FIELD

The present invention relates to methods of determining whether or not an animal carries a genetic marker linked to dwarfism, and particularly, but not exclusively, to methods for animal evaluation and for selecting breeding animals.

BACKGROUND

Dwarfism results in animals with a significantly smaller birth size than average. These animals are also smaller from birth to maturity than unaffected animals. In cattle, for example, the weights of calves with dwarfism may vary from 18 to 27kg at birth compared to an average of 40 kg for an unaffected Holstein-Friesian calf. Typically, when calves with dwarfism reach two years of age they are also markedly smaller and have been described as being the size of an animal that is only one year of age.

In bovine animals, dwarfism predominantly affects holstein friesian breeds, and to a lesser extent crossbred animals. In New Zealand, the incidence of the syndrome has been assessed to be around one to two affected calves in the average 400 cow herd per year.

Production animals affected by dwarfism will typically perform poorly. For example, lactating cows that have been identified as having dwarfism may have an average lactation worth that is 155 points lower than what is expected based from their genetic merit (breeding worth).

There has been no effective means of screening animals to identify whether they are carriers of dwarfism. Similarly, other than physical observation based on size, which could be a result of a number of underlying issues which may be unrelated to dwarfism, there is no effective means of determining whether or not an animal has dwarfism.

Bibliographic details of the publications referred to herein are collected at the end of the description. OBJECT

It is an object of the present invention to provide a method of determining whether or not an animal, one or more cells or embryo carries a genetic marker linked to dwarfism, and/or to provide other related methods, or at least to provide the public with a useful choice.

STATEMENT OF INVENTION

The inventors have identified that alteration or variation in the GALNT2 gene is associated with dwarfism. Alterations or variations can be used as genetic markers to determine whether or not an animal is a carrier for dwarfism. They may also be used to diagnose dwarfism in an animal. Such information may be used in methods for selecting or screening cells or embryos, selecting, screening and/or breeding animals, farm management, and for estimating an animal's worth to a particular industry, for example.

In a first aspect, the invention provides a method for determining whether or not an animal, one or more cells or an embryo carries a genetic marker linked to dwarfism, the method comprising at least the step of analysing a nucleic acid from said animal, one or more cells or embryo to determine whether or not it includes one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith, wherein where the nucleic acid from the animal, one or more cells or embryo includes one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith, the animal, one or more cells or embryo is determined to carry a genetic marker linked to dwarfism.

In one embodiment, the nucleic acid is analysed to determine the nucleotide present at a position corresponding to position 1312334 of chromosome 28 of Bos Taurus and/or the nucleotide sequence of one or more genetic marker in linkage disequilibrium therewith.

In one embodiment, the presence of an A at the position corresponding to position 1312334 and/or the presence of one or more genetic marker in linkage disequilibrium therewith, indicates that the animal, one or more cells or embryo carries a genetic marker linked to dwarfism. In a second aspect, the invention provides a method for determining whether or not an animal is a carrier for or has dwarfism, the method comprising at least the step of analysing a nucleic acid from said animal to determine whether or not it includes one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith, wherein where the nucleic acid includes one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith, it is determined to be a carrier of or to have dwarfism.

In one embodiment, where the animal is heterozygous for one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith, the animal is identified as a carrier for dwarfism. In another embodiment, where the animal is homozygous for one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith, the animal is identified as having dwarfism.

In one embodiment, the invention provides a method for determining whether or not an animal is a carrier for or has dwarfism, the method comprising at least the step of analysing a nucleic acid from said animal to determine the nucleotide present at a position corresponding to position 1312334 of chromosome 28 of Bos Taurus and/or the nucleotide sequence of one or more genetic marker in linkage disequilibrium therewith.

In one embodiment, the presence of an A at the position corresponding to position 1312334 and/or the presence of one or more genetic marker in linkage disequilibrium therewith, indicates that the animal is a carrier for or has dwarfism. In one embodiment, where the animal is heterozygous for A at this position and/or one or more genetic marker in linkage disequilibrium therewith the animal is identified as a carrier. In one embodiment, where the animal is homozygous for A at this position and/or one or more genetic marker in linkage disequilibrium therewith, the animal is identified as having dwarfism. In one embodiment, the presence of a G at the position corresponding to position 1312334 and/or one or more genetic marker in linkage disequilibrium therewith, indicates that the animal does not have dwarfism.

In a third aspect the invention provides a method for selecting or rejecting an animal, the method comprising at least the step of analysing a nucleic acid from said animal to determine whether or not it includes one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith, wherein the presence of one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith indicates that the animal has or is a carrier for dwarfism.

In one embodiment, where the animal is heterozygous for one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith, the animal is identified as a carrier for dwarfism. In another embodiment, where the animal is homozygous for one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith, the animal is identified as having dwarfism.

In one embodiment, an animal is selected if it is identified not to be a carrier for or have dwarfism. In one embodiment, an animal is rejected if it is identified to have or be a carrier for dwarfism.

In one embodiment, the invention provides a method for selecting or rejecting an animal, the method comprising at least the step of analysing a nucleic acid from said animal to determine the nucleotide present at a position corresponding to position 1312334 on chromosome 28 of Bos taurus and/or the nucleotide sequence of one or more genetic marker in linkage disequilibrium therewith, wherein the nucleotide present at the position corresponding to position 1312334 and/or the nucleotide sequence of one or more genetic marker in linkage disequilibrium therewith indicates whether or not the animal has or is a carrier for dwarfism. In one embodiment, the method comprises at least the steps of:

a) analysing a nucleic acid from the animal to determine the nucleotide present at the position corresponding to position 1312334 and/or the nucleotide sequence of one or more genetic marker in linkage disequilibrium therewith; and, b) selecting or rejecting an animal based on the nucleotide present at the position corresponding to position 1312334 and/or the nucleotide sequence of one or more genetic marker in linkage disequilibrium therewith.

In one embodiment, the animal is rejected if it has an A at the position corresponding to position 1312334 and/or one or more genetic marker in linkage disequilibrium therewith. In one embodiment, the animal is selected if it has a G at the position corresponding to position 1312334 and/or one or more genetic marker in linkage disequilibrium therewith. In one embodiment, the animal is selected if it is homozygous for G at the position corresponding to position 1312334 and/or one or more genetic marker in linkage disequilibrium therewith.

In one embodiment, the method is performed for the purpose of selecting or rejecting an animal for milking purposes. In one embodiment, the method is performed for the purpose of selecting or rejecting an animal for beef farming. In another embodiment, the method is performed for the purpose of selecting or rejecting an animal for breeding purposes. In one embodiment, the method is performed for the purpose of selecting or rejecting an animal for inclusion in a herd.

In a fourth aspect, the invention provides a method for estimating the worth of an animal and/or its offspring, the method comprising at least the step of analysing a nucleic acid from said animal to determine whether or not it includes one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith, wherein the presence of one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith indicates that the animal has or is a carrier for dwarfism. In one embodiment, where the animal is heterozygous for one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith, the animal is identified as a carrier for dwarfism. In another embodiment, where the animal is homozygous for one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith, the animal is identified as having dwarfism.

In one embodiment, the invention provides a method for estimating the worth of an animal and/or its offspring, the method comprising at least the step of analysing a nucleic acid from the animal to determine the nucleotide present at a position corresponding to position 1312334 of chromosome 28 of Bos taurus and/or the nucleotide sequence of one or more genetic marker in linkage disequilibrium thereof, wherein the nucleotide present at the position corresponding to position 1312334 and/or the nucleotide sequence of one or more genetic marker in linkage disequilibrium therewith indicates whether or not the animal is a carrier for or has dwarfism.

In one embodiment, the presence of an A at the position corresponding to position 1312334 and/or the presence of one or more genetic marker in linkage disequilibrium therewith, indicates that the animal is a carrier for or has dwarfism. In one embodiment, where the animal is heterozygous for A at this position and/or one or more genetic marker in linkage disequilibrium therewith the animal is identified as a carrier. In one embodiment, where the animal is homozygous for A at this position and/or one or more genetic marker in linkage disequilibrium therewith, the animal is identified as having dwarfism.

In one embodiment, the presence of a G at the position corresponding to position 1312334 and/or one or more genetic marker in linkage disequilibrium therewith, indicates that the animal does not have dwarfism.

In a fifth aspect, the invention provides a method for selecting or rejecting one or more cells or embryo, the method comprising at least the step of analysing a nucleic acid from said one or more cells or embryo or from an animal from which the one or more cell or embryo is derived, to determine whether or not it includes one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith, and selecting or rejecting one or more cells or embryo.

In one embodiment, where the nucleic acid includes one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith, the one or more cells or embryo is rejected.

In one embodiment, where the nucleic acid does not include one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith, the one or more cells or embryo is selected.

In one embodiment, the method comprises the step of analysing a nucleic acid from the one or more cells or embryo or from an animal from which the one or more cell or embryo is derived, to determine the nucleotide present at a position corresponding to position 1312334 on chromosome 28 of Bos taurus and/or the nucleotide sequence of one or more genetic marker in linkage disequilibrium therewith.

In one embodiment, the method comprises at least the steps of:

a) analysing a nucleic acid from one or more cells or embryo or from an animal from which the one or more cells or embryo is derived, to determine the nucleotide present at the position corresponding to position 1312334 and/or the nucleotide sequence of one or more genetic marker in linkage disequilibrium therewith; and,

b) selecting or rejecting one or more cells or embryo based on the nucleotide present at the position corresponding to position 1312334 and/or the nucleotide sequence of one or more genetic marker in linkage disequilibrium therewith.

In one embodiment, the one or more cells or embryo is rejected if it, or an animal from which the one or more cell or embryo is derived, has an A at the position corresponding to position 1312334 and/or one or more genetic marker in linkage disequilibrium therewith. In one embodiment, the one or more cells or embryo is selected if it, or an animal from which the one or more cells or embryo is derived, has a G at the position corresponding to position 1312334 and/or one or more genetic marker in linkage disequilibrium therewith.

In one embodiment, the one or more cells or embryo is selected if it, or an animal from which the one or more cell or embryo is derived, is homozygous for G at the position corresponding to position 1312334 and/or one or more genetic marker in linkage disequilibrium therewith.

In one embodiment, the method is conducted for the purpose of selecting or rejecting one or more cell or embryo for use in cloning an animal and/or breeding an animal. In one embodiment, breeding an animal may involve IVF.

In a sixth aspect, the invention provides a method for breeding animals to produce offspring, the method comprising at least the step of selecting a first animal that is heterozygous for one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith or does not include one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith and mating said first animal with a second animal to produce offspring.

In one embodiment, the invention provides a method for breeding animals to produce offspring, which comprises selecting at least a first animal that has a G at a position corresponding to position 1312334 on chromosome 28 of Bos Taurus and mating said first animal with a second animal to produce offspring.

In one embodiment, the method further comprises the step of selecting the second animal on the basis that it is heterozygous for one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith or it does not include one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith. In one embodiment, the method comprises the step of selecting the second animal on the basis that it has a G at a position corresponding to position 1312334 on chromosome 28 of Bos Taurus.

In one embodiment, the first animal and/or the second animal are selected as a result of performing a method of any one of the first to fourth aspects of the invention.

In seventh aspect, the invention provides a method of breeding an animal to produce offspring, the method comprising at least the step of selecting a first gamete that is heterozygous for one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith or that does not include one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith and fusing said first gamete with a second gamete to form a zygote.

In one embodiment, the method further comprises selecting the second gamete on the basis that is heterozygous for one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith or that it does not include one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith.

In one embodiment, the method comprises selecting the first and/or second gamete on the basis that it has a G at a position corresponding to position 1312334 on chromosome 28 of Bos Taurus.

In one embodiment, the first and/or second gamete are selected as a result of performing a method of the first or fifth aspects of the invention.

In an eighth aspect, the invention provides a method of breeding an animal to produce offspring, the method comprising at least the step of selecting an embryo that is heterozygous for one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith or that does not include one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith.

In one embodiment, the method comprises selecting an embryo where it has a G at a position corresponding to position 1312334 on chromosome 28 of Bos Taurus.

In one embodiment, the embryo is selected as a result of performing a method of the first or fifth aspects of the invention.

In a ninth aspect, the invention provides a method of cloning an animal to produce offspring, the method comprising at least the step of selecting one or more cell that is heterozygous for one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith or that does not include one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith.

In one embodiment, the method comprises selecting one or more cell where it has a G at a position corresponding to position 1312334 on chromosome 28 of Bos Taurus.

In one embodiment, the one or more cell is selected as a result of performing a method of the first or fifth aspects of the invention.

In a tenth aspect, the invention provides a method of forming a herd the method comprising at least the steps of:

a. performing a method of any one or more of the first to fourth aspects of the

invention;

b. selecting or rejecting an animal, wherein an animal is selected for inclusion in a herd where it is identified to be heterozygous for a genetic marker linked to dwarfism or not to include a genetic marker linked to dwarfism, where it is determined to be a carrier for dwarfism or not to have dwarfism, and/or where it is heterozygous for one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith or does not include one or more genetic variation which disrupts the GALNT2 gene or one or more genetic marker in linkage disequilibrium therewith; and,

c. forming a herd of selected animals.

In one embodiment, an animal is rejected if it determined to be a carrier for dwarfism.

In one embodiment, the animal is rejected if it has an A at the position corresponding to position 1312334 and/or one or more genetic marker in linkage disequilibrium therewith. In one embodiment, the animal is selected if it has a G at the position corresponding to position 1312334 and/or one or more genetic marker in linkage disequilibrium therewith. In one embodiment, the animal is selected if it is homozygous for G at the position corresponding to position 1312334 and/or one or more genetic marker in linkage disequilibrium therewith.

In one embodiment, the methods of any of the first to tenth aspects involve analysing a nucleic acid to determine whether or not it includes one or more genetic variation which disrupts the GALNT2 gene alone or in combination with one or more genetic marker in linkage disequilibrium therewith.

In one embodiment, the methods of any of the first to tenth aspects involve analysing a nucleic acid to determine the nucleotide present at the position corresponding to position 1312334 of chromosome 28 of Bos Taurus alone or in combination with one or more genetic marker in linkage disequilibrium therewith.

In one embodiment, the methods of any of the first to tenth aspects further involve analysing one or more additional biological markers. In one embodiment, the one or more biological markers is one or more genetic markers.

Preferably, analysis of a nucleic acid in accordance with the invention occurs using one or more of: polymerase chain reaction (PCR); gel electrophoresis; Southern blotting; nucleic acid sequencing; restriction fragment length polymorphism (RFLP); single-strand confirmation polymphism (SSCP); LCR (ligase chain reaction); denaturing gradient gel electrophoresis (DGGE); allele-specific oligonucleotides (ASOs); proteins which recognize nucleic acid mismatches; RNAse protection; oligonucleotide array hybridisation; denaturing HPLC (dHPLC); high resolution melting (HRM); and, matrix-assisted laser desorption/ionisation time-of-flight mass spectroscopy (MALDI-TOF MS).

In one embodiment of the methods of any of the first to tenth aspects the animal is a mammal. In one particular embodiment, the animal is from the Bovidae family. In one embodiment, the animal is bovine. In a particular embodiment the bovine animal is Bos taurus or Bos indicus. In a particular embodiment, the animal is chosen from the group consisting Jersey, Holstein, Friesian or crossbred dairy cattle.

In an eleventh aspect, the invention provides isolated nucleic acids encompassing a G/A transition at a position corresponding to position 1312334 of chromosome 28 of Bos Taurus.

The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

FIGURES

These and other aspects of the present invention, which should be considered in all its novel aspects, will become apparent from the following description, which is given by way of example only, with reference to the accompanying figures, in which:

Figure 1: Illustrates Manhattan plots of the genome of test animals indicating

chromosome 28 had the most significant affect.

Figure 2: Illustrates the main peak of potential markers on chromosome 28 were located between 0-lOmb. Figure 3: Illustrates that the animals that do not have the genetic variation of interest but were identified as small do not have a strong genetic component to the phenotype as shown by no significant effects over the genome.

Figure 4: Illustrates that the animals that do not have the genetic variation of interest but were identified as small do not have a strong genetic component to the phenotype from the region of interest on chromosome 28.

Figure 5: Illustrates the genetic region surrounding the G/A variation at position 1312334 of chromosome 28 of Bos Taurus. 250bp upstream and downstream sequence surrounding the SNP is provided. The target SNP is provided in square brackets. Any other known polymorphisms are indicated with a trailing underscore - eg NNNNT_NNNN, where the "T" is the other known polymorphism.

SEQUENCE LISTING

A sequence listing accompanies this application. The sequence listing includes 2 sequences which represent the alternative forms of a region of the GALNT2 gene surrounding the G/A variation at position 1312334, as follows:

SEQ Π) No. 1: nucleic acid sequence of a region of GALNT2 gene including A at position 1312334 which is linked to dwarfism; and,

SEQ ID No. 2: nucleic acid sequence of a region of GALNT2 gene including G at position 1312334 which is not linked to dwarfism.

PREFERRED EMBODIMENT(S)

The following is a description of the present invention, including preferred embodiments thereof, given in general terms. The invention is further elucidated from the disclosure given under the section "Examples" which provides, inter alia, experimental data supporting the invention.

The inventors have surprisingly identified that particular alleles of a novel genetic marker located in the GALNT2 (polypeptide N-acetylgalactosaminyltransferase 2) gene in a region of chromosome 28 in Bos taurus indicate that an animal is a carrier for, or has, dwarfism. The genotype of this particular genetic marker may therefore be used to identify carriers for dwarfism and also diagnose dwarfism. The inventors also contemplate that the analysis of one or more genetic marker which is in linkage disequilibrium with the specific marker they have identified may also be used for the same purpose. In addition, haplotypes including the genetic marker of the invention may also be used for this purpose.

This is the first time that alterations in GALNT2 have been associated with dwarfism, and thus the first time a direct link between GALNT2 and dwarfism has been made.

Accordingly, the inventors contemplate that any genetic alteration which disrupts this gene may be used as a marker to identify carriers and diagnose dwarfism and to assist in selecting or rejecting animals, cells or embryos. Accordingly, while the description which follows may focus on the analysis of the nucleotide sequence at a particular position in this gene, it should be understood to extend to the analysis of the sequence at any other position within the gene.

Analysis of a genetic marker in accordance with the invention (including in the alternative or in addition one or more markers in linkage disequilibrium therewith) may assist in: predicting phentotypic performance, including use in production management systems known as Marker Assisted Selection; the selection or rejection of animals for breeding purposes; the selection or rejection of animals for milk production and/or meat production purposes; managing animals in order to maximise their individual potential performance and value; estimating the worth or economic value of an animal; improving profits related to selling animals and/or products produced from the animals; improving the genetics of a population of animals by selecting and breeding desirable animals; generating and maintaining herds of animals; cloning animals likely to have or not have a specific trait; the selection or rejection of cells or embryos of use in breeding or cloning animals;

predicting the suitability of an animal and/or its offspring to use in different industries and/or breeding programmes or cloning. Animals more or less suitable for a particular production system or industry can be tested or screened to predict life time performance and segregated or managed to suit their genotype and therefore predicted phenotype. Animals may be tested or screened at any time during their life, including but not limited to early at birth, as gametes, zygotes, embryos, foetuses.

Definitions

The term "genetic marker" as used herein refers to nucleic acids or specific genetic loci (including specific nucleotide positions) that are polymorphic or contain sequence alterations or variations within a population, the alleles of which can be detected and distinguished by one or more analytic methods. The term "genetic marker" further includes within its scope a plurality of genetic markers co-segregating, in the form of a "haplotype". In this context, the term "haplotype" refers to a plurality of genetic markers that are generally inherited together. Typically, genetic markers within a haplotype are in linkage disequilibrium. Reference herein to analysing a nucleic acid to determine the nucleotide sequence of a "genetic marker" or at a particular genetic position should be taken to include analysing and determining the nucleotide sequence on either strand of the nucleic acid.

The term "single nucleotide polymorphism" (SNP) refers to nucleic acid sequence variations that occur when a single nucleotide in the genome sequence is altered. A single nucleotide polymorphism may also be a single nucleotide insertion or deletion. The different nucleotides within a SNP are referred to as an allele.

The term "genotype" as used herein means the genetic constitution or nucleotide sequence at one or more genetic locus, in particular the nucleotide sequence of an allele of a genetic locus.

A genetic alteration or variation which "disrupts" the GALNT2 gene, may be any genetic change which has an affect on the expression or activity of the GALNT2 gene product. By way of example, it may decrease the level of expression or alter the structure or function of the gene product. The term "disrupts" should not be taken to imply that there is substantially no expression or activity, although this may be preferred, but rather taken to encompasses any change in the level of expression or activity. A genetic alteration or variation may include substitution of one or more nucleotide, addition of one or more nucleotide, and/or deletion of one or more nucleotide.

One can readily determine whether a variation disrupts the GALNT2 gene using standard assays known in the art. However, by way of example, expression levels can be measured using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), northern blotting, microarray analysis, RNA sequencing for measuring cDNA levels and western blotting, PAGE, mass spectrometry, and immunoprecipitation for measuring protein levels and GALNT2 activity can be measured using in vitro peptide glycosylation analysis.

Reference to the "GALNT2 gene" should be taken to include reference to the coding and non-coding regions of the gene, including upstream and downstream regulatory elements. Exemplary sequence information for GALNT2 is provided on NCBI databases using the reference NM_001193103.1 (amino acid and mRNA) and ac_000185.1 (genomic sequence of gene including 5' and 3' UTRs).

"Linkage disequilibrium" should be taken broadly to refer to the tendency of the presence of an allele at one genetic locus to predict the presence of an allele at one or more other genetic loci (for example a distinct genetic marker). The genetic loci need not necessarily be on the same chromosome. However, in a preferred embodiment, the genetic loci are located on the same chromosome.

One measure of linkage disequilibrium is DELTA ² , which is calculated using the formula described by Devlin et al (Genomics 29 (2):311-22 (1995)), and is a measure of how well an allele X at a first genetic loci predicts the occurrence of an allele Y at a second genetic loci. A DELTA ² value of 1.0 indicates the prediction is perfect (for example, if Y is present then X is present). It should be appreciated that reference to linkage disequilibrium herein should not be taken to imply a DELTA ² value of 1.0. In particular embodiments, the linkage disequilibrium between an allele at one genetic locus and an allele at a second genetic locus, has a DELTA ² value of at least 0.75, at least 0.80, at least 0.85, at least 0.90, at least 0.95, and most preferably 1.0. Skilled persons will readily appreciate methods for determining whether any two alleles are in linkage disequilibrium. However, by way of example, see Genetic Data Analysis II, Weir, Sinauer Associates, Inc. Publishers, Sunderland, Mass., 1996.

Where reference is made herein to analysing a nucleic acid to determine whether or not it includes a genetic marker in linkage disequillibrium with one or more genetic variation which disrupts the GALNT2 gene, it should be appeciated that such a genetic marker may be native to the genome of the animal or one or more cell, for example, or it may have been artificially generated or inserted (ie introduced in to the genome).

While the inventors have identified the genetic marker of the invention in bovine animals, they contemplate that it is equally applicable to a variety of different mammals. Various mammals can suffer from "dwarfism". Accordingly, the term "animal" is used herein primarily in reference to mammals. In one particular embodiment, the mammal is one within within the Bovidae family. In particular embodiments, the animal is a bovine animal. More particularly the animal is Bos taurus or Bos indicus. In one particular embodiment the animal is a beef or dairy breed. By way of further example, the animal may be chosen from the group of animals including, but not limited to, Jersey, Holstein, Friesian, Ayrshire, crossbred dairy cattle, Angus, Hereford, Simmental and crossbred beef cattle.

"Dwarfism" should be taken to refer collectively to a group of syndromes or disorders which results in animals that are significantly smaller than the normal or average size expected for a particular animal. Animals may be smaller at birth and/or to maturity.

As used herein the "worth" of an animal refers to an index used to evaluate the value of an animal, for breeding purposes, inclusion in a herd, herd management, for example. The "worth" is the sum of the estimated value of one or more characteristics which may be associated with the animal, typically weighted by an economic value. Exemplary characteristics include milk fat, protein, milk volume, liveweight, fertility, milk somatic cells, growth rate, and feed conversion efficiency. The term "worth" should be taken to encompass "breeding worth" and other known indexes used to assess the value of an animal. Persons skilled in the art to which the invention relates will readily appreciate methods and formulae suitable for estimating breeding worth on the basis of any number of different characteristics. However, in one particular embodiment, the following calculation may be used:

It should be appreciated that where methods of the invention relate to breeding animals, any appropriate breeding methods may be utilised including for example natural insemination, artificial insemination and in vitro fertilisation (IVF). Accordingly, the word "mating" should be construed broadly and not limited to the physical pairing of two animals.

The methods of the invention may be used to identify animals for cloning. They may also be used during cloning processes, to determine whether or not one or more cell, embryo or cloned animal has a genetic variation in the GALNT2 gene and the animal (or an animal derived from the one or more cell of embryo) is a carrier for or has dwarfism. Any appropriate cloning method could be used. However, by way of example, such cloning techniques include somatic cell nuclear transfer, chromatin transfer, and embryo splitting. Persons of general skill in the art will readily appreciate appropriate somatic cell nuclear transfer and chromatin transfer methodologies. However, by way of example, the methods described in the following publications may be used: Bovine somatic cell nuclear transfer, Ross PI and Cibelli, IB 2010. Methods in Molecular Biology 636: 155-177; and, Influence of cloning by chromatin transfer on placental gene expression at Day 45 of pregnancy in cattle. Mesquita FS, Machado SA, Drnevich I, Borowicz P, Wang Z, Nowak RA. Anim Reprod Sci. 2013 Ian 30;136(4):231-44. doi: 10.1016/j.anireprosci.2012.10.030. Epub 2012 Nov 8.

Where IVF is employed in the context of the invention, any appropriate IVF methodology may be used, as will be apparent to persons of general skill in the art to which the invention relates. However, by way of example, appropriate methods are described, for example, in: Imai K, Tagawa M, Yoshioka H, Matoba S, Narita M, et al. (2006) The efficiency of embryo production by ovum pick-up and in vitro fertilization in cattle. J Reprod Dev 52: 19-29. "Embryo" should be taken broadly to include an organism from the first division of the zygote. In certain embodiments, an embryo is an organism between the first division of the zygote until the time it becomes a foetus. Reference to an "embryo" should be taken to include reference to an organism at different developmental stages, including a blastula, blastocyst, gastrula, and morula for example.

In one aspect, the invention provides methods for the selection or rejection of one or more cells. In certain embodiments, such "cells" may include a gamete (for example, sperm or ovum) or zygote. Selection of such cells may be of use in an IVF program, for example. In other embodiments, such "cells" may be somatic cells, embryonic cells, embryonic stem cells, cells in a cell line, one or more cells of use in cloning, for example. Selection of these cells may be of use in cloning procedures, or preparing of cells lines for use in cloning and other procedures, for example.

In certain embodiments, the methods of the invention involve taking a "sample" from an animal to be tested. The sample may be any appropriate tissue or body fluid sample. In one embodiment, the sample may comprise one or more cell, blood, muscle, bone, somatic cell(s), saliva, liver, brain, placenta, amniotic fluid and/or semen. A "sample" can be taken from the animal using standard techniques known in the art. It should be appreciated that a sample may be taken from an animal at any stage of life, including prior to birth; by way of non-limiting example, a zygote, an embryo, a foetus.. Individual gametes could also be tested using the methods of the invention. This may assist in breeding and/or cloning programmes. Accordingly, "sample" should be taken to include a zygote, embryonic tissue, foetal tissue and gametes. A "sample" may also be taken after the death of an animal. The samples are analysed using techniques which allow for the observation or analysis of nucleic acids, including the sequence of a particular nucleic acid, as will be described further herein after.

It should be appreciated that analysis of a nucleic acid of an animal in accordance with a method of the invention may be conducted during gestation. In this case, the analysis could be conducted by analysing nucleic acid or one or more cell of that animal that may be present in the maternal blood supply, placenta, amniotic fluid or any other maternal tissue or fluid prior to birth of the animal. Accordingly, reference to "analysing a nucleic acid from an animal" should be taken to include reference to analysing nucleic acid from that animal that may be present in a maternal tissue or fluid.

Reference may be made herein to a "desirable genotype". This should be taken broadly to include a genotype which is not linked to dwarfism or which does not result in dwarfism. In certain embodiments, a "desirable genotype" could include a genotype associated with being a carrier for dwarfism, although this is not necessarily preferred.

Certain aspects and embodiments of the invention may be described herein with reference to "fusing a first and second gamete" to form a zygote. This phrase should be taken broadly to include fertilisation processes, such as may be used in in vitro fertilisation processes. Skilled persons will readily appreciate standard means of "fusing" gametes to form a zygote.

Markers

The specific marker identified by the inventors is a G/A SNP located in the GALNT2 gene at nucleotide position 1312334 of chromosome 28 of Bos Taurus. The sequence and position is based on the genomic sequence of chromosome 28 in bovine build UMD3.1 (gi!2585133391ref IAC_000185.11 in the GenBank database http://www.ncbi.nlm.nih.gov/). The position of the genetic marker should be read in accordance with base position being the start site of the polymorphism, given that the first nucleotide in the sequence (gil258513339lreflAC_000185.ll) is denoted as position one. Further sequence information in the region of the genetic marker is provided herein before (with reference to NCBI database reference sequences), in the sequence listing and in Figure 5 herein after.

It will be appreciated that the precise location of the genetic marker of the invention may vary slightly from genome to genome; for example, in a different species of animal, or different breed of animal, the location of the marker may vary. However, persons of skill in the art to which the invention relate will be able to readily identify the marker in different genomes through routine sequence alignment and with knowledge that it resides in the GALNT2 gene. To account for this variation in the location of the genetic marker across genomes the marker is described herein as being "at a position corresponding to position 1312334 of chromosome 28 of Bos Taurus" in the UMD3.1 genome build.

While the inventors have observed that a G/A transition at the genetic marker is indicative of dwarfism, they contemplate that any variation in the nucleotide at this genetic locus may be indicative of dwarfism. For example, a T or C at this position may indicate an animal is a carrier for or has dwarfism. The invention should be interpreted accordingly.

The inventors note that the alteration at position 1312334 which is linked to dwarfism is recessive in nature. Accordingly, for example, where an animal is identified as being heterozygous for the above nucleotides at position 1312334 it is considered a carrier and where an animal is identified as being homozygous for A (or T or C) at position 1212334 it will be considered to have dwarfism.

It should also be appreciated that in methods of the invention one could analyse the nucleic acid sequence of either strand of the nucleic acid to identify the sequence at a particular genetic locus or position; for example, instead of analysing the strand associated with the sequence variant listed above, the nucleotide sequence of the opposite or complementary strand of DNA could be analysed. Persons of skill in the art will readily appreciate nucleic acid sequence variations on such opposite strand which correlate with the genotypes mentioned above, having regard to the information contained herein and nucleic acid base pairing principles (ie, A pairs with T and C pairs with G).

As noted herein before, this is the first time that alterations in GALNT2 have been associated with dwarfism, and thus the first time a direct link between GALNT2 and dwarfism has been made. Accordingly, the inventors contemplate that any genetic alteration which disrupts this gene may be used as a marker to identify carriers and diagnose dwarfism and to assist in selecting or rejecting animals, cells or embryos.

Methods of the invention As described herein before, the invention provides various methods. The methods may be used, for example, for: determining whether or not an animal, one or more cells or an embryo carries a genetic marker linked to dwarfism; determining whether or not an animal is a carrier for or has dwarfism; selecting or rejecting an animal; estimating the worth of an animal and/or its offspring; selecting or rejecting one or more cells or embryo. It should be appreciated that where methods of the invention involve selecting an animal, one or more cell or embryo (including for example in methods of breeding, cloning, herd formation), in one preferred embodiment animals, cells or embryos are selected where they do not include one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith. However, it should be appreciated that in certain cases animals, cells or embryos which are heterozygous for one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium thereof may be selected.

The methods involve the analysis of a nucleic acid (including one or more nucleic acid) from an animal or one or more cells or embryo to determine the presence or absence of one or more genetic marker associated with dwarfism.

In one embodiment, these methods involve the analysis of a nucleic acid (including one or more nucleic acid) to determine whether or not it includes one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith. Where a nucleic acid from an animal, cell or embryo is found to include one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith, it is determined to carry a genetic marker linked to dwarfism.

As noted above, the invention encompasses use of one or more genetic markers which are in linkage disequilibrium with a marker of the invention. Such markers may be analysed instead of or in addition to a genetic marker of the invention.

In one embodiment of the methods of the invention, where an animal is heterozygous for one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith, the animal is identified as a carrier for dwarfism. In another embodiment, where an animal is homozygous for one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith, the animal is identified as having dwarfism.

In one embodiment, the methods involve the analysis of a nucleic acid (including one or more nucleic acid) to determine the nucleotide present at a position corresponding to position 1312334 of chromosome 28 of Bos taurus. Alternatively, or in addition, the methods may involve analysising the nucleotide sequence of a nucleic acid to determine the nucleotide sequence of one or more genetic marker in linkage disequilibrium with the genetic marker at the position corresponding to position 1312334. In one particular embodiment, the methods of the invention may involve analysing the nucleotide sequence of a nucleic acid to determine the haplotype of an animal, one or more cell or embryo, wherein the haplotype includes the genetic marker at the position corresponding to position 1312334.

In one embodiment of the methods of the invention, the presence of an A at the position corresponding to position 1312334 of chromosome 28 of Bos Taurus and/or the presence of one or more genetic marker in linkage disequilibrium therewith, indicates that the animal, one or more cells or embryo carries a genetic marker linked to dwarfism.

In one embodiment, the presence of an A at the position corresponding to position 1312334 and/or the presence of one or more genetic marker in linkage disequilibrium therewith, indicates that an animal is a carrier for or has dwarfism. In one embodiment, where an animal is heterozygous for A at this position and/or one or more genetic marker in linkage disequilibrium therewith the animal is identified as a carrier. In one embodiment, where an animal is homozygous for A at this position and/or one or more genetic marker in linkage disequilibrium therewith, the animal is identified as having dwarfism.

In one embodiment, the presence of a G at the position corresponding to position 1312334 and/or one or more genetic marker in linkage disequilibrium therewith, indicates that the animal does not have dwarfism. In one embodiment, heterozygosity for G and A at this position and/or one or more genetic marker in linkage disequilibrium therewith, indicates that the animal is a carrier for dwarfism. In one embodiment, homozygous G at this position and/or one or more genetic marker in linkage disequilibrium therewith, indicates that the animal does not have dwarfism.

In methods of the invention involving the selection or rejection of an animal, one or more cells or embryos, the animal, one or more cells or embryo is selected if it is identified not to include a genetic marker linked to dwarfism and the animal (or in some embodiments if it is identified to be heterozygous for a genetic marker linked to dwarfism), one or more cells or embryo is rejected if it is identified to include a genetic marker linked to dwarfism; ie the animal, one or more cells or embryo is selected if it does not include one or more genetic variation which disrupts the GALNT2 gene (or in some embodiments if it is identified to be heterozygous for a genetic marker linked to dwarfism) and is rejected if it includes one or more genetic variation which disrupts the GALNT2 gene. In one embodiment, an animal is selected if it is identified not to be a carrier for or have dwarfism. In one embodiment, an animal is rejected if it is identified to have or to be a carrier for dwarfism.

Methods of the invention which involve the selection or rejection of animals may be used for the purpose of selecting or rejecting an animal for milking, beef farming, breeding, inclusion in a herd, for example. The methods for the selection or rejection of one or more cells may be used for the purpose of selecting or rejecting cells or embryos for cloning and/or breeding purposes, for example.

Nucleic acids can be analysed to determine the genotype/sequence of the genetic markers described herein according to any appropriate technique. Such techniques include for example polymerase chain reaction (PCR), including allele-specific PCR, gel electrophoresis, the use of oligonucleotide probe hybridisation, Southern blotting, direct sequencing, restriction digestion, restriction fragment length polymorphism (RFLP), single-strand confirmation polymorphism (SSCP), LCR (ligase chain reaction), denaturing gradient gel electrophoresis (DGGE), the use of allele-specific oligonucleotides (ASOs), the use of proteins which recognize nucleic acid mismatches, such as E.coli mutS protein, RNAse protection assays, oligonucleotide array hybridisation (for example microarray), denaturing HPLC (dHPLC), fluorescence quenching PCR (TaqMan™ Applied Biosystems, CA 94404, USA), High Resolution Melting (HRM), and matrix-assisted laser desorption/ionisation time-of-flight mass spectroscopy (MALDI-TOF MS). Combinations of two or more of such techniques may be used. Such combination may increase the sensitivity of the analysis being conducted.

The technique(s) used will depend on the nature of the marker to be detected as will be appreciated by skilled persons. For example, single nucleotide polymorphisms (SNPs), may be analysed using those techniques capable of resolving a single nucleotide difference between sequences; for example, direct sequencing or LCR, allele-specific PCR, RFLP, SSCP, DGGE, using allele-specific oligonucleotides (ASOs), or proteins which recognize nucleic acid mismatches, oligonucleotide array hybridisation, dHPLC, fluorescence quenching PCR and matrix MALDI-TOF MS.

Any one or more of the techniques mentioned hereinbefore (including for example, SSCP, RFLP, DGGE, dHPLC and direct sequencing) may be used to analyse genetic markers which may include insertion or deletion of one or more nucleotide.

It should be appreciated that certain of the techniques of use in analysing a genetic marker in accordance with the invention will utilise one or more oligonucleotides which hybridise to a genetic region encompassing the marker, adjacent to the marker, or flanking the marker. Such oligonucleotides may be DNA, RNA or derivatised forms thereof and include nucleic acid primers, such as PCR and LCR primers, and nucleic acid probes.

Persons of ordinary skill in the art to which the invention relates will readily appreciate appropriate oligonucleotides of use in the invention having regard to one or more of the nucleic acid sequence of chromosome 28, particularly in the genetic regions proximal to a the genetic marker, the nature of the genetic marker to be analysed, and the general principles of nucleic acid hybridisation. The nucleic acids will be capable of hybridising in a specific manner to a target nucleic acid and in the case of primers they will be capable of priming a PCR or like reaction. While such nucleic acids will preferably have 100% complementarity to their target region of genomic DNA, mRNA or cDNA of interest, they may contain one or more non-complementary nucleotides at a particular position while still substantially retaining specificity for the target nucleic acid to which they are designed to bind. By way of example, the nucleic acids may have approximately 80%, approximately 90%, approximately 95%, or approximately 99% complementarity or homology to its target. By way of further example, in certain cases, the oligonucleotides may be designed such that a mismatch at a particular nucleotide position is indicative of the nature of the genetic marker being analysed (for example, a SNP). By way of example, a mismatch in the nucleotide present at the 3' end of an LCR primer will inhibit the reaction providing an indication of the nature of the nucleotide at that position. Mismatches may similarly be utilised in techniques including RNAse protection assays and allele-specific PCR, as well as in fluorescence quenching PCR, for example. Typically, the nucleic acids will hybridise to their target nucleic acid under stringent hybridisation conditions (see for example, Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).

The oligonucleotide probes or primers may be of any length as is appropriate for a particular application, having regard to the sequence of the genetic region to which they are designed to bind. A probe or primer will typically be capable of forming a stable hybrid with the complementary sequence to which it is designed to hybridise. Accordingly, the length is dependent on the nucleic acid composition and percent homology between the oligonucleotide and its complementary sequence, as well as the hybridisation conditions which are utilised (for example, temperature and salt concentrations). Such hybridisation factors are well known in the art to which the invention relates. By way of example, oligonucleotides of use in the present invention may be from 2 to 500 nucleotides in length. In one embodiment, particularly where they are used as primers, the oligonucleotides may be of approximately 15 nucleotides to 30 nucleotides in length.

Oligonucleotide probes and primers of use in the invention may be prepared by any number of conventional nucleic acid synthesis methods including recombinant techniques and chemical synthesis, or they may be purchase commercially. It will be appreciated that the usefulness of any probe or primer may be evaluated, at least notionally, using appropriate software and sequence information for the nucleic acid encoding the protein of interest. For example, software packages such as Primer3

(http://primer3.sourceforge.net/), PC 01igo5 (National Bioscience Inc), Amplify (University of Wisconsin), and the PrimerSelect program (DNAStar Inc) may be used to design and evaluate primers.

Where amplification techniques (for example PCR) are used in methods of the invention amplification may be conducted according to conventional procedures in the art to which this invention relates, such as described in US Patent No 4,683,202. By way of example PCR reactions will generally include 0.1μΜ-1μΜ of each primer, 200μΜ each dNTP, 3- 7mM MgCl ₂ , and 1U Taq DNA polymerase. Further, exemplary PCR cycling conditions include: denaturation at a temperature of approximately 94°C for 30 to 60 seconds, annealing at a temperature calculated on the basis of the sequence and length of the primer (as herein after discussed) for 30 to 60 seconds, and extension at a temperature of approximately 70°C to 72°C for 30 to 60 seconds. By way of example, between 25 and 45 cycles are run.

It will be appreciated by those of ordinary skill in the art that any amplification conditions provided herein are merely exemplary and may be varied so as to optimise conditions where, for example, alternative PCR cyclers or DNA polymerases are used, where the quality of the template DNA differs, or where variations of the primers not specifically exemplified herein are used, without departing from the scope of the present invention. The PCR conditions may be altered or optimised by changing the concentration of the various constituents within the reaction and/or changing the constituents of the reaction, altering the number of amplification cycles, the denaturation, annealing or extension times or temperatures, or the quantity of template DNA, for example. Those of skill in the art will appreciate there are a number of other ways in which PCR conditions may be optimised to overcome variability between reactions. It will be understood that whilst not specifically exemplified herein, appropriate annealing temperatures for any primer within the scope of the present invention may be derived from the calculated melting temperature of that primer. Such melting temperatures may be calculated using standard formulas, such as that described in Sambrook and Russell, 2001. As will be understood by those of ordinary skill in the art to which this invention relates annealing temperatures may be above or below the melting temperature but generally an annealing temperature of approximately 5°C below the calculated melting temperature of the primer is suitable.

Oligonucleotides used for detection and/or analysis of genetic markers in accordance with the invention may be modified to facilitate such detection. Similarly, nucleic acid products obtained using techniques such as PCR may be modified to facilitate detection and/or analysis. For example, the nucleic acid molecules may be labelled to facilitate visual identification using techniques standard in the art. By way of example nucleic acids may be radio-labelled using P ³² as may be described in Sambrook and Russell, 2001. Further, nucleic acids may be appropriately labelled for use in colorigenic, fluorogenic or chemiluminescence procedures.

It will be appreciated that the methods of the invention may employ one or more control samples. Such control samples may be positive or negative controls for a particular genetic marker. The type of control samples used may vary depending on such factors as the nature of the genetic marker being analysed and the specific technique being used for such detection and analysis. Positive controls may include samples having known nucleic acid sequences. Negative controls may include samples having no nucleic acid present. By way of general example, in analysing a SNP positive control samples could include nucleic acids known to have a particular nucleotide at the relevant position.

In order to facilitate detection of a genetic marker in accordance with the invention, a sample, one or more cells or embryo may be processed prior to analysis where appropriate. For example, a sample, one or more cells or embryo may be processed to isolate nucleic acid to be analysed or to amplify a specific genetic region to be analysed. In one embodiment, nucleic acid is isolated or extracted from a sample, one or more cells or embryo prior to analysis. In one embodiment, genomic DNA is isolated or extracted. In an alternative embodiment, mRNA may be isolated or extracted. In such a case, the mRNA may be converted to cDNA using reverse transcription techniques known in the art. Techniques for isolating nucleic acids from samples, one or more cells and embryos will be readily appreciated by skilled persons. By way of Example, methods of use in isolating nucleic acids are described in Sambrook and Russell, 2001.

In an alternative form of this embodiment of the invention analysis of the nucleic acid may occur in situ obviating the need to extract nucleic acid. This may be done using PCR for example. Skilled persons will readily appreciate appropriate techniques and methodology to this end (see for example, Sambrook and Russell, 2001).

The methods of the invention may be combined with one or more other methods of use in assessing genotype, predicting phenotype, selecting an animal, one or more cells or embryo based on certain characteristics, estimating breeding values or estimating worth and the like. Accordingly, the methods of the invention may include, in addition to analysis of a genetic marker identified herein, analysis of additional genetic markers, and/or the level of expression of certain genes/proteins, and/or one or more phenotypic traits, for example.

Breeding and cloning

As described herein before, the invention also provides a method for breeding animals to produce offspring. The method comprises selecting at least a first animal that is heterozygous for one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith or that does not include one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith and mating said first animal with a second animal to produce offspring. In certain embodiments, the method also comprises the step of selecting the second animal on the basis that it is heterozygous for one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith or it does not include one or more genetic variation which disrupts the GALNT2 gene and/or one or more genetic marker in linkage disequilibrium therewith. In one embodiment, the first animal and/or the second animal is selected if it has a G at a position corresponding to position 1312334 on chromosome 28 of Bos Taurus and/or one or more genetic marker in linkage disequilibrium therewith. In one

embodiment the first and/or second animal is selected if it is heterozygous for G at this position and/or one or more genetic marker in linkage disequilibrium thereof. In one embodiment the first and/or second animal is selected if it is homozygous for G at this position and/or one or more genetic marker in linkage disequilibrium thereof.

In certain embodiments, the first and/or second animal may be selected using a method of the invention as herein before described; for example, a method for determining whether or not an animal is a carrier for or has dwarfism, a method for determining whether or not an animal carries a genetic marker linked to dwarfism, a method of selecting or rejecting an animal, a method for estimating the worth of an animal. Accordingly, a method of breeding animals may involve first conducting a method of the first to fourth aspects of the invention described herein before, selecting the first and/or second animal based on the results of such method, and then mating the first and second animal.

The animals may be mated using any appropriate methods including naturally, artificial insemination or IVF. In some cases, individual gametes may be selected for use in the process. Such gametes may be selected using a method of the invention; for example, a method of the invention may be used to identify animals that have a desirable genotype and gametes from those animals selected for use in a breeding program or process. In one particular embodiment, a method of selecting or rejecting one or more cells or embryos (accordingly to the fifth aspect of the invention described herein before, for example) or a method of the first or second aspect of the invention could be used to select the first and/or second animal and their gametes used in IVF. Following selection of male and female gametes, the female gamete is fertilised in vitro. At the relevant time, one or more embryo is transferred to a gestational carrier.

In another embodiment, in vitro fertilisation of a female gamete may occur and then a method of the invention used to determine whether or not an embryo has a desired genotype and should be selected or rejected for further use in a breeding programme. This might occur where individual gametes, or animals from which they have been obtained or derived, have not been tested to determine if they carry one or more genetic marker linked to dwarfism, prior to fertilisation (accordingly, the invention should be taken to include methods of breeding where the first and/or second animal and/or gametes are not selected on the basis of such a test, but a resulting embryo or offspring is tested and selected on the basis it is not a carrier or does not have dwarfism). Alternatively, a method of the invention could be used where the individual gametes or animals from which they have been obtained or derived have been tested and selected on the basis of having a desirable genotype, for quality control purposes or to double check that the resulting embryos have the same desirable genotype.

Optionally, following mating of the animals, one or more method of the invention may be used to determine whether or not any offspring carries a marker linked to dwarfism, is a carrier or has dwarfism as herein before described. Such testing may occur at any time during the life of the offspring, including before birth; by way of example only, testing an embryo, a foetus, amniotic fluid, placenta, maternal blood, at birth.

In certain cases, cloning may be used to generate an animal. In such cases, the method may comprise identifying at least one first animal that has a desirable genotype (using one or more method as described herein) and using the nucleus or chromatin from one or more cell of that animal in a cloning procedure (such as somatic cell nuclear transfer, chromatin transfer techniques, and embryo splitting). Such cloning methods are described, for example, in Bovine somatic cell nuclear transfer Ross PJ and Cibelli, JB 2010. Methods in Molecular Biology 636: 155-177. At the relevant time during the cloning procedure, one or more embryo will be transferred to a gestational carrier.

In certain embodiments, a cloning procedure may utilise a cell derived from a cell line and a method of the invention may be used to select such a cell which is, or cell line whose cells are, capable of being used to generate an animal which is not a carrier of or will not have dwarfism. In one embodiment, the cell line may be an embryonic cell line. One or more cell of use in cloning may be selected using a method of the invention. For example, a method of the invention may be used to identify animals that have a desirable genotype and cells from those animals selected for use in a cloning process. Similarly, a method of the invention may be used to identify cells from a cell line or other source which have a desirable genotype and may be of use in generating an animal which is not a carrier of or will not have dwarfism. In one embodiment, a method of the first or fifth aspects of the invention may be used for such purposes. Methods of the invention (such as, for example, methods of the first to fifth aspects described herein before) could also be used to identify animals whose cells could be used to generate cell lines for cloning purposes.

Optionally, at various stages during the cloning procedure, one or more method of the invention may be used to determine whether or not any cloned animal carries a marker linked to dwarfism, is a carrier or has dwarfism as herein before described. Such testing may occur at any time during the life of the cloned animal, including before birth; by way of example only, testing of an embryo, a foetus, amniotic fluid, placenta, maternal blood, at birth.

In addition, a cloning method of the invention may involve selecting desirable cells without testing those cells or the animals or cell line from which they came for the presence or absence of a genetic variation which disrupts GALNT2. The cloning procedure can be initiated and then a method of the invention used to determine whether an embryo, foetus or animal resulting from the cloning procedure has a variation which disrupts GALNT2 and an embryo, foetus or animal selected where it has a desirable genotype.

Forming a Herd

The invention also provides methods for forming a herd of animals. Such methods may comprise determining whether or not an animal carries a genetic marker linked to dwarfism according to the first aspect of the invention described herein, determing whether or not an animal is a carrier for or has dwarfism according to the second aspect of the invention described herein, selecting or rejecting an animal according to the third aspect of the invention as described herein, and/or estimating the worth of an animal according to the fourth aspect of the invention as described herein. Animals may be selected or rejected for inclusion in the herd based on the results of those methods. Where an animal is identified not to carry a genetic marker linked to dwarfism, not to be a carrier for or to have dwarfism, and/or to have a desirable "worth", it may be selected for inclusion in the herd. In certain embodiments, animals identified to be carriers for dwarfism may be selected for inclusion in a herd, although, this may not be prefered.

Accordingly, methods of this aspect of the invention involving testing one or more animals in accordance with a method of any one or more of the first to fourth aspects of the invention, selecting animals having a desirable genotype or worth and forming a herd with the selected animals. The invention should also be taken to include a herd formed by such a method.

The herd of animals may be formed for any desirable reason. However, by way of example only, it may desirable to form a herd for beef farming or milk production.

Nucleic acids

The invention also provides nucleic acids carrying genetic markers of the invention. For example, isolated nucleic acids ecompassing a region of a GALNT2 gene in which the G/A polymorphism resides are encompassed by the invention. In one particular embodiment, the invention provides cDNA comprising the G/A polymorphism.

The invention also encompasses nucleic acids which can hydridise, preferably under stringent conditions (as herein before described), to a region of a GALNT2 gene in which the G/A polymorphism resides. Such nucleic acids may be used as probes or primers or otherwise in analysis of genetic markers of the invention, as herein before described.

Nucleic acids of the invention may have 100% sequence identity, homology or

complementarity to the relevant region of a GALNT2 gene (for example, the GALNT2 gene sequence provided in Figure 5), but may also have some sequence variation. For example, nucleic acids of the invention may have approximately 80%, approximately 90%, approximately 95% or approximately 99% sequence identity, homology or

complementarity.

These nucleic acids may be of any appropriate length. In one embodiment, they are at least 4 nucleotides in length, or at least 10, 20, 30, 40, 50, 60, 70, 80 or more nucleotides in length. In one embodiment, a nucleic acid of the invention will comprise the sequence provided in Figure 5.

Kits

The invention also relates to kits which are of use in a method of the invention.

In one embodiment, the kit comprises at least one or more reagents suitable for analysis of one or more genetic marker in accordance with the invention. Reagents suitable for analysis of one or more of the markers include one or more nucleic acid probes and/or primers as herein before described.

Reagents of use in processing samples for analysis may also be contained in the kits of the invention. The kits may also comprise one or more standard and/or other controls including nucleic acids whose sequence or genotype at a particular position is known. Further, kits of the invention can also comprise instructions for the use the components of the kit as well as printed charts or the like that could be used as standards against which results obtained from test samples could be compared. Reagents may be held in any suitable container.

EXAMPLES Example 1

52 animals where identified by farmers as being significantly smaller than normal. The animals were either Holstein-Friesian breed or crossbred (Holstein-Friesian x Jersey) and both males and females were represented in the dataset.

DNA samples from were taken from the animals and were genotyped over the Illumina Bovine SNP50 Genotyping Bead chip (50K)

(http://www.illumina.com/Documents/products/datasheets/da tasheet_bovine_snp50.pdf). Statistical analysis

In addition to the 52 "affected" animals, 650 control animals were genotyped over the Illumina Bovine SNP50 Genotyping Bead chip (50K). The control animals were a selection of bulls that were born in 2011 and were Holstein-Friesian. This ensured the case and control animals were age and breed matched.

The 50K genotypes were imputed to 711,678 SNP markers using Beagle software (Browning and Browning 2007). The 711,678 markers are a subset of the Illumina High- Density Genotyping panel (Matukumalli et al 2009) and are autosomal SNP markers as the phenotype has shown not to be sex-specific.

Whole genome association analysis was initially undertaken using PLINK software (Purcell 2007) using a case-control setting. PLINK undertakes a single SNP analysis. Plots were generated using -logio(p-values) that were generated from the PLINK analysis. Manhatten plots of the genome identified that chromosome 28 had the most significant effect (Figure 1).

On chromosome 28 the main peak was between 0-10Mb (Figure 2) with the 20 most significant markers listed in Table 1.

Table 1

-LOG10

Chromosome marker Bp p-value pvalue

28 BovineHD2800000584 1585173 2.82E-33 32.55036735

28 BovineHD2800000081 187472 7.37E-32 31.13265038

28 BovineHD2800000545 1421696 1.18E-30 29.92738252

28 BovineHD2800000854 2577607 5.13E-29 28.28954414

28 BovineHD4100018457 3777859 4.92E-27 26.30821148

28 BovineHD2800000552 1440986 4.92E-26 25.30838813

28 BovineHD2800001088 3662323 1.33E-25 24.87484417

28 BTA-64283-no-rs 8192374 1.95E-25 24.71018816

28 BovineHD2800002428 8194292 1.95E-25 24.71018816

28 BovineHD2800001341 4650836 1.38E-23 22.85949196

28 BovineHD2800000825 2421308 6.67E-23 22.17587417

28 BovineHD2800000777 2250654 2.44E-22 21.6129663

28 BovineHD2800001017 3354704 4.38E-20 19.35832763

28 BovineHD2800001596 5388745 6.16E-20 19.2104898

28 BovineHD2800001589 5365117 7.90E-20 19.10259286

28 BovineHD2800001845 6194710 2.01E-18 17.69766907

28 BovineHD2800000900 2893694 3.92E-18 17.40660316

28 BovineHD2800000566 1509105 6.86E-18 17.16367588

28 BovineHD2800001500 5168795 8.76E-18 17.05749589

28 BovineHD2800001501 5171247 1.62E-17 16.79155864

Haplotype analysis for the region on chromosome 28 was undertaken utilizing Beagle. In addition, to the case and control animals one more animal was included in the dataset. This animal had been implicated as the "founder" in the population based on common ancestry in the pedigrees of affected animals.

The BEAGLE haplotype analysis identified the same marker as the PLINK analysis as being the most significant (Table 2). Table 2:

Marker allelic_p

BovineHD2800000584 6.02E-22

BovineHD2800000081 4.19E-21

BovineHD2800000545 1.54E-20

BovineHD4100018457 2.51E-20

BovineHD2800000552 4.63E-20

BovineHD2800000854 1.37E-19

BovineHD2800001088 1.62E-19

BTA-64283-no-rs 7.93E-18

BovineHD2800002428 7.93E-18

BovineHD2800001341 2.06E-17

BovineHD2800001017 3.17E-17

BovineHD2800000825 8.93E-17

BovineHD2800000777 1.81E-16

BovineHD2800001327 3.12E-15

BovineHD2800001589 3.61E-15

BovineHD2800001596 4.04E-15

BovineHD2800001536 5.20E-15

BovineHD2800002672 5.71E-15

ARS-BFGL-NGS-36783 5.71E-15

BovineHD2800001533 5.77E-15

The haplotypes for the affected animals generated from Beagle at BovineHD2800000584 were investigated in conjunction with the "founder" animal and the control animals.

The hypothesis constructed was that the mode of transmission for the small calves was autosomal recessive. Under this hypothesis one would expect that for the haplotypes generated from Beagle at BovineHD2800000584:

Affecteds -homozygous for a particular haplotype

Founder - heterozygous with one of the haplotypes being that which the affecteds are homozygous for. Controls - not to be homozygous for the same haplotype as the affecteds.

Analysis of the haplotypes revealed that of the 52 affected animals, 20 were homozygous for the same haplotype. The hypothesis tested was whether the animals that were not homozygous for the haplotype were significant for another haplotype in the region or on another chromosome. To test this, the 32 non-homozygous animals and the control animals were analysed using PLINK. From this dataset there was no genetic signal detected in the genome (Figure 3) and more specifically on chromosome 28 there was no genetic signal (Figure 4).

Therefore based on this analysis it was determined that the 32 non-homozygous animals were not controlled by the genetic variation of interest and thus were removed from the dataset.

The "founder" was heterozygous with one of the haplotypes being that for which the 20 affecteds were homozygous.

In the 650 control animals there were two animals that were identified as being homozygous for the same haplotype as the affecteds. The control animals were assumed to be of normal stature as they were being considered for inclusion in a dairy cattle breeding programme and thus had been physically observed by the farmers to be of suitable physical appropriateness for inclusion. These two animals had not been purchased by the breeding organization and the owners of the two animals were contacted and both described the animals as being significantly smaller than the normal.

Narrowing the region

Taking the phased genotypes (haplotypes) of the 20 affecteds plus the two affected controls one can identify boundaries where the causative genetic variation is. This is on the basis of identifying where all 22 animals have the same haplotypes. Where one animal starts to have a different haplotype one can exclude that region as the animals are now not all homozygous. Utilising the common homozygozity the region that harbours the genetic variation was narrowed to 1.3 to 1.9 Mb on chromosome 28.

Sequencing

The inventors undertook whole genome sequencing for 502 animals. These 502 animals included the founder animal.

Library Construction: DNA was extracted and sent to Illumina FastTrack for library construction. Most libraries generated were 100 base pair paired end reads.

Sequencing: All libraries were sequenced on Illumina HiSeq. Twenty three phasel animals sequenced in 2011 ran on the V2 chemistry and were sequenced to an average 28X coverage. The 477 remainder animals ran on the newer MuminaV3 chemistry.

Mapping: Reads were aligned using the RTG version 2.7.2 (Real Time Genomics, Inc. San Bruno, CA) against version 3.1 of the University of Maryland Bovine genome reference assembly (Zimin et al 2009). Median mapping coverage (X) is 6, mean is 10X and maximum is 138X.

Genotypes: Genotypes were called using RTG pipelines. Identifying carrier sires that have been sequenced

The 502 animals were phased using Beagle for the chromosome 28 region and haplotypes as generated from Beagle at BovineHD2800000584 were analysed. Of the 502 animals, 40 (including founder) animals were identified as being heterozygous heterozygous with one of the haplotypes being that for which the 20 affecteds were homozygous.

The sequence data was interrogated for the region of interest between 1.3 to 1.9 Mb on chromosome 28 to find genetic variations in the sequence data where the 40 carriers are all heterozygous for the locus and the other 462 animals are homozygous for one of the variants. One variant was identified which also was identified to be a high impact variation based on snpEFF (Cingolani 2012). The variation sits in the GALNT2 gene at base pair 1312334 and is predicted to be a splice site variant. The gene GALNT2 is associated with blood lipid levels, insulin regulation, placental development, but the biological mode of action is yet to be determined.

Validating the SNP and haplotype

1330 animals were genotyped for the GALNT2 SNP and also had haplotypes generated using Beagle at BovineHD2800000584. Animals of all 3 genotype classes were genotyped and there was 100% concordance between the haplotype and genotype.

Genotype test

In Figure 5, the target SNP is illustrated with 250bp of upstream and downstream sequence with the target SNP indicated in square brackets eg NNNN[A/G]NNNN. Any other known polymorphisms are indicated with a trailing underscore eg NNNNNT_NNNNN where the is the other known polymorphism. The target SNP position references the UMD3.1 genome.

Example 2

Herds of cattle or individual animals have blood or tissue samples (for example, an ear punch) taken and DNA extracted for genotyping to establish whether an animal carries a marker linked to dwarfism and in one embodiment whether it carries one or two copies of a marker linked to dwarfism. Once the genotype (and for example, carrier status for dwarfism) of individual animals is established, then decisions can be made regarding culling animals from the herd or regarding their use in breeding programs. In the latter cases, decisions could be made, for example, to proceed with breeding from a heterozygous sire but only to cows that were not carriers of the dwarfism trait, thus avoiding expression of the dwarf characteristics. Another example, could be culling of a cow or bull shown to have two copies of the genetic marker.

The invention has been described herein, with reference to certain preferred embodiments, in order to enable the reader to practice the invention without undue experimentation. However, a person having ordinary skill in the art will readily recognise that many of the components and parameters may be varied or modified to a certain extent or substituted for known equivalents without departing from the scope of the invention. It should be appreciated that such modifications and equivalents are herein incorporated as if individually set forth. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. Furthermore, titles, headings, or the like are provided to enhance the reader's comprehension of this document, and should not be read as limiting the scope of the present invention.

The entire disclosures of all applications, patents and publications, cited above and below, if any, are hereby incorporated by reference. However, the reference to any applications, patents and publications in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

Throughout this specification and any claims which follow, unless the context requires otherwise, the words "comprise", "comprising" and the like, are to be construed in an inclusive sense as opposed to an exclusive sense, that is to say, in the sense of "including, but not limited to".

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S R Browning and B L Browning (2007) Rapid and accurate haplotype phasing and missing data inference for whole genome association studies using localized haplotype clustering. Am J Hum Genet 81:1084-1097.

Matukumalli LK, Lawley CT, Schnabel RD, Taylor JF, Allan MF, Heaton MP, O'Connell J, Moore SS, Smith TP, Sonstegard TS, Van Tassell CP.

PLoS One. 2009;4(4):e5350. doi: 10.1371/journal.pone.0005350. Epub 2009 Apr 24.

Purcell S, Neale B, Todd-Brown K, Thomas L, Ferreira MAR, Bender D, Mailer J, Sklar P, de Bakker PIW, Daly MJ & Sham PC (2007) PLINK: a toolset for whole-genome association and population-based linkage analysis. American Journal of Human Genetics, 81.

Zimin A, Delcher A, Florea L, Kelley D, Schatz M, Puiu D, Hanrahan F, Pertea G, Van Tassell C, Sonstegard T, Marcais G, Roberts M, Subramanian P, Yorke J , Salzberg S (2009) Genome Biology 10:R42

A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain wll l8; iso-2; iso-3.", Cingolani P, Platts A, Wang le L, Coon M, Nguyen T, Wang L, Land S J, Lu X, Ruden DM. Fly (Austin). 2012 Apr-Jun;6(2):80-92. PMJX>: 22728672 [PubMed - in process]