WO2008014550A1 - Markers for pigmentation - Google Patents

Markers for pigmentation Download PDF

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Publication number
WO2008014550A1
WO2008014550A1 PCT/AU2007/001070 AU2007001070W WO2008014550A1 WO 2008014550 A1 WO2008014550 A1 WO 2008014550A1 AU 2007001070 W AU2007001070 W AU 2007001070W WO 2008014550 A1 WO2008014550 A1 WO 2008014550A1
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Prior art keywords
animal
genetic marker
pigmentation
tyrosinase
marker
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PCT/AU2007/001070
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French (fr)
Inventor
Herman Raadsma
Karen Fullard
Jamie Hernan Gongora Romero
Imke Tammen
Colin Cavanagh
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The University Of Sydney
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Priority claimed from AU2006904111A external-priority patent/AU2006904111A0/en
Application filed by The University Of Sydney filed Critical The University Of Sydney
Priority to AU2007281024A priority Critical patent/AU2007281024B2/en
Publication of WO2008014550A1 publication Critical patent/WO2008014550A1/en

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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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • 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
    • 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
    • C12Q2600/172—Haplotypes

Definitions

  • the present invention relates to genetic markers for pigmentation in skin and fleece areas of animals.
  • the present invention relates to methods, systems and kits for predicting pigmentation and pigmented fibre contamination in animals using genetic markers, to associated methods, systems and kits for selecting animals using marker assisted selection, and to associated methods and systems for animal breeding and prediction of lifetime performance.
  • the present invention is predicated on the development of a genetic marker shown to be associated with pigmentation and pigmented fibre contamination in animals.
  • a method for predicting pigmentation in an animal comprising analysing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase- related protein gene, wherein said genetic marker is predictive of pigmentation in said animal.
  • the pigmentation may include pigmented skin or dark fibre contamination.
  • the tyrosinase-related protein may be TYRP-I .
  • the presence or absence of the polymorphism may be analysed through the use of nucleic acid sequences flanking the polymorphism, such as PCR primers.
  • nucleic acid sequences flanking the polymorphism such as PCR primers.
  • a method for selecting an animal using marker assisted selection comprising:
  • a system for predicting pigmentation in an animal comprising: (a) means for analyzing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene, wherein said genetic marker is predictive of pigmentation in said animal.
  • a system for selecting an animal using marker assisted selection comprising: (a) means for analyzing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene, wherein the genetic marker is predictive of pigmentation in said animal; and
  • a system for breeding an animal using marker assisted selection comprising: (a) means for analyzing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene, wherein the genetic marker is predictive of pigmentation in said animal; and (b) means for breeding said animal based on the presence or absence of the genetic marker, and
  • (c) means for selecting progeny of said animal based on the presence or absence of the genetic marker.
  • kit for predicting pigmentation in an animal wherein said kit comprises:
  • kit for selecting an animal using marker assisted selection comprising:
  • kits for breeding an animal using marker assisted selection comprising: (a) means for analyzing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene, wherein said genetic marker is predictive of pigmentation in said animal; and
  • a polypeptide comprising a tyrosinase-related protein (TYRP).
  • TYRP tyrosinase-related protein
  • the TYRP may be TYRP-I.
  • the TYRP-I may comprise the amino acid sequence set forth in SEQ ID NOS: 36, 39, 42, 45, 47, 74, 77 and 80.
  • a polynucleotide encoding the polypeptide of the eleventh aspect.
  • a vector comprising the polynucleotide of the twelfth aspect.
  • a host cell transformed with the vector of the thirteenth aspect.
  • an animal product derived from the animal of the fifteenth aspect is provided.
  • the animal product may be selected from the group comprising hair, wool, skin, cashmere, and mohair.
  • the animal product may be wool.
  • a method of sampling fibres to determine non-visual pigmentation comprises (a) genotyping a plurality of animals based on the presence or absence of at least one genetic marker in the tyrosinase-related protein gene, wherein the genetic marker is predictive of pigmentation in said animal;
  • the pigmentation is present in the form of pigmented fibres.
  • the pigmented fibres may be in the form of pigmented wool, hair, mohair or cashmere.
  • the pigmented fibres are rare and/or isolated.
  • the proportion of pigmented fibres may comprise less than
  • 0.001% of total fibre count less than 0.0001% of total fibre count, less than 0.00001% of total fibre count, less than 0.000001% of total fibre count, less than 0.0000001% of total fibre count, less than 0.00000001% of total fibre count, or less than 0.0000000001% of total fibre count.
  • QTL location for Pigmented Fibre on Chromosome 26 I. QTL location for Hoof Pigmentation on Chromosome 2; J. QTL location for Hoof Pigmentation on Chromosome 18; K. QTL location for Binary score hornsite and leg Fibre score on Chromosome 2; L. QTL location for Binary score hornsite and leg fibre score on Chromosome 8; M. QTL location for Binary score hornsite and leg fibre score on Chromosome 19.
  • FIG. 4 Black and white (top), and negative picture (bottom) of PCR products for TYRP-I exons 2, 3, 4 and 5 electrophoresed on a 2% agarose gel.
  • M marker
  • - negative control
  • C cattle
  • H human
  • M mouse
  • S sheep. Number in brackets indicates the ⁇ size of the PCR product in relation to the molecular weight of the marker.
  • Figure 5 Black and white (top), and negative photograph (bottom) of PCR products for TYRP-I exons 6, 7, 8 and 8-1 (using R2 primers described below) electrophoresed on a 2% agarose gel.
  • Figure 6. Black and white photograph of PCR products of TYRP-I exons 1 and upstream region electrophoresed on a 2% agarose gel. Primers are indicated by number according to Tables 7-9
  • Genotype '1' is significantly more associated with a higher Fibre Pigmentation Score (PigFibre) than genotype '2'.
  • FIG. 8 Parental haplotypes inherited from the Merino granddam (C7_M), and the Awassi grandsire (C7 AW), and other (partial) haplotypes. In relation thereto, animals with haplotype "34" also had a confirmed high isolated pigmented fibre count using the AWTA test.
  • Figure 9 Histogram figure of Pigmented Fibre score out of 500 for pigmented fibre, against haplotypes predictive of HIGH and LOW risk of pigmentation respectively from the 1997 mapping family
  • primer means a single-stranded oligonucleotide capable of acting as a point of initiation of template-directed DNA synthesis.
  • oligonucleotide is a single-stranded nucleic acid typically ranging in length from 2 to about 500 bases. The precise length of a primer will vary according to the particular application, but typically ranges from 15 to 30 nucleotides. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize to the template.
  • gene means the genetic constitution of an organism. This may be considered in total, or with respect to the alleles of a gene(s) or genetic marker(s).
  • homozygote refers to an organism that has identical alleles at a given locus on homologous chromosomes.
  • heterozygote refers to an organism in which different alleles are found on homologous alleles for a given locus.
  • linkage disequilibrium describes a situation in which some combinations of alleles of two or more different loci (haplotypes) occur more or less frequently within a population than would be expected by random chance alone.
  • pigmentation as used herein includes skin and fibre pigmentation, and in particular includes skin and fibre pigment contamination.
  • skin may include nose, lips, hoofs, eyelids, udder, and fibre may include hair, wool, fleece, and eyelashes.
  • the term “pigmentation” may include isolated or partial fleece colouration, also known as “white spotting", piebald, or any other form of discolouration or full- fleece colouration.
  • the term “pigmentation” may include fibres containing a degree of colouration that is not or substantially not visible to the eye.
  • dark fibre and “pigmentation” are synonymous.
  • the terms “pigment” and “pigmented” have corresponding meanings.
  • the term "genetic marker” refers to a variant or polymorphism at DNA sequence level linked to a specific chromosomal location unique to an individual's genotype, inherited in a predictable manner, and measured as a direct DNA sequence variant or polymorphism, such as at least one Single Nucleotide Polymorphism (SNP), Restriction Fragment Length Polymorphism (RFLP), or Short Tandem Repeat (STR), or as measured indirectly as a DNA sequence variant (eg. Single-strand conformation polymorphism (SSCP), Denaturing Detergent Gradient Gel Electrophoresis (DDGE).
  • SNP Single Nucleotide Polymorphism
  • RFLP Restriction Fragment Length Polymorphism
  • STR Short Tandem Repeat
  • DNA sequence variant eg. Single-strand conformation polymorphism (SSCP), Denaturing Detergent Gradient Gel Electrophoresis (DDGE).
  • single nucleotide polymorphisms or "SNP” or “SNPs”, as used herein, refers to common DNA sequence variations among subjects.
  • the DNA sequence variation is typically a single base change or point mutation resulting in genetic variation between individuals.
  • the single base change can be an insertion or deletion of a base.
  • base pair means a pair of nitrogenous bases, each in a separate nucleotide, in which each base is present on a separate strand of DNA and the bonding of these bases joins the component DNA strands.
  • a DNA molecule contains four bases; A (adenine), G (guanine), C (cytosine), and T (thymidine).
  • a and G are purine bases, typically designated by the letter “R”
  • C and T are pyrimidine bases, typically designated by the letter "Y”. Where A or T may occupy a single position it is typically designated by the letter W. Where G or C may occupy a single position it is typically designated by the letter S.
  • a or C may occupy a single position it is typically designated by the letter M.
  • G or T may occupy a single position it is typically designated by the letter K.
  • A, T or C may occupy a single position it is typically designated by the letter H.
  • G, C or T may occupy a single position it is typically designated by the letter B.
  • G, A or T may occupy a single position it is typically designated by the letter D.
  • G, C or A may occupy a single position it is typically designated by the letter V.
  • G, C, A or T may occupy a single position it is typically designated by the letter N.
  • base pair is abbreviated to "bp”
  • kilobase pair is abbreviated to kb.
  • animal refers to an individual at any stage of life, or after death, including an entity prior to birth such as a fertilised ovum, either before fusion of the male and female pro-nucleus or after the pro-nuclei have fused to form a zygote, an embryo (created by any means including somatic cell nuclear transfer) or an individual cell (N, 2N or greater); for the avoidance of doubt, this also includes a cell or a cluster of cells including stem cells and stem cell-like cells, cell line, haploid gametes and their progenitor cell lines, as well as products resulting from the gametes, including embryos.
  • chromosomes which contained genes contributing to skin and fibre pigmentation in sheep, detailed in Table 6.
  • One of the major regions with strongest significance was found on chromosome 2, and was significant for 6 skin and fibre traits.
  • mapping progeny was sufficient to account for over 50% of the differences in pigmentation, and indicated that the colour phenotype originated from the Awassi allele (Figure 7).
  • This region was shown to contain a very strong candidate gene, namely tyro sine-related protein- 1 (TYRP- 1 - Figure 3).
  • TYRP-I was sequenced by alignment of TYRP-I sequence from all known species, with conserved sequence sites used to design DNA primers for ovine sequence. A predicted ovine gene structure was then identified from comparative mapping, with ovine sequence confirming close alignment to bovine TYRP-I .
  • Genetic assay-assisted selections for animal breeding are important in that they allow selections to be made without the need for raising and phenotypic testing of progeny. In particular, such tests allow selections to occur among related individuals that do not necessarily exhibit the trait in question and that can be used in introgression strategies to select both for the trait to be introgressed and against undesirable background traits.
  • Non- limiting examples of methods for identifying markers corresponding to genetic polymorphisms between members of a population include: restriction-fragment-length polymorphism (RFLP) Bostein et al (1980) Am J Hum Genet 32:314-331; single-strand conformation polymorphism (SSCP) Fischer et al. (1983) Proc Natl Acad Sci USA 80:1579-1583, Orita et al. (1989) Genomics 5:874-879; amplified fragment-length polymorphism (AFLP) Vos et al.
  • RFLP restriction-fragment-length polymorphism
  • SSCP single-strand conformation polymorphism
  • AFLP amplified fragment-length polymorphism
  • Primers that may be used in this regard may, for example, comprise sequences set out in Tables 7, 8 and 9 and complements thereof.
  • selections may be unambiguously made based on genotypes assayed at any time after a nucleic acid sample can be collected from an individual, such as an infant animal, or even earlier in the case of testing of embryos in vitro, or testing of foetal offspring. Any source of nuclear DNA may be analyzed for scoring of genotype.
  • nucleic acids are screened that have been isolated from the blood or semen of the animal analyzed.
  • DNA from the animal to be assessed may be extracted by a number of suitable methods known to those skilled in the art. Most typically, DNA is extracted from a blood or semen sample, and in particular from peripheral blood leucocytes. A sufficient amount of cells are obtained to provide a sufficient amount of DNA for analysis, although only a minimal sample size will be needed where scoring is by amplification of nucleic acids.
  • the DNA can be isolated from the blood cells by standard nucleic acid isolation techniques known to those skilled in the art.
  • Nucleic acids used as a template for amplification may be isolated from cells, tissues or other samples according to standard methodologies. Such embodiments may find particular use with the invention, for example, in the detection of repeat length polymorphisms, such as microsatellite markers. In certain embodiments of the invention, amplification analysis is performed on whole cell or tissue homogenates or biological fluid samples without substantial purification of the template nucleic acid.
  • the amplification product may be detected or quantified.
  • the detection may be performed by visual means.
  • the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical and/or thermal impulse signals.
  • scoring of repeat length may be performed by visual means.
  • PCR polymerase chain reaction
  • the genetic marker(s) in the tyrosinase related protein gene useful in the invention may be selected from, but is not limited to, the group of polymorphisms shown in Table 1 -0 below.
  • amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods. Amplification products isolated in this way may be eluted from an excised portion of the gel for further manipulation.
  • the nucleic acid may be removed from the excised portion of an agarose gel by heating the gel in a chao tropic solution, followed by extraction of the nucleic acid.
  • Separation and isolation of nucleic acids may also be performed by chromatographic techniques known in art. Numerous kinds of chromatography may be used in the present invention, including adsorption, partition, ion-exchange, hydroxylapatite, gel-filtration (molecular sieve), and reverse-phase. These may be performed in a number of formats including, column, paper, thin-layer, and gas chromatography as well as by batch HPLC or FPLC. In certain embodiments, the amplification products are visualized. A typical visualization method involves staining of a gel with ethidium bromide and visualization of bands under UV light.
  • a labeled nucleic acid probe is brought into contact with the amplified marker sequence.
  • the probe preferably is conjugated to a chromophore but may be radiolabeled or otherwise labeled to facilitate detection.
  • the probe is conjugated to a molecule, such as an antibody or biotin, or another molecule carrying a moiety to facilitate detection.
  • detection is by nucleic acid blotting and hybridization with a labeled probe.
  • the techniques involved in nucleic acid blotting are well known to those skilled in the art.
  • the nucleic acid blotting and hybridization may be performed under stringent conditions.
  • stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 3O 0 C for short probes (e.g., 10 to 50 nucleotides) and at least about 6O 0 C for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
  • Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37°C, and a wash in 1.
  • Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37 0 C, and a wash in 0.5x to IxSSC at 55 to 5O 0 C.
  • Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37 0 C, and a wash in 0. IxSSC at 60 to 65 0 C.
  • Kits All the essential materials and/or reagents required for screening animals for genetic marker genotype in accordance with the invention may be assembled together in a kit.
  • This generally will comprise a probe or primers designed to hybridize specifically to individual nucleic acids of interest in the practice of the present invention, for example, primer sequences such as those for amplifying TYRP-I.
  • enzymes suitable for amplifying nucleic acids including various polymerases, deoxynucleotides and buffers to provide the necessary reaction mixture for amplification.
  • Such kits also may include enzymes and other reagents suitable for detection of specific nucleic acids or amplification products.
  • Such kits generally will comprise, in suitable means, distinct containers for each individual reagent or enzyme as well as for each probe or primer pair.
  • the invention also can provide for a kit which can be used to determine the TYRP-I genotype of genetic material, for example the kit may include a set of primers used for amplifying the genetic material.
  • a kit can contain a primer including a nucleotide sequence for amplifying a region of the genetic material containing one of the polymorphisms described herein.
  • the 177 offspring were male backcrossed progeny from mating between a single Awassi/Merino Fl sire (C7/A95.0453) and unselected Merino ewes, giving a progeny of 75% Merino and 25% Awassi. Animals were born from May 1997, and separated into two management cohorts according to birth dates. Table 3; Pi mentation traits and trait descri tion
  • ColourScore transformed to a binary trait
  • NoseLPig was arcsin transformed
  • LHSFibre was transformed to a binary trait.
  • a full genome scan was conducted with 204 microsatellite markers.
  • a framework map was constructed using the open source mapping programs Carthagene and Mapmaker, which was used to inform marker positions.
  • a full genome scan was conducted using a maximum likelihood analysis written in R, developed by the inventors (QTL-MLE).
  • the QTL-MLE algorithm was developed to match the particular experimental design and genotyping characteristics of the study. These included (1) incorporation of known linkage phase information resulting from the backcross design, (2) utilisation of all the relevant genotypic information, and (3) valid modelling the marker-QTL associations by means of a finite mixture model. Item (2) involved development of an algorithm to utilise information from heterozygous marker genotypes, using estimates of marker allele frequencies in the merino population to. assist this. The QTL model is fitted using a maximum likelihood routine at each putative QTL position along the length of the target chromosome (interval mapping).
  • the probability of each backcross offspring inheriting either allele from the Fl sire is provided, in terms of a posterior probability. This allows animals with a high probability of receiving the "favourable" QTL genotype to be identified.
  • a 1- LOD support interval is provided to give an indication of the precision of estimating the location of the QTL.
  • TGLAlO was found to be the closest linked mapped genetic marker and tyrosinase-related protein- 1 (TYRP-I) was identified as the most significant positional candidate gene by comparative mapping to human, cattle and rat using the Comparative Predictions of Orthologues software at the Oxford Grid Project (http://oxgrid.angis.org.au).
  • TYRP-I exons from sample C7 were amplified by PCR using the conditions described in Tables 10 and 12 using TIi DNA polymerase (Promega) or 11 and 13 using Invitrogen Taq DNA polymerase. PCR products were gel-purified using UltraClean DNA purification kits (Mo Bio Laboratories, Inc.), or were enzymatically cleaned using ExoSAP-IT (USB). PCR products were directly sequenced using the SUPAMAC (Sydney University Prince Alfred Macromolecular Analysis Centre) commercial sequencing service on an ABI PRISM® 3700 platform (Applied Biosystems, Foster City), or were sequenced using an ABI BigDyeV3 kit, and run in-house on an ABI PRISM® 3100 platform. The DNA and amino acid (based on nuclear code) TRYP-I sequences obtained from the upstream region, and exons 2 to 8 (as per Table 2) .
  • TGTA(T)A is not recognised by the enzyme.
  • BsrGI PCR-RFLP of TYRP-I Upstream 604 using the restriction enzyme BsrGI for C7 was shown to be heterozygous containing the cut product (123bp) and the uncut product (326bp).
  • C4 is a homozygote for the uncut product (326bp)
  • Cl 7 is a heterozygote for the cut product (123bp) and the uncut product (326bp).
  • the variant G(A)AAAAATTC is recognised and digested by the Xmn I while the variant G(-)AAAAATTC is not recognised by the enzyme.
  • TYRPl Upstream 917, insertion deletion mutation identified via Xmn I PCR-RFLP of TYRP-I Upstream 604 product for C7 was shown to be heterozygous for the cut product (426bp) and the uncut product (149bp).
  • C4 is a homozygote for the cut product (149bp)
  • Cl 7 is a heterozygote for the cut product (149bp) and the uncut product (426bp).
  • the variant A(T)TAAT is recognised and digested by the Ase I while the variant
  • A(C)TAAT is not recognised by the enzyme.
  • Ase I PCR-RFLP of TYRP-I Intron 1 1830 for C7 was shown to be heterozygous for the cut product (347bp) and the uncut product (127bp).
  • C4 is a heterozygote for the cut product (127bp) and the uncut product (347bp)
  • C17 is a homozygote for the cut product (127bp).
  • the variant GG(T)GATAGCCGAC is recognised and digested by the Hph I while the variant GG(C)GATAGCCGAC is not recognised by the enzyme.
  • Hph I PCR-RFLP of TYRP-I Exon 2 2404 after electrophoresis on a 4% agarose gel reflects.
  • C7 as heterozygous for the cut product (339bp) and the uncut product (453bp).
  • C4 is a heterozygote for the cut product (339bp) and the uncut product (453bp)
  • Cl 7 is a homozygote for the uncut product (453bp).
  • the variant GTA(C)G is recognised and digested by the Rsa I while the variant GTA(T)G is not recognised by the enzyme.
  • PCR-RFLP of TYRPl exon 6 using restriction enzyme Rsa I on a 4% agarose gel indicates C7 as heterozygous, containing both the cut allele (158bp) and the uncut allele (186bp).
  • C4 is homozygous for the uncut allele (186bp)
  • C17 is homozygous for the cut allele (158bp).
  • PCR PCR product without digestion
  • DIG PCR product after incubation with the enzyme restriction.
  • TYRPl Exon 8 63 the C/A variation was tested using restriction enzyme Ace II
  • the variant (C)GCG is recognised and digested by the Ace II while the variant (A)GCG is not recognised by the enzyme.
  • PCR-RFLP of TYRP-I exon 8 63 using the restriction enzyme Ace II on a 4% agarose gel indicates C7 to be heterozygous, containing both the cut allele (244bp) and the uncut allele (180bp).
  • C4 is homozygous for the cut allele (180bp)
  • Cl 7 is homozygous for the uncut allele (244bp).
  • Samples C7, C4 and Cl 7 were amplified using PCR conditions described above, with 10 ul of PCR products mixed with 0.5 - 2 units of restriction enzyme, 3 ul of Enzyme Buffer and 7 ul OfH 2 O and incubated for 3 hours at 37 0 C (BstUI at 65 0 C).
  • microsatellite marker for C7 was shown to be heterozygous at position 357, with both 4x 'GA' (l l lbp) and 5x 'GA' repeats (113bp).
  • C4 is a homozygote for 5x 'GA' (113bp) repeat
  • C17 is a homozygote for 4x 'GA' (11 lbp) repeat.
  • insertion deletion mutation marker for C7 was shown to be homozygous for a TCA insertion (179bp). C4 and Cl 7 are heterozygotes, with both the insertion (179bp) and deletion (176bp) fragment lengths. Further, for marker TYRPl Exon 2 2131, insertion deletion mutation marker for C7 was shown to be homozygous for a lbp deletion product size (453bp). C4 is a homozygote for the deletion product size (453bp) while Cl 7 is a heterozygote, with both the 'A' insertion (454bp) and deletion (453bp) fragment lengths.
  • a second indel fragment length (455bp) is also identified by this PCR.
  • C7 was shown to be heterozygous, containing both the cut allele (158bp) and the uncut allele (186bp).
  • C4 is homozygous for the uncut allele (186bp)
  • C17 is homozygous for the cut allele (158bp).
  • PCR PCR product without digestion, DIG - PCR product after incubation with the enzyme restriction.
  • microsatellite marker for C7 was shown to be heterozygous, with product size 300bp and 302bp.
  • C4 is a heterozygote, with product size 295bp and 300bp, while Cl 7 is a heterozygote, with product size 302bp and 302bp.
  • PCR (10 ul) and restriction enzyme digestion conditions as described above were then used to genotype all available Awassi sheep progeny, including the backross mapping population, complex crosses, and other sire families, for seven loci.
  • the families include the following mating designs. In addition to the 177 males of the mapping population, a further 336 animals were generated using the same mating design (backcrossed progeny from mating between a single Awassi/Merino Fl sire (C7) and unselected Merino ewes). Advanced intercrosses were made by crossing C7 to the progeny of three independent Awassi/merino Fl sires (sires A96.0463, A96.0473,
  • Table 14 show summarised genotyping results with the seven loci genotyped, although some loci are currently incompletely genotyped, and secondary (gap filling) genotyping needs to be completed.
  • the TGLA 1C locus was identified as being closest to the estimated QTL location.
  • Progeny were allocated a genotypic score of "1” if the allele passed on by the sire originated from the grandsire, and a genotypic score of "2" if the sire donated an allele originating from the granddam. Phase unknown inheritance was indicated as a "12".
  • the "1" allele was of Awassi origin
  • the "2" allele was of Merino origin.
  • Example 4 Statistical association analysis of TYRP-1 markers and pigmentation Following genotyping of all of the described Awassi families for the seven loci, analysis was conducted to test for an association between known TYRPl haplotypes and PigFibre or ColourScore (1-9). Initially, the same individuals used in the previous analysis (Example 3) were used, with ANOVA results confirming that TYRPl haplotypes ("Combined") explained a large proportion of the variance of the PigFibre trait ( Figure 9, Table 16). Table 16. ANOVA results for PigFibre Trait, with sex, management group and haplotype ("Combined”) as fixed effects

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Abstract

The present invention relates to a genetic marker for distinguishing animals that have a trait of pigmentation, wherein said marker is a polymorphism in the tyrosinase- related protein gene, and use thereof in prediction, selection, breeding and identification of pigmentation.

Description

Markers for Pigmentation
Technical Field
The present invention relates to genetic markers for pigmentation in skin and fleece areas of animals. In particular, the present invention relates to methods, systems and kits for predicting pigmentation and pigmented fibre contamination in animals using genetic markers, to associated methods, systems and kits for selecting animals using marker assisted selection, and to associated methods and systems for animal breeding and prediction of lifetime performance.
Background Art
The presence of pigmented wool fibres grown in white wool fleece for use in apparel presents a serious quality assurance problem in wool production and processing. Risk of such pigment contamination, especially non- visual contamination, can arise when apparently white wool animals grow isolated pigmented fibres, or when white wool and coloured wool sheep are mixed in grazing or in shearing sheds. The former risk is the most serious since isolated pigmented fibres are not easily detected, whereas in relation to the latter risk good management practices can ensure separation of coloured sheep from white sheep. In addition the prediction of coloured animals may not be evident from breeding apparently white animals, since some fleece colouration characteristics -either full, partial or in isolated areas, maybe inherited in a recessive manner.
Diagnostic tests that are currently available for determining dark pigmented fibre contamination in wool are based on evidence of pigmented spots on key locations of the body, or evidence of pigmented hair (as distinct from wool) on eye lashes and horn sites. Scoring animals for these traits is laborious and often cannot be done until later in the life of the animals. Hence the accurate assessment of animals which are either potentially affected by or free from problematic pigmentation is difficult to achieve. Furthermore, in the absence of extensive progeny testing, there is no easy or reliable means by which to predict the breeding quality of sires and dams, so as to predict the quality of possible progeny. Currently there exists no DNA test which is capable of predicting the genetic profile for pigmentation.
The present invention is predicated on the development of a genetic marker shown to be associated with pigmentation and pigmented fibre contamination in animals. Summary of the Invention
According to a first aspect of the present invention, there is provided a genetic marker for distinguishing animals that have a trait of pigmentation, wherein said marker is a polymorphism in the tyrosinase-related protein gene. The tyrosinase-related protein gene includes associated intronic and regulatory sequences.
According to a second aspect of the present invention, there is provided a method for predicting pigmentation in an animal, said method comprising analysing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase- related protein gene, wherein said genetic marker is predictive of pigmentation in said animal.
The pigmentation may include pigmented skin or dark fibre contamination.
The fibre may be selected from the group comprising wool or hair. The wool may be wool or cashmere. The hair fibre may be mohair. The animal may be an artiodactyl species. The animal may be sheep, alpaca, lama or goat.
The tyrosinase-related protein (TYRP) may be TYRP-I .
The presence or absence of the polymorphism may be analysed through the use of nucleic acid sequences flanking the polymorphism, such as PCR primers. For example, reference is made to the nucleic acid sequences set forth in SEQ ID NOS: 1-33 and 48-71.
According to a third aspect of the present invention, there is provided a method for selecting an animal using marker assisted selection, wherein said method comprises :
(a) analysing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene, wherein the genetic marker is predictive of pigmentation in an animal; and
(b) selecting an animal based on the presence or absence of said genetic marker. According to a fourth aspect of the present invention, there is provided a method for breeding an animal using marker assisted selection, wherein said method comprises:
(a) analyzing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene, wherein said genetic marker is predictive of pigmentation in said animal;
(b) breeding from said animal based on the presence or absence of said genetic marker; and (c) selecting progeny of said animal based on the presence or absence of said genetic marker.
According to a fifth aspect of the present invention, there is provided a system for predicting pigmentation in an animal, wherein said system comprises: (a) means for analyzing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene, wherein said genetic marker is predictive of pigmentation in said animal.
According to a sixth aspect of the present invention, there is provided a system for selecting an animal using marker assisted selection, wherein said system comprises: (a) means for analyzing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene, wherein the genetic marker is predictive of pigmentation in said animal; and
(b) means for selecting said animal based on the presence or absence of the genetic marker. According to a seventh aspect of the present invention, there is provided a system for breeding an animal using marker assisted selection, wherein said system comprises: (a) means for analyzing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene, wherein the genetic marker is predictive of pigmentation in said animal; and (b) means for breeding said animal based on the presence or absence of the genetic marker, and
(c) means for selecting progeny of said animal based on the presence or absence of the genetic marker.
According to an eighth aspect of the present invention, there is provided a kit for predicting pigmentation in an animal, wherein said kit comprises:
(a) means for analyzing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene, wherein the genetic marker is predictive of pigmentation in said animal.
According to a ninth aspect of the present invention, there is provided a kit for selecting an animal using marker assisted selection, wherein said kit comprises:
(a) means for analyzing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene, wherein the genetic marker is predictive of pigmentation in said animal; and (b) means for selecting said animal based on the presence or absence of the genetic marker.
According to a tenth aspect of the present invention, there is provided a kit for breeding an animal using marker assisted selection, wherein said kit comprises: (a) means for analyzing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene, wherein said genetic marker is predictive of pigmentation in said animal; and
(b) means for breeding said animal based on the presence or absence of the genetic marker, and (c) means for selecting progeny of said animal based on the presence or absence of the genetic marker.
According to an eleventh aspect of the present invention, there is provided a polypeptide comprising a tyrosinase-related protein (TYRP).
The TYRP may be TYRP-I. The TYRP-I may comprise the amino acid sequence set forth in SEQ ID NOS: 36, 39, 42, 45, 47, 74, 77 and 80.
According to a twelfth aspect of the present invention, there is provided a polynucleotide encoding the polypeptide of the eleventh aspect.
According to a thirteenth aspect of the present invention, there is provided a vector comprising the polynucleotide of the twelfth aspect. According to a fourteenth aspect of the present invention, there is provided a host cell transformed with the vector of the thirteenth aspect.
According to a fifteenth aspect of the present invention, there is provided an animal selected by the method of the third aspect, the system of the sixth aspect or using the kit of the ninth aspect. According to a sixteenth aspect, there is provided an animal bred by the method of the fourth aspect, the system of the seventh aspect or using the kit of the tenth aspect.
According to a seventeenth aspect of the present invention, there is provided an animal product derived from the animal of the fifteenth aspect.
The animal product may be selected from the group comprising hair, wool, skin, cashmere, and mohair. The animal product may be wool.
According to an eighteenth aspect of the present invention, there is provided a method of sampling fibres to determine non-visual pigmentation, wherein said method comprises (a) genotyping a plurality of animals based on the presence or absence of at least one genetic marker in the tyrosinase-related protein gene, wherein the genetic marker is predictive of pigmentation in said animal;
(b) grouping said animals according to genotype; (c) sampling fibres from animals grouped as having said genetic marker(s); and
(d) determining a likelihood of contamination of said fibre samples with pigmented fibres.
Tests for sampling dark or pigmented fibres are well known in the art. For example, Hansford, Literature review for managing the risk of dark/and or medullated fibre contamination. Australian Wool Innovation project EC573, describes a range of methods. According to a nineteenth aspect of the present invention, there is provided a method for identifying pigmentation in an animal, wherein said method comprises:
(a) analyzing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene wherein the presence of the genetic marker is indicative of pigmentation in said animal.
In an embodiment of the nineteenth aspect of the present invention, the pigmentation is present in the form of pigmented fibres. Further, the pigmented fibres may be in the form of pigmented wool, hair, mohair or cashmere.
In a further embodiment of the present invention, the pigmented fibres are rare and/or isolated. For example, the proportion of pigmented fibres may comprise less than
10% of total fibre count, less than 5% of total fibre count, less than 1% of total fibre count, less than 0.1% of total fibre count, less than 0.01% of total fibre count, less than
0.001% of total fibre count, less than 0.0001% of total fibre count, less than 0.00001% of total fibre count, less than 0.000001% of total fibre count, less than 0.0000001% of total fibre count, less than 0.00000001% of total fibre count, or less than 0.0000000001% of total fibre count.
In a further embodiment of all aspects of the present invention, the genetic marker for distinguishing animals that have a trait of pigmentation includes polymorphisms in the tyrosinase-related protein gene together with one or more genetic markers in linkage disequilibrium thereto. For example, genetic markers in linkage disequilibrium to the tyrosinase-related protein gene capable of distinguishing animals that have a trait of pigmentation may be TGLAlO, bracketed by the markers CSAP 16E or MCM252. Brief Description of the Drawings
The present invention will now be described, by way of example only, with reference to the following drawings.
Figure 1. QTL location for Binary Colour Score on Chromosome 2. Figure 2. A. QTL location for Pigmented Skin on Chromosome 2; B. QTL location for Pigmented Skin on Chromosome 19; C. QTL location for Arc Sine transformed NoseLipsPigmented skin on Chromosome 2; D. QTL location for Arc Sine transformed NoseLipsPigmented skin on Chromosome 3; E. QTL location for Arc Sine transformed NoseLipsPigmented skin on Chromosome 19; F. QTL location for Arc Sine transformed NoseLipsPigmented skin on Chromosome 25; G. QTL location for Pigmented Fibre on Chromosome 2; H. QTL location for Pigmented Fibre on Chromosome 26; I. QTL location for Hoof Pigmentation on Chromosome 2; J. QTL location for Hoof Pigmentation on Chromosome 18; K. QTL location for Binary score hornsite and leg Fibre score on Chromosome 2; L. QTL location for Binary score hornsite and leg fibre score on Chromosome 8; M. QTL location for Binary score hornsite and leg fibre score on Chromosome 19.
Figure 3. Schematic of TYRPl gene layout
Figure 4. Black and white (top), and negative picture (bottom) of PCR products for TYRP-I exons 2, 3, 4 and 5 electrophoresed on a 2% agarose gel. Symbols, M = marker, - = negative control, C = cattle, H = human, M = mouse, S = sheep. Number in brackets indicates the ~ size of the PCR product in relation to the molecular weight of the marker.
Figure 5. Black and white (top), and negative photograph (bottom) of PCR products for TYRP-I exons 6, 7, 8 and 8-1 (using R2 primers described below) electrophoresed on a 2% agarose gel. Figure 6. Black and white photograph of PCR products of TYRP-I exons 1 and upstream region electrophoresed on a 2% agarose gel. Primers are indicated by number according to Tables 7-9
Figure 7. Association between genotype and phenotype at Marker TGLAlO. Genotype '1' is significantly more associated with a higher Fibre Pigmentation Score (PigFibre) than genotype '2'.
Figure 8. Parental haplotypes inherited from the Merino granddam (C7_M), and the Awassi grandsire (C7 AW), and other (partial) haplotypes. In relation thereto, animals with haplotype "34" also had a confirmed high isolated pigmented fibre count using the AWTA test. Figure 9. Histogram figure of Pigmented Fibre score out of 500 for pigmented fibre, against haplotypes predictive of HIGH and LOW risk of pigmentation respectively from the 1997 mapping family
Definitions In the context of this specification, the term "comprising" means "including principally, but not necessarily solely". Furthermore, variations of the word "comprising", such as "comprise" and "comprises", have correspondingly varied meanings.
The term "primer" as used herein means a single-stranded oligonucleotide capable of acting as a point of initiation of template-directed DNA synthesis. An
"oligonucleotide" is a single-stranded nucleic acid typically ranging in length from 2 to about 500 bases. The precise length of a primer will vary according to the particular application, but typically ranges from 15 to 30 nucleotides. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize to the template.
The term "genotype" as used herein means the genetic constitution of an organism. This may be considered in total, or with respect to the alleles of a gene(s) or genetic marker(s).
The term "homozygote" refers to an organism that has identical alleles at a given locus on homologous chromosomes.
The term "heterozygote" refers to an organism in which different alleles are found on homologous alleles for a given locus.
The term "linkage disequilibrium" describes a situation in which some combinations of alleles of two or more different loci (haplotypes) occur more or less frequently within a population than would be expected by random chance alone.
The term "pigmentation" as used herein includes skin and fibre pigmentation, and in particular includes skin and fibre pigment contamination. In this context, skin may include nose, lips, hoofs, eyelids, udder, and fibre may include hair, wool, fleece, and eyelashes. The term "pigmentation" may include isolated or partial fleece colouration, also known as "white spotting", piebald, or any other form of discolouration or full- fleece colouration. In this context, the term "pigmentation" may include fibres containing a degree of colouration that is not or substantially not visible to the eye. Also, the term "dark fibre" and "pigmentation" are synonymous. The terms "pigment" and "pigmented" have corresponding meanings. As used herein, the term "genetic marker" refers to a variant or polymorphism at DNA sequence level linked to a specific chromosomal location unique to an individual's genotype, inherited in a predictable manner, and measured as a direct DNA sequence variant or polymorphism, such as at least one Single Nucleotide Polymorphism (SNP), Restriction Fragment Length Polymorphism (RFLP), or Short Tandem Repeat (STR), or as measured indirectly as a DNA sequence variant (eg. Single-strand conformation polymorphism (SSCP), Denaturing Detergent Gradient Gel Electrophoresis (DDGE). A marker can also be a variant at the level of a DNA derived product such as RNA polymorphism/abundance, protein polymorphism or cell metabolite polymorphism, or any other biological characteristics which have a direct relationship with the underlying DNA variants or gene product.
As used herein, the term "single nucleotide polymorphisms" or "SNP" or "SNPs", as used herein, refers to common DNA sequence variations among subjects. The DNA sequence variation is typically a single base change or point mutation resulting in genetic variation between individuals. The single base change can be an insertion or deletion of a base.
The term "base pair" as used herein means a pair of nitrogenous bases, each in a separate nucleotide, in which each base is present on a separate strand of DNA and the bonding of these bases joins the component DNA strands. Typically a DNA molecule contains four bases; A (adenine), G (guanine), C (cytosine), and T (thymidine). A and G are purine bases, typically designated by the letter "R", whereas C and T are pyrimidine bases, typically designated by the letter "Y". Where A or T may occupy a single position it is typically designated by the letter W. Where G or C may occupy a single position it is typically designated by the letter S. Where A or C may occupy a single position it is typically designated by the letter M. Where G or T may occupy a single position it is typically designated by the letter K. Where A, T or C may occupy a single position it is typically designated by the letter H. Where G, C or T may occupy a single position it is typically designated by the letter B. Where G, A or T may occupy a single position it is typically designated by the letter D. Where G, C or A may occupy a single position it is typically designated by the letter V. Where G, C, A or T may occupy a single position it is typically designated by the letter N. The term "base pair" is abbreviated to "bp", and the term "kilobase pair" is abbreviated to kb.
It will be understood that the term "animal" as used herein refers to an individual at any stage of life, or after death, including an entity prior to birth such as a fertilised ovum, either before fusion of the male and female pro-nucleus or after the pro-nuclei have fused to form a zygote, an embryo (created by any means including somatic cell nuclear transfer) or an individual cell (N, 2N or greater); for the avoidance of doubt, this also includes a cell or a cluster of cells including stem cells and stem cell-like cells, cell line, haploid gametes and their progenitor cell lines, as well as products resulting from the gametes, including embryos.
Best Mode of Performing the Invention
A comprehensive gene mapping experiment identified chromosomes which contained genes contributing to skin and fibre pigmentation in sheep, detailed in Table 6. One of the major regions with strongest significance was found on chromosome 2, and was significant for 6 skin and fibre traits.
Association between the closest linked marker TGLAlO, and 177 mapping progeny was sufficient to account for over 50% of the differences in pigmentation, and indicated that the colour phenotype originated from the Awassi allele (Figure 7). This region was shown to contain a very strong candidate gene, namely tyro sine-related protein- 1 (TYRP- 1 - Figure 3). TYRP-I was sequenced by alignment of TYRP-I sequence from all known species, with conserved sequence sites used to design DNA primers for ovine sequence. A predicted ovine gene structure was then identified from comparative mapping, with ovine sequence confirming close alignment to bovine TYRP-I .
Novel DNA sequence was generated for TYRP in sheep and DNA variants were discovered based on single nucleotide polymorphisms (SNPs) or short tandem repeat sequences, insertion deletion mutations or similar. These DNA variants were discovered in the mapping sire known to be heterozygous for a major gene linked to skin and fibre pigmentation (Sire C7). A screen of additional advanced cross progeny with 7 selected phase known markers (Figure 8) confirmed an association between the parental C7_AW parental/individual TYRPl haplotype and pigmentation (Figure 9), thereby suggesting that markers linked to variants located in and around the TYRPl gene represent a powerful means for predicting the genetic pigmentation status of animals, both for their own performance and as breeding candidates. To test this hypothesis, surviving animals from the initial backcross strategy, and advanced repeat cross animals were shorn, and their fleece allocated to bins according to two broad parental haplotypes (C7_M, C7 AW) to be tested for fleece colour. Other QTL regions of significance for pigmentation were found to be located on chromosome 19 (LOD>5), possibly harbouring a second major gene for pigmentation which may account for the remainder of the variation seen in pigmentation. QTLs on remaining chromosomes appeared to be of lesser significance.
Detection of genetic markers
Genetic assay-assisted selections for animal breeding are important in that they allow selections to be made without the need for raising and phenotypic testing of progeny. In particular, such tests allow selections to occur among related individuals that do not necessarily exhibit the trait in question and that can be used in introgression strategies to select both for the trait to be introgressed and against undesirable background traits.
It is readily apparent that, having identified a polymorphism for a particular associated trait, there are an essentially infinite number of ways to genotype animals for this polymorphism. The design of such alternative tests merely represents a variation of the techniques provided herein and is thus within the scope of this invention as fully described herein.
Non- limiting examples of methods for identifying markers corresponding to genetic polymorphisms between members of a population include: restriction-fragment-length polymorphism (RFLP) Bostein et al (1980) Am J Hum Genet 32:314-331; single-strand conformation polymorphism (SSCP) Fischer et al. (1983) Proc Natl Acad Sci USA 80:1579-1583, Orita et al. (1989) Genomics 5:874-879; amplified fragment-length polymorphism (AFLP) Vos et al. (1995) Nucleic Acids Res 23:4407-4414; microsatellite or single-sequence repeat (SSR) Weber J L and May P E (1989) Am J Hum Genet 44:388-396; rapid-amplified polymorphic DNA (RAPD) Williams et al (1990) Nucleic Acids Res 18:6531-6535; sequence tagged site (STS) Olson et al. (1989) Science 245:1434-1435; genetic-bit analysis (GBA) Nikiforov et al (1994) Nucleic Acids Res 22:4167-4175; allele-specific polymerase chain reaction (ASPCR) Gibbs et al. (1989) Nucleic Acids Res 17:2437-2448, Newton et al. (1989) Nucleic Acids Res 17:2503- 2516; nick-translation PCR (e.g., TAQMAN.TM.) Lee et al. (1993) Nucleic Acids Res 21 :3761-3766; and allele-specific hybridization (ASH) Wallace et al. (1979) Nucleic Acids Res 6:3543-3557, (Sheldon et al. (1993) Clinical Chemistry 39(4):718-719) and gene chip analysis such as the Affymetrix or ParAllele systems, among others. Each technology has its own particular basis for detecting polymorphisms in DNA sequence but all are applicable to the detection of SNP's.
In particular, techniques employing PCR. detection are advantageous in that detection is more rapid, less labour intensive and requires smaller sample sizes. Primers that may be used in this regard may, for example, comprise sequences set out in Tables 7, 8 and 9 and complements thereof.
Once an assay format has been selected, selections may be unambiguously made based on genotypes assayed at any time after a nucleic acid sample can be collected from an individual, such as an infant animal, or even earlier in the case of testing of embryos in vitro, or testing of foetal offspring. Any source of nuclear DNA may be analyzed for scoring of genotype.
In one embodiment of the invention, nucleic acids are screened that have been isolated from the blood or semen of the animal analyzed. DNA from the animal to be assessed may be extracted by a number of suitable methods known to those skilled in the art. Most typically, DNA is extracted from a blood or semen sample, and in particular from peripheral blood leucocytes. A sufficient amount of cells are obtained to provide a sufficient amount of DNA for analysis, although only a minimal sample size will be needed where scoring is by amplification of nucleic acids. The DNA can be isolated from the blood cells by standard nucleic acid isolation techniques known to those skilled in the art.
Amplification of nucleic acids
Nucleic acids used as a template for amplification may be isolated from cells, tissues or other samples according to standard methodologies. Such embodiments may find particular use with the invention, for example, in the detection of repeat length polymorphisms, such as microsatellite markers. In certain embodiments of the invention, amplification analysis is performed on whole cell or tissue homogenates or biological fluid samples without substantial purification of the template nucleic acid.
Pairs of primers designed to selectively hybridize to nucleic acids are contacted with the template nucleic acid under conditions that permit selective hybridization. Depending upon the desired application, high stringency hybridization conditions may be selected that will only allow hybridization to sequences that are completely complementary to the primers. In other embodiments, hybridization may occur under reduced stringency to allow for amplification of nucleic acids containing one or more mismatches with the primer sequences. Once hybridized, the template-primer complex is contacted with one or more enzymes that facilitate template-dependent nucleic acid synthesis. Multiple rounds of amplification, also referred to as "cycles", are conducted until a sufficient amount of amplification product is produced.
The amplification product may be detected or quantified. In certain applications, the detection may be performed by visual means. Alternatively, the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of incorporated radiolabel or fluorescent label or even via a system using electrical and/or thermal impulse signals. Typically, scoring of repeat length
10 polymorphisms will be done based on the size of the resulting amplification product.
A number of template dependent processes are available to amplify the oligonucleotide sequences present in a given template sample. One of the best known amplification methods is the polymerase chain reaction (PCR) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159, each of which is incorporated
I5 herein by reference in their entirety.
Genetic markers
The genetic marker(s) in the tyrosinase related protein gene useful in the invention may be selected from, but is not limited to, the group of polymorphisms shown in Table 1 -0 below.
Table 1: Selection of markers within and upstream of the TYRPl gene
Figure imgf000013_0001
Figure imgf000014_0001
The means for analyzing the nucleic acid sample may be selected from the group of oligonucleotides set forth in SEQ ID NOS 1-33 and 48 - 71.
Table 2: Sequence Identification Number
Figure imgf000014_0002
Detection of polymorphsim
After amplification, it may be desirable to separate the amplification product from the template and/or the excess primer and other unused reagents. In one embodiment, amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods. Amplification products isolated in this way may be eluted from an excised portion of the gel for further manipulation. The nucleic acid may be removed from the excised portion of an agarose gel by heating the gel in a chao tropic solution, followed by extraction of the nucleic acid.
Separation and isolation of nucleic acids may also be performed by chromatographic techniques known in art. Numerous kinds of chromatography may be used in the present invention, including adsorption, partition, ion-exchange, hydroxylapatite, gel-filtration (molecular sieve), and reverse-phase. These may be performed in a number of formats including, column, paper, thin-layer, and gas chromatography as well as by batch HPLC or FPLC. In certain embodiments, the amplification products are visualized. A typical visualization method involves staining of a gel with ethidium bromide and visualization of bands under UV light. Alternatively, if the amplification products are integrally labeled with radio- or fiuorometrically-labeled nucleotides, the separated amplification products can be exposed to x-ray film or visualized under light of the appropriate excitatory wavelength.
In one embodiment, following separation of amplification products, a labeled nucleic acid probe is brought into contact with the amplified marker sequence. The probe preferably is conjugated to a chromophore but may be radiolabeled or otherwise labeled to facilitate detection. In another embodiment, the probe is conjugated to a molecule, such as an antibody or biotin, or another molecule carrying a moiety to facilitate detection.
In particular embodiments, detection is by nucleic acid blotting and hybridization with a labeled probe. The techniques involved in nucleic acid blotting are well known to those skilled in the art. For example, the nucleic acid blotting and hybridization may be performed under stringent conditions.
Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 3O0C for short probes (e.g., 10 to 50 nucleotides) and at least about 6O0C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37°C, and a wash in 1. times, to 2 x SSC (20xSSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 550C Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 370C, and a wash in 0.5x to IxSSC at 55 to 5O0C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 370C, and a wash in 0. IxSSC at 60 to 650C.
Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl, Anal. Biochem., 138:267-284 (1984): Tm =81.50C +16.6 (log M)+0.41 (% GQ-0.61 (% form)- 500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about I0C for each 1% of mismatching; thus, Tm, hybridization and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with 90% identity are sought, the Tm can be decreased 1O0C. Generally, stringent conditions are selected to be about 50C lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 40C lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 1O0C lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 2O0C lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 450C (aqueous solution) or 320C (formamide solution) it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology- Hybridization with Nucleic Acids Probes, Part I, Chapter 2, Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, New York (1995).
Kits All the essential materials and/or reagents required for screening animals for genetic marker genotype in accordance with the invention may be assembled together in a kit. This generally will comprise a probe or primers designed to hybridize specifically to individual nucleic acids of interest in the practice of the present invention, for example, primer sequences such as those for amplifying TYRP-I. Also included may be enzymes suitable for amplifying nucleic acids, including various polymerases, deoxynucleotides and buffers to provide the necessary reaction mixture for amplification. Such kits also may include enzymes and other reagents suitable for detection of specific nucleic acids or amplification products. Such kits generally will comprise, in suitable means, distinct containers for each individual reagent or enzyme as well as for each probe or primer pair. In certain embodiments, the invention also can provide for a kit which can be used to determine the TYRP-I genotype of genetic material, for example the kit may include a set of primers used for amplifying the genetic material. A kit can contain a primer including a nucleotide sequence for amplifying a region of the genetic material containing one of the polymorphisms described herein. The present invention will now be further described in greater detail by reference to the following specific examples, which should not be construed as in any way limiting the scope of the invention.
Examples Example 1 : QTL mapping of pigmentation traits
Six pigmentation traits, being (1) tail wool colour (ColourScore), (2) face skin colouration (PigmSkin), (3) nose and lip skin pigmentation (NoseLPig), (4) face, legs and hooves fibre colouration (PigFibre), (5) hoof pigmentation alone (HoofPigm), and (6) horn and leg fibre (LHSFibre), as set out in Table 3, were assessed in a quantitative trait loci (QTL) gene mapping experiment with n = 177 individuals. The 177 offspring were male backcrossed progeny from mating between a single Awassi/Merino Fl sire (C7/A95.0453) and unselected Merino ewes, giving a progeny of 75% Merino and 25% Awassi. Animals were born from May 1997, and separated into two management cohorts according to birth dates. Table 3; Pi mentation traits and trait descri tion
Figure imgf000018_0001
For all traits detailed in Table 3 except ColourScore, the fixed effect of management cohort was significant, and traits were adjusted accordingly. The fixed effect of sex was not significant. Traits were transformed as follows: ColourScore transformed to a binary trait; NoseLPig was arcsin transformed, and LHSFibre was transformed to a binary trait. A full genome scan was conducted with 204 microsatellite markers. A framework map was constructed using the open source mapping programs Carthagene and Mapmaker, which was used to inform marker positions. A full genome scan was conducted using a maximum likelihood analysis written in R, developed by the inventors (QTL-MLE).
The QTL-MLE algorithm was developed to match the particular experimental design and genotyping characteristics of the study. These included (1) incorporation of known linkage phase information resulting from the backcross design, (2) utilisation of all the relevant genotypic information, and (3) valid modelling the marker-QTL associations by means of a finite mixture model. Item (2) involved development of an algorithm to utilise information from heterozygous marker genotypes, using estimates of marker allele frequencies in the merino population to. assist this. The QTL model is fitted using a maximum likelihood routine at each putative QTL position along the length of the target chromosome (interval mapping). In addition to the estimated QTL location and effects, the probability of each backcross offspring inheriting either allele from the Fl sire (Q or q) is provided, in terms of a posterior probability. This allows animals with a high probability of receiving the "favourable" QTL genotype to be identified. In addition, a 1- LOD support interval is provided to give an indication of the precision of estimating the location of the QTL. The procedure (QTL-MLE) has been coded using the open source statistical program R (also compatible with the commercial version, S=PLUS).
The above analysis identified QTL regions with log of odds (LOD) scores >2 (P<0.05) on chromosomes 2, 3, 8, 18, 19, 25 and 26. The most significant effect was seen on chromosome 2, with a QTL LOD >20 for all 6 traits, as shown below in Tables 4 and 5 and Figures 1 and 2.
Table 4: Colour Score Trait
Figure imgf000019_0001
Table 5: Pi ment Traits
Figure imgf000019_0002
*: from interaction model; ': ArcSine transformation; 2: Binary trait
Shown below in Table 6 are the summarized QTL positions on each chromosome for the different traits based on the data shown above in Tables 4 and 5. As can be seen from Table 6, one of the major regions with strongest significance was on chromosome 2.
Figure imgf000020_0001
*: from interaction model; : ASin transformation; : Binary trait
Based on the results of this QTL mapping experiment, TGLAlO was found to be the closest linked mapped genetic marker and tyrosinase-related protein- 1 (TYRP-I) was identified as the most significant positional candidate gene by comparative mapping to human, cattle and rat using the Comparative Predictions of Orthologues software at the Oxford Grid Project (http://oxgrid.angis.org.au).
In particular, a GenBank database search was performed for the Ovis aries TYRP-I DNA sequences generated in this study using the Basic Local Alignment Search Tool (BLAST) on human, sheep and mouse genomes at the NCBI (http://www.ncbi.nlm.nih.gov/BLAST). This search identified that orthologous sequences of the Ovis aries TYRP-I DNA map to chromosomes 8, 9, and 5 in cattle, human and rat respectively. For comparative predictions of orthologues between sheep versus cattle, human and rat, the OXGRID program (http://oxgrid.angis.org.au) was used. OXGRID results indicate that the orthologous position of cattle, human and rat TYRP-I gene corresponds to the chromosome number 2 in sheep {Ovis aries), which is consistent with the QTL mapping described above. Example 2: Characterisation of sheep TYRP-1 2.1 Primer Design for sheep TYRP-I
At the time of designing primers for exonic regions, the human TYRP-I gene was the only available full length DNA sequence for the gene that included all exonic and intronic regions. The human sequence, together with the available exonic sequences from cattle and goat, was used as a sequence model to design primers and estimate the expected size of TYRP-I exons and introns in sheep. Primers for certain of the upstream regions were designed based on the Bos taurus chromosome 8 genomic contig, whole genome shotgun sequence NW 932015.1.
Primers as shown below in Tables 7-9 were designed based on the entire Genbank human sequence as well as partial and mRNA bovine, ovine, horse and dog sequences (Figure 4). Sequence line ups showing primer design and individual species sequence are shown in Figures 5 - 13. One primer pair was positioned at the beginning and end of each exon. In certain cases, other primers were chosen to flank the exon from intronic regions, all of which is detailed in Figure 3.
Table 7. Primer pairs used to amplify exonic regions 2-8. Forward and reverse primer sequences start at the first and last position respectively of each exon.
Figure imgf000021_0001
Exon 8 F GGTCGGAGTTTTAGTATTCCTGA (SEQ ID NO: 13) 5' lbp
Rl ♦TGATTCAAATGCATATGAGAATTTAC (SEQ ID NO : 14) 5" 240bp
R2 ATTCACTTATGACCACCTTATACAGTTCT (SID NO: 15) 5" 125 lbp
M13(-29) sequence added for the purposes of fluorescent dye incorporation
Table 8. Additional primers used to amplify and sequence upstream region
Figure imgf000022_0001
: M13(-29) sequence added for the purposes of fluorescent dye incorporation
Table 9. Additional primers used to amplify genotyping markers
Figure imgf000022_0002
Figure imgf000023_0001
* M13(-29) sequence added for the purposes of fluorescent dye incorporation
2.2 PCR amplification of sheep TYRP-I
PCR cycle and reaction conditions are described in Tables 10, 11 and 12, 13 respectively.
Figure imgf000023_0002
Table 11. PCR cycle conditions for marker amplification
Figure imgf000024_0001
Table 12. PCR reaction com onents - 25ul volume
Figure imgf000024_0002
Table 13. PCR reaction com onents - lOul volume
Figure imgf000024_0003
DNA samples from grandparents (C4 and C 17) and the sire (C7) from the Awassi sheep family were used to test the TYRP-I exonic and intronic primers. In addition, negative controls and other DNA species such as cattle, human and mouse were used. Results of the PCR are shown in Figures 4, 5 and 6.
2.3 PCR of sire C7 for direct sequencing
TYRP-I exons from sample C7 were amplified by PCR using the conditions described in Tables 10 and 12 using TIi DNA polymerase (Promega) or 11 and 13 using Invitrogen Taq DNA polymerase. PCR products were gel-purified using UltraClean DNA purification kits (Mo Bio Laboratories, Inc.), or were enzymatically cleaned using ExoSAP-IT (USB). PCR products were directly sequenced using the SUPAMAC (Sydney University Prince Alfred Macromolecular Analysis Centre) commercial sequencing service on an ABI PRISM® 3700 platform (Applied Biosystems, Foster City), or were sequenced using an ABI BigDyeV3 kit, and run in-house on an ABI PRISM® 3100 platform. The DNA and amino acid (based on nuclear code) TRYP-I sequences obtained from the upstream region, and exons 2 to 8 (as per Table 2) .
For all variant sites the chromatograms showed ambiguous sites (N), with two collocated peaks of significant height, thereby indicating potential SNP sites. Insertion deletion mutations were recognised from a sudden and repeatable loss of sequencing readability. These ambiguities were corroborated with the alternate sequencing experiments of the other two individuals, and by sequencing from the other end of the PCR fragment.
2.4 PCR-RFLP approach
Six of the potential SNPs were tested by restriction fragment length polymorphism (RFLP) analysis to determine whether they were consistent with restriction enzyme sites.
For marker TYRPl Upstream 604, the C/T variation was tested using restriction enzyme BsrGI (NEB) which recognises:
T'GTACA ACATGAT The variant TGTA(C)A is recognised and digested by the BsrGI while the variant
TGTA(T)A is not recognised by the enzyme. For example, BsrGI PCR-RFLP of TYRP-I Upstream 604 using the restriction enzyme BsrGI for C7 was shown to be heterozygous containing the cut product (123bp) and the uncut product (326bp). C4 is a homozygote for the uncut product (326bp), while Cl 7 is a heterozygote for the cut product (123bp) and the uncut product (326bp).
For marker TYRPl Upstream 917, the AJ- variation was tested using restriction enzyme Xmn I (NEB) which recognises:
GAANN'NNTTC CTTNN.NNAAG
The variant G(A)AAAAATTC is recognised and digested by the Xmn I while the variant G(-)AAAAATTC is not recognised by the enzyme. For example, TYRPl Upstream 917, insertion deletion mutation identified via Xmn I PCR-RFLP of TYRP-I Upstream 604 product for C7 was shown to be heterozygous for the cut product (426bp) and the uncut product (149bp). C4 is a homozygote for the cut product (149bp), while Cl 7 is a heterozygote for the cut product (149bp) and the uncut product (426bp).
For marker TYRPl Intron 1 1830, the C/T variation was tested using restriction enzyme Ase I (NEB) which recognises:
AT'TAAT TAAT.TA
The variant A(T)TAAT is recognised and digested by the Ase I while the variant
A(C)TAAT is not recognised by the enzyme. For example,, Ase I PCR-RFLP of TYRP-I Intron 1 1830 for C7 was shown to be heterozygous for the cut product (347bp) and the uncut product (127bp). C4 is a heterozygote for the cut product (127bp) and the uncut product (347bp), while C17 is a homozygote for the cut product (127bp).
For marker TYRPl Exon 2 2404, the C/T variation was tested using restriction enzyme Hph I (NEB) which recognises:
GGTGANNNNNNNN' CCACTNNNNNNN^
The variant GG(T)GATAGCCGAC is recognised and digested by the Hph I while the variant GG(C)GATAGCCGAC is not recognised by the enzyme. For example, , Hph I PCR-RFLP of TYRP-I Exon 2 2404 after electrophoresis on a 4% agarose gel reflects. C7 as heterozygous for the cut product (339bp) and the uncut product (453bp). C4 is a heterozygote for the cut product (339bp) and the uncut product (453bp), while Cl 7 is a homozygote for the uncut product (453bp).
For Exon 6, the C/T variation in position 30 of this exon was tested using restriction enzyme Rsa I (Promega) which recognises GT'AC CAATG
The variant GTA(C)G is recognised and digested by the Rsa I while the variant GTA(T)G is not recognised by the enzyme. For example, PCR-RFLP of TYRPl exon 6 using restriction enzyme Rsa I on a 4% agarose gel indicates C7 as heterozygous, containing both the cut allele (158bp) and the uncut allele (186bp). C4 is homozygous for the uncut allele (186bp), while C17 is homozygous for the cut allele (158bp). PCR = PCR product without digestion, DIG = PCR product after incubation with the enzyme restriction. For TYRPl Exon 8 63, the C/A variation was tested using restriction enzyme Ace II
(Amersham Biosciences) or BstU I (NEB) which recognises:
Figure imgf000027_0001
The variant (C)GCG is recognised and digested by the Ace II while the variant (A)GCG is not recognised by the enzyme. For example, PCR-RFLP of TYRP-I exon 8 63 using the restriction enzyme Ace II on a 4% agarose gel indicates C7 to be heterozygous, containing both the cut allele (244bp) and the uncut allele (180bp). C4 is homozygous for the cut allele (180bp), while Cl 7 is homozygous for the uncut allele (244bp). Samples C7, C4 and Cl 7 were amplified using PCR conditions described above, with 10 ul of PCR products mixed with 0.5 - 2 units of restriction enzyme, 3 ul of Enzyme Buffer and 7 ul OfH2O and incubated for 3 hours at 370C (BstUI at 650C).
Similarly, for marker TYRPl Upstream 357, microsatellite marker for C7 was shown to be heterozygous at position 357, with both 4x 'GA' (l l lbp) and 5x 'GA' repeats (113bp). C4 is a homozygote for 5x 'GA' (113bp) repeat, while C17 is a homozygote for 4x 'GA' (11 lbp) repeat.
Also, for marker TYRPl Upstream 1621, insertion deletion mutation marker for C7 was shown to be homozygous for a TCA insertion (179bp). C4 and Cl 7 are heterozygotes, with both the insertion (179bp) and deletion (176bp) fragment lengths. Further, for marker TYRPl Exon 2 2131, insertion deletion mutation marker for C7 was shown to be homozygous for a lbp deletion product size (453bp). C4 is a homozygote for the deletion product size (453bp) while Cl 7 is a heterozygote, with both the 'A' insertion (454bp) and deletion (453bp) fragment lengths. Note: A second indel fragment length (455bp) is also identified by this PCR. Also, with respect to TYRPl exon 6, using restriction enzyme Rsa I on a 4% agarose gel C7 was shown to be heterozygous, containing both the cut allele (158bp) and the uncut allele (186bp). C4 is homozygous for the uncut allele (186bp), while C17 is homozygous for the cut allele (158bp). PCR = PCR product without digestion, DIG - PCR product after incubation with the enzyme restriction.
Still further for marker TYRPl Intron 5, microsatellite marker for C7 was shown to be heterozygous, with product size 300bp and 302bp. C4 is a heterozygote, with product size 295bp and 300bp, while Cl 7 is a heterozygote, with product size 302bp and 302bp.
2.5 Genotype analysis of Awassi sheep families
PCR (10 ul) and restriction enzyme digestion conditions as described above were then used to genotype all available Awassi sheep progeny, including the backross mapping population, complex crosses, and other sire families, for seven loci.
The families include the following mating designs. In addition to the 177 males of the mapping population, a further 336 animals were generated using the same mating design (backcrossed progeny from mating between a single Awassi/Merino Fl sire (C7) and unselected Merino ewes). Advanced intercrosses were made by crossing C7 to the progeny of three independent Awassi/merino Fl sires (sires A96.0463, A96.0473,
A96.0474) mated to merino ewes (n=64). Finally C7/merino backcross progeny were double backcrossed to the C7 sire (n=119), and likewise, sire A96.0463 and sire
A96.0474 were double backcrossed to their own backcrossed progeny, or to the backcrossed progeny of A96.0473 (n=88 and 53, respectively).
The data in Table 14 show summarised genotyping results with the seven loci genotyped, although some loci are currently incompletely genotyped, and secondary (gap filling) genotyping needs to be completed.
Table 14. Summary of genotyping results
Position in TYRP1 sequence
357 917 1621 1830 Intron 5 Exon 6 Exon 8 63
Allele N I Allele N Allele N I Allele N I Allele N I Allele N I Allele ' N
Awassi origin 113 319 146 1188 347 334 300 380 186 393 181 831
Merino origin 111 803 424 426 127 1123 302 723 158 517 239 669
Unknown origin 176 431 295 495
179 621 298 6
Example 3: Detection of QTL
Following QTL analysis, the TGLA 1C locus was identified as being closest to the estimated QTL location. Male sheep from the mapping population (Sire C7 backcrossed to merino sheep) with genotypic information at the TGLAlO locus, and phenotypic information for PigFibre (Table 3) were selected (n=177). Progeny were allocated a genotypic score of "1" if the allele passed on by the sire originated from the grandsire, and a genotypic score of "2" if the sire donated an allele originating from the granddam. Phase unknown inheritance was indicated as a "12". In the mapping population, the "1" allele was of Awassi origin, and the "2" allele was of Merino origin. Animals were binned according to genotypic score, and "12" animals were discarded as having unclear inheritance. A histogram of the PigFibre trait for animals in the "1" bin (n=70) and animals in the "2" bin (n=68), showed distinct differentiation in the trait range, in that animals with an Awassi inheritance had no animals without pigmentation, while 16.17% of animals with the Merino allele were scored as 0 (colourless) (Figure 7). A two sample t test assuming unequal variances revealed significant support for phenotypic differentiation between the two classes (p(two tailed)<5.95xlOE23) as shown in Table 15.
Table 15: T test- two sample assuming unequal variances
t-Test: Two-Sample Assuming Unequal Variances
Variable 1 Variable 2
Mean 279.70 74.03
Variance 12224.27 8493.97
Observations 71.00 69.00
Hypothesized Mean Difference 0.00 df 135.00 t Stat 11.97
P(T<=t) one-tail 0.00 t Critical one-tail 1.66
P(T<=t) two-tail 0.00 t Critical two-tail 1.98
Example 4: Statistical association analysis of TYRP-1 markers and pigmentation Following genotyping of all of the described Awassi families for the seven loci, analysis was conducted to test for an association between known TYRPl haplotypes and PigFibre or ColourScore (1-9). Initially, the same individuals used in the previous analysis (Example 3) were used, with ANOVA results confirming that TYRPl haplotypes ("Combined") explained a large proportion of the variance of the PigFibre trait (Figure 9, Table 16). Table 16. ANOVA results for PigFibre Trait, with sex, management group and haplotype ("Combined") as fixed effects
Factor Type Levels Values Sex fixed 2 ewe, wet Group fixed 2 1, 2
Combined fixed 21 10_34, 11_34, 15_33, 15_34, 18_34, 29_34, 3_10, 3_15,
3_18, 3_20, 3_23, 3_26, 3_29, 3_3 , 3_34, 3_4 , 3_9, 4_11, 4_29, 6_11, 9_34
Analysis of Variance for PigFibre, using Adjusted SS for Tests
Source DF Seq SS Ad] SS Adj MS F P
Sex 1 26855 6531 6531 0 .65 0 .420
Group 1 23867 11074 11074 1 .11 0 .294
Combined 20 1735625 1735625 86781 8 .68 0 .000
Error 138 1379497 1379497 9996
Total 160 3165845
S = 99.9818 R-Sq = 56.43% R-Sq(adj) = 49.48%
Subsequently, animals from the complex crossing strategies detailed in section 2.5 were used in an ANOVA analysis, confirming that TYRPl hap Io types ("Combined") explained a large proportion of the variance of the ColourScore trait (Table 17).
Table 17. ANOVA results for ColourScore Trait, with year of birth, sex and haplotype ("Combined") as fixed effects
General Linear Model: ColourScore versus Year, Sex, Combined
Factor Type Levels Values
Year fixed 5 1999, 2000, 2002, 2004, 2005 Sex fixed 2 ewe, wet
Combined fixed 55 *, 10_15, 10_23, 10_34, 11_13 , 11_15, 11_18, 11_34,
15_15, 15_17, 15_18, 15_20, 15_21, 15_23, 15_29, 15_33, 15_34, 18_18, 18_22, 18_29, 18_34, 1_15, 26_34, 29_34, 2_18, 30_34, 34_34, 34_36, 3_10, 3_11, 3_15, 3_18, 3_20, 3_22, 3_29, 3_3 , 3_34, 3_37, 3_4 , 3_9,
4_10, 4_11, 4_34, 4_35, 5_22, 6_15, 6_34, 7_15, 7_18, 7_20, 7_28, 7_29, 8_34, 9_18, 9_34
Analysis of Variance for ColourNumberOnly, using Adjusted SS for Tests
Source DF Seq SS Adj SS Adj MS F P
Year 4 153.184 34.461 8.615 1 .37 0 .246
Sex 1 21.742 0.823 0.823 0 .13 0 .718
Combined 54 603.921 603.921 11.184 1 .77 0 .002
Error 242 1525.527 1525.527 6.304
Total 301 2304.374
S = 2 . 51074 R- Sq = 33 . 80 % R- Sq ( adj ) = 17 . 66 %

Claims

1. A genetic marker for distinguishing animals that have a trait of pigmentation, wherein said marker is a polymorphism in the tyrosinase-related protein gene.
2. A method for predicting pigmentation in an animal, said method comprising analysing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene, wherein said genetic marker is predictive of pigmentation in said animal.
3. The method of claim 1 or 2, wherein the pigmentation comprises pigmented skin, pigmented fibre or partial fleece colouration or full fleece colouration.
4. The method of claim 3, wherein the fibre is selected from the group comprising wool, hair or cashmere.
5. The method of claim 4, wherein the fibre is hair or mohair.
6. The method of claim 5, wherein the fibre is wool or cashmere.
7. The method of any one of claims 1-6, wherein the animal is an artiodactyl species.
8. The method of claim 7, wherein the animal is selected from the group comprising sheep, alpaca, lama and goat.
9. The method of claim 7, wherein the animal is a sheep.
10. The method of any one of claims 1 -9, wherein the tyrosinase-related protein is tyrosinase-related protein - 1.
11. A method for selecting an animal using marker assisted selection, wherein said method comprises
(a) analysing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene, wherein the genetic marker is predictive of pigmentation in an animal; and
(b) selecting an animal based on the presence or absence of said genetic marker.
12. A method for breeding an animal using marker assisted selection, wherein said method comprises:
(a) analyzing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene, wherein said genetic marker is predictive of pigmentation in said animal;
(b) breeding from said animal based on the presence or absence of said genetic marker; and (c) selecting progeny of said animal based on the presence or absence of said genetic marker.
13. A system for predicting pigmentation in an animal, wherein said system comprises: (a) means for analyzing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene, wherein said genetic marker is predictive of pigmentation in said animal.
14. A system for selecting an animal using marker assisted selection, wherein said system comprises: (a) means for analyzing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene, wherein the genetic marker is predictive of pigmentation in said animal; and
(b) means for selecting said animal based on the presence or absence of the genetic marker.
15. A system for breeding an animal using marker assisted selection, wherein said system comprises:
(a) means for analyzing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene, wherein the genetic marker is predictive of pigmentation in said animal; and (b) means for breeding said animal based on the presence or absence of the genetic marker, and
(c) means for selecting progeny of said animal based on the presence or absence of the genetic marker.
16. A kit for predicting pigmentation in an animal, wherein said kit comprises: (a) means for analyzing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene, wherein the genetic marker is predictive of pigmentation in said animal.
17. A kit for selecting an animal using marker assisted selection, wherein said kit comprises: (a) means for analyzing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene, wherein the genetic marker is predictive of pigmentation in said animal; and
(b) means for selecting said animal based on the presence or absence of the genetic marker.
18. A kit for breeding an animal using marker assisted selection, wherein said kit comprises:
(a) means for analyzing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene, wherein said genetic marker is predictive of pigmentation in said animal; and
(b) means for breeding said animal based on the presence or absence of the genetic marker, and
(c) means for selecting progeny of said animal based on the presence or absence of the genetic marker.
19. A polypeptide comprising a sheep tyrosinase-related protein (TYRP).
20. The polypeptide of claim 19, wherein said polypeptide is sheep tyrosinase- related protein- 1 (TYRP-I).
21. The polypeptide of claim 19, wherein TYRP-I may comprise the amino acid sequence set forth in SEQ ID NO: 36, 39, 42, 45, 47, 74, 77 and 80.
22. A polynucleotide encoding the polypeptide of any one of claims 19 to 21.
23. A vector comprising the polynucleotide of claim 22.
24. A host cell transformed with the vector of claim 23.
25. An animal selected by the method of claim 11, or the system of claim 14 or the kit of claim or 17.
26. An animal bred by the method of claim 12, or the system of claim 15 or the kit of claim or 18.
27. An animal product derived from the animal of claim 25 or 26.
28. A method of sampling fibres to determine non-visual pigmentation, wherein said method comprises (a) genotyping a plurality of animals based on the presence or absence of at least one genetic marker in the tyrosinase-related protein gene, wherein the genetic marker is predictive of pigmentation in said animal;
(b) grouping said animals according to genotype;
(c) sampling fibres from animals grouped as having said genetic marker(s); and (d) determining a likelihood of contamination of said fibre samples with pigmented fibres.
29. A method for identifying pigmentation in an animal, wherein said method comprises: (a) analyzing a nucleic acid sample from said animal for the presence of at least one genetic marker in the tyrosinase-related protein gene, wherein the presence of the genetic marker is indicative of pigmentation in said animal.
30. The method of claim 29, wherein the pigmentation is present in the form of pigmented fibres.
31. The method of claim 29, wherein the pigmented fibres are in the form of pigmented wool, hair, mohair or cashmere.
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KR101768417B1 (en) 2011-08-03 2017-08-16 주식회사 엘지생활건강 Genetic polymorphic markers for determining type of white skin and use thereof
KR101768414B1 (en) 2011-08-03 2017-08-16 주식회사 엘지생활건강 Genetic polymorphic markers for determining type of white skin and use thereof
KR101768415B1 (en) 2011-08-03 2017-08-16 주식회사 엘지생활건강 Genetic polymorphic markers for determining type of white skin and use thereof
KR101768424B1 (en) 2011-08-03 2017-08-16 주식회사 엘지생활건강 Genetic polymorphic markers for determining type of white skin and use thereof
KR101768423B1 (en) 2011-08-03 2017-08-16 주식회사 엘지생활건강 Genetic polymorphic markers for determining type of white skin and use thereof
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