; CHCHD7; RDHE2; SDR16C6; and, PENK; in the second animal, and selecting the second animal where there is an increase in the expression of one or more of said proteins, precursors, isoforms, fragments and/or nucleic acids encoding same compared to a standard.
  • the invention provides a method for breeding animals to produce offspring, the method comprising observing the level of one or more of the following proteins, precursors thereof, isoforms thereof, fragments thereof, and/or nucleic acids encoding the proteins: RPS20; MOS; PLAG1; CHCHD7; RDHE2; SDR16C6; and, PENK; in at least a first animal, selecting the first animal where there is a decrease or substantially no increase in the expression of one or more of said proteins, precursors, isoforms, fragments and/or nucleic acids encoding same compared to a standard, and mating said first animal with a second animal to produce offspring.
  • the method further comprises observing the level of one or more of the following proteins, precursors thereof, isoforms thereof, fragments thereof, and/or " nucleic acids encoding the proteins:. RPS20; MOS; PLAG1; CHCHD7; RDHE2; SDR16C6; and, PENK; in the second animal, and selecting the second animal where there is a decrease or substantially no increase in the expression of one or more of said proteins, precursors, isoforms, fragments and/or nucleic acids encoding same compared to a standard.
  • RDHE2, SDR16C6, and, PENK a precursor thereof, an isoform thereof, a fragment thereof, and a nucleic acid encoding any one or more thereof in the sample;
  • At least an approximately 1.2 fold increase in the level of one or more of the proteins, a precursor thereof, an isoform thereof, a fragment thereof, and/or a nucleic acid encoding same is indicative of a higher growth rate potential and/or a higher liveweight potential of the animal and/or its offspring.
  • the methods further involve analysis of one or more additional biological markers.
  • the level of the one or more proteins, precursors, fragments, isoforms and nucleic acids encoding same is determined using an
  • the immunoassay separation based on characteristics such as molecular weight and isoelectric point, gel electrophoresis, Western Blotting or mass spectroscopy.
  • the immunoassay is an ELISA.
  • the gel electrophoresis is 2D gel electrophoresis or gel-free systems based on microfluidics technologies.
  • the animal is bovine.
  • the bovine animal is Bos taurus or Bos indicus.
  • the animal is chosen from the group consisting Jersey, Holstein, Friesian or crossbred dairy cattle.
  • the animal is Simmental.
  • 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 Structure of the Holstein-Friesian (HF) x Jersey (J) intercross generated for QTL mapping. Following conventional genetic formalism matings are described mentioning females first and males second. Hence "HF x J" corresponds to a mating between HF dams and J sires. Males are labeled with squares/rectangles, females with circles/ovals. Number of individuals of a given genotype are given in parentheses.
  • HF x J corresponds to a mating between HF dams and J sires.
  • Males are labeled with squares/rectangles, females with circles/ovals. Number of individuals of a given genotype are given in parentheses.
  • the black dotted line corresponds to the 5% genome-wide significance threshold determined with a permutation test (Churchill & Doerge, 1994).
  • the lower horizontal bar (CI) corresponds to the 95% CI for the QTL location determined by bootstrapping (Visscher et al., 1996).
  • the higher horizontal bar (SIAG) shows the position of the 1.1 MB critical interval defined by Mizoshita et al. (2005).
  • C Highest chromosome-wide log(l/p) values obtained within each of the six sire families for height (white bars) and live weight (black bars). The corresponding map positions are given above the bars. Note the very distinct map positions for sire 4.
  • the black dotted line marks the 5% chromosome-wide significance threshold.
  • FIG. 3 (A) Black line (labeled "HSQM”): Linkage-based QTL analysis of live weight in the HFxJ intercross using HSQM (Coppieters et al., 1998) and a 56 microsatellite marker map (cfr. Fig. 2B). Results are expressed as F-values (right axis). Dark dots (labeled "SP-T”): Single-point linkage + LD analysis of live weight in the HFxJ intercross using Dualphase (Druet & Georges, 2009) and a 925 SNP + 56 microsatellite marker map. Results are expressed as a likelihood ratio (LR) test (21nLR)(left axis).
  • LR likelihood ratio
  • Black line (labeled "MP-T”): Haplotype-based linkage + LD analysis of live weight in the HFxJ intercross using Dualphase and a 925 SNP + 56 microsatellite marker map. Results are expressed as a likelihood ratio (LR) test (21nLR)(left axis).
  • C Black line (labeled "OUTBRED”): Haplotype-based linkage + LD analysis of breeding value for live weight in 2,700 progeny-tested sires from the New Zealand outbred dairy cattle population using Dualphase and 293 SNP markers from the USDA 50K SNP chip (Van tassel et al., 2008) spanning a 15 Mb BTA14 segment spanning the QTL location.
  • Results are expressed as a likelihood ratio (LR) test (21nLR)(left axis).
  • D Dark dots (labeled "TAQ"): Single-point linkage + LD analysis of live weight in the HFxJ intercross using Dualphase and 11 candidate QTN identified by resequencing the 750 Kb critical interval in the six Fl sires.
  • A-D The X-axis corresponds to the chromosomal position in base pairs. The lower horizontal line corresponds to the QTL critical defined by Mizoshita et al. (2005), the higher horizontal line to the 750 Kb segment sequenced in the present study. Every graph shows the results of all analysis in gray watermarks to facilitate cross comparison.
  • Figure 4 (A) Dark gray line: Single QTL haplotype-based combined L+LD analysis of live weight in the HFxJ intercross (cfr. Fig. 3B). Black line: Two QTL haplotype-based combined L+LD analysis of live weight conducted with the same mixed model augmented with a random effect corresponding to the hidden state at the most likely QTL position obtained in the single QTL analysis (black arrow). The black line measures the increase in LRT (above that of the single-QTL analysis at the position of the black arrow) by adding a second QTL at the corresponding position.
  • Black line Two QTL haplotype-based combined L+LD analysis of breeding value for live weight in the outbred New Zealand dairy cattle population with the same mixed model augmented with a random effect corresponding to the hidden state at the most likely QTL position obtained in the single QTL analysis (black arrow).
  • the black line measures the increase in LRT (above that of the single-QTL analysis at the position of the arrow) by adding a second QTL at the corresponding position.
  • an additional QTL within the interval defined by Mizoshita et al. (2005) is observed (gray arrow).
  • Figure 5 Effect on live weight (in Kgs; X-axis) and frequency (number of observations in the studied population; Y-axis) for the 20 hidden haplotype states modeled with Dualphase (Druet and Georges, 2009) in the New Zealand outbred dairy cattle population. Shades of gray distinguish the breed origin of the corresponding animals: Holstein-Friesian (black), Jersey (gray), crossbred (white). The number of haplotype states in each class is given above the corresponding bars.
  • Figure 6 Upper track (labeled "Genes"): A. Organization of the eight genes mapping to the 750Kb critical region LYN, RPS20, MOS, PLAG1, CHCHD7, RDHE2, SDR16C6 and PENK).
  • SNP Quantitative Trait Nucleotides
  • QTN Quantitative Trait Nucleotides
  • CHCHD7 half height
  • Brown track labeled “Phastcons conserved elements, 5-way Vertebrate Multiz Alignment”: Location of "Phastcons” multispecies conserved elements.
  • B Orthologous locus in zebrafish (D. rerio)
  • Figure 7 Combined linkage + LD analysis of weight at birth (A), weight at 6 months (B), weight at 12 months (C), weight at 18 months (D) and height at 18 months (E) using 925 SNPs + 56 microsatellites in single point analysis (gray dots), 925 SNPs + 56 microsatellites in haplotype-based analysis (grey line), and 10 of the 14 candidate QTN (black circled dots, labeled).
  • the higher and lower horizontal lines respectively mark (i) the 750 Kb critical region sequenced in this work, and (ii) the interval defined by Mizoshita et al. (2005).
  • Figure 8 (A) Identification of the Q (grey), q (darker grey), and "recombinant” (grey + darker grey) haplotypes in a bovine diversity panel. Breeds and corresponding numbers of genotyped animals are given. Breed-specific haplotype frequencies are given and highlighted with a black box on white text when > 0.03. (B) Haplotype effect on birth weight in Simmental, assuming that the "R" haplotype (cfr. A) is q (left panel) or Q (right panel). Animals are sorted by predicted QTL genotype as shown. Smaller dots (with a shadow) correspond to individual observation. Larger dots correspond to genotype means. Arrows correspond to the 95% confidence intervals of the genotype mean (mean + 1.96*SEM).
  • Figure 10 Effect of pQTN genotype on the expression level of the eight genes mapping to 750-kb CI for the QTL in fetal liver, bone, muscle and brain, estimated by QRTPCR (mid-darkness bars), an allelic imbalance test using 3'UTR SNPs (darkest bars) or an allelic imbalance test using an intronic SNP (lightest bars).
  • the X-axis measures the slope of the regression (QRTPCR) or the ratio of Q over q allele (allelic imbalance tests) on a log 2 -scale.
  • the vertical black lines correspond to the absence of an effect of pQTN genotype on expression. #: pO.10; *:0.01 ⁇ p ⁇ 0.05; **p ⁇ 0.01. ND: not done. NE: no detectable expression.
  • Figure 11 (A) QRT-PCR results for LYN, RPS20, MOS, PLAG1, CHCHD7, RDHE2, SDR16C6 and PENK and MOS in fetal bone, muscle, brain and liver samples. Small grey circles correspond to the expression level in a given animal relative to the mean expression level across all animals. Relative expression levels are expressed on a log 2 -scale, and correspond to the mean relative expression level estimated with one or more amplicons (the number of tested amplicons is given in the upper left corner of each graph). Animals were sorted by pQTN genotype as indicated at the top of the figure (qq, Qq and QQ).
  • Ratios of firefly to renila luminescence obtained after transfection of Cosl cells with (i) a promoterless pGL4 vector, (ii) two distinct, sequence-verified preparations of the pGL4 vector endowed with the TK promoter, (iii) pairs of sequence-verified preparations of the vector endowed with the q or Q version, of the long or short fragment, cloned either (as denoted under figure) in forward or reverse orientation.
  • Error bars correspond to standard errors of the mean computed from replicates.
  • Figure 13 Representative results of EMS A experiments conducted with radiolabeled 29- mer (sQ and sq) and 74/80-mer probes (LQ and Lq) spanning the FJX_PLAPROTRI_l : 1 and FJX_PLAPROSNP_l:l pQTN.
  • sCtrl corresponds to an umelated 25-mer control duplex used as control competitor. Results shown were obtained with nuclear extracts derived from fetal bone (A) and C2C12 cells (B&C).
  • Figure 14 (A) Mapping eQTL influencing CHCHD7 expression levels in liver (lighter and upper line) and adipose (darker and lower line line) using a 56 microsatellite BTA14 map and HSQM (Coppieters et al., 1998). Location scores correspond to F-values. The black dotted line corresponds to the 5% chromosome- wide significance threshold determined with a permutation test (Churchill & Doerge, 1994). Inlet: Results of the analysis conducted within each of the six sire families separately.
  • the p-values obtained on BTA14 for each of the six families is indicated by the height of the first (liver) or second (adipose) bars, and indicate segregation of the eQTL in pedigree "1" only (arrow).
  • B Haplotype cluster-based association analysis conducted as described in M&M for CHCHD7 expression in liver (lighter and upper line) and adipose (darker and lower line). Markers considered are the 925 BTA14 SNPs + 56 BTA14 microsatellites. Location scores correspond to log(l/p), where p corresponds to the p-value of the corresponding F- test.
  • C Single SNP association analysis conducted as described in M&M for CHCHD7 expression in liver (lighter dots) and adipose (darker dots). Markers considered were the 925 BTA14 SNPs + 56 BTA14 microsatellites. The arrows point towards the p-values obtained for the CHCHD7 FJX_303486_1:1 splice site variant.
  • D Two SNP SNP association analysis conducted as described in M&M for CHCHD7 expression in liver (lighter dots) and adipose (darker dots). Markers considered were the 925 BTA14 SNPs + 56 BTA14 microsatellites.
  • Log(l/p) values correspond to the p-value of adding the corresponding SNP in a model already including the CHCHD7 FJX_303486_1 :1 splice site variant.
  • A-D The position of the sequenced 750 Kb interval containing CHCHD7 is marked by the black horizontal line.
  • Figure 15 (A) Mapping eQTL influencing RPS20 expression levels in liver (lighter and upper line) and adipose (darker and lower line) using a 56 microsatellite BTA14 map and HSQM (Coppieters et al., 1998b). Location scores correspond to F- values. The black dotted line corresponds to the 5% chromosome-wide significance threshold determined with a permutation test (Churchill & Doerge, 1994). Inlet: Results of the analysis " conducted within each of the six sire families separately.
  • the p- values obtained on BTA14 for each of the six families is indicated by the height of the first (liver) or second (adipose) bars, and indicate segregation of the eQTL in pedigree "1", "5" and "6" (arrows).
  • B Haplotype cluster-based association analysis conducted as described in M&M for RPS20 expression in liver (upper and darker line) and adipose (lower and lighter line). Markers considered are the 925 BTA14 SNPs + 56 BTA14 microsatellites. Location scores correspond to log(l/p), where p corresponds to the p-value of the corresponding F-test. The position of the sequenced 750 Kb interval containing CHCHD7 is marked by the black horizontal line.
  • Figure 16 (A) Agarose gel of RT-PCR products obtained from AA, AT and TT individuals for the CHCHD7 FJX_303486_1 :1 splice site variant.
  • the homozygous mutant ⁇ AA) animal shows three bands shown (by sequencing) to correspond to mRNAs with structures D and E as depicted, i.e. having lost exon 3. The largest band correspond to a D/E heteroduplex.
  • the homozygous wild-type (TT) animals show three bands corresponding to mRNAs with structures A, B and C as depicted. The largest band is a mixture of B/C heteroduplex plus band A.
  • Heterozygous (AT) animals show all fragments (A-E) plus additional heteroduplexes.
  • the inventors have identified that particular alleles of a number of genetic markers in a region of chromosome 14 in Bos taurus correlate with the weight and/or height of an animal.
  • the inventors note a particular correlation to weight at birth, at 6 months of age, 12 months of age, 18 months of age and 24 months of age.
  • the genotype of one or more of these particular genetic markers may therefore be used to infer or predict the size potential of an animal and/or its offspring.
  • the size of an animal may contribute to its value to a particular industry. For example, typically a larger animal or an animal with a high growth rate is preferable for beef farming and efficiencies can be gained in dairy farming by using smaller animals which require less feed.
  • the inventors also note that there is a correlation between increases in the expression of certain genes and the size of an animal. Thus, observing the level of expression of one or more of these genes may also be used to infer or predict the size potential of an animal and/or its offspring.
  • analysis of the genetic markers of the invention may assist in: predicting phentotypic performance; identifying animals more or less likely to have a desired trait; the selection or rejection of animals for breeding 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; cloning animals likely to have a specific trait; predicting the suitability of an animal and/or its offspring to use in different industries.
  • genetic marker refers to nucleic acids or specific genetic loci (including specific nucleotide positions) that are polymorphic or contain sequence 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.
  • 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.
  • VNTR variable number tandem repeat
  • allele refers to a tandem repeat of a nucleic acid sequence at a genetic locus in which the number of repeated DNA segments varies between individuals in a population.
  • the different repeat numbers within a VNTR at a particular locus may be 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.
  • isoform(s) should be taken broadly and includes those generated by posttranslational modification (for example, glycosylation), and proteolytic processing.
  • precursor(s) should also be interpreted broadly and includes for example preproteins, prepeptides, preproproteins and prepropeptides including an N-termihal leader sequence.
  • size should be taken broadly, and includes total size, weight (including liveweight), height, and/or growth rate.
  • Growth rate as used herein is intended to mean the average daily weight gain of an animal.
  • the phrase "infer size potential”, and like phrases, should be taken broadly to mean that the presence of a particular allele of a genetic marker indicates an increased likelihood that an animal and/or its offspring has the capacity to have a certain size characteristic (at one or more time points in its life; for example, at birth, or at an age at which it will be put to productive use in a particular industry such as the beef or dairy industry) compared to if it had a different allele of the genetic marker.
  • the phrase should not be taken to imply that the actual size of an animal is predicted.
  • the presence of a particular allele of a genetic marker of the invention provides an indication that the animal and/or its offspring is more likely than not to have a particular size characteristic relative to if it had a different allele.
  • an animal with one copy of a Q allele of a genetic marker (as outlined herein) will have from an approximately 60% to approximately 70% probability of having a larger size than if it had a different allele of that genetic marker.
  • an animal with one copy of a Q allele of a genetic marker (as outlined herein) will have-an approximately 65% probability of having a larger size than if it had a different allele of that genetic marker.
  • an animal with two copies of a Q allele of a genetic marker will have from an approximately 60% to approximately 70% probability of having a larger size than if it only had a single copy of the Q allele of that genetic marker. In one particular embodiment, an animal with two copies of a Q allele of a genetic marker (as outlined herein) will have an approximately 65% probability of having a larger size than if it only had a single copy of the Q allele of that genetic marker.
  • an animal with two copies of a Q allele of a genetic marker (as outlined herein) will have from an approximately 73% to approximately 83% probability of having a larger size than if it had two copies of a different allele of that genetic marker.
  • an animal having two copies of a Q allele of a genetic marker (as outline herein) will have an approximately 78% probability of having a larger size than if it had two copies of a different allele of that genetic marker.
  • low(er)”, “high(er)”, “small(er)”, and “large(er)” when used herein in relation to size, including weight, height, and/or growth rate, are intended to mean that an animal will have a size that is “low(er)”, “high(er)”, “small(er)”, “large(er)” than if it had a different allele of a genetic marker (or a different level of expression of a specified protein).
  • the terms indicate that the animal may have a size that is "low(er)", “high(er)”, “small(er)”, “large(er)” than another animal or the average in a population.
  • the following values are used as reference points: a height average at 24 months of approximately 1.25 metres with a standard deviation (SD) of approximately 3.7cm; weight average at 24 months of approximately 437 kg with a SD of approximately 40kg; average growth rate from birth to 24 months of approximately 0.58 kg/day with a SD of approximately 0.05 kg/day; wherein the population is a population of Holstein Fresian/Jersey crossbreed cattle.
  • SD standard deviation
  • animal is used herein primarily in reference to mammals 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 "worth” of an animal refers to an index used to evaluate the value of an animal, for breeding purposes or herd management, for example.
  • the “worth” is the sum of the estimated value of one or more characteristics which may be associated with the animal, typically weighted by an economic value. Exemplary characteristics include milk fat, protein, milk volume, liveweight, fertility, and milk somatic cells.
  • the term “worth” should be taken to encompass “breeding worth” and other known indexes used to assess the value of an animal. Persons skilled in the art to which the invention relates will readily appreciate methods and formulae suitable for estimating breeding worth on the basis of any number of different characteristics. Further details of parameters and methods for calculating the worth of an animal are provided in the "Examples" section herein after.
  • the methods of the invention allow for "selection” or “rejection” of an animal based on the genetic marker present or the level of a specified protein (including precursors, fragments and/or isoforms thereof) and/or nucleic acids encoding said protein, precursor, fragment and/or isoform.
  • a specified protein including precursors, fragments and/or isoforms thereof
  • nucleic acids encoding said protein, precursor, fragment and/or isoform.
  • an animal is selected for purposes relating to the beef industry or rejected for purposes relating to the dairy industry where the genetic marker or level of protein (including precursors, fragments and/or isoforms thereof) and/or nucleic acid encoding same infers a larger size potential for the animal.
  • an animal is selected for purposes relating to the dairy industry or rejected for purposes relating to the beef industry where the genetic marker or level of protein (including precursors, fragments and/or isoforms thereof) arid/or nucleic acid encoding same infers a smaller size potential for the animal.
  • the methods of the invention relate generally to inferring the size potential of an animal and/or its offspring. In one particular embodiment, the methods relate to inferring the liveweight potential of an animal and/or its offspring. In another particular embodiment, the methods relate to inferring the growth rate of an animal and/or its offspring. As mentioned hereinbefore, such information can be used, for example, to select animals for breeding purposes and/or estimate an animal's worth.
  • the invention may also be used to predict bull or cow phenotype performance and as such can be used in production management systems known as Marker Assisted Selection. Animals more or less suitable for a particular production system can be screened early at birth or as embryo's to predict life time performance and segregated or managed to suit their genotype and therefore predicted phenotype.
  • the methods involve the analysis of a nucleic acid from the animal to determine the nucleotide sequence of at least one genetic marker as described herein, wherein the sequence infers the size potential of the animal and/or its offspring.
  • the methods involve the analysis of the level of a specified protein or nucleic acid encoding it, wherein the level infers the size potential of the animal and/or its offspring.
  • the methods involve taking a sample from an animal to be tested.
  • the sample may be any appropriate tissue or body fluid sample.
  • the sample is blood, muscle, bone, somatic cell(s), saliva, or semen.
  • the sample is taken from the liver or brain.
  • 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; for example, an embryo, feotus, blastocyst. Individual gametes could also be tested using the methods of the invention. This may assist in breeding and/or cloning programmes. 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 levels of nucleic acid and/or the sequence of a particular nucleic acid, as will be described further herein after.
  • the methods of the invention may be combined with one or more 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 the markers 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.
  • methods of the invention involve analysing one or more genetic markers to identify the sequence or genotype of the genetic marker(s).
  • Genetic markers of use in the invention are listed in Table 1 below, along with the genotypes which the inventors have identified to be indicative of a smaller size (q allele), or a larger size (Q allele).
  • the sequence and position provided for each genetic marker is based on the genomic sequence of chromosome 14 in bovine build Btau 4.0
  • nucleotide variations listed in Table 1 provide specific examples of markers of use in the methods of the invention, the inventors contemplate that any variation in a nucleotide or the nucleotide sequence at any one or more the genetic loci referred to may be indicative of size potential.
  • the marker(s) are chosen from the group outlined in Table 8 herein after. In another particular embodiment, the marker(s) are chosen from
  • FJX_PLAPROTRI_l 1 and FJX PLAPROSNP 1 : 1.
  • nucleic acid sequence of either strand of the nucleic acid could be analysed.
  • nucleic acid sequence variations on such opposite strand which correlate with the genotypes of Table 1, having regard to the information contained herein and nucleic acid base pairing principles (ie, A pairs with T and C pairs with G).
  • the invention also encompasses use of one or more genetic markers which are in linkage disequilibrium with one or more of the markers mentioned herein before. Such markers may be analysed instead of or in addition to the genetic markers mentioned herein before.
  • Linkage disequilibrium should be taken broadly to refer to the tendency of the presence of an allele at one genetic loci (for example a genetic marker of Table 1) 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 linkage disequilibrium
  • 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.
  • 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 microarray), denaturing HPLC (dHPLC), fluorescence quenching PCR (TaqManTM, Applied Biosystems, CA 94404, USA), High Resolution Melting (HRM), and matrix-
  • SNPs single nucleotide polymorphisms
  • RFLP allele-specific PCR
  • SSCP SSCP
  • DGGE allele-specific oligonucleotides
  • ASOs allele-specific oligonucleotides
  • proteins which recognize nucleic acid mismatches oligonucleotide array hybridisation, dHPLC, fluorescence quenching PCR and matrix MALDI-TOF MS.
  • Any one or more of the techniques mentioned hereinbefore may be used to analyse the 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 14 (as detailed herein before), particularly in the genetic regions proximal to a particular marker, the nature of the genetic markers 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 purchase 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 01igo5 (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 01igo5 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 0.1 ⁇ -1 ⁇ of each primer, 200 ⁇ each dNTP, 3- 7mM MgCk, 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.
  • amplification conditions 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 the genetic markers of 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.
  • nucleic acids may be appropriately labelled for use in colorigenic, fluorogenic or chemiluminescence procedures. Specific examples are provided herein after, in the "Examples" section.
  • 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.
  • Negative controls may include samples having no nucleic acid present.
  • SNP positive control samples could include nucleic acids known to have a particular nucleotide at the relevant position.
  • 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.
  • 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 methods of the invention involve observing the level of one or more of the proteins RPS20, MOS, PLAG1, CHCHD7, RDHE2, SDR16C6 and PENK, including any one or more isoforms, precursors and fragments thereof, and/or any nucleic acids encoding one or more of the foregoing.
  • the methods of this embodiment of the invention will typically involve taking a sample from an animal, observing the level of expression of one or more of the proteins (including isoforms, precursors or fragments thereof) or nucleic acids encoding same, and comparing the level detected against one or more standard. Any difference in the level of expression between the sample and the standard infers the size potential of an animal and/or its offspring, including growth rate potential and/or liveweight potential.
  • the sample is a muscle, bone, brain or liver sample.
  • At least an approximately 1.2-fold difference in the level of one or more of the proteins, a precursor, fragment, or isoform thereof and/or nucleic acid encoding them compared to the standard is indicative of size potential.
  • a standard is a control sample having a known level of one or more of the proteins (including isoforms, precursors and fragments thereof) or a nucleic acid encoding same, which is tested concurrently with the sample from an animal to be tested.
  • the standard could be a printed chart or electronic information or the like containing previously generated data considered to provide an appropriate standard and which the test samples could be compared to on the basis of colour, fluorescence levels, or numerical values, for example.
  • the standard is preferably a level of one or more of the proteins (including isoforms, precursors or fragments thereof) or a nucleic acid encoding same, which is associated with an expression level in an animal or animals having a particular size (including growth rate and liveweight).
  • the inventors have identified that an increase in expression of one or more of the above proteins correlates with a larger size.
  • any increase in the level of expression one or more of these proteins may be considered to infer size potential of an animal, in a one embodiment, an increase in the level of expression of at least approximately 1.2 fold infers an increase in the size potential of an animal and/or its offspring.
  • a decrease or substantially no increase in the level of expression compared to a standard may also infer a lower size (including growth rate and/or liveweight) potential for the animal and/or its offspring.
  • the one or more proteins (including precursors, fragments and 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.
  • protein purification methods immunological techniques, separation of proteins based on characteristics such as molecular weight and isoelectric point including gel electrophoresis and microfluidics- based technologies as for example in gel-free protein separation techniques, and mass spectroscopy (MS) utilizing isobaric label based MS such as iTRAQ or label-free approaches such as multiple reaction monitoring (MRM) may be employed.
  • MS mass spectroscopy
  • 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
  • Nucleic acid-based techniques of use in the invention include differential display procedures, Northern Blotting, and competitive PCR. 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 RPS20, MOS, PLAG1, CHCHD7, RDHE2, SDR16C6 and PENK.
  • the nucleic acids will be capable of hybridising in a specific manner to a mRNA or cDNA associated with RPS20, MOS, PLAG1 , CHCHD7, RDHE2, SDR16C6 and PENK 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 RPS20, MOS, PLAG1, CHCHD7, PvDHE2, SDR16C6 and PENK or nucleic acids encoding RPS20, MOS, PLAG1, CHCHD7, RDHE2, SDR16C6 and PENK 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.
  • a detection molecule such as an antibody or nucleic acid probe or primer
  • 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 one or more of RPS20, MOS, PLAG1, CHCHD7, RDHE2, SDR16C6 and PENK and/or nucleic acids 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 may be removed from the sample.
  • high abundance proteins which have the potential to make it difficult to analyse such as detect and/or measure the level of RPS20, MOS, PLAG1, CHCHD7, RDHE2, SDR16C6 and/or PENK
  • MERS Multi affinity removal system
  • 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 one or more of RPS20, MOS, PLAG1, CHCHD7, RDHE2, SDR16C6 and PENK. 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 one or more of RPS20, MOS, PLAG1, CHCHD7, RDHE2, SDR16C6 and PENK 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. In one embodiment, mRNA may be observed in situ.
  • 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 the sequence of one or more of the genetic markers referred to herein.
  • Reagents suitable for analysis of one or more of the variants include one or more nucleic acid probes and/or primers as herein before described.
  • the kit comprises at least one or more reagents suitable for detection of one or more of RPS20, MOS, PLAG1, CHCHD7, RDHE2, SDR16C6 and PENK (including precursors, isoforms and fragments thereof), and/or nucleic acids encoding same.
  • the kit may comprise one or more antibody specific to one or more of the proteins (including precursors, isoforms and fragments thereof).
  • ELISA is used and the kit comprises one or more capture and/or detection antibody for one or more of the proteins (including precursors, isoforms and fragments thereof).
  • a method of the invention involves detection of the level of nucleic acids encoding one or more of the proteins (including precursors, isoforms and fragments thereof), it may comprise one or more nucleic acid probes and/or primers which have specificity for the target nucleic acids.
  • the kit comprises at least one reagent suitable for analysis of the sequence of the one or more genetic markers and at least one reagent suitable for detection of one or more of RPS20, MOS, PLAG1, CHCHD7, RDHE2, SDR16C6 and PENK (including precursors, isoforms and fragments thereof), and/or nucleic acids encoding same.
  • Reagents of use in processing samples for analysis may also be contained in the kits of the invention.
  • kits may also comprise one or more standard and/or other controls including nucleic acids whose sequence or genotype at a particular position is known, or containing known levels of one or more of RPS20, MOS, PLAG1, CHCHD7, RDHE2, SDR16C6 and PENK (including precursors, isoforms and fragments thereof) or nucleic acids encoding same.
  • 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. Breeding
  • the invention also relates to methods of breeding animals to produce offspring.
  • the method involves selecting at least a first animal that has one or more of the following genotypes 22986260T, 23186380T, 23186648A, 23187238A, 23205280A, 23211989(deletion), 23215765C, 23218552T, 23228064G, 23232324A, 23235239C, 23243588 (deletion), 23243734A, 23264810(CCG)11, 23264854G, 23272034A, and 2327347 IT and mating said first animal with a second animal to produce offspring, wherein the offspring has a smaller size potential.
  • the second animal is selected on the basis that it also has one or more of the following genotypes 22986260T, 23186380T, 23186648A, 23187238A, 23205280A,
  • the method involves selecting at least a first animal that has one or more of the following genotypes 22986260T, 23186380T, 23186648A, 23187238A, 23205280A, 23211989(deletion), 23215765C, 23218552T, 23228064G, 23232324A, 23235239C, 23243588 (deletion), 23243734A, 23264810(CCG)11, 23264854G,
  • the second animal is selected on the basis that it also has one or more of the following genotypes 22986260T, 23186380T, 23186648 A, 23187238 A, 23205280A, 23211989(deletion), 23215765C, 23218552T, 23228064G, 23232324A, 23235239C, 23243588 (deletion), 23243734A, 23264810(CCG)11, 23264854G, 23272034A, and 23273471T
  • the method involves observing the level of one or more of the following proteins, precursors thereof, isoforms thereof, fragments thereof, and/or nucleic acids encoding the proteins: RPS20; MOS; PLAG1; CHCHD7; RDHE2; SDR16C6; and, PENK; in at least a first animal, selecting the first animal where there is an increase in the expression of one or more of said proteins, precursors, isoforms, fragments and/or nucleic acids encoding same compared to a standard, and mating said first animal with a second animal to produce offspring.
  • the method further comprises observing the level of one or more of the following proteins, precursors thereof, isoforms thereof, fragments thereof, and/or nucleic acids encoding the proteins: RPS20; MOS; PLAG1; CHCHD7; RDHE2; SDR16C6; and, PENK; in the second animal, and selecting the second animal where there is an increase in the expression of one or more of said proteins, precursors, isoforms, fragments and/or nucleic acids encoding same compared to a standard.
  • the first and/or second animals are selected for breeding for beef farming where the level of expression is increased compared to a standard.
  • the method involves observing the level of one or more of the following proteins, precursors thereof, isoforms thereof, fragments thereof, and/or nucleic acids encoding the proteins: RPS20; MOS; PLAG1; CHCHD7; RDHE2; SDR16C6; and, PENK; in at least a first animal, selecting the first animal where there is substantially no increase in the expression of one or more of said proteins, precursors, isoforms, fragments and/or nucleic acids encoding same compared to a standard, and mating said first animal with a second animal to produce offspring.
  • the method further comprises observing the level of one or more of the following proteins, precursors thereof, isoforms thereof, fragments thereof, and/or nucleic acids encoding the proteins: RPS20; MOS; PLAG1; CHCHD7; RDHE2; SDR16C6; and, PENK; in the second animal, and selecting the second animal where there is substantially no increase in the expression of one or more of said proteins, precursors, isoforms, fragments and/or nucleic acids encoding same compared to a standard.
  • the first and/or second animals are selected for breeding for dairy farming where there is no substantial increase in the level of expression compared to a standard.
  • an animal is rejected for breeding for the purposes of dairy farming where there is an increase in the level of expression compared to the standard. In one embodiment, the animal is rejected for breeding for the purposes of beef farming wherein there is substantially no increase in the level of expression compared to the standard.
  • breeding methods may be utilised in this embodiment of the invention including natural insemination, artificial insemination and in vitro fertilisation. Accordingly, the word “mating” should be construed broadly and not limited to the physical pairing of two animals.
  • the methods herein before described may be used to determine the genotype of one or both potential parents and/or the level of expression of one or more specified protein.
  • HF Holstein-Friesian
  • J Jersey line-cross comprising 864 F2 cows
  • Fig. 1 36 HF bulls were mated with 430 J dams, and 24 J bulls with 366 HF dams, yielding six Fl sires (three HFxJ and three JxHF) and 796 Fl dams (430 HFxJ and 366 JxHF).
  • Fl animals were mated in order to obtain approximately equal proportions of the four possible genotypes ((HfxJ)x(HFxJ); (HfxJ)x(JxHF); (JxHF)x(HFxJ); (JxHF)x(JxHF)).
  • This experimental design primarily targets QTL segregating between HF and J, but - if analyzed appropriately - also allows the identification of QTL segregating within breeds. More than 500-hundred traits were measured on the F2 animals (Spelman et al, 2007). More than twenty of these pertained to body size, including weight at birth, 6, 8, 12, 18 and > 24 (- 'live weight”) months, as well as height at withers at 18 months. All animals of the pedigree were genotyped for 294 microsatellite markers spanning the bovine genome. When applying a line-cross model (i.e.
  • Combined linkage and linkage disequilibrium (LD) analysis in the HFxJ intercross and outbred dairy cattle population positions the QTL within a ⁇ 750 Kb chromosome segment
  • the BTA14 marker density was increased focusing0 on the region spanning the QTL peak.
  • the entire HFxJ cross was genotyped for 925 SNP markers corresponding to (i) 629 public domain SNPs, and (ii) 296 novel SNPs. The latter were identified by resequencing coding exons as well as highly conserved sequence elements in the six Fl sires.
  • Combined linkage and linkage disequilibrium analyses e.g. Blott et al., 2003; Druet & Georges, 2009
  • live weight i.e. the most significant trait
  • chromosome 14 was then evaluated in the New-Zealand outbred5 dairy cattle population.
  • the data were analyzed using the same mixed model as above (extracting both linkage and linkage disequilibrium information) with addition of a regression on percentage Jersey blood given available pedigree information.
  • The0 obtained location scores are shown in Fig. 3C.
  • a maximum LRT of 156 (corresponding to a lod score of 34.0) was obtained at position 23.425.845, i.e.
  • FIG. 5 shows the effect on breeding value for live weight of the different haplotype clusters as well as their respective frequencies in the New Zealand dairy cattle population.
  • the bimodal distribution of haplotype effects supports a bi-allelic QTL.
  • the q allele associated with lower weight/height is virtually fixed in the Jersey population, while the Q allele associated with higher weight/height predominates in the Holstein Friesian population.
  • Massive parallel resequencing of the -750Kb critical region identifies a cluster of 16 candidate QTN.
  • a 103 long-range PCR products spanning the entire 750 Kb interval was generated. PCR products of the predicted size were obtained from HF and J genomic DNA thereby (i) confirming the accuracy of the local sequence assembly, and (ii) indicating that both HF and J alleles could be amplified with comparable efficacy.
  • the same 103 long-range PCR products were then amplified from genomic DNA of the six Fl sires, pooled, size- fractionated by nebulization, and appended with adaptors allowing for massive parallel resequencing on a Roche FLX instrument.
  • the adaptors included individual specific multiplex identifiers (MIDs) allowing pooling and simultaneous sequencing of the eight libraries (HF, J and six Fl sires).
  • the obtained sequence traces were analyzed using the GS Reference Mapper software from Roche. The average sequence depth of non repetitive sequences was ⁇ 20-fold per individual. Mining of DNA sequence polymorphisms using GS Reference Mapper revealed a total of 8,851 putative DNA sequence polymorphisms (DSP) corresponding to an average nucleotide diversity of 0.3%.
  • DSP putative DNA sequence polymorphisms
  • the causative "Quantitative Trait Nucleotide" (hereafter referred to as pQTN for phenotypic QTN) has to be heterozygous in the four Fl sires that segregate for the QTL, homozygous in the two non-segregating Fl sires, and homozygous for alternate alleles in the HF and J samples. Applying this filter to the 8,851 DSP yielded only 17 candidate pQTN: 16 SNPs and one VNTR marker (Table 2).
  • DNA samples from 44 unrelated Simmental animals with birth weight information were collected and genotyped for the 12 polymorphisms.
  • the phase of the 44 animals was manually determined.
  • the frequency of the Q, q and R haplotypes in this sample was 22%, 35% and 34%, respectively.
  • the remaining 8% of the chromosomes corresponded to four minor haplotypes.
  • Fetuses were genotyped for the candidate pQTN, two homozygous "QQ” and two homozygous "qq” fetus were selected, and RT-PCR reactions using overlapping amplicons that would jointly span the complete MOS, PLAGl and CHCHD7 were performed. No evidence for genotype-specific RT-PCR products (Fig. 9) was obtained. These findings indicate that the pQTN affect the expression profile of the causative gene(s) rather than their structure, i.e. they are regulatory pQTN.
  • the pQTN affect expression of a regulon including RPS20, MOS, PLAGl, CHCHD7, RDHE2, SDR16C6 andPENK.
  • the pQTN could not only affect the expression of the spanned MOS, PLAGl and CHCHD7, but also of more distant genes.
  • the effect of pQTN genotype on the expression level of all eight genes in the 750 Kb interval in the fetal tissue samples described above was examined.
  • First expression by QRT-PCR, using up to three amplicons per gene was assayed. Data were normalized using from two to five housekeeping control genes selected from eight candidates using geNorm (Vandesompele et al. 2002). Normalized expression levels were expressed on a log 2 -scale, relative to the mean of all animals. For a given gene, the individual's average relative expression across amplicons was computed.
  • Reporter and electrophoretic mobility shifty assays support the causality of two pQTN in the PL A G 1-CHCHD 7 bidirectional promoter
  • FJX_PLAPROTRI_l :l (phastcons score: 0.999) is a (CCG) n trinucleotide repeat with either 9 (q) or 11 (Q) copies located immediately upstream of the presumed human PLAG1 transcriptional start site, while FJX_PLAPROSNP_l :l (phastcons score: 0.996) is a A (q) to G (g) SNP 12-bp upstream from FJX_PLAPROTRI_l : 1.
  • PLAGl and CHCHD7 are positioned head-to-head, separated by only ⁇ 500-bp supposed to encompass a bidirectional promoter (Trinklein ND et al. 2004).
  • FJX_250879_1 :1 also affects a conserved element (phastcons score: 0.905) located in the 3'UTR of PLAGl (Table 8).
  • FJX PLAPROTRIJ 1 and FJX_PLAPROSNP_l :l DSP on the presumed bidirectional promoter activity of the PLAG1-CHCHD7 intergenic region was tested.
  • allelic forms (Q and q) of a 378-bp and 659-bp fragment centered around the two pQTN were cloned in both orientations in the pGL4 luciferase vector (Fig. 12 A).
  • Cosl cells were transfected and measured luciferase activity after 24 hours.
  • both fragments indeed increased luciferase activity: ⁇ 9-fold (short) and ⁇ 20-fold (long) in the PLAG1 direction, and ⁇ 90-fold (short) and 44-fold (long) in the CHCHD7 direction (Fig. 12B).
  • the level of luciferase activity was systematically higher with the Q constructs than with the q constructs, the difference being ⁇ 1.5-fold, i.e. a magnitude comparable to that observed in vivo (Fig. 12B).
  • FJX_PLAPROTRI_l : 1 and FJX_PLAPROSNP_l : 1 variants might reflect differential binding of trans-act g factors
  • electrophoretic mobility shift assays were performed.
  • radiolabeled double-stranded 29-mers centered around the FJX PLAPROSNP lrl and nuclear extracts from Cosl, C2C12 and ATDC5 cells, as well as from fetal bone, brain, muscle and liver were used. With all extracts, a number of complexes were observed, some with the same mobility across tissues and some specific, that were systematically 1.2 to 3 x more abundant with the q than with the Q duplex.
  • cold q duplex tended to be more proficient than cold Q duplex in displacing either of the radio labelled probes (Fig. 13). Then 74/80-mer probes encompassing both the FJX_PLAPROTRI_l:l and FJX_PLAPROSNP_l:l variants (Cosl and C2C12 cells) were used. As with the 29-mer oligonucleotide, large complexes that were ⁇ 2.5 x more abundant with the q than with the Q probe were observed. As these would be displaced by cold 29-mers (q more effective than Q), they were assumed to be related to the complexes detected with the 29-mer sequence alone.
  • RPS20 encodes ribosomal protein S20.
  • RPS20 mutations cause p53-mediated skin darkening with pleiotropic effects including reduced body size (McGowan et al. 2008).
  • MOS encodes a protein kinase that is specifically expressed in oocytes where it is involved in the control of meiotic maturation. However, ectopic expression of MOS in somatic cells induces oncogenic transformation (Sagata 1997).
  • PLAG1 (Pleomorphic Adenoma Gene 1) was discovered as an oncogene whose ectopic expression due to translocation-induced promoter swapping with ubiquitously expressed genes (such as ⁇ -catenin), causes pleomorphic adenomas especially of the salivary gland (Van Dyck et al. 2007).
  • PLAG1 encodes a transcription factor that is strongly and broadly expressed during foetal development (particularly in the anterior pituitary), yet severely downregulated after birth.
  • PLAG1 has been shown to regulate transcription of several growth factors including IGF2, a key regulator of body size (Van Dyck et al. (2007) Voz et al. (2004)).
  • CHCHD7 encodes a widely expressed protein of unknown function, named for the coiled-coil-helix-coiled-coil-helix (CHCH) domain it contains. It was identified as a PLAG1 fusion partner in tumors of the salivary gland (Van Dyck et al. 2007).
  • RDHE2 (alias SDR16C5) and SDR16C6 encode members 5 and 6 of the 16C family of short-chain alcohol dehydogenases/reductases. RDHE2 catalyzes the first and rate-limiting step generating retinal from retinol.
  • PENK encodes the precursor of met- and leu-enkephalins, which play a role in pain perception and response to stress (Kiefer et al. 2002). While the candidacy of PLAG1 seem strongest, supported by GWAS signals maximizing on top of the same gene in human, available evidence neither proves PLAGVs causality, nor disproves a contribution of one or more of the other genes.
  • genome-wide expression data generated for 429 of the HFxJ F2 animals was used by hybridizing liver and adipose cDNA (sampled at 60 or 70 months of age) on Affymetrix Bovine 24K expression arrays.
  • the array included probes interrogating LYN, RPS20, MOS, CHCHD7 and RDHE2 but not PLA Gl and SDR16C6.
  • QTL mapping using HSQM (Coppieters, et al. 1998) and the 56 microsatellite BTA14 map revealed chromosome-wide significant eQTL effects for CHCHD7 in liver and adipose, and for RPS20 in liver.
  • Fine-mapping using the 925 SNP map positioned both eQTL in the resequenced 750-Kb segment, strongly suggesting cis-acting eQTL (Fig. 14 & 15).
  • FJX_303486_1:1 is the causative "eQTN" (Fig. 14C).
  • FJX_303486_1:1 in the association model caused marked drops in the significance of all other markers. Nevertheless, several of them remained significant, including the pQTN (Fig. 14D).
  • FJX_303486_1:1 affects splicing
  • the 79 fetuses were genotyped: 61 were homozygous TT, 17 were heterozygous AT and one was homozygous AA (without obvious phenotype) (Table 9).
  • RT-PC experiments were conducted on RNA samples from fetuses of the three genotypes using primers spanning the entire CHCHD7 transcript. RT-PCR products from AA individuals were shortened by 70 bp when compared to TT individuals, which was shown by direct sequencing to result from the skipping of exon 3. The ensuing mutant transcripts have truncated ORF closing within exon four (of five) (Fig.
  • the FJX_303486_1:1 should have an independent effect on body size. This hypothesis was tested by estimating the effect of the FJX_303486_1 : 1 SNP on height/weight in the HFxJ line-cross using a mixed model including the pQTN and an individual animal effect. There was no evidence for the slightest residual effect of FJX_303486_1:1 on body size when including the pQTN in the model (data not shown).
  • Genotyping Genotyping the 56 microsatellite markers on the HFxJ F2 pedigree was performed using standard procedures labeling the PCR products with fluorescent primers and size fractionating them on a ABI36730 capillary sequencer (e.g. Coppieters et al., 1998a). Genotyping the 925 BTA14 SNP markers on the HFxJ F2 pedigree was performed with a custom-made Golden Gate assay on an Illumina Beadstation500 using standard procedures. Genotyping the pQTN was done using Taqman assays-by-design using standard procedures (ABI, Foster City, CA). The primers used to analyse the genetic markers are listed in Table 7. below.
  • the mixed model includes a mean (fixed), a haplotype effect (random), an animal effect (random) and an error term (random).
  • LRT is asymptotically distributed as a chi-squared variable with ⁇ one degree of freedom.
  • two QTL positions were fitted simultaneously.
  • Evidence in favor of a QTL at position 2 was then evaluated from the increase in the LRT achieved by adding this second QTL in the model, (iv)
  • Association analysis was conducted using a mixed model including a mean, a regression on the number of alleles "1" for the considered SNPs (fixed), an animal effect (random) and an error (random). Variance components and effects were estimated by REML. The significance of the SNP effect was estimated using an F-test. In some instances, association analysis only considered the marker allele inherited from the mother.
  • Transcriptomic data (i) eQTL mapping in the HFxJ population under a half-sib pedigree model was conducted with HSQM (Coppieters et al., 1998b) as for the phenotypic data, (ii) Association analysis was conducted using a linear model including a mean, an experiment (1 or 2) effect (fixed), a regression on the number of alleles "1" for the considered SNPs (fixed), and an error term. The significance of the SNP effect was estimated using an F-test. In some instances two distinct SNP effects were fitted simultaneously. The significance of the second SNP effect conditional on the first one was estimated by an F-test. Haplotype-based association analysis was conducted by multiple regression against the number of copies of each of the 20 HMM-defined haplotype clusters (Druet & Georges, 2009)
  • FJX 21374 1 1 (35) CCTTCAAGAAGGTGGAGAAGAAAACA FJX_21374-161 R (36) GACTGAACTGAACTGAACTGAACTG
  • FJX 221494 (37) AAACCAGCAATCGCAAAACACTT FJX_221494-FJX2R (38) CAGAAGGGAGAAGCAGAAACCT
  • FJX 221762 (39) AGTCATGTATGGGTGTAAGAGTTGGA FJX_221762-2195R (40) CAACACCACAGTTCAAAAGCATCAA
  • FJX 222352 1 1 (41) TA FJX_222352-FJX3R (42) GTCCCTTTTCCAGTTATCTGCATGA
  • FJX 253666 (45) AAAACACTGAATGAGAAAGATACCACAGT FJX_253666-FJX9R (46) CACCAAAAGCAGAAAACAGAACAAC
  • Taqman FJX_263178-FJX4F GCTAATTAAGGGCAAAAGCTATCATAATC ATGTTGACATTTTCTTAATTGTTTACTAATGAGT
  • Taqman FJX_270353-FJX5F TTAAATCTTTATTTCTTGG I I I I I CTCCTTACAT
  • FJX 270353 1 1 (51) TGTTGTAGAAATAGGTACTCAGAGCCT FJX_270353-FJX5R (52) TTTTAGAA
  • FJX 278702 1 1 (53) AAAA FJX_278702-FJX6R (54) CACTGAACTGACTGACTGACTGATA
  • NP 1 :1 ABI3730 5UTR_SNP3F (85) AGAACTCACCGCGGGGCTTTAACAT 5UTR_SNP2R (86) GGAGGAGCGCGCGGGGAAGG
  • FJX 307148 1 (57) CAGATAGTTTGTGTCCCTTCTCTTCAT FJX_307148-3031 R (58) ACGAGCTGGACAGTTTGTGT
  • FJX 308585 1 :1 AGTCCAAAGGTTAACATCTGTGTTTCT FJX_308585-FJX7R (60) TGGTGGGCTGCCATCTATG
  • FJX_21374_1 :1 161 2 (61) IC TCTGCCAGACTCTCC FJX 21374-161 M2 (62) FAM TGCCAGGCTCTCC Reverse
  • FJX_221494 FJX2V1 (63) IC TGTGCATCTCACCCCCT (64) FA CTGTGCATCTTACCCCCT Forward
  • FJX_221762 2195V1 (65) VIC AAGAACACTAAGCACCAAAG (66) FAM AACACTAAGCGCCAAAG Forward
  • FJX_253666 FJX9V2 (71) VIC CCGGCTCACCAACCA (72) FAM CGGCTCGCCAACCA Reverse
  • FJX_267438_2:2 RPTV1 (75) VIC CATCTGCCACATCCCA (76) FAM TCTGCCGCATCCCA Forward
  • FJX_278702_1 :1 FJX6V2 (79) VIC TGACATATCTCTCAAGTCTATT (80) FAM TGACATATCTCTCGTCTATT Reverse
  • FJX_307148_1 :1 3031 2 (81) VIC CAGCAGCCGTTGTAAT (82) FAM CAGCAGCCGTCGTAAT Reverse
  • the effect of the C allele for the FJX_250879_1:1 SNP has been demonstrated to increase live weight in mature dairy cattle by approximately 10-20 kg in comparison to the G allele.
  • the inventors have demonstrated this effect in 3 breeds of cattle (Holstein- Friesian, Jersey and Ayrshire). Live weight has a negative relative economic value in the New Zealand dairy breeding index; Breeding Worth (BW). This reflects that a larger cow requires more energy for maintenance. Animals that are smaller but produce the same output of milk solids are desirable.
  • Analysis of the genotype of the FJX_250879_1:1 SNP may assist in calculations of the BW of an animal.
  • the effect of the genotype of the FJX_250879_1 :1 SNP for 34 dairy traits that are of economic or farmer importance was studied.
  • Genotype results of the 2150 HF sires identified that the A allele frequency for FJX_307148_1:1 is 0.87. There were only 38 sires that have the GG genotype for the FJX_307148_1:1 marker. For the statistical analysis the allelic effects were estimated between animals with the AA genotype (1678 animals) and AC genotype (479 animals).
  • the model fitted included the genotype as a fixed effect and also fitted year of birth and percent of North American genetics as covariates.
  • the effect of the genotype of the FJX_307148_1 :1 SNP for 34 dairy traits is shown in Table 3 below.
  • Other traits that are significant at the 5% level are; body condition score, calving difficulty, capacity, dairy conformation, fat percent, fat persistency, fore udder, gestation length, legs, milk volume, overall opinion, protein yield, protein persistency, rear teat, rump angle and width, somatic cell, stature and temperament.
  • Table 3 Allelic effect of the G allele compared to the A allele for marker FJX_307148_1:1 for 34 dairy traits.
  • EBVs are an estimate of a bull or cow's genetic merit for any given trait. Such genetic merit may also reflect somatic cell and/or germ cell characteristics, depending on the trait(s) being examined. EBVs can therefore not only predict phenotype performance and herd lifetime productivity (that is, provide a genetic diagnostic of phenotype) but also provide predictive insight into the ability of an animal to pass on superior genetic material (such as increased protein yield) to its offspring.
  • Breeding values are calculated/estimated for production traits (expressed in units of measurements, that is, litres of milk and kilograms of fat and protein), fertility traits, conformation traits, as well as other health, management and survival traits; for example, individual type traits, liveweight, somatic cell count, daughter fertility, calving difficulty and gestation length.
  • 'Best' is a reference to the method which gives the best estimates for the breeding value, or to put it more precisely, this minimizes the variance of difference between the estimates and the true breeding values.
  • 'Linear' means that there is a linear relation between parameters in the statistical model.
  • 'Unbiased' means that the breeding value estimates are central, that they are expected to be normally distributed with the true breeding value as mean value.
  • 'Prediction' normally refers to the future. But prediction also refers to the estimation of realized values of a random variable drawn from a population with known variance- and co-variance-structure.
  • a simpler and less precise formulation: 'prediction' is estimation of a given value of a random variable. It is better to use the expression 'prediction of breeding values', but 'breeding value estimates' are commonly used. With BLUP the breeding values are calculated for all animals simultaneously, and at the same time environmental effects can be corrected for. Animal Evaluation
  • Zealand national breeding objective is to identify animals whose progeny will be the most efficient converters of feed into farmer profit.
  • the evaluation system is designed to identify the most profitable and efficient sires regardless of breed.
  • the BVs reported from these models are calculated as the simple average of the four 270- day lactation BVs.
  • genetic groups were assigned by breed, sex of missing parent, birth year and country of origin. Four breed classes were assigned genetic grouping, namely, Holstein-Friesian, Jersey, Ayrshire-Red, and other breeds. Genetic groups were assigned in five year intervals from 1960 to 1980 then yearly, with the first birth year group being prior to 1960. Country of origin was - defined as New Zealand, North American and Other. Missing male parent of New Zealand origin had two categories: (i) unknown male but known from mating records to be an artificial insemination proven sire, or (ii) completely unknown male.
  • Liveweight The statistical model for liveweight is a repeated record, single trait, additive effects repeatability model. It includes effects for herd-year-season-age contemporary group, age at calving in months (nested within breed), stage of lactation when weighed (nested within age), heterosis, genetic merit of the animal, and random non-additive genetic and permanent environment effects.
  • Cow Fertility The objective for herd reproductive performance in most New Zealand herds is to achieve high pregnancy rates in a short time period following the planned start of mating, and to maintain calving intervals very close to 365 days. In this system successful reproduction depends on two factors which display genetic variation.
  • the first factor is the ability of the cow to resume cycling soon after calving, and to be mated early in the herd's mating period.
  • the second factor is the cow's ability to conceive, sustain a pregnancy and calve early in the herd's subsequent calving period.
  • Animal Evaluation has developed a genetic evaluation of cow fertility that incorporates both these aspects of successful reproductive performance in seasonal dairying. Mating records - Being presented for mating in the first 21 days of the herd's mating period (PM21) is scored 1 for the cows that are mated in this period, and 0 for cows which fail to be mated.
  • CR42 standing for Calving Rate in the first 42 days of the herd's calving period.
  • CR42 is scored 1 for cows that successfully re-calve in the first 42 days, and 0 for cows that fail to re-calve in this period.
  • CR42 is coded "missing" for cows which leave the herd prior to the re- calving period for reasons other than low fertility, or which have not yet had the opportunity to re-calve to the recorded mating.
  • Somatic Cells in milk - Test day records for somatic cell counts are transformed into somatic cell scores (SCS) by taking the log (base 2) of test day SCC/1000.
  • SCS is analysed in a multiple trait random regression animal model.
  • the two traits are first lactation SCS — and second and third lactation SCS analysed as repeated observations of a single trait.
  • the statistical model for analysis of a cow with SCS records includes effects for herdyear- season-age-testday contemporary groups, induced lactation, heterosis, age at calving (in months nested within breed class), stage of lactation, genetic group, genetic merit of the animal, random non-additive genetic and permanent environment effects, and random residual effects. Results for breeding values averaged over the two traits are reported.
  • Herd-life - Herd-life is defined as the interval from the date a cow has her first calf to the date when she has her last herd test, and is recorded in days. It is evaluated using a single- trait animal model.
  • the multiple-trait (MT) animal model used for the national genetic evaluation of survival contains 4 survival traits (SV12, SV13, SV14, SV15) and 9 predictor traits. The model can cope with missing data on any combination of traits.
  • yyki is the record for ith trait
  • hysij is the jth herd-year-season fixed effect for a cow's first lactation for trait i, with season referring to spring or autumn calving period,
  • htis is the linear regression coefficient for the sth heterosis effect for trait i
  • Whns is the sth heterosis covariate for animal n
  • ai k is the random additive genetic effect of animal k for trait i
  • eyki is the random residual associated with record yijkl.
  • Residual Survival For inclusion in the total economic merit index called Breeding Worth (BW), Residual Survival has been defined in a way that ensures that herd-life is counted only once in the index. Residual Survival is defined as "Herd-life after accounting for the genetic effects of production, liveweight, fertility and milk somatic cells on herd life.”
  • the Residual Survival trait included in BW is calculated from the following equation, where EBV stands for Estimated Breeding Value.
  • EBV(Total Long'ty) 5.434*EBV(Milkfat) + 4.408 *EBV(Protein) + 0.03815*EBV(Milk) - 0.489*EBV(Liveweight) + 27.847*EBV(Fertility) - 65,13 l*EBV(Somatic Cells) + Residual Survival
  • the estimated Residual Survival Breeding Values calculated by this method are uncorrelated with the production, liveweight and fertility traits included in the BW index. This property is important in order to ensure that effects on herd life are counted only once in the BW index.
  • the models for the linear management and conformation traits are single record, multiple trait, additive genetic effects models.
  • the statistical model for analysis of a cow with linear type scores includes effects for herd-year-season contemporary group, stage of lactation class when scored and age at first calving class (in months nested within breed), heterosis, genetic group, animal genetic merit and the random residual.
  • the four farmer-scored management traits (Adaptability to milking, Shed temperament, Milking speed, Overall opinion) are evaluated together in a multiple trait evaluation that takes the genetic correlations amongst the four traits into account.
  • the inspector-scored traits associated with body conformation are evaluated together in a multiple trait evaluation that takes the genetic correlations amongst these six traits into account.
  • the inspector-scored traits associated with udder conformation (but excluding Udder overall) are analysed together in a multiple trait evaluation. These traits are Udder support, Front udder, Rear udder, Front teat placement, and Rear teat placement. They are evaluated together in a multiple trait evaluation that takes the genetic correlations amongst the five traits into account.
  • Udder overall is evaluated in a single trait model. Breeding Values for individual traits are expressed in the units in which the trait is measured.
  • Calving Difficulty - Calving Difficulty Breeding Values are supplied to help Artificial Breeding organisations assess the suitability of bulls for mating with yearling heifers; and to give farmers knowledge about bulls which cause higher than usual rates of calving assistance when mated to their cows and heifers.
  • a sire's Calving Difficulty Breeding Value predicts the percentage of assisted calvings expected when he is mated to yearling heifers. The higher the BV, the higher the expected percentage of assisted calvings. The average BV of sires born in 1985 is set to zero. All breeds have been evaluated together for the calculation of Calving Difficulty BVs.
  • the Calving Difficulty BV is expressed in terms of assisted births in first-calving heifers, the BV can also be used to identify bulls that are expected to increase rates of calving assistance for cows carrying the bulls' calves.
  • the records used for the analysis comprise over 5 million records of calving assistance collected from New Zealand herds since 1994. The records were extracted in January 2006.
  • the BV for Calving Difficulty was estimated with a multiple-trait sire model using BLUP methodology (best linear unbiased prediction). The two traits were: - assistance for calves born to heifers, and - assistance for calves born to cows.
  • the statistical model used in the estimation was:
  • Body Condition Score Breeding Values - Body condition score (BCS) is commonly used as a method to assess body energy reserves.
  • BCS Body Condition Score
  • breeders are interested in knowing about sires that transmit lower or higher BCS for their daughters in early lactation when cows' body reserves are being mobilised for lactation at the same time as the cows are expected to get back in calf. Consequently Estimated Breeding Values for day 60 of first lactation heifers have been calculated for all AE Enrolled sires.
  • Persistency Breeding Values have been calculated for milkfat, protein, and volume.
  • the Persistency BVs are estimates of the genetic ability of cows to maintain production subsequent to peak of lactation. They are reported on a scale where higher values correspond to greater persistency of lactation after the peak.
  • Cows with persistent lactation curves can have high total lactation yields, average total lactation yields or low total lactation yields.
  • the persistency measure is designed to be independent of total yield - so farmers ought not to use high Persistency Breeding Values in an attempt to select for higher yields.
  • Persistency Breeding Values are reported in the same units as the dairy production traits - kilograms of milkfat, kilograms of protein, and litres of volume. To a close approximation, a Persistency BV of +5 kg can be interpreted as the genetic predisposition to yield 5 kg more in the lactation period after the 115th day of lactation compared to the earlier lactation period.
  • Persistency BV of -5 kg can be interpreted as the genetic pre-disposition to yield 5 kg less in the lactation period after the 115th day of lactation compared to the earlier lactation period.
  • the total lactation period used for the evaluation is 270 days.
  • Gestation length - Gestation length is the number of days from date of insemination to the date of parturition.
  • the average number of days for Gestation length in dairy bovine is 282 days.
  • a breeding value of -10 can be interpreted as an animal that will leave progeny on average 5 days shorter than the dairy bovine average.
  • the BW is the sum of the Breeding Values for milkfat, protein, milk volume, liveweight, fertility, milk somatic cells and residual survival each weighted by an economic value.
  • the BW economic values for each trait represent the expected net income per unit of genetic change (per unit of feed) from breeding
  • the economic values are calculated using a bio-economic devised to value technological change.
  • the model includes income streams from milk production, cull cows and bobby calf sales and cost streams associated with maintaining and growing cows and replacements, the feed required for production and dairy cash expenses. Predictions of future milk component prices are taken into account. The prices and costs used in the farm model are reviewed annually.
  • the unit of feed adopted for reporting economic values is 4.5 tonnes of dry matter of feed containing 10.5 megajoules of metabolisable energy per kilogram.
  • the economic values used in the BW are given in the following table.
  • Breeding Worth BW ($) - The expected ability of an animal to breed replacements which are efficient converters of feed into profit.
  • a Breeding Worth of 206 indicates the bull is expected to generate an extra $206 profit per year per unit of feed, through breeding replacements, compared with using a bull with a BW of zero.
  • Reliability - Associated with the BW, the reliability figure is the amount of confidence we can place in the figure. The more information included in the evaluation, the greater the reliability and less likely it is to change with additional records. Reliability ranges from 0%, meaning we know nothing about the animal or any of its ancestors, to 99%.
  • the base averages are:
  • the Traits Other than Production (TOP) Evaluation System is a national scheme for assessment of non-productive characteristics of dairy cattle (bulls and cows) based on linear assessment. Its main objective is to provide bull and herd owners with accurate and easy-to-use information for decision making.
  • Daughters are evaluated for 16 traits using a linear assessment on a scale from 1 to 9 where 1 and 9 represent the biological extremes. The traits are scored across breed and are defined as follows: Traits scored by the herd owner:
  • Adaptability to milking - describes how soon the animal settled into the milking routine after calving.
  • Shed Temperament describes the temperament of the animal in the shed while being handled and milked. It is a different trait to adaptability to milking and should be assessed once animals have settled into the milking routine.
  • Milking Speed - describes the milking speed of the animal, i.e. the time from putting cups on to the time milk flow stops or the cups are taken off.
  • Stature - describes the height at the shoulders of the animal. Each score represents 5 cm height at the withers.
  • Capacity - describes the capacity of the animal as a combination of strength and depth of chest and body as viewed from side, rear and front in relation to the physical size of the animal.
  • Rump Angle - describes the angle of a line between the centre of the hips and the top of the pins.
  • Pins high 1 5 9 Pins low/Sloping Rump Width - describes the width of the pins, hips and thurls relative to the size of the animal.
  • Narrow 1 -— 5 -— 9 Wide Legs - describes the straightness or curvature of the back legs from an imaginary line between the thurls and the mid hoof while the animal is walking.
  • Udder Support - describes the strength of the suspensory ligament as viewed from the rear. It also includes udder depth relative to the hocks.
  • Front Udder - describes how well the front udder is attached to the body wall.
  • Rear Udder - describes the height and width of the rear udder attachment, as distinct from udder support.
  • High Front Teat Placement describes the placement of the front teats (at the point of attachment to the udder) relative to the centre of the quarter as viewed from the rear.
  • Rear Teat Placement - describes the placement of the rear teats (at the point of attachment to the udder) relative to the centre of the quarter as viewed from the rear.
  • Dairy Conformation All traits pertaining to dairy conformation including those body traits that have been linearly scored, but excluding all the udder traits. Undesirable 1 5 9 Desirable
  • Table 5 Genotyic effects on live weight in the Ayrshire population for marker FJX_307148_1 : 1 relative to the CC genotype.
  • Genotyping was conducted using Taqman assays-by-design using standard procedures (ABI, Foster City, CA). The assays were performed using the relevant primers referred to herein before in Example 1 (Table 7).
  • the statistical model fitted for each of the phenotypes was a linear model with genotype as a fixed effect and year of birth fitted as a covariate.
  • Gudbjartsson DF Walters GB, Thorleifsson G, Stefansson H, Halldorsson BV, Zusmanovich P, Sulem P, Thorlacius S, Gylfason A, Steinberg S, Helgadottir A, Ingason A, Steinthorsdottir V, Olafsdottir EJ, Olafsdottir GH, Jonsson T, Borch-Johnsen K, Hansen T, Andersen G, Jorgensen T, Pedersen O, Aben KK, Witjes JA, Swinkels DW, den Heijer M, Franke B, Verbeek AL, Becker DM, Yanek LR, Becker LC, Tryggvadottir L, Rafnar T, Gulcher J, Kiemeney LA, Kong A, Thorsteinsdottir U, Stefansson K. (2008) Many sequence variants affecting diversity of adult human height. Nat Genet. 40:609-615.
  • PLAG1 the prototype of the PLAG gene family: versatility in tumour development. Int J Oncol. 30:765-774.

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Abstract

La présente demande de brevet concerne des procédés visant à développer la taille potentielle d'un animal et/ou de sa descendance et, en particulier, mais pas exclusivement, des procédés d'identification et de sélection d'animaux sur la base de leur poids vif et/ou de leur vitesse de croissance potentiels. L'invention concerne également des marqueurs biologiques utilisables dans le cadre desdits procédés, ces marqueurs étant placés dans une région du chromosome bovin 14 comprenant les gènes RPS20, MOS, PLAGl, CHCHD7, RDHE2, SDRl 6C6 and PENK.
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CN108866202A (zh) * 2018-06-13 2018-11-23 西北农林科技大学 中国黄牛PLAG1基因19-bp重复缺失多态性的检测方法及其应用

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WO2014193247A1 (fr) * 2013-05-31 2014-12-04 Livestock Improvement Corporation Limited Marqueurs génétiques du nanisme et leur utilisation
MX2016005379A (es) 2013-10-25 2017-02-15 Livestock Improvement Corp Ltd Marcadores geneticos y usos de los mismos.
CN109811061B (zh) * 2019-02-20 2023-05-09 新疆农业大学 Coil基因特异snp标记、和田乔达红羊产羔数性状的检测方法及其应用
CN111411159A (zh) * 2020-04-23 2020-07-14 西北民族大学 一种绵羊chchd7基因插入/缺失多态性的检测方法及其应用

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CN108866202A (zh) * 2018-06-13 2018-11-23 西北农林科技大学 中国黄牛PLAG1基因19-bp重复缺失多态性的检测方法及其应用

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