Genetic markers and methods related thereto

Abstract

The invention broadly relates to methods for marker assisted identification and / or selection of subjects having one or more genetic markers associated with a phenotype of interest. More particularly, the invention relates to methods of marker assisted identification and / or selection or rejection of non-human subjects, and particularly bovine subjects, for one or more traits associated with productivity and / or worth, for example fertility such as sperm and / or semen quality, by determining the presence of one or polymorphisms associated with the one or more productivity and / or worth traits.

Description

[0001] GENETIC MARKERS AND METHODS RELATED THERETO

[0002] TECHNICAL FIELD

[0003] The invention broadly relates to methods for marker assisted identification and / or selection of subjects having one or more genetic markers associated with a phenotype of interest. More particularly, the invention relates to methods of marker assisted identification and / or selection or rejection of non-human subjects for one or more traits associated with productivity and / or worth by determining the presence of one or polymorphisms associated with the one or more productivity and / or worth traits.

[0004] BACKGROUND OF THE INVENTION

[0005] The following includes information that may be useful in understanding the present inventions. It is not an admission that any of the information provided herein is prior art, or relevant, to the presently described or claimed inventions, or that any publication or document that is specifically or implicitly referenced is prior art. Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0006] The genetic bases of animal production are of great significance to agriculture, food production, food security, and the meat and dairy industries. Accordingly, production animals, and particularly dairy animals, are assessed to determine their suitability (or otherwise) for a particular purpose including breeding, inclusion in a herd, and meat and milk production, on the basis of a number of different traits relating to productivity and worth. In New Zealand, for example, up to 25 different traits are currently assessed. These traits include production traits (also referred to as production efficiency traits) such as milk protein yield, milk fat yield, milk volume, and liveweight, robustness traits such as fertility, functional survival, gestation length, body condition score, udder overall, and somatic cell score (SCS), also referred to as somatic cell count (SCC), and traits other than production (TOP) such as milking speed and temperament, for example. In various combinations, assessment of these traits is used to determine or estimate an animal's value or worth to a breeder or a farmer. In New Zealand, for example, assessment of and scoring an animal on the basis of a combination of such traits is used to determine or estimate Breeding Worth and / or Production Worth, for example. Similar evaluation systems are used in other markets.

[0007] Traditional methods of assessment, herd formation and breeding programmes for livestock have focussed on assessment and selection of animals based on the presence of certain phenotypic characteristics. More recent methods of assessment and selection have incorporated criteria based on genotypic characteristics associated with one or more desirable traits, or assessment and rejection of animals based on genotypic characteristics associated with one or more undesirable traits. Selection or rejection of animals on the basis of genotypic characteristics allows for earlier, more certain, and more specific identification of animals of interest than does selection on the basis of phenotypic characteristics.

[0008] A number of genetic markers are known to be associated (either positively or negatively) with productivity and / or worth in dairy animals and are used to assess animals, calculate breeding values, determine Breeding Worth, production efficiency, and / or robustness, and to make or inform selection decisions. However, there is an ongoing need to identify markers which can be used to improve identification and selection methods, for example to increase the speed and accuracy of assessment and selection methods.

[0009] Marker assisted selection, which provides the ability to follow a specific favourable genetic allele, involves the identification of a molecular marker or markers, typically DNA markers, that segregate(s) with a gene or group of genes associated with or which in part defines a trait. DNA markers have several advantages. They are relatively easy to measure and are unambiguous, and as DNA markers are usually co-dominant, heterozygous and homozygous animals can be distinctively identified. Once a marker system is established, selection decisions are able to be made easily, as DNA markers can be assayed at any time after a DNA-containing sample has been collected, for example from an individual subject, whether embryonic, infant or adult.

[0010] The present invention relates to one or more methods for marker assisted identification and / or selection of a non-human subject having one or more desired traits, such as one or more productivity and / or worth traits including for example desired fertility, and / or for identification and / or rejection of a non-human subject having one or more undesired traits, such as undesired fertility, and / or to one or more subjects (including one or more non-human animals, and / or one or more cells, nuclei, or embryos) identified or selected using such methods, and / or to one or more related methods such as a method of selecting and forming a herd of animals identified by a method as described herein, or to at least providing the public with a useful choice.

[0011] SUMMARY OF THE INVENTION

[0012] This invention relates to the determination of the role of one or more polymorphisms in the Bos taurus genome associated with one or more productivity and / or worth traits, and particularly, the determination of one or more variant alleles at these polymorphisms that are associated with a deleterious effect on one or more productivity and / or worth traits, particularly if present in a homozygous state.

[0013] The invention further relates to the identification of one or more polymorphisms in bovine associated with one or more deleterious effects on productivity and / or worth in bovine, and in particular a deleterious effect on one or more of disease or disease risk in bovine, such as an increased incidence of a disease or condition or an increased risk of a disease or condition, including a late onset disease or condition.

[0014] In various examples, the deleterious effect is a deleterious effect on fertility.

[0015] In one example, the invention relates to the determination of one or more variant alleles at these polymorphisms that are associated with a deleterious effect particularly if present in a homozygous state.

[0016] In specifically contemplated examples, the invention relates to the association for the first time of the variant allele at one or more of the polymorphisms identified in Table 1 herein with one or more deleterious effects on productivity and / or worth, and particularly on fertility, in bovine.

[0017] In particular examples, the invention relates to the association of the variant allele at one or more of the polymorphisms identified in Table 1 herein with a deleterious effect on fertility, for example, a deleterious effect on sperm quality and / or on sperm quantity, such as but not limited to decreased sperm motility, and / or decreased sperm production, and / or abnormal sperm morphology.

[0018] This gives rise to numerous, and separate, aspects of the invention.

[0019] In one aspect the invention relates to a method of determining the genetic status of a nonhuman animal subject, cell, embryo, gamete, or nucleus, the method comprising determining the allelic profile of the subject, cell, embryo, gamete, or nucleus, and determining the genetic status of the subject, cell, embryo, gamete, or nucleus with respect to one or more productivity and / or worth traits, or with respect to capability of producing progeny that will have one or more productivity and / or worth traits, on the basis of the allelic profile.

[0020] In one example, the one or more productivity and / or worth traits is a robustness trait. In one example, the robustness trait is fertility, for example, a fertility trait.

[0021] In one example, the fertility trait is sperm quality. In one example, the fertility trait is sperm quantity.

[0022] In various examples, fertility is selected from the group consisting of sperm motility, sperm production, and sperm morphology - for example, the fertility trait is selected from the group consisting of sperm motility, sperm production, and sperm morphology. Accordingly, a deleterious effect on fertility will in certain examples comprise decreased sperm motility, or decreased sperm production, or abnormal sperm morphology, or any combination thereof.

[0023] In various examples, the sperm motility trait is selected from the group consisting of % total motile, % progressive motile, % non-motile (i.e. , 100 - total motile), and speed. For example, a deleterious effect on sperm motility and / or a deleterious effect on a sperm motility trait is selected from the group consisting of a deleterious effect on % total motile sperm, a deleterious effect on % progressive motile sperm, a deleterious effect on % non-motile sperm, and a deleterious effect on sperm speed.

[0024] In various examples, the sperm production trait is selected from the group consisting of ejaculate volume, sperm concentration, total sperm number, and total live sperm (e.g., total sperm number multiplied by % total motile). For example, a deleterious effect on sperm production and / or a deleterious effect on a sperm production trait is selected from the group consisting of a deleterious effect on ejaculate volume, a deleterious effect on sperm concentration, a deleterious effect on total sperm number, and a deleterious effect on total live sperm.

[0025] In various examples, the sperm morphology trait is selected from the group consisting of head morphology, and tail morphology. For example, a deleterious effect on sperm morphology and / or a deleterious effect on a sperm morphology trait is selected from the group consisting of a deleterious effect on head morphology, and a deleterious effect on tail morphology.

[0026] In various examples, head morphology comprises one or more of head shape, acrosome, free heads, and / or vacuoles. For example, a deleterious effect on head morphology or a deleterious effect on a head morphology trait is selected from the group consisting of a head shape abnormality, abnormal acrosome, increased free heads, and increased or abnormal vacuoles. In various examples, tail morphology comprises one or more of mid-piece morphology, principal piece morphology, and / or cytoplasmic droplets. For example, a deleterious effect on tail morphology or a deleterious effect on a tail morphology trait is selected from the group consisting of a mid-piece abnormality, a principal piece abnormality, and the presence of or increased cytoplasmic droplets.

[0027] Accordingly, in one example, the genetic status of the subject, cell, embryo, gamete, or nucleus with respect to one or more productivity and / or worth traits is genetic status with respect to fertility.

[0028] Accordingly, in one example the method is a method of determining the genetic status of a nonhuman animal subject, cell, embryo, gamete, or nucleus, the method comprising determining the allelic profile of the subject, cell, embryo, gamete, or nucleus, and determining the genetic status of the subject, cell, embryo, gamete, or nucleus with respect to fertility, or with respect to capability of producing progeny that will have desired fertility, on the basis of the allelic profile.

[0029] In another example, the genetic status of a bovine subject, cell, embryo, gamete, or nucleus with respect to capability of producing progeny that will have one or more productivity and / or worth traits is capability of producing progeny that will have desired fertility.

[0030] Any of the examples described herein can relate to any of the aspects presented herein.

[0031] In various examples, the genetic status of the non-human subject, cell, embryo, gamete, or nucleus is determined to facilitate the identification and / or selection or rejection of a subject, cell, embryo, gamete, or nucleus having one or more desired or undesired traits, such as one or more desired or undesired productivity and / or worth traits, such as those set out above.

[0032] Accordingly, in one example the method is for identifying, and / or selecting or rejecting a non- human animal subject, cell, embryo, gamete, or nucleus, the method comprising determining the allelic profile of the non-human animal subject, cell, embryo, gamete, or nucleus, and identifying, and / or selecting or rejecting said non-human animal subject, cell, embryo, gamete, or nucleus, on the basis of the determination.

[0033] In one example, the method is for identifying and / or selecting a non-human animal subject, cell, embryo, gamete, or nucleus, the method comprising determining the allelic profile of the non- human animal subject, cell, embryo, gamete, or nucleus, and identifying and / or selecting said non- human animal subject, cell, embryo, gamete, or nucleus having one or more desired productivity and / or worth traits.

[0034] In one example, the method is for identifying and / or rejecting a non-human animal subject, cell, embryo, gamete, or nucleus, the method comprising determining the allelic profile of the non- human animal subject, cell, embryo, gamete, or nucleus, and identifying and / or rejecting said non- human animal subject, cell, embryo, gamete, or nucleus having one or more undesired productivity and / or worth traits, on the basis of the determination.

[0035] Accordingly, in one example the invention relates to a method for identifying, and / or selecting or rejecting a bovine subject, cell, embryo, gamete, or nucleus, the method comprising determining the allelic profile of the bovine subject, cell, embryo, gamete, or nucleus, and identifying and / or selecting said bovine subject, cell, embryo, gamete, or nucleus having one or more desired productivity and / or worth traits, or that is capable of producing progeny that will have one or more desired productivity and / or worth traits, or rejecting said bovine subject, cell, embryo, gamete, or nucleus having one or more undesired productivity and / or worth traits, or that is capable of producing progeny that will have one or more undesired productivity and / or worth traits, on the basis of the determination.

[0036] As used herein, the phrase "allelic profile" contemplates data indicative of the presence or absence of one or more alleles at one or more polymorphisms identified in any one of Tables 1 - 3 herein. In certain examples, the allelic profile comprises data indicative of the presence or absence of one or more alleles at one or more polymorphisms which affect expression from a gene in which one or more of the polymorphisms identified in any one of Tables 1 - 3 is present, or which are associated with variation in the expression from such a gene or with variation in the expression or activity of a gene product of such a gene.

[0037] In specifically contemplated examples, the allelic profile comprises data indicative of the presence or absence of one or more alleles at one or more polymorphisms associated with a beneficial or deleterious effect on one or more productivity and / or worth traits.

[0038] For example, in various specifically contemplated examples, the allelic profile of the subject, cell, embryo, gamete, or nucleus comprises data indicative of: a) the presence or absence of the variant allele at one or more of the polymorphisms presented in any one or more of Tables 1 - 3; and / or b) the presence or absence of the wild-type allele at one or more of the polymorphisms presented in any one or more of Tables 1 - 3; and / or c) the presence or absence of a polymorphism in linkage disequilibrium with any one or more of the alleles referred to in (a) or (b) above, such as the presence or absence of a polymorphism in greater than R2= 0.7 linkage disequilibrium, such as 100% linkage disequilibrium R2= 1.0), with any one or more of the alleles referred to in (a) or (b) above; and / or d) any combination of any two or more of a) to c) above.

[0039] In various examples, the polymorphism in linkage disequilibrium with any one or more of the alleles referred to in (a) or (b) above is one or more of the polymorphisms presented in any one or more of Tables 4 - 9.

[0040] In other examples, the allelic profile comprises data indicative of the presence or absence of one or more alleles at one or more polymorphisms in the promoter of a gene in which one or more of the polymorphisms identified in any one of Tables 1 - 3 is present, or in a regulatory region of a gene in which one or more of the polymorphisms identified in any one of Tables 1 - 3 is present, or in an intron of a gene in which one or more of the polymorphisms identified in any one of Tables 1 - 3 is present.

[0041] In one example, the allelic profile comprises data indicative of the presence or absence of one or more alleles which affect expression from a gene in which one or more of the polymorphisms identified in any one of Tables 1 - 3 is present. In one example, the allelic profile comprises data indicative of the presence or absence of one or more alleles which affect the expression or activity of a gene product of a gene in which one or more of the polymorphisms identified in any one of Tables 1 - 3 is present.

[0042] In one example, the allelic profile comprises data indicative of the presence or absence of one or more alleles which are associated with variation in the expression from a gene in which one or more of the polymorphisms identified in any one of Tables 1 - 3 is present. In one example, the allelic profile comprises data indicative of the presence or absence of one or more alleles which are associated with variation in the expression or activity of a gene product of a gene in which one or more of the polymorphisms identified in any one of Tables 1 - 3 is present.

[0043] In one example, the allelic profile comprises data indicative of the presence or absence of the T allele at the Chr2: 101396615 G>T (rs433745406) polymorphism in the bovine SPAG16 gene. In various examples, the T allele at the Chr2: 101396615 G>T (rs433745406) polymorphism in the bovine SPAG16 gene is associated with one or more deleterious fertility traits selected from the group consisting of decreased sperm motility, for example, decreased Forward Progress Motility, and / or decreased Live Sperm Percentage Motility; and decreased sperm quality, for example increased culling and / or increased non-return rate.

[0044] In one example, the allelic profile comprises data indicative of the presence or absence of the A allele at the Chr4:30657607 G>A (rs457253516) polymorphism in the bovine DNAH11 gene. In various examples, the A allele at the Chr4:30657607 G>A (rs457253516) polymorphism in the bovine DNAH11 gene is associated with one or more deleterious fertility traits selected from the group consisting of decreased sperm motility, for example decreased Forward Progress Motility and / or decreased Live Sperm Percentage Motility; increased abnormal sperm morphology, for example increased abnormal Tail Percentage, increased Distal Reflex without Protoplasmic Droplet, increased Distal Reflex with Protoplasmic Droplet, decreased Normal Percentage; and decreased sperm quality, for example increased culling.

[0045] In one example, the allelic profile comprises data indicative of the presence or absence of the C allele at the Chr4:30411361 CA>C (rs465963699) polymorphism in the bovine DNAH11 gene. In various examples, the C allele at the Chr4:30411361 CA>C (rs465963699) polymorphism in the bovine DNAH11 gene is associated with one or more deleterious fertility traits selected from the group consisting of decreased sperm motility, for example decreased Forward Progress Motility and / or decreased Live Sperm Percentage Motility; increased abnormal sperm morphology, for example increased Distal Reflex with Protoplasmic Droplet, decreased Normal Percentage; and decreased sperm quality, for example increased culling.

[0046] In one example, the allelic profile comprises data indicative of the presence or absence of the A allele at the Chr8:98463916 G>A (rs459888392) polymorphism in the bovine ACTL7B gene. In various examples, the A allele at the Chr8:98463916 G>A (rs459888392) polymorphism in the bovine ACTL7B gene is associated with one or more deleterious fertility traits selected from the group consisting of increased abnormal sperm morphology, for example increased abnormal Acrosome Percentage, abnormal Head Percentage, decreased Normal Percentage; and decreased sperm quality, for example increased culling. In one example, the allelic profile comprises data indicative of the presence or absence of the T allele at the Chrl7:63363268 TG>T (rs434709552) polymorphism in the bovine TCHP gene. In various examples, the T allele at the Chrl7:63363268 TG>T (rs434709552) polymorphism in the bovine TCHP gene is associated with one or more deleterious fertility traits selected from the group consisting of decreased sperm motility, for example decreased Forward Progress Motility and / or decreased Live Sperm Percentage Motility, decreased Normal Percentage; decreased Initial Concentration; and decreased sperm quality, for example increased culling and / or increased non-return rate.

[0047] In one example, the allelic profile comprises data indicative of the presence or absence of the C allele at the Chr25: 1194914 G>C (rs433354487) polymorphism in the bovine IFT140 gene. In various examples, the C allele at the Chr25: 1194914 G>C (rs433354487) polymorphism in the bovine IFT140 gene is associated with one or more deleterious fertility traits selected from the group consisting of increased abnormal sperm morphology, for example increased abnormal Acrosome Percentage, decreased Normal Percentage; and decreased sperm quality, for example increased culling and / or increased non-return rate.

[0048] It will further be appreciated that the allelic profile may comprise information correlating the presence or absence of one or more polymorphisms as described above with one or more productivity and / or worth traits.

[0049] In one example, the allelic profile is determined using nucleic acid obtained from said subject, cell, embryo, gamete, or nucleus, preferably DNA obtained from said subject, cell, embryo, gamete, or nucleus, or alternatively, said allelic profile is determined using RNA obtained from said subject, cell, embryo, gamete, or nucleus.

[0050] Conveniently, in said method the presence or absence of nucleic acid encoding a wild-type gene product of a gene in which one or more of the polymorphisms identified in any one of Tables 1 - 3 is present in said subject, cell, embryo, gamete, or nucleus is determined, directly or indirectly, for example using an expressed gene product.

[0051] Alternatively, in said method the presence or absence of at least one nucleotide difference from the nucleotide sequence encoding a wild-type gene product of a gene in which one or more of the polymorphisms identified in any one of Tables 1 - 3 is present in said subject, cell, embryo, gamete, or nucleus or from the wild-type gene in which one or more of the polymorphisms identified in any one of Tables 1 - 3 is present is determined, directly or indirectly.

[0052] In various examples, the method comprises determining an allelic profile of a bovine subject, such as a bovine, bovine cell, bovine embryo, or nucleus, the allelic profile comprising information relating to the identity of one or more alleles present at any one or more of the polymorphisms identified in Tables 1 - 3 herein.

[0053] More specifically, in various examples of said method, the presence or absence of one or more wild-type alleles and / or one or more variant alleles at one or more of the polymorphisms identified in any one of Tables 1 - 3 is determined, directly or indirectly.

[0054] For example, the presence of a wild-type allele at one of the polymorphisms identified in any one of Tables 1 - 3 is determined directly, for example by sequencing at the polymorphism. In another example, the presence of a wild-type allele at one of the polymorphisms identified in any one of Tables 1 - 3 is determined indirectly, for example by using a polymorphism in linkage disequilibrium with said wild-type allele. In various examples, a polymorphism in linkage disequilibrium is selected from one or more of the polymorphisms presented in any one or more of Tables 4 - 9.

[0055] In another example, the presence of a variant allele at one of the polymorphisms identified in any one of Tables 1 - 3 is determined directly, for example by sequencing at the polymorphism. In another example, the presence of a variant allele at one of the polymorphisms identified in any one of Tables 1 - 3 is determined indirectly, for example by using a polymorphism in linkage disequilibrium with said variant allele.

[0056] In various examples, indirect determination includes determination via imputation, and / or determination via a marker in linkage disequilibrium with the target allele, genotype, haplotype, or polymorphism.

[0057] In one example, the method includes ascertaining, from a sample of material containing DNA obtained from the subject, cell, embryo, gamete, or nucleus, whether a sequence of the DNA encoding a protein "(A)" having biological activity of the wild-type protein encoded by a gene comprising one of the polymorphisms identified in Table 1 is present, or whether a sequence of the DNA encoding an allelic protein "(B)" at least partially lacking the activity of (A) is present, or whether a sequence of the DNA encoding (A) and a sequence of the DNA encoding (B) are both present. The absence of the DNA encoding (A) and the presence of the DNA encoding (B) indicates an association with decreased production efficiency and / or robustness, such as decreased production efficiency. The reverse association holds true, where the presence of the DNA encoding (A) and the absence of the DNA encoding (B) indicates an association with increased production efficiency and / or robustness, such as increased production efficiency.

[0058] As used herein, biological activity of a wild-type protein refers to both expression levels and activity characteristic of the protein encoded by the wild-type gene - that is, the gene comprising the wild-type allele at the polymorphism identified in Table 1.

[0059] In another example, the method includes ascertaining, from a sample of material containing DNA obtained from the subject, cell, embryo, gamete, or nucleus, whether the wild-type gene sequence is present. In still another example, the method includes ascertaining, from a sample of material containing DNA obtained from the subject, cell, embryo, gamete, or nucleus, the expression or activity of a gene product, for example by determining the presence or absence of one or more polymorphisms associated with decreased or increased expression or activity, for example one or more promoter polymorphisms associated with increased or decreased expression, or one or more polymorphisms associated with increased or decreased activity.

[0060] In another example, the method includes ascertaining whether mRNA encoding a protein "(A)" having biological activity of the wild-type protein encoded by a gene comprising one of the polymorphisms identified in Table 1 is present, or whether mRNA encoding a protein "(B)" at least partially lacking the activity of (A) is present, or whether mRNA encoding (A) and mRNA encoding (B) are both present. The absence of the mRNA encoding (A) and the presence of the mRNA encoding (B) again indicates an association with decreased production efficiency and / or robustness, such as decreased production efficiency. The reverse association again holds true, where the presence of the mRNA encoding (A) and the absence of the mRNA encoding (B) again indicates an association with increased production efficiency and / or robustness, such as increased production efficiency.

[0061] In another example, the method includes ascertaining the amount of mRNA encoded by a gene comprising one of the polymorphisms identified in Table 1 present in a sample of material containing mRNA obtained from the subject, cell, embryo, gamete, or nucleus. In another example, the method includes ascertaining the expression profile of a gene comprising one of the polymorphisms identified in Table 1, for example including ascertaining the mRNA expression profile of a gene comprising one of the polymorphisms identified in Table 1 in the subject, cell, embryo, gamete, or nucleus.

[0062] In yet a further example, the allelic profile is determined with reference to the protein encoded by a gene comprising one of the polymorphisms identified in Table 1, for example, the amino acid sequence of expressed protein obtained from said subject, cell, embryo, gamete, or nucleus. In another example, the allelic profile is determined with reference to the size, amount or activity of protein obtained from said subject, cell, embryo, gamete, or nucleus.

[0063] In one example, the method includes ascertaining whether a protein "(A)" having biological activity of a wild-type protein encoded by a gene comprising one of the polymorphisms identified in Table 1 is present, or whether a protein "(B)" at least partially lacking the activity of (A), for example completely lacking the activity of (A), is present, or whether (A) and (B) are both present. The absence of (A) and the presence of (B) again indicates an association with decreased production efficiency and / or robustness, such as decreased production efficiency. The reverse association again holds true, where the presence of (A) and the absence of (B) again indicates an association with increased production efficiency and / or robustness, such as increased production efficiency.

[0064] In another example, the method includes ascertaining the amount or activity of the wild-type protein encoded by a gene comprising one of the polymorphisms identified in Table 1 is present in a sample of material containing protein obtained from the subject, cell, embryo, gamete, or nucleus.

[0065] Accordingly, in one example the invention relates to a method for identifying, and / or selecting or rejecting a non-human animal subject, cell, embryo, gamete, or nucleus, the method comprising determining the expression or activity of a gene product encoded by a gene comprising one of the polymorphisms identified in Table 1, and identifying, and / or selecting said non-human animal subject, cell, embryo, gamete, or nucleus having one or more desired productivity and / or worth traits, or identifying and / or rejecting said non-human animal subject, cell, embryo, gamete, or nucleus having one or more undesired productivity and / or worth traits, on the basis of the determination.

[0066] Accordingly, in one example the invention relates to a method for identifying, and / or selecting or rejecting a bovine subject, cell, embryo, gamete, or nucleus, the method comprising determining the expression or activity of a gene product encoded by a gene comprising one of the polymorphisms identified in Table 1 is present, and identifying and / or selecting said bovine subject, cell, embryo, gamete, or nucleus having one or more desired productivity and / or worth traits, or that is capable of producing progeny that will have one or more desired productivity and / or worth traits, or rejecting said bovine subject, cell, embryo, gamete, or nucleus having one or more undesired productivity and / or worth traits, or that is capable of producing progeny that will have one or more undesired productivity and / or worth traits, on the basis of the determination.

[0067] It will be appreciated that methods comprising determining the expression or activity of a gene product encompass determining expression from or of the gene.

[0068] In one example, expression or activity of the gene product is determined using mRNA, for example by determining the presence or amount of mRNA encoded by a gene comprising one of the polymorphisms identified in Table 1. In other examples, expression or activity of a gene product is determined using protein, for example by determining the amount of protein encoded by a gene comprising one of the polymorphisms identified in Table 1, or by determining the activity of a protein, for example the activity of a protein encoded by a gene comprising one of the polymorphisms identified in Table 1 that is present in a sample obtained from the subject, cell, embryo, gamete, or nucleus, or by determining the amount of a protein variant, derivative or fragment.

[0069] Likewise, it will be understood that reference to a gene product includes full length gene products and precursors, fragments, and isoforms thereof. Accordingly, reference to a protein encoded by a gene comprising one of the polymorphisms identified in Table 1 includes full length protein and precursors, fragments, and isoforms thereof.

[0070] In the context of this disclosure, specifically contemplated proteins include any one of the variant proteins encoded by a gene in which one or more of the polymorphisms identified in any one of Tables 1 - 3 is present, such as a variant protein encoded by a gene comprising one of the polymorphisms identified in Table 1 and comprising the corresponding amino acid(s) substitution as set out in Table 1.

[0071] Accordingly, in the context of this invention, the presence of such a variant protein and / or of a gene encoding said variant protein, are examples of data comprising an allelic profile associated with a deleterious effect on one or more productivity and / or worth traits, such as production efficiency and / or robustness, as contemplated herein.

[0072] In still other examples, the expression or activity of a gene product encoded by a gene comprising one of the polymorphisms identified in Table 1 is determined using DNA, preferably by determining the presence or absence of one or more polymorphisms described herein, or one or more polymorphisms associated with decreased or increased expression or activity of said gene product, for example one or more or the polymorphisms associated with increased or decreased expression or activity as described herein.

[0073] Thus, in one aspect the invention relates to a method for determining the genotype of a nonhuman animal subject at any one or more polymorphisms identified in any one of Tables 1 - 3, as may be desirable to know for breeding purposes.

[0074] In one example, the method includes ascertaining, with reference to a sample of material containing nucleic acid obtained from the subject and uncontaminated by heterologous nucleic acid, whether the sample contains (I) nucleic acid molecule comprising a wild-type gene in which one or more of the polymorphisms identified in any one of Tables 1 - 3 is present, or nucleic acid encoding a gene product such as a protein having biological activity of a wild-type protein encoded by a gene comprising one of the polymorphisms identified in Table 1; and optionally ascertaining whether the sample contains an (ii) allelic nucleic acid molecule comprising a variant of said gene or encoding a variant gene product such as a protein lacking biological activity of the wild-type protein.

[0075] In another example, the method includes ascertaining, with reference to a sample of material containing protein obtained from the subject and uncontaminated by heterologous protein, whether the sample contains (i) a protein having biological activity and / or amino acid sequence and / or size of a wild-type protein encoded by a gene comprising one of the polymorphisms identified in Table 1; and optionally ascertaining whether the sample contains (ii) a protein lacking biological activity and / or amino acid sequence and / or size of said wild-type protein.

[0076] In a further aspect, the invention relates to a method of determining genetic status of a nonhuman animal subject with respect to one or more productivity and / or worth traits which comprises providing the allelic profile of the subject, optionally together with the allelic profile of the subject at one or more genetic loci associated with one or more productivity and / or worth traits, and determining the genetic status on the basis of the allelic profile(s).

[0077] In various examples, the allelic profile comprises data indicative of the presence or absence of one or more alleles at one or more polymorphisms identified in any one of Tables 1 - 3.

[0078] In one example, the one or more genetic loci is one or more polymorphisms in one or more genes associated with one or more productivity and / or worth traits.

[0079] In one example, the allelic profile comprises data indicative of the presence or absence of the variant allele at one or more polymorphisms identified in any one of Tables 1 - 3.

[0080] In one example, the allelic profile comprises data indicative of the presence or absence of the variant allele at one or more polymorphisms identified in Table 2.

[0081] In one example, the allelic profile comprises data indicative of the presence or absence of the variant allele at two or more polymorphisms identified in Table 2. In one example, the allelic profile comprises data indicative of the presence or absence of the variant allele at three or more polymorphisms identified in Table 2. In one example, the allelic profile comprises data indicative of the presence or absence of the variant allele at each of the polymorphisms identified in Table 2.

[0082] In one example, the allelic profile comprises data indicative of the presence or absence of the variant allele at one or more polymorphisms identified in Table 3. In one example, the allelic profile comprises data indicative of the presence or absence of the variant allele at two or more polymorphisms identified in Table 3.

[0083] In one example, the genetic status of the non-human subject, cell, embryo, gamete, or nucleus is determined to facilitate the estimation of the worth of the non-human subject, cell, embryo, gamete, or nucleus and / or its progeny.

[0084] Accordingly, the invention relates to a method for estimating the worth of a non-human animal subject, cell, embryo, gamete, or nucleus, or any progeny thereof, the method comprising determining the allelic profile of the non-human animal subject, cell, embryo, gamete, or nucleus, and estimating the worth of the animal, cell, embryo, gamete, or nucleus and / or any progeny thereof on the basis of the determination. In one example, the allelic profile is determined for the purpose of selecting or rejecting a nonhuman animal for production purposes. In one example, the allelic profile is determined for the purpose of selecting or rejecting a non-human animal, such as a bovine, for milking purposes. In one example, the allelic profile is determined for the purpose of selecting or rejecting a non-human animal for meat production, such as a bovine for beef farming. In another example, the allelic profile is determined for the purpose of selecting or rejecting a non-human animal for breeding purposes. In one example, the allelic profile is determined for the purpose of selecting or rejecting a non-human animal for inclusion in a herd.

[0085] In other examples the allelic profile is determined for the purpose of selecting or rejecting a non-human cell, embryo, gamete, or nucleus for use in cloning a non-human animal and / or breeding a non-human animal. In one example, breeding an animal may involve IVF.

[0086] Accordingly, the invention relates to a method for breeding or producing a non-human animal, wherein the method comprises selecting: a) at least a first non-human animal; and / or b) a first non-human gamete; and / or c) a non-human embryo; and / or d) one or more non-human cells or nuclei; wherein the selection is performed on the basis of allelic profile.

[0087] In one example, the method comprises mating said first animal with a second non-human animal.

[0088] In one example, the method comprises fusing said first gamete with a second non-human gamete to form a zygote. In one example, the zygote is maintained under conditions conducive to cellular development and / or differentiation, for example, as are typically employed in IVF.

[0089] In various examples, the second non-human animal or second gamete has been selected on the basis of its allelic profile.

[0090] In one example, the embryo is maintained under conditions conducive to development and / or differentiation. In one example, the method comprises transferring the embryo to a gestational carrier.

[0091] In one example, the method comprises nuclear transfer of said one or more nuclei. In one example, the one or more selected cells or nuclei are used to clone an animal.

[0092] Accordingly, the invention also relates to a method of cloning a non-human animal, the method comprising at least the step of selecting one or more non-human cells on the basis of its allelic profile, and maintaining the one or more cells under conditions conducive to cellular development, replication, and / or differentiation or subsequent cellular development, replication, and / or differentiation.

[0093] In a further aspect, the invention relates to a method of forming a herd of non-human animals, the method comprising selecting two or more animals at least in part on the basis of their allelic profile as described herein, and forming a herd comprising the animals thereby selected.

[0094] In various examples, the herd is segregated from animals a) for which the allelic profile is not known, and / or b) having a different allelic profile from the selected herd animals. In one example, the different allelic profile is a different genotype at one or more polymorphisms identified in any one of Tables 1 - 3.

[0095] In a further aspect, the invention relates to a method for generating a non-human animal subject, the method comprising at least the step of introducing into at least one of the genes comprising one of the polymorphisms identified in any one of Tables 1 - 3 that is present in a cell, embryo, gamete, or nucleus a genetic alteration, wherein said genetic alteration is responsible for at least partially restoring the function of a gene product encoded by said gene comprising one of the polymorphisms identified in Table 1, whereby the cell, embryo, gamete, or nucleus carrying the genetic alteration is capable of being used to generate said non-human animal subject.

[0096] In another aspect, the invention relates to a method for generating a non-human cell, embryo, gamete, or nucleus, the method comprising at least the step of introducing into at least one of the genes comprising one of the polymorphisms identified in Table 1 which is present in said cell, embryo, gamete, or nucleus a genetic alteration at least partially restoring the function of a gene product encoded by said gene. In one example, prior to said introduction the cell, embryo, gamete or nucleus comprises at least one variant allele at one or more of the polymorphisms identified in any one of Tables 1 - 3.

[0097] In one example, the cell is a gamete. In an example of this example, the gamete is a sperm or an oocyte.

[0098] In various examples, the genetic alteration is introduced via a gene editing technique, such as a CRISPR or CRISPR / CAS technique.

[0099] It will be appreciated that the methods described herein, including those directed to the genetic modification of non-human animals, cells, embryos, gametes, and nuclei, are amenable to use where improvements in productivity and / or worth traits, whether at an individual or population (e.g., herd) level, are desired. Such methods can thus be employed to, for example, increase the likelihood that a non-human animal or population thereof will have one or more improved productivity and / or worth traits.

[0100] In one example of any one or more aspects as described herein, the deleterious effect on productivity and / or worth includes a deleterious effect on robustness, such as a deleterious effect on fertility.

[0101] Typically, said deleterious effects are undesirable.

[0102] Regarding bovine productivity and / or worth, low production efficiency and / or robustness contemplates production efficiency and / or robustness (such as fertility, sperm production, sperm motility, or sperm morphology, for example) that is below the average for the reference group, for example, the national herd for the subject breed. Low production efficiency and / or robustness is undesirable. For example, as shown herein in the Examples, bovine carrying the variant allele at any one of the polymorphisms identified in any one of Tables 1 - 3 have substantially lower fertility than those bovine homozygous for the wild-type allele.

[0103] In various examples of the methods described herein, selecting or rejecting a non-human animal subject, cell, embryo, gamete, or nucleus on the basis of its allelic profile comprises analysing a sample obtained from the subject, cell, embryo, gamete, or nucleus and determining the allelic profile. In other examples of the methods described herein, selecting or rejecting a non-human animal subject, cell, embryo, gamete, or nucleus on the basis of its allelic profile comprises providing data relating to the allelic profile, and determining the allelic profile on the basis of said data, for example on the basis of an analysis of said data.

[0104] In various examples, the allelic profile of the subject, cell, embryo, gamete, or nucleus is determined together with the allelic profile of the subject, cell, embryo, gamete, or nucleus at one or more other genetic loci associated with one or more productivity and / or worth traits. In one example, the one or more genetic loci is one or more polymorphisms in one or more genes associated with one or more productivity and / or worth traits. In one example, the one or more productivity and / or worth trait(s) are the same - that is, the productivity and / or worth trait(s) associated with the allelic profile and the productivity and / or worth trait(s) associated with the one or more other loci or polymorphisms are the same. In other examples, the one or more productivity and / or worth trait(s) are different.

[0105] The one or more polymorphisms can be detected directly or by detection of one or more polymorphisms which are in linkage disequilibrium with said one or more polymorphisms.

[0106] In various examples, the allelic profile at one or more other loci is determined by reference to a marker associated with a productivity and / or worth trait.

[0107] In other aspects, the invention relates to:

[0108] An animal, cell, embryo, gamete, or nucleus selected by a method as described herein;

[0109] Progeny or an animal produced by a method as described herein;

[0110] A herd formed using at least one animal identified using a method as described herein, such as a herd formed using two or more animals identified using a method as described herein;

[0111] An animal, cell, embryo, gamete, or nucleus generated by a method as described herein;

[0112] In still other aspects, the invention relates to:

[0113] Use of an animal, cell, embryo, gamete, or nucleus selected according to a method as described herein to breed or clone a non-human animal;

[0114] Use of an animal selected or generated according to a method as described herein to form a herd;

[0115] Use of the results of a method as described herein to identify whether or not an animal is suitable for (or at least more or less suitable for) for inclusion in a herd, production purposes and / or breeding purposes.

[0116] A method to identify whether or not an animal is suitable for (or at least more or less suitable for) inclusion in a herd, for production purposes and / or breeding purposes, the method comprising predicting the animal's suitability based on the results of a method as described herein;

[0117] Use of the results of a method as described herein to identify whether or not a cell or embryo is suitable for (or at least more or less suitable for) generating an animal which is suitable for inclusion in a herd, production purposes and / or breeding purposes. A method to identify whether or not a cell or embryo is suitable for (or at least more or less suitable for) generating an animal which is suitable for inclusion in a herd, for production purposes and / or breeding purposes, the method comprising predicting the animal's suitability based on the results of a method as described herein;

[0118] A method to identify whether or not a cell or embryo is suitable for (or at least more or less suitable for) generating an animal which is suitable for inclusion in a herd, for production purposes and / or breeding purposes, the method comprising: a) performing a method as described herein and b) predicting the animal's suitability based on the results of a);

[0119] A method for estimating the worth of an animal, the method comprising the step of incorporating information on genetic status of the animal, for example, its allelic profile, in a calculation of worth; and / or

[0120] A method for selecting or rejecting an animal, cell, embryo, gamete, or nucleus, the method comprising the step of selecting or rejecting an animal, cell or embryo on the basis of its allelic profile.

[0121] In specifically contemplated examples of any of the aspects described herein, the allelic profile is determined, wherein the allelic profile comprises information relating to the presence or absence of a variant allele at one or more of the polymorphisms identified in any one of Tables 1 - 3. In certain examples, the allelic profile comprises information relating to the presence or absence of two variant alleles at any one of the polymorphisms identified in any one of Tables 1 - 3 - that is, the allelic profile comprises information relating to whether or not a subject is homozygous for the variant allele at any one or more of the polymorphisms identified in any one of Tables 1 - 3.

[0122] In certain examples, said allelic profile is determined in a genotyping method. In another specifically contemplated example, said allelic profile is determined on the basis of data obtained in a genotyping method.

[0123] In specifically contemplated examples of any of the aspects described herein, the identification, determination, selection or rejection is on the basis of an allelic profile as herein contemplated, wherein said allelic profile is determined in, and / or on the basis of data obtained in, a genotyping method, wherein the genotyping method comprises the hybridisation of nucleic acid obtained from the subject with one or more immobilised nucleic acid molecules, such as multiple nucleic acid molecules present on a SNP chip.

[0124] In a specifically contemplated example, the identification, determination, selection or rejection is on the basis of an allelic profile as herein contemplated, wherein said allelic profile is determined on the basis of data obtained in a genotyping method, wherein the genotyping method comprises the hybridisation of nucleic acid obtained from the subject with two or more immobilised nucleic acid molecules, such as multiple nucleic acid molecules present on a SNP chip.

[0125] Accordingly, in one example the invention relates to a method of determining genetic status of a bovine with respect to one or more one or more productivity and / or worth traits, or with respect to capability of producing progeny predisposed to or with one or more one or more productivity and / or worth traits, the method comprising: providing data about the allelic profile of said bovine, wherein the data is or has been obtained in a genotyping method, wherein the genotyping method comprises the hybridisation of nucleic acid obtained from the subject with two or more immobilised nucleic acid molecules, such as multiple nucleic acid molecules present on a SNP chip; and determining the genetic status of the bovine on the basis of the data.

[0126] In one example, the data about the allelic profile comprises data representative of the presence or absence of one or more of the variant alleles at one or more of the polymorphisms identified in any one of Tables 1 - 3.

[0127] In one example, one or more of the immobilised nucleic acid molecules comprises a nucleotide sequence corresponding to the nucleotide sequence comprising or surrounding one or more of the polymorphisms identified in any one of Tables 1 - 3.

[0128] In another specifically contemplated example, the nucleotide sequence comprising or surrounding the polymorphism is not present in any of the immobilised nucleic acid molecules to which the nucleic acid obtained from the subject is hybridised. Rather, the identity of the allele(s) at one or more of the polymorphisms identified in any one of Tables 1 - 3 is imputed, for example, imputed by comparison to a reference sequence.

[0129] In one example, the invention relates to a SNP chip comprising two or more immobilised nucleic acids, wherein at least one of the two or more of the immobilised nucleic acid molecules comprises a nucleotide sequence corresponding to the nucleotide sequence comprising or surrounding one or more of the polymorphisms identified in any one of Tables 1 - 3.

[0130] In another example, the invention relates to a SNP chip comprising one or more or two or more immobilised nucleic acids, wherein none of the immobilised nucleic acids present on the SNP chip comprise the polymorphism. In one example, the SNP chip comprises multiple immobilised nucleic acids, wherein none of the immobilised nucleic acids present on the SNP chip comprise a polymorphism identified in any one of Tables 1 - 3, but one or more nucleic acids are present from which the allelic profile at one or more of the polymorphisms identified in any one of Tables 1 - 3 can be imputed, for example, one or more nucleic acids comprising a polymorphism in linkage disequilibrium with one of the alleles present at one or more of the polymorphisms identified in any one of Tables 1 - 3 are present on the SNP chip.

[0131] In another aspect, the invention relates to a method for identifying an amino acid mutation or variation in a protein encoded by a gene comprising one of the polymorphisms identified in Table 1 which is linked to a deleterious effect on productivity and / or worth of an animal.

[0132] In another aspect, the invention relates to an isolated, purified, recombinant or synthetic nucleic acid comprising the variant allele at one of the polymorphisms identified in any one of Tables 1 - 3 and / or a genetic marker in linkage disequilibrium therewith, and / or a nucleic acid which is complementary to or can hybridise to said nucleic acids under stringent conditions.

[0133] In various examples, the isolated, purified, recombinant or synthetic nucleic acid comprising the variant allele comprises a nucleic acid having a nucleotide sequence corresponding to that of any one of SEQ ID NO.s: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21, wherein the wild-type allele present in the nucleotide sequence is substituted with the variant allele as set out in Table 1.

[0134] In another aspect, the invention relates to an isolated, purified, synthesised, or recombinant polypeptide consisting essentially of or consisting of 10 or more contiguous amino acids encoded by a variant gene comprising one of the polymorphisms identified in Table 1. In one aspect, the invention relates to an isolated, purified, synthesised, or recombinant polypeptide comprising, consisting essentially of or consisting of an amino acid sequence encoded by a variant gene comprising one of the polymorphisms identified in Table 1.

[0135] In another aspect, the invention provides other isolated nucleic acids, including oligonucleotides, probes, and primers, and / or peptides and / or proteins as may be described herein. In one example, the nucleic acid is an isolated, purified, synthesised, or recombinant nucleic acid comprising one or more of the sequences presented herein and a heterologous nucleic acid sequence. In one example, the nucleic acid is an isolated, purified, synthesised, or recombinant nucleic acid comprising one or more of the sequences presented herein operatively linked to a heterologous promoter and / or terminator.

[0136] It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7). These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

[0137] Those skilled in the art will appreciate the meaning of various terms of degree used herein. For example, as used herein in the context of referring to an amount (e.g., "about 9%"), the term "about" represents an amount close to and including the stated amount that still performs a desired function or achieves a desired result, e.g., "about 9%" can include 9% and amounts close to 9% that still perform a desired function or achieve a desired result. For example, the term "about" can refer to an amount that is within less than 10% of, within less than 5% of, within less than 1% of, within less than 0.1% of, or within less than 0.01% of the stated amount. It is also intended that where the term "about" is used, for example with reference to a figure, concentration, amount, integer or value, the exact figure, concentration, amount, integer or value is also specifically contemplated.

[0138] Other aims, aspects, features and advantages of the present invention will become apparent from the following description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred examples of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0139] The invention is exemplified in the following non limiting examples and discussion.

[0140] DETAILED DESCRIPTION

[0141] There is a need for methods of determining the genetic status of non-human animal subjects, particularly agriculturally important animals, such as bovine subjects, including, for example, for production animals their genetic status with respect to one or more traits associated with productivity and / or worth.

[0142] The present invention recognises that one or more bovine polymorphisms are associated with variations that impact productivity and / or worth, such as fertility, including for example a polymorphism that affects and / or is associated with a deleterious effect on sperm quality and / or on sperm quantity.

[0143] Accordingly, one or more of such polymorphisms are useful in the identification and selection, including the selective breeding, of bovine having a desirable productivity and / or worth phenotype or a capability of producing progeny having a desirable productivity and / or worth phenotype. Conversely, one or more of such polymorphisms are useful in the identification and rejection, including rejection from breeding, of bovine having an undesirable productivity and / or worth phenotype or a capability of producing progeny having a desirable productivity and / or worth phenotype.

[0144] This invention relates to the identification of polymorphisms associated with variations in productivity and / or worth, such as one or more robustness traits, such as fertility, in bovine. As described herein, and as elucidated in the disclosure of data presented herein in the Examples, genetic variations in the bovine genome have been identified that, when present and particularly when present in a homozygous state, have a deleterious effect on one or more productivity and / or worth traits, such as one or more robustness traits, and particularly as disclosed herein on fertility.

[0145] A deleterious effect on productivity and / or worth, such as a deleterious effect on robustness, such as reduced fertility, is generally undesirable. Furthermore, without wishing to be bound by any theory, the inventors believe that one or more of the variant alleles identified herein may be associated with one or more of increased disease, disease severity, and / or disease susceptibility such as an increased likelihood of developing a disease, such as a late onset disease, and / or increased incidence and / or risk of a condition, such as a late onset condition. In some examples, the inventors believe, without wishing to be bound by any theory, that one or more of the variant alleles identified herein may be associated with one or more of a deleterious effect on acceptability, for example to a farmer or breeder, and the like.

[0146] Relatedly, and again without wishing to be bound by any theory, the inventors believe that decreased production efficiency and / or robustness, such as decreased production efficiency, particularly the late onset effect identified herein, may be a proxy for one or more of disease and / or disease susceptibility, including an increased likelihood of developing a disease, and / or decreased acceptability, for example, particularly late onset occurrences of these traits.

[0147] The polymorphisms of interest as discussed herein are presented below in Table 1.

[0148] Table 1. Polymorphisms of interest

[0149] Selected definitions

[0150] The term "and / or" can mean "and" or "or".

[0151] Those skilled in the art will appreciate the meaning of various terms of degree used herein. For example, as used herein in the context of referring to an amount (e.g., "about 9%"), the term "about" represents an amount close to and including the stated amount that still performs a desired function or achieves a desired result, e.g., "about 9%" can include 9% and amounts close to 9% that still perform a desired function or achieve a desired result. For example, the term "about" can refer to an amount that is within less than 10% of, within less than 5% of, within less than 1% of, within less than 0.1% of, or within less than 0.01% of the stated amount. It is also intended that where the term "about" is used, for example with reference to a figure, concentration, amount, integer or value, the exact figure, concentration, amount, integer, or value is also specifically contemplated.

[0152] The term "comprising" as used in this specification means "consisting at least in part of". When interpreting each statement in this specification that includes the term "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises", and the terms "including", "include" and "includes" are to be interpreted in the same manner.

[0153] The term "consisting essentially of" when used in this specification refers to the features stated and allows for the presence of other features that do not materially alter the basic characteristics of the features specified.

[0154] The term "consisting of" as used herein means the specified materials or steps of the claimed invention, excluding any element, step, or ingredient not specified in the claim.

[0155] As used herein, a "gene" includes coding sequences encoding one or more products of the gene, non-coding sequences such as introns, as well as all nucleotide regions which regulate the production of the one or more gene products, whether or not such regulatory sequences are adjacent to coding and / or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions.

[0156] 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.

[0157] The term "polynucleotide(s)" as used herein, means a single or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length, and include as non-limiting examples, coding and non-coding sequences of a gene, sense and antisense sequences, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polynucleotides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers, fragments, genetic constructs, vectors and modified polynucleotides. Reference to nucleic acids, nucleic acid molecules, nucleotide sequences and polynucleotide sequences is to be similarly understood. It will be appreciated that a wide variety of synthetic and / or non- naturally occurring nucleotide analogues are available, such that polynucleotides comprising one or more of said synthetic or non-naturally occurring nucleotide analogues can be prepared. The use of such polynucleotides in the methods and compositions described herein is likewise contemplated.

[0158] While the inventors have identified the biological markers of the invention in bovine animals, they contemplate that it is equally applicable to a variety of different mammals. Various mammals can suffer from decreased productivity and / or worth. Accordingly, the term "animal" is used herein primarily in reference to mammals. In one particular example, the mammal is a ruminant. In another example, the mammal is one within the Bovidae family. In particular examples, the animal is a bovine animal. More particularly the animal is Bos taurus or Bos indicus. In one particular example 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.

[0159] A "subject" as used herein is an animal, usually a mammal, including a mammalian agricultural animal or a companion animal or a human. Particularly contemplated subjects are non-human animals. Representative agricultural animals include caprine, ovine, bovine, cervine, and porcine. Representative companion animals include feline, equine, and canine.

[0160] Accordingly, the term "animal" is used herein primarily in reference to mammals. In one particular example, the mammal is a ruminant. In particular examples, the animal is a bovine animal. More particularly the animal is Bos taurus. In one particular example the animal is a meat or dairy breed.

[0161] Broadly, the invention relates to methods of assessing the genetic status of a non-human animal (and related entities, including for example cells, gametes, or nuclei from said animal) with respect to one or more productivity and / or worth traits, particularly those associated with an allelic profile. One such method comprises the step of determining the allelic profile of said bovine. Another such method comprises the step of determining the level (including, for example, the concentration, amount, or activity) of a gene product of said bovine.

[0162] The invention also relates to methods for identifying, and / or selecting or rejecting a non-human animal subject (and related entities) with a genotype indicative of one or more desired productivity and / or worth traits.

[0163] It is contemplated that methods involving the determination of whether or not the variation is present, for example in a nucleic acid sample from a subject, is useful to, inter alia, determine the genetic status of an animal, cell, embryo, nucleus, and the like with respect to such production and / or worth phenotypes, for example to identify whether or not an animal, cell, embryo, gamete, or nucleus carries a genetic marker linked to a beneficial, neutral, or deleterious effect on productivity and / or worth. This can be used to infer or predict the phenotype of an animal, for example, identify whether or not an animal is suitable for (or at least more or less suitable for) production and / or breeding purposes, identify whether a cell, nucleus or embryo is suitable for use (or at least more or less suitable) in producing an animal which is suitable for production and / or breeding purposes, and to calculate the worth of an animal.

[0164] Those skilled in the art will understand that the analysis of one or more genetic markers in linkage disequilibrium with the specific marker identified herein will also be informative for the same purposes. In addition, haplotypes including the genetic marker identified herein and / or one or more marker in linkage disequilibrium therewith will likewise be useful for this purpose.

[0165] The specific genetic variations identified herein include nucleotide substitutions which result in an amino acid substitution and thus a variant protein, or which result in a premature stop codon resulting in truncation of the encoded protein, or which result in splicing variants, including those resulting in a variant protein. Accordingly, certain examples of methods contemplated herein comprise the analysis of the encoded polypeptide, fragments, isoforms, and / or precursors thereof, and / or of the levels (including size, sequence, amount, expression and activity levels) of the encoded protein. As discussed above, examples of the methods described herein also comprise the analysis of the nucleotide sequence of a transcript or a fragment thereof.

[0166] The applicant believes that this is the first time that the polymorphisms identified in any one of Tables 1 - 3 have been associated with a deleterious effect on productivity and / or worth in a nonhuman animal, and consequently the worth of the animal, and thus the first time a link between these polymorphisms and the variant genes in which they are present, and productivity and / or worth, has been made. Accordingly, it is contemplated that any genetic alteration which disrupts this gene may be deleterious to the productivity and / or worth of an animal, particularly if the animal is homozygous for such a variant or elsewise carries two copies of a defective gene. As such, the invention should be taken to encompass methods involving the analysis of nucleic acid from an animal to determine whether or not it includes any such alteration or variation in the gene (including reference to the transcript), analysis of the size or amino acid sequence of the encoded protein (including reference to fragments, isoforms, and / or precursors thereof) to determine whether or not it includes any alteration that disrupts the encoded protein, and / or the level (including the amount and level of expression, for example) and / or activity of the encoded protein, to identify whether or not it has a variation linked to a deleterious effect on productivity and / or worth, for example to assess whether or not an animal is suitable for (or at least more or less suitable for) production purposes, breeding purposes, and / or inclusion in a herd, or whether or not a cell or embryo is suitable for (or at least more or less suitable for) producing an animal that is suitable for production purposes, breeding purposes and / or inclusion in a herd, for example.

[0167] While the description which follows may focus on the analysis of the nucleotide sequence or amino acid sequence at a particular position, it should be understood to extend to the analysis of the sequence at any other position within the relevant gene, transcript, or the encoded protein.

[0168] Table 1 and Table 3 herein provide identifiers, such as Gene IDs for the gene in which the polymorphism occurs, and identifies the genomic position of the variant according to the reference sequence for the relevant bovine chromosome in the ARS-UCD 1.2 bovine genome build.

[0169] By way of example, the Chr2: 101396615 G>T (rs433745406) polymorphism in the SPAG16 gene (see Table 1 and Table 3) is located at nucleotide position 101396615 of the bovine chromosome 2 reference sequence in the ARS-UCD 1.2 genome build. The wild-type allele is Chr2: 101396615G, or the G allele at the Chr2:101396615 G>T (rs433745406) polymorphism. The variant allele is Chr2: 101396615T, or the T allele at the Chr2:101396615 G>T (rs433745406) polymorphism. As shown in Table 3, the variant allele is a nonsense variant and is predicted to result in the replacement of the glutamic acid at position 257 of the wild-type SPAG16 protein with a premature termination codon and thus a truncated protein.

[0170] Those skilled in the art will recognise that the remaining polymorphisms identified herein, for example in any one of Tables 1 - 3, can be interpreted and identified accordingly with reference to the information in the Tables presented herein, and in Table 1 and Table 3 in particular.

[0171] By way of further example, the Chr4:30657607 G>A (rs457253516) polymorphism (see Table 1 and Table 3) is located at nucleotide position 30657607 of the bovine chromosome 4 reference sequence in the ARS-UCD 1.2 genome build. The wild-type allele is Chr4:30657607G, or the G allele at the Chr4:30657607 G>A (rs457253516) polymorphism. The variant allele is Chr4:30657607A, or the A allele at the Chr4:30657607 G>A (rs457253516) polymorphism. As shown in Table 3, the variant allele is a missense variant and is predicted to result in the replacement of the valine amino acid at position 4118 of the wild-type DNAH11 protein with a methionine and thus a variant protein.

[0172] Similarly, in a further example, the Chr4:30411361 CA>C (rs465963699) polymorphism (see Table 1 and Table 3) is located at nucleotide position 30411361 of the bovine chromosome 4 reference sequence in the ARS-UCD 1.2 genome build. The wild-type allele is Chr4: 30411361CA, or the CA allele at the Chr4:30411361 CA>C (rs465963699) polymorphism. The variant allele is Chr4: Chr4:30411361C, or the C allele at the Chr4:30411361 CA>C (rs465963699) polymorphism. As shown in Table 3, the variant allele (resulting in the variation in codons ACA GAC [T]>ACG ACT) is a frameshift mutation and is predicted to result in the replacement of the aspartic acid amino acid at position 1791 (in the ENSBTAT00000061103.4 sequence), and all subsequent amino acids, of the wild-type DNAH11 protein with, as a result of the frameshift, a variant sequence of 61 amino acids beginning with a threonine (see the C-terminal 61 amino acids of SEQ ID NO. : 10) followed by a termination codon, and thus a variant truncated protein.

[0173] In a further example, the Chr8:98463916 G>A (rs459888392) polymorphism (see Table 1 and Table 3) is located at nucleotide position 98463916 of the bovine chromosome 8 reference sequence in the ARS-UCD 1.2 genome build. The wild-type allele is Chr8:98463916G, or the G allele at the Chr8:98463916 G>A (rs459888392) polymorphism. The variant allele is Chr8:98463916A, or the A allele at the Chr8:98463916 G>A (rs459888392) polymorphism. As shown in Table 3, the variant allele is a missense variant and is predicted to result in the replacement of the alanine amino acid at position 53 of the wild-type ACTL7B protein with a valine and thus a variant protein.

[0174] By way of further example, the Chrl7:63363268 TG>T (rs434709552) polymorphism (see Table 1 and Table 3) is located at nucleotide position 63363268 of the bovine chromosome 17 reference sequence in the ARS-UCD 1.2 genome build. The wild-type allele is Chrl7:63363268TG, or the TG allele at the Chrl7:63363268 TG>T (rs434709552) polymorphism. The variant allele is Chrl7:63363268T, or the T allele at the Chrl7:63363268 TG>T (rs434709552) polymorphism. As shown in Table 3, the variant allele is a frameshift mutation and is predicted to result in the replacement of the glutamine acid at position 44 of the wild-type TCHP protein with, as a result of the frameshift, a variant sequence of 30 amino acids beginning with an arginine (see the C-terminal 30 amino acids of SEQ ID NO. : 18) followed by a termination codon, and thus a variant truncated protein.

[0175] In a further example, the Chr25: 1194914 G>C (rs433354487) polymorphism (see Table 1 and Table 3) is located at nucleotide position 1194914 of the bovine chromosome 25 reference sequence in the ARS-UCD 1.2 genome build. The wild-type allele is Chr25: 1194914G, or the G allele at the Chr25: 1194914 G>C (rs433354487) polymorphism. The variant allele is Chr25: 11949140, or the C allele at the Chr25: 1194914 G>C (rs433354487) polymorphism. As shown in Table 3, the variant allele is a missense variant and is predicted to result in the replacement of the glutamine amino acid at position 810 of the wild-type IFT140 protein with a glutamic acid and thus a variant protein.

[0176] Determination of genetic status, and / or of allelic profile in accordance with this disclosure may assist in: predicting phenotypic performance, including use in production management systems known as Marker Assisted Selection; the selection or rejection of animals for breeding and / or other purposes; managing animals in order to maximise their individual potential performance and value; estimating the worth or economic value of an animal; improving profits related to selling animals and / or products produced from the animals; improving the genetics of a population of animals by selecting and breeding desirable animals; generating and maintaining herds of animals; cloning animals likely to have or not have a specific trait; predicting the suitability of an animal and / or its progeny to use in different industries and / or breeding programmes or cloning; avoiding carrier x carrier matings; removing an adverse genetic variant from a population; gene editing to remove deleterious genetic variations from cells or embryos used to generate animals; to increase or optimise productivity of an animal or a herd; for example. It should be appreciated that animals may be tested or screened any time during their life, including early at birth or as embryos or foetuses, to predict lifetime performance and segregated or managed to suit their genotype and therefore predicted phenotype. Any time during their life includes, but is not limited to, early at birth, as gametes, zygotes, embryos, foetuses. Similarly, the invention finds application to the determination of genetic status and / or allelic profile of isolated cells and / or nuclei, for example, for subsequent cloning methods.

[0177] As will be apparent from the above, various methods described herein are directed to the determination of genetic status with respect to one or more productivity traits, and / or one or more traits associated with worth. For example, certain examples of methods are used to identify whether an animal, cell, embryo, gamete, or nucleus has an allelic profile associated with a deleterious effect on productivity and / or worth.

[0178] Various aspects of the invention are described in further detail in the following subsections. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control. Although methods and materials similar or equivalent to those described herein can be used in the practice of the invention, examples of suitable methods and materials are described below. The materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

[0179] "Productivity" as used herein refers to the level and / or efficiency of production of a particular product by an animal. In one example, it includes efficiency of production per unit liveweight of an animal. For example, the product may be meat, or milk. Such raw products may be further processed into consumer products such as milk powder, cheeses, whey protein, specialty beverages and meat products. Productivity can be measured based on an assessment of one or more parameters, having regard to the product to be produced, as will be understood by persons skilled in the art. However, by way of example, where the product is milk (or a derivative thereof), milk yield, protein yield, and / or fat yield can be assessed, alone or in combination with one or more other animal assessment parameters as described herein. By way of further example, where the product is meat (or a derivative thereof), liveweight can be assessed, alone or in combination with one or more other animal assessment parameters as described herein.

[0180] As used herein the "worth" of an animal refers to an index used to evaluate the value of an animal, for breeding purposes, production purposes, inclusion in a herd, herd management, for example. The "worth" is the sum of the estimated value of one or more characteristics (or animal assessment parameters) which may be associated with the animal, typically weighted by an economic value. Exemplary assessment parameters may include those which may be referred to as "production traits" and those which may be referred to as "traits other than production" or "TOP"; as described in "Evaluation System for Traits Other Than Production (TOP) for Dairy Cattle in New Zealand" published in August 2014 by Advisory Committee on Traits Other than Production at dairynz.co.nz / media / 1205535 / TOP_Booklet_A4.pdf, "Your Index Your Animal Evaluation System (Second Edition)" published by NZ Animal Evaluation Unit at dairynz.co.nz / media / 581307 / Your_Index_Your_AE_System.pdf, and at www.dairynz.co.nz / animal / animal-evaluation / interpreting-the-info / .

[0181] Examples of such traits and their assessment are presented herein in the Examples, however, persons skilled in the art to which the invention relates will readily appreciate alternative or additional assessment parameters which may be included in any calculation to estimate an animal's worth.

[0182] The term "worth" should be taken to encompass "breeding worth", "lactation worth", "production worth" and other known traits and indexes used to assess the value of an animal. Breeding worth can comprise the productivity traits (also referred to as production efficiency traits) including milk protein, milk fat yield, milk volume, and liveweight, and the robustness traits, including somatic cell count, fertility, gestation length, functional survival, body condition score and udder overall. Breeding worth, lactation worth and production worth, are described in "Your Index Your Animal Evaluation System (Second Edition)" and at www.dairynz.co.nz / animal / animal- evaluation / interpreting-the-info / , as detailed above, for example. Other exemplary indexes include the Balanced Performance Index (BPI) in Australia, and the Economic Breeding Index (EBI) applied in Ireland. These are described, for example in "Your Herd. Your Test. Your Future. National Breeding Objective - Final Report" published by Dairy Australia and the Australian Dairy Herd Improvement Scheme at adhis.com.au / v2 / downv2.nsf / (ContentByKey) / d356f55a05cac8f0ca257c78000a2677 / $file / nbo%20fina l%20short%20report.pdf?open and "Understanding the Economic Breeding Index (EBI)" published by the Agricultural and Food Development Authority in Ireland at www.icbf.com / wp- content / uploads / 2023 / 01 / U nderstandinq-EBI-18.01.23-l.pdf.

[0183] Other productivity and / or worth traits are likewise amenable to assessment, and polymorphisms associated with variation in these traits accordingly amenable to identification and use in selection methods such as those contemplated herein. One such example is the trait dairy conformation, while another is the trait 'days after planned calving date in heifers' (see the Examples recited herein), the latter being defined as the number of days from the planned start of calving date to calving for a given herd-year. This is a continuous trait expressed in days, and ranges from -50 to 150 days.

[0184] Notwithstanding the inclusion of fertility as one of the traits comprising the Breeding Worth index referred to above, "fertility" in the context of the present disclosure in the main relates to male fertility, both directly, for example the association of the variant allele with one or more undesirable effects on sperm quality and / or on sperm quantity, and indirectly, for example the productivity and / or worth of progeny. Accordingly, as used herein desired fertility contemplates but is not limited to the production of sperm having a high percentage of live sperm, of high-motility sperm, or sperm lacking morphological abnormalities, each of which is desirable not least as male fertility impacts successful breeding outcomes.

[0185] Those skilled in the art will recognise that artificial insemination (Al) is common in the livestock industry, and is a technology well-disposed to the targeted use (when required) of semen from high genetic merit males. For example, cattle genetics companies will generally have a number of high genetic merit bulls from which semen is collected and made available for breeding, typically via Al. Semen samples are routinely assessed for sperm volume, sperm motility, sperm concentration, semen morphological factors. Genetics companies will also assess the fertility of the bulls from insemination and pregnancy data of the cows with which they have been mated.

[0186] Accordingly, for most bovine agriculture systems productivity and / or worth for bulls is based in part on the productivity and / or worth traits they pass on to progeny - with the focus in dairying systems being on the productivity and / or worth traits of their daughters - and in part on their own fertility traits. Bulls with low fertility are generally undesirable, and impact profitability. For example, in dairying systems if breeding is unsuccessful, insemination needs to be attempted on the next oestrus cycle, and calving and hence milk production is delayed.

[0187] Persons skilled in the art to which the invention relates will readily appreciate methods and formulae suitable for estimating worth on the basis of any number of different characteristics. However, by way of example the methods described in the documents listed in this and the preceding paragraphs could be used. Results, data and / or information generated by one or more method of the invention may be used in calculations for estimating "worth". In examples of the invention, an animal may be selected or rejected for a particular purpose (for example, inclusion in a herd, for production purposes and / or for breeding purposes) based on its worth.

[0188] In certain examples, the methods of the invention are described in terms of estimating the worth of an animal "and / or its progeny". This should be taken to mean that the worth of the progeny of an animal or pair of animals can be estimated indirectly based on evaluation of one or both parents without the need to directly assess the progeny. Such evaluation would typically involve determining the genetic status of, and / or the allelic profile of, one or both parents. For example, the method may comprise identifying whether or not a parent animal has allelic profile associated with a deleterious effect on productivity and / or worth of an animal and using this information to in a calculation to estimate the worth of the progeny. Such methods may be used to estimate the potential worth of progeny if a pair of chosen animals was mated, for example. Alternatively, it could be used to estimate the worth of existing progeny. Such methods may later be followed up with direct analysis or observation of the progeny, if desired. Persons skilled in the art will readily appreciate methods for estimating the worth of progeny according to this example having regard to the description provided herein and known methods for estimating worth, as hereinbefore described, for example.

[0189] In one example, worth is calculated taking into consideration the animal assessment parameter (or trait) survival, alone or in combination with one or more other animal assessment parameters. Any combination of traits can be assessed in a calculation of worth.

[0190] A "deleterious effect on productivity" should be understood to be mean any level of detrimental effect on the level and / or efficiency of production of one or more product by an animal. In one example, it means the animal has or is predicted to have a reduced level of production compared to an animal or animals having at least a desirable level of productivity or the same animal if it did not have a genetic variation which disrupts a gene associated with productivity. In one example, there is at least a 5% level of reduction, at least a 10% level of reduction, at least a 15% level or reduction or at least a 20% level of reduction in the production of one or more product by the animal compared to an animal or animals having at least a desirable level of productivity or the same animal if it did not have a genetic variation which disrupts a gene associated with productivity, for example.

[0191] What is considered "desired productivity" or a "desirable level of productivity" or grammatical equivalents may differ from one animal or breed or farm or herd or country to another, for example. It may also differ depending on the product which an animal is being used to produce. Skilled persons will readily appreciate a benchmark desirable level of productivity having regard to the product to be produced, information on breed, the herd or population of animals used and one or more assessment criteria known in the art. In one example, a desirable level of productivity is the mean of a population of animals. In another example, a desirable level of productivity is the mean of a population of animals that is homozygous for the wild-type allele at a polymorphism associated with productivity. In another example, a desirable level of productivity is the level of a chosen highly productive animal.

[0192] A "deleterious effect on worth" or like phrases is intended to mean any level of detrimental effect on the estimated worth of an animal. In one example, it means the animal has or is predicted to have a lower worth compared to an animal or animals having a desirable worth or the worth of same animal if it did not have a genetic variation which disrupts a gene associated with worth, such as a gene comprising one of the polymorphisms identified in Table 1, Table 2, or Table 3. In some examples, there is at least a 5% level of reduction, at least a 10% level of reduction, or at least a 15% level of reduction in the worth of an animal compared to an animal or animals having a desirable worth or the worth of same animal if it did not have a genetic variation which disrupts a gene comprising one of the polymorphisms identified in Table 1, Table 2, or Table 3, for example.

[0193] What is considered a "desirable worth" or grammatical equivalents may differ from one animal or breed or farm or herd or country to another, for example. It may also differ depending on the purpose for which an animal is to be used, the industry in which an animal is to be used, and / or the product which an animal is being used to produce. Persons skilled in the art will readily be able to appreciate or calculate a benchmark desirable worth having regard to such factors and using one or more known methods to calculate worth of an animal (for example, those herein before described). In one example, a desirable worth is the mean of a population of animals. In another example, a desirable worth is the mean of a population of animals that is homozygous for the wild-type allele at one of the polymorphisms identified in Table 1, Table 2, or Table 3. In another example, a desirable worth is the worth of a chosen animal.

[0194] In one example, "a deleterious effect on productivity and / or worth" includes reference to a deleterious effect on one or more animal assessment parameters (or traits) representative of productivity or worth, such as those used in worth estimations. These traits variously include production traits, production efficiency traits, "traits other than production" or "TOP", and worth traits such as the robustness traits described above, as described herein before.

[0195] In one example, the one or more animal assessment parameters representative of productivity and / or worth includes the robustness trait fertility.

[0196] As will be appreciated in the context of this disclosure, a deleterious effect on productivity and / or a deleterious effect on worth will typically be considered an undesired productivity and / or worth trait when determining genetic status. Similarly, a beneficial effect on productivity and / or a beneficial effect on worth will typically be considered a desirable productivity and / or worth trait when determining genetic status.

[0197] Persons of general skill in the art to which the invention relates will readily understand the meaning and nature of the animal assessment parameters discussed and exemplified herein, including, for example, "production efficiency and / or robustness". However, these traits are described, for example, in "Evaluation System for Traits Other Than Production (TOP) for Dairy Cattle in New Zealand" and "Your Index Your Animal Evaluation System (Second Edition)", and at www.dairynz.co.nz / animal / animal-evaluation / interpreting-the-info / , as referred to herein. When assessing an animal, the animal assessment parameters may be assigned a measure or a score. In one example, when assessing dairy conformation (and for dairy conformation the scores of the component traits stature, capacity, rump angle, rump width, and legs) may be assigned a score on a scale. Persons of skill in the art will readily appreciate appropriate scales of use. However, in one specifically contemplated example, the scales described in "Evaluation System for Traits Other Than Production (TOP) for Dairy Cattle in New Zealand", typically being a score of 1 - 10, are used. In certain examples, liveweight is assigned a measure in Kg, (or other appropriate units of measure), for example.

[0198] Markers, Linkage, and Alleles

[0199] The term "marker" as used herein should be taken broadly and includes, for example, genetic markers such as those specifically described herein, the level of a protein (including reference to a fragment thereof, a precursor thereof, an isoform thereof) or a nucleic acid encoding a protein (including reference to a fragment thereof, a precursor thereof, an isoform thereof), the level of expression of a gene or protein (including reference to a fragment thereof, a precursor thereof, an isoform thereof), the level of activity of a protein (including reference to a fragment thereof, a precursor thereof, an isoform thereof) and / or variation in the amino acid sequence of a protein (including reference to a fragment thereof, a precursor thereof, an isoform thereof) which may include observation of the size of a protein (including reference to a fragment thereof, a precursor thereof, an isoform thereof) or nucleic acid (including for example a transcript). 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.

[0200] Specifically contemplated genetic markers include the polymorphisms identified in Table 1, Table 2, or Table 3 herein. The a I lele(s) present at any one or more of these polymorphisms can be detected directly or by detection of one or more polymorphisms which are in linkage disequilibrium with one or other of the alleles at this polymorphism.

[0201] It will be understood that in the context of the present invention the term "polymorphism" means the occurrence together in the same population at a rate greater than that attributable to random mutation (usually greater than 1%) of two or more alternate forms (such as alleles or genetic markers) of a chromosomal locus that differ, for example, in nucleotide sequence or have variable numbers of repeated nucleotide units. Accordingly, the term "polymorphisms" as used herein contemplates genetic variations, including single nucleotide substitutions, insertions and deletions of nucleotides, repetitive sequences (such as microsatellites), and the total or partial absence of genes (eg. null mutations). As used herein, the term "polymorphisms" also includes genotypes and haplotypes. A genotype is the genetic composition at a specific locus or set of loci. A haplotype is a set of closely linked genetic markers present on one chromosome which are not easily separable by recombination, tend to be inherited together, and can be in linkage disequilibrium. A haplotype can be identified by patterns of polymorphisms such as single nucleotide polymorphisms (SNPs).

[0202] Similarly, the term "single nucleotide polymorphism" or "SNP" in the context of the present invention includes single base nucleotide substitutions and short deletion and insertion polymorphisms.

[0203] A common measure of linkage (or association) is the frequency with which traits cosegregate. This can be expressed as a percentage of cosegregation (recombination frequency) or, also commonly, in centiMorgans (cM), being a reciprocal unit of recombination frequency and a unit of measure of genetic recombination frequency. One cM is equal to a 1% chance that a trait at one genetic locus will be separated from a trait at another locus due to recombination in a single generation (meaning the traits segregate together 99% of the time). Because chromosomal distance is approximately proportional to the frequency of recombination events between traits, there is an approximate physical distance that correlates with recombination frequency. For example, in humans, 1 cM correlates, on average, to about 1 million base pairs (1 Mbp).

[0204] Marker loci are themselves traits and can be assessed according to linkage analysis by tracking the marker loci during segregation. Thus, in the context of the present invention, one cM is equal to a 1% chance that a marker locus will be separated from another locus (which can be any other trait, e.g., another marker locus, or another trait locus that encodes a QTL for one or more productivity and / or worth traits or a variation that is associated with one or more productivity and / or worth traits), due to recombination in a single generation. The markers described and exemplified herein can correlate with one or more productivity and / or worth traits. This means that the markers comprise or are sufficiently proximal to a QTL or variation for one or more productivity and / or worth traits that they can be used as a predictor for the trait itself. This is extremely useful in the context of selection, for example for selective breeding programs.

[0205] The polymorphisms associated with one or more productivity and / or worth traits, such as one or more alleles associated with decreased lactation, for example, have been identified as described herein as being more prevalent in case animals versus control animals. Any marker that is linked to a trait locus of interest (e.g., in the present case, a QTL, variant, or identified linked marker locus for one or more productivity and / or worth traits) can be used as a marker for that trait. Thus, in addition to the markers specifically identified herein, other markers closely linked to these markers can also usefully predict the presence of the identified marker alleles, and thus, the relevant phenotypic trait. Such linked markers are particularly useful when they are sufficiently proximal to a given locus so that they display a low recombination frequency with the given locus. These closely linked markers are specifically contemplated for use in the methods described herein. In certain examples, closely linked loci display a recombination frequency with a given marker of about 20% or less (the given marker is within 20 cM of the given marker) - that is, closely linked loci co-segregate at least 80% of the time. In certain examples, the recombination frequency is 10% or less (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.25%, or 0.1% or less, including, for example, 0% recombination frequency). In one typical class of examples, closely linked loci are within 1 cM or less of each other.

[0206] As one of skill in the art will recognize, recombination frequencies (and, as a result, map positions) can vary depending on the map used (and the markers that are on the map). Additional markers that are closely linked to (e.g., within about 20 cM, for example within about 10 cM, such as within 5 cM of, or within 1 cM of) the markers identified herein may readily be used for identification of QTL for one or more desired productivity and / or worth traits and / or a variation that is associated with one or more productivity and / or worth traits.

[0207] Marker loci are especially useful in the present invention when they are closely linked to target loci (e.g., QTL for or variation associated with one or more productivity and / or worth trait phenotypes, or, alternatively, simply other marker loci that are, themselves linked to such QTL or variation) that they are being used as markers for. The more closely a marker is linked to a target locus that encodes or affects a phenotypic trait, the better an indicator for the target locus that marker is (due to the reduced cross-over frequency between the target locus and the marker). Thus, in one example, closely linked loci such as a marker locus and a second locus display an inter-locus cross-over frequency of about 20% or less, e.g., 15% or less, preferably 10% or less, more preferably about 9% or less, still more preferably about 8% or less, yet more preferably about 7% or less, still more preferably about 6% or less, yet more preferably about 5% or less, still more preferably about 4% or less, yet more preferably about 3% or less, and still more preferably about 2% or less. In certain examples, the relevant loci (e.g., a marker locus and a target locus such as a QTL or a variation that is associated with one or more productivity and / or worth traits) display a recombination a frequency of about 1% or less, e.g., about 0.75% or less, more preferably about 0.5% or less, or yet more preferably about 0.25% or 0.1% or less. Thus, the loci are about 20 cM, 19 cM, 18 cM, 17 cM, 16 cM, 15 cM, 14 cM, 13 cM, 12 cM, 11 cM, 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.75 cM, 0.5 cM, 0.25 cM, 0.2 cM, or 0.1 cM or less apart. Put another way, two loci that are localized to the same chromosome, and at such a distance that recombination between the two loci occurs at a frequency of less than 20% (e.g., about 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, 0.1% or less, including, for example, 0% recombination frequency) are said to be "proximal to" each other.

[0208] In one example, linked markers are within 1000 kb (which correlates in humans to about 1 cM, depending on local recombination rate), e.g., within 500 kb, or within 200 kb or less, of each other. In one example, linked markers are within 100 kb (which correlates in humans to about 0.1 cM, depending on local recombination rate), e.g., 50 kb, or even 20 kb or less, of each other.

[0209] In one example, linked markers are in linkage disequilibrium. For example, in one example the one or more polymorphisms in linkage disequilibrium with one or more of the polymorphisms specified herein is within about 1000 kb of the specified polymorphism. In certain examples, the one or more polymorphisms in linkage disequilibrium with one or more of the polymorphisms specified herein is within about 500 kb of the specified polymorphism, for example is within about 400 kb, within about 300 kb, within about 200 kb, or within about 150 kb, of the specified polymorphism. In another example, the one or more polymorphisms in linkage disequilibrium with one or more of the polymorphisms specified herein is within about 100 kb of the specified polymorphism. In certain examples, the one or more polymorphisms in linkage disequilibrium with one or more of the polymorphisms specified herein is within about 50 kb of the specified polymorphism, for example is within about 40 kb, within about 30 kb, within about 20 kb, within about 10 kb, or within about 5 kb, of the specified polymorphism.

[0210] Linkage disequilibrium is a phenomenon in genetics whereby two or more mutations or polymorphisms are in such close genetic proximity that they are co-inherited. This means that in genotyping, detection of one polymorphism as present implies the presence of the other. (Reich DE et al; Linkage disequilibrium in the human genome, Nature 2001, 41 1: 199-204.)

[0211] Various degrees of linkage disequilibrium are possible. In one example, the one or more polymorphisms in linkage disequilibrium with one or more of the polymorphisms specified herein are in greater than about 60% linkage disequilibrium, are in about 70% linkage disequilibrium, about 75%, about 80%, about 85%, about 90%, about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% linkage disequilibrium with one or more of the polymorphisms specified herein.

[0212] Those skilled in the art will appreciate that in various examples, when expressed with reference to the deviation from the expected of the observed frequency of a pair of alleles cosegregating, linkage disequilibrium is denoted by the measure D. Accordingly, in certain examples the phrase two alleles are in LD or similar means that D does not equal 0. Contrariwise, "linkage equilibrium" denotes the case D = 0.

[0213] Commonly, the scaled version of D, D', which ranges between -1 to +1 or conveniently its modulus | D'|, is used. Thus, in certain examples, linkage disequilibrium is determined using the linkage disequilibrium measure |D' |, which give a quantitative measure of the extent of linkage disequilibrium (LD) between two genetic elements, such as two polymorphic markers such as an allele at one polymorphic locus and an allele at another polymorphic locus. Certain numerical values of these measures for particular markers are indicative of the markers being in linkage disequilibrium, as described further herein. When utilising this measure, in various examples the one or more polymorphisms in LD with the one or more polymorphisms specified herein are in LD of |D' | > 0.2, of |D' | > 0.3, of |D' | > 0.4, of |D' | > 0.5, of |D' | > 0.6, of |D' | > 0.7, of |D' | > 0.75, of |D' | > 0.8, of |D' | > 0.85, of |D' | > 0.9, of |D' | > 0.91 , of |D' | > 0.92, of |D' | > 0.93, of |D' | > 0.94, of |D' | > 0.95, of |D' | > 0.96, of |D' | > 0.97, of |D' | > 0.98, of |D' | > 0.99, or of about |D' | =1.0.

[0214] Those skilled in the art will understand that D' (whether or not it is expressed as its modulus | D'| ) is particularly useful in examples where the frequency of the subject alleles is similar. D' as a measure of LD can be less informative, however, when one allele is rare, or when only a small sample size is assessed. In such circumstances, an alternative measure of linkage disequilibrium, R2, will frequently be used.

[0215] In certain examples, linkage disequilibrium is determined using the linkage disequilibrium measure R2. In one example of the invention, linkage disequilibrium between markers contemplates an R2> 0.1. In another example, linkage disequilibrium contemplates R2> 0.2. In other examples, the one or more polymorphisms in linkage disequilibrium with the one or more polymorphisms specified herein are in LD of R2> 0.25, R2> 0.3, R2> 0.35, R2> 0.4, R2> 0.45, R2> 0.5, R2> 0.55, R2> 0.6, R2> 0.65, R2> 0.7, R2> 0.75, R2> 0.8, R2> 0.85, R2> 0.9, R2> 0.91, R2> 0.92, R2> 0.93, R2> 0.94, R2> 0.95, R2> 0.96, R2> 0.97, R2> 0.98, or R2> 0.99.

[0216] Those skilled in the art will recognise that when either D' = 1, or when R2= 1, the two polymorphisms are in complete linkage disequilibrium.

[0217] In certain examples, the one or more polymorphisms in linkage disequilibrium with the one or more polymorphisms specified herein fulfil two criteria of R2and |D' |, such as R2> 0.2 and |D' | > 0.8. Various other combinations of values for R2and |D' | are also possible and are contemplated herein, including but not limited to the values for these parameters as described above and / or as exemplified herein in the Examples.

[0218] In certain examples, the degree of linkage disequilibrium will be calculated in the same population in which the subject belongs. For example, in certain examples relating to the assessment of bovine subjects, the degree of linkage disequilibrium is calculated with reference to the subject's breed, such as the local breeding population of the subject's breed. In a representative example, the degree of linkage disequilibrium is calculated for the local population of Holstein-Friesian bovine when the subject for which the allelic profile is to be determined is itself Holstein-Friesian. In a further representative example, the degree of linkage disequilibrium is calculated for the local population of Jersey bovine when the subject for which the allelic profile is to be determined is itself Jersey.

[0219] In certain examples, the one or more polymorphisms in linkage disequilibrium with the one or more polymorphisms specified herein is identified in any one of Tables 4 to 9.

[0220] A "haplotype" as contemplated herein refers to a combination of genetic markers, for example "alleles" such as those set forth in the Examples herein, and such haplotypes are useful in the methods described herein to identify subjects having a desired genetic characteristic, and via the association studies described herein one or more desired productivity and / or worth phenotypes. In a certain example, the haplotype can comprise one or more alleles (e.g., a haplotype containing a single SNP), two or more alleles, three or more alleles, four or more alleles, or five or more alleles. The genetic markers are particular alleles at polymorphic sites associated with a productivity and / or worth phenotype, such that the haplotypes useful herein, e.g., having markers such as those shown in the Examples, have predictive value for detecting subjects having or likely to have a particular productivity and / or worth phenotype, or likely to produce progeny having a particular productivity and / or worth phenotype. Haplotypes will in some cases comprise a combination of various genetic markers, e.g., SNPs and microsatellites. Therefore, detecting haplotypes can be accomplished by methods known in the art for detecting sequences at polymorphic sites, such as the methods described herein.

[0221] There are numerous standard methods known in the art for analysing a nucleic acid or nucleic acid sequence, for example to determine whether a particular nucleotide sequence is present in a sample. Thus, reference herein to "analysing" a nucleic acid or nucleic acid sequence of an animal, cell, embryo, gamete, or nucleus to identify whether or not it includes a given genetic marker such as one of the polymorphisms identified in Table 1, Table 2, or Table 3 herein, and / or a genetic marker in linkage disequilibrium therewith should be taken to include the physical, biological and / or chemical analysis of a nucleic acid from an animal, cell, embryo, gamete, or nucleus, as well as the analysis of data already available for the nucleic acid of a particular animal, cell, embryo, gamete, or nucleus .

[0222] References to "analysing" a nucleic acid should be taken to include analysis of DNA or RNA (for example, an mRNA transcript) and either or both strands of a double-stranded nucleic acid. Further, reference to nucleotide or nucleic acid "sequence" should be taken broadly to include both a single nucleotide as well as two or more nucleotides. In addition, with reference to nucleic acids which may have a double-stranded state, reference to determining or identifying whether or not a nucleic acid includes a specific genetic marker (such as a wild-type allele or a variant allele at one of the polymorphisms identified herein) should be read to encompass determining or identifying the genetic marker by observing the nucleotide or sequence on either strand of a DNA molecule.

[0223] In one example of the invention, the step of "analysing" a nucleic acid of an animal, cell, embryo, gamete, or nucleus to identify whether or not it includes a given genetic marker (e.g., a specified marker or a marker in linkage disequilibrium therewith) may comprise imputing a genotype of said animal. In one example, the step of analysing may comprise directly genotyping (for example, involving physical, biological, or chemical analysis of a nucleic acid) the animal, cell, embryo, gamete, or nucleus for a marker and comparing the genotype of the animal, cell, embryo, gamete, or nucleus to the genotype of a family member for which an additional marker has been genotyped. In one example, the step may comprise identifying a genotype of the animal, cell, embryo, gamete, or nucleus for a marker (for example, involving analysis of genetic data already available for a particular animal, cell, embryo, gamete, or nucleus) and comparing the genotype of the animal, cell, embryo, gamete, or nucleus to the genotype of a family member for which an additional marker has been genotyped. A genetic marker such as one of the polymorphisms identified in Table 1, Table 2, or Table 3 herein and / or a marker in linkage disequilibrium is imputed to be present or absent from the animal, cell, embryo, gamete, or nucleus based on the comparison.

[0224] In another example, the step of "analysing" may comprise directly genotyping the animal, cell, embryo, gamete, or nucleus for a marker and comparing the genotype of the animal, cell, embryo, gamete, or nucleus to the genotype(s) of a reference population comprising apparently unrelated individuals for which an additional marker has been genotyped. In another example, the step may comprise identifying a genotype of the animal, cell, embryo, gamete, or nucleus for a marker (for example, involving analysis of genetic data already available for a particular animal, cell, embryo, gamete, or nucleus) and comparing the genotype of the animal, cell, embryo, gamete, or nucleus to the genotype(s) of a reference population comprising apparently unrelated individuals for which an additional marker has been genotyped. A genetic marker as contemplated herein and / or a marker in linkage disequilibrium is imputed to be present or absent from the animal, cell, embryo, gamete, or nucleus based on the comparison. In one example, a reference population may be assembled from animals of a similar breed or crossbreed composition, or animals otherwise expected to share a similar haplotype structure, for example. Skilled persons will readily appreciate other appropriate reference populations.

[0225] Imputation methods are described, for example, in Li, Y., Wilier, C., Sanna, S., & Abecasis, G. (2009). Genotype imputation. Annual review of genomics and human genetics, 10, 387. Computational, statistical and / or in silico methods for genotype imputation will be readily known to those skilled in the art to which the invention relates. In some examples, these methods may be practised using readily available algorithms, software packages and tools such as, by way of nonlimiting example, IMPUTE, MACH, fastPHASE, BIMBAM, PLINK, TUNA, WHAP, FImpute and / or BEAGLE.

[0226] It should be appreciated that determining the genetic status of an animal, cell, embryo, gamete, or nucleus with respect to one or more productivity and / or worth traits, and / or determining the allelic profile of an animal, cell, embryo, gamete, or nucleus, will in certain examples comprises determination by imputation. Similarly, reference to an animal, cell, embryo, gamete, or nucleus for which the genetic status or allelic profile has been determined should also be taken to include it having been determined by imputation. Similar references to "identify", "identifying", "determining", and the like should be construed in the same way, unless the context requires otherwise.

[0227] Reference to "analysing" an amino acid sequence or a protein, a precursor thereof, an isoform thereof, and / or a fragment thereof of an animal, cell, embryo, gamete, or nucleus to identify whether or not it includes a variation which disrupts the protein should be taken to include the physical, biological and / or chemical analysis of the protein, a precursor thereof, an isoform thereof, and / or a fragment thereof from an animal, cell, embryo, gamete, or nucleus, as well as the analysis of data already available for, a precursor, an isoform, a fragment of a particular animal, cell, embryo, gamete, or nucleus.

[0228] In one example, analysing a protein contemplates assessing the level, such as the expression level, the amount or concentration, or the activity, of the protein. It will be apparent that the term "activity" may refer both to the inherent activity of a single molecule of the protein, which may be wild-type activity or may be less or greater than wild-type activity as may depend, for example on the amino acid sequence, the presence of any amino acid substitutions, the availability of co-factors, and the like, as well as to the total activity of the population of molecules of the protein present (for example, in a bovine or in a sample taken from a bovine), as may depend on both the activity of each molecule present and the level of expression (for example, how many such molecules are present). As used herein, such as when used in reference to an allelic protein lacking the activity of wildtype protein, the phrase "lacking the activity of (A)" contemplates activity both greater than that of (A) and less than that of (A). For example, an allelic protein lacking the activity of a protein encoded by a gene comprising a variant allele may be a variant protein of greater or lesser activity than that of wild-type protein. Typically however, most variant alleles that result in a variant protein will exhibit activity that is less than that of the wild-type protein.

[0229] Methods to assay the expression or activity of a protein are well known in the art. For example, Northern analysis, RT-PCR, or immunostaining for one or more of these proteins may be used. Other exemplary methods utilise HPLC, such as reversed-phase HPLC, mass spectrometry, ELISA, and the like.

[0230] In general, where reference is made to an "increase" or "decrease" in the level or activity of a protein, an isoform thereof, a fragment thereof, a precursor thereof, and / or a nucleic acid encoding any one or more thereof, or for example "greater activity" or "lesser activity", it should be taken broadly to include any increase or decrease in said level or activity compared to a reference animal or animals or a standard. In one particular example, the reference animal or animals are those having wild-type protein. In another example, the reference animal or animals are those having a genetic or amino acid variation which is linked to or associated with a deleterious effect on productivity and / or worth (for example, a variation which disrupts the protein). Reference may also be made herein to a "higher" or a "lower" level or activity of a protein, an isoform thereof, a fragment thereof, a precursor thereof, and / or a nucleic acid encoding any one or more thereof compared to a reference animal or animals or a standard. This should not be taken to imply a particular level or activity of the protein, an isoform thereof, a fragment thereof, a precursor thereof, and / or a nucleic acid encoding any one or more thereof. One can readily determine whether a variation results in an increase or decrease in the level or activity of a protein, such as a higher or lower level or activity of the protein compared to a standard using standard assays known in the art, including via those techniques described herein.

[0231] In one example, the level or level of activity of a protein in an animal, cell, embryo, gamete, or nucleus having a variation which disrupts the protein or the gene encoding it will be at least approximately 20%, at least approximately 30%, at least approximately 40%, at least approximately 50%, at least approximately 60%, at least approximately 70%, at least approximately 80%, at least approximately 90%, or at least approximately 95% lower than an animal or animals which do not have a genetic variation which disrupts the protein and / or the gene encoding it.

[0232] In one example, for example, in animals homozygous for the Chr2: 101396615T allele at the Chr2: 101396615T (rs433745406) polymorphism in the SPAG16 gene, it is predicted (without wishing to be bound by any theory) that there would be substantially no functional SPAG16 activity.

[0233] The polymorphisms recited herein in Tables 1 - 3 have been identified in bovine animals. However, the predicted loss of function resulting from the variant allele at any one of these polymorphisms supports the applicability of polymorphisms in the gene in which one of the polymorphisms identified in Tables 1 - 3 is located having a comparable effect on protein or gene product activity to a variety of different mammals. Various mammals are used for product productions purposes and / or may be assessed using a worth calculation.

[0234] Those skilled in the art will recognise that determining genetic status as contemplated herein is conveniently, and increasingly cost-effectively achieved by sequencing a sample comprising nucleic acid. Thus, in one example, the step of determining genetic status, such as allelic profile or whether or not the specified nucleotides are present in a nucleic acid derived from a subject, includes the step of sequencing the nucleic acid. Methods for nucleotide sequencing are well known to those skilled in the art.

[0235] For example, in examples of methods for determining the genetic status of a bovine with respect to one or more productivity and / or worth traits, the method includes ascertaining, from a sample of material containing DNA obtained from the bovine, whether a sequence of the DNA encoding (a) a protein having biological activity of wild-type protein encoded by a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located is present, and whether a sequence of the DNA encoding (b) an allelic protein lacking the activity of (a) is present.

[0236] An example of another art standard method known for determining whether a particular DNA sequence is present in a sample is the Polymerase Chain Reaction (PCR). A preferred aspect of the invention thus includes a step in which ascertaining whether a sequence of the DNA encoding (a) is present, and whether a sequence of the DNA encoding (b) is present includes amplifying the DNA in the presence of primers based on a nucleotide sequence encoding a protein having biological activity of wild-type protein, and / or in the presence of a primer containing at least a portion of a polymorphism known to naturally occur and which when present results in high relative protein levels, and / or in the presence of a primer containing at least a portion of a polymorphism known to naturally occur and which when present results in low relative protein levels.

[0237] A primer of the present invention, used in PCR for example, is a nucleic acid molecule sufficiently complementary to the sequence on which it is based and of sufficient length to selectively hybridise to the corresponding portion of a nucleic acid molecule intended to be amplified and to prime synthesis thereof under in vitro conditions commonly used in PCR. Likewise, a probe of the present invention, is a molecule, for example a nucleic acid molecule of sufficient length and sufficiently complementary to the nucleic acid molecule of interest, which selectively binds under high or low stringency conditions with the nucleic acid sequence of interest for detection in the presence of nucleic acid molecules having differing sequences.

[0238] Accordingly, a preferred example of the invention thus includes the step of amplifying a polynucleotide in the presence of at least one primer comprising a nucleotide sequence of or complementary to, the gene or flanking sequence thereof within, and / or in the presence of a such a primer comprising sequence corresponding to or flanking the allele-specific nucleotides described herein.

[0239] PCR methods are well known by those skilled in the art (Mullis et al., 1994.) The template for amplification may be selected from genomic DNA, mRNA or first strand cDNA derived from a sample obtained from the bovine under test (Sambrook et al., 1989). Primers suitable for use in PCR based methods of the invention should be sufficiently complementary to the gene sequence, such as that set forth in any one of SEQ ID NO.s: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21, or flanking sequence thereof, and of sufficient length to selectively hybridise to the corresponding portion of a nucleic acid molecule intended to be amplified and to prime synthesis thereof under in vitro conditions commonly used in PCR. Such primers should comprise at least about 12 contiguous bases of or complementary to the recited sequence(s), or naturally occurring flanking sequences thereof. Examples of such PCR primers may be derived from any one or more of the sequences presented herein as SEQ ID NO.s: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21.

[0240] Suitable PCR primers may in certain examples include a variant allele-specific nucleotide or a wild-type allele-specific nucleotide. Generation of a corresponding PCR product, or the lack of product, will in certain examples constitute a test for the presence or absence of the specified nucleotides in the relevant gene of the test subject.

[0241] Other methods for determining whether a particular nucleotide sequence is present in a sample may include the step of restriction enzyme digestion of nucleotide sample. Separation and visualisation of the digested restriction fragments by methods well known in the art, may form a diagnostic test for the presence of a particular nucleotide sequence. The nucleotide sequence digested may be a PCR product amplified as described above.

[0242] Still other methods for determining whether a particular nucleotide sequence is present in a sample include a step of hybridisation of a probe to a sample nucleotide sequence. Thus, methods for detecting one or more of the allele-specific nucleotides described herein may comprise the additional steps of hybridisation of a probe derived from the sequences disclosed herein. Such probes should comprise a nucleic acid molecule of sufficient length and sufficiently complementary to the gene sequence, to selectively bind under high or low stringency conditions with the nucleic acid sequence of a sample to facilitate detection of the presence or absence of the allele-specific nucleotides described herein. With respect to polynucleotide molecules greater than about 100 bases in length, typical stringent hybridisation conditions are no more than 25 to 30° C (for example, 10° C) below the melting temperature (Tm) of the native duplex (see generally, Sambrook et al., 1989; Ausubel et al., 1987). Tm for polynucleotide molecules greater than about 100 bases can be calculated by the formula Tm = 81. 5 + 0. 41% (G + C-log (Na+). With respect to polynucleotide molecules having a length less than 100 bases, exemplary stringent hybridisation conditions are 5 to 10° C below Tm. On average, the Tm of a polynucleotide molecule of length less than 100 bp is reduced by approximately (500 / oligonucleotide length) <0> C.

[0243] Such a probe may be hybridised with genomic DNA, mRNA, or cDNA produced form mRNA, derived from a sample taken from a bovine under test. Such probes would typically comprise at least 12 contiguous nucleotides of or complementary to one of the sequences recited herein, such as the sequence presented in any one of the reference sequences for the relevant bovine chromosome in the ARS-UCD 1.2 genome build, and may comprise sequence corresponding to the allele-specific nucleotides described herein.

[0244] Such probes may additionally comprise means for detecting the presence of the probe when bound to sample nucleotide sequence. Methods for labelling probes such as radiolabelling are well known in the art (see for example, Sambrook et al., 1989). In another example, the method for determining the genetic status of bovine with respect to one or more productivity and / or worth traits is performed with reference to a sample of material containing mRNA obtained from the bovine. In one example this method includes ascertaining whether a sequence of the mRNA encoding (A) a protein having biological activity of wild-type protein is present, and whether a sequence of the mRNA encoding (B) a protein at least partially lacking the activity of (A) is present, and may include determining the amount of mRNA. The absence of the mRNA encoding (A) and the presence of the mRNA encoding (B) indicates an association with decreased production efficiency and / or robustness. The reverse association again holds true.

[0245] Again, if an amplification method such as PCR is used in ascertaining, whether a sequence of the mRNA encoding (A) is present, and whether a sequence of the mRNA encoding (B) is present, the method includes amplifying the mRNA, for example in the presence of a pair of primers complementary to a nucleotide sequence encoding a protein having wild-type biological activity, or in the presence of a pair of primers complementary to a nucleotide sequence encoding a variant protein. It will be appreciated that in examples of the invention reliant on assessing the amount of mRNA present in a sample, quantitative amplification methods well known in the art may be employed, for example quantitative RT-PCR, microarray analysis, and other methods described herein.

[0246] Other methods to quantitate or otherwise assess the amount of nucleic acid, particularly the amount of mRNA, are well known in the art. These include Northern analysis using probes able to hybridise to the target mRNA. Such probes should comprise a nucleic acid molecule of sufficient length and sufficiently complementary to the coding sequence to selectively bind under high or low stringency conditions with the nucleic acid sequence of a sample to facilitate detection and assessment of the amount of target mRNA present. As is evident to the person skilled in the art, such quantitative methods generally utilise an internal control, for example in the case of Northern analysis quantitation may be done with reference to, for example, rRNA present in the sample.

[0247] In a further aspect, the invention relates to a method of determining genetic status of a bovine with respect to one or more productivity and / or worth traits which comprises determining the allelic profile of said bovine, together with determining the allelic profile of said bovine at one or more genetic loci associated with one or more productivity and / or worth traits.

[0248] Exemplary methods of the invention are reliant on genetic information such as that derived from methods suitable to the detection and identification of polymorphisms, particularly single nucleotide polymorphisms (SNPs) associated with the qualitative trait for which an assessment is desired.

[0249] 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 that may be present at a SNP are referred to as an allele.

[0250] For the sake of convenience the following discussion refers particularly to SNPs, yet the art- skilled worker will appreciate that the methods discussed are amenable to the detection and identification of other genetic polymorphisms, such as triplet repeats or microsatellites. A SNP is a single base change or point mutation resulting in genetic variation between individuals that can occur in coding or non-coding regions. A number of databases have been constructed of known SNPs, and for some such SNPs, the biological effect associated with a SNP. For example, the NCBI SNP database "dbSNP" is incorporated into NCBI's Entrez system and has records for many millions of refSNPs mapped onto the human genome sequence. Another example is the Ensembl database, which can be conveniently searched for sequences and variations, including using the 'rs' identifiers specified herein. Similar databases exist for other mammalian genomes.

[0251] Genotyping approaches to analyse nucleic acids, for example to detect SNPs, well-known in the art include DNA sequencing, methods that require allele specific hybridisation of primers or probes, including for example on "SNP chips" or microarrays, allele specific incorporation of nucleotides to primers bound close to or adjacent to the polymorphisms (often referred to as "single base extension", or "mini-sequencing"), allele-specific ligation (joining) of oligonucleotides (ligation chain reaction or ligation padlock probes), allele-specific cleavage of oligonucleotides or PCR products by restriction enzymes (restriction fragment length polymorphisms analysis or RFLP) or chemical or other agents, resolution of allele-dependent differences in electrophoretic or chromatographic mobilities, by structure specific enzymes including invasive structure specific enzymes, the use of proteins which recognize nucleic acid mismatches, such as E.coli mutS protein, nucleotide protection assays, denaturing HPLC (dHPLC), fluorescence quenching PCR (TaqMan™, Applied Biosystems, CA 94404, USA), High Resolution Melting (HRM), matrix-assisted laser desorption / ionisation time-of-flight mass spectroscopy (MALDI-TOF MS), or mass spectrometry. Analysis of amino acid variation is also possible where the SNP lies in a coding region and results in an amino acid change.

[0252] DNA sequencing allows the direct determination and identification of SNPs. Mini-sequencing involves allowing a primer to hybridise to the DNA sequence adjacent to the SNP site on the test sample under investigation.

[0253] A number of sequencing methods and platforms are particularly suited to large- scale implementation and are amenable to use in the methods of the invention. These include pyrosequencing methods, such as that utilised in the Genome Sequencer™ FLX pyrosequencing platform available from 454 Life Sciences (Branford, CT) which can generate -400 million nucleotide data in a 10 hour run with a single machine, solid-state sequencing methods, such as that utilised in the SOLiDcl M> sequencing platform (Applied Biosystems, Foster City, CA), second-generation synthetic sequencing technologies such as the TruSeq™ massively parallel terminator-based sequencing platform (Illumina, San Diego, CA), the PacBio RS realtime single molecule sequencing system (Pacific Biosciences, CA), the PostLight™ semiconductor-based sequencing platform (Ion Torrent, Guilford, CT), nanopore-based sequencing technologies including exonuclease-associated nanopore sequencing (Oxford Nanopore Technologies, Oxford, UK), and the tSMS™ single molecule sequencing flow cell-based platform (Helicos Bioscience Corporation, Cambridge, MA).

[0254] Genotyping by sequencing (also referred to as GBS) has become increasing cost effective through the development of high throughput whole genome sequencing technologies. However, the data analysis requirements to achieve high depth, high density representations across a whole genome from multiple sequence reads is currently not usually cost effective at scale. The cost of high confidence GBS (e.g., at high depth and high density) will be justified in the context of this disclosure in certain circumstances where high confidence data and / or direct genotyping is required - for example, to genotype individuals and lineages having high value or highly deleterious genotypes. In the context of large-scale population genotyping, however, the current cost is typically prohibitive.

[0255] Reduced representation approaches, such as whole genome skim sequencing, are however envisaged as being employable at scale in the near future. Here, sequence information is derived from fewer genomic reads - for example, 0.5 - 1 reads across the genome, as opposed to the ~30 reads typically employed in high depth, high density GBS approaches. Bioinformatic approaches, such as imputation, are employed to allow the presence of variants that have not been directly genotyped to be inferred on the basis of comparisons of the sequence that has been determined to one or more reference sequence(s). These genotyping approaches reduce the time, complexity and cost associated with assembling high confidence sequence data from multiple reads, thereby allowing for large-scale implementation.

[0256] A number of methods currently used for SNP detection involve site-specific and / or allele-specific hybridisation. These methods are largely reliant on the discriminatory binding of oligonucleotides to target sequences containing the SNP of interest. The techniques of Illumina (San Diego, CA), Affymetrix (Santa Clara, CA.) and Nanogen Inc. (San Diego, Calif.) are particularly well-known, and utilize the fact that DNA duplexes containing single base mismatches are much less stable than duplexes that are perfectly base- paired. The presence of a matched duplex is usually detected by fluorescence. A number of whole-genome genotyping products and solutions amenable or adaptable for use in the present invention are now available, including those available from the above companies.

[0257] Methods to detect or identify SNPs by site-specific hybridisation frequently require target amplification by methods such as PCR to increase sensitivity and specificity (see, for example U.S. Pat. No. 5,679,524, PCT publication WO 98 / 59066, PCT publication WO 95 / 12607). US Patent Application Publication No. US 20050059030 (incorporated herein by reference in its entirety) describes a method for detecting a SNP in total human DNA without prior amplification or complexity reduction to selectively enrich for the target sequence, and without the aid of any enzymatic reaction. The method utilises a single-step hybridisation involving a hybridisation of a first portion of the target sequence to a capture probe, and hybridisation of a second portion of said target sequence to a detection probe.

[0258] It will be appreciated that hybridisation methods need not target the specific sequence comprising or surrounding the genotype of interest to be informative. For example, SNP chips comprising a number of sequences can be informative even when the specific polymorphism or polymorphisms of interest are not present in the sequences on the chip. Rather, as described above in relation to whole genome skim sequencing, provided a sufficient number of other polymorphisms are present on the chip and a reference sequence or sequences are available, the identity of an allele at a target polymorphism or polymorphisms can be imputed. The use of SNP chips in conjunction with imputation methods to identify the allele present at the polymorphism of interest are specifically contemplated herein.

[0259] The technique of Lynx Therapeutics (Hay ward, Calif.) using MEGATYPE™ technology can genotype very large numbers of SNPs simultaneously from small or large pools of genomic material. Two populations are compared using fluorescently labelled probes, enabling detection and recovery of SNPs that distinguish the two populations.

[0260] Other methods for detecting and identifying SNPs include mass spectrometry. A preferred example is the use of mass spectrometric determination of a nucleic acid sequence which comprises the polymorphisms of the invention (whether the coding sequence or a complementary sequence). Such mass spectrometric methods are known to those skilled in the art, and the genotyping methods of the invention are amenable to adaptation for the mass spectrometric detection of the polymorphisms of the invention.

[0261] SNPs can also be determined by ligation-bit analysis, while a large number of methods reliant on the conformational variability of nucleic acids have been developed to detect SNPs. For example, Single Strand Conformational Polymorphism (SSCP, Orita et al., PNAS 1989 86:2766-2770), and various modifications of SSCP as are well known in the art. These include the use of differing gel running conditions, such as for example differing temperature, or the addition of additives, and different gel matrices, RNA-SSCP, restriction endonuclease fingerprinting-SSCP, dideoxy fingerprinting (a hybrid between dideoxy sequencing and SSCP), bi-directional dideoxy fingerprinting (in which the dideoxy termination reaction is performed simultaneously with two opposing primers), and Fluorescent PCR-SSCP (in which PCR products are internally labelled with multiple fluorescent dyes, may be digested with restriction enzymes, followed by SSCP, and analysed on an automated DNA sequencer able to detect the fluorescent dyes).

[0262] Other methods which utilise the varying mobility of different nucleic acid structures include Denaturing Gradient Gel Electrophoresis (DGGE), Temperature Gradient Gel Electrophoresis (TGGE), Heteroduplex Analysis (HET), and capillary electrophoresis. Denaturing High Pressure Liquid Chromatography (HPLC) is yet a further method utilised to detect SNPs, using HPLC methods to detect, for example, homoduplexes and heteroduplexes which elute from the HPLC column at different rates, thereby enabling detection of mismatch nucleotides and thus SNPs.

[0263] Yet further methods to detect SNPs rely on the differing susceptibility of single stranded and double stranded nucleic acids to cleavage by various agents, including chemical cleavage agents and nucleolytic enzymes. Further examples include the Protein Translation Test (PTT), used to resolve stop codons generated by variations which lead to a premature termination of translation and to protein products of reduced size, and the use of mismatch binding proteins. US Patent 6,821,733 (incorporated herein in its entirety) describes methods to detect differences in the sequence of two nucleic acid molecules.

[0264] Protein- and proteomics-based approaches are also suitable for polymorphism detection and analysis. These approaches typically require separation of the various proteins within a sample, by, for example, gel electrophoresis or HPLC, and identification of said proteins or peptides derived therefrom, for example by NMR or protein sequencing such as chemical sequencing or more prevalently mass spectrometry. Proteomic methodologies are well known in the art, and have great potential for automation. For example, integrated systems, such as the ProteomlQ™ system from Proteome Systems, provide high throughput platforms for proteome analysis combining sample preparation, protein separation, image acquisition and analysis, protein processing, mass spectrometry and bioinformatics technologies. The majority of proteomic methods of protein identification utilise mass spectrometry, including ion trap mass spectrometry, liquid chromatography (LC) and LC / MSn mass spectrometry, gas chromatography (GC) mass spectroscopy, Fourier transform-ion cyclotron resonance-mass spectrometer (FT-MS), MALDI-TOF mass spectrometry, and ESI mass spectrometry, and their derivatives. Mass spectrometric methods are also useful in the determination of post-translational modification of proteins, such as phosphorylation or glycosylation, and thus have utility in determining polymorphisms that result in or are associated with variation in post-translational modifications of proteins.

[0265] Associated technologies are also well known, and include, for example, protein processing devices such as the "Chemical Inkjet Printer" comprising piezoelectric printing technology that allows in situ enzymatic or chemical digestion of protein samples electroblotted from 2-D PAGE gels to membranes by jetting the enzyme or chemical directly onto the selected protein spots. After in-situ digestion and incubation of the proteins, the membrane can be placed directly into the mass spectrometer for peptide analysis.

[0266] Suitable polypeptide-based analyses include those able to discriminate between full-length and truncated protein products, and may include but are not limited to, the following: Native polyacrylamide gel electrophoresis (PAGE), isoelectric focussing, 2D PAGE, or Western blotting with specific antibodies. Mass spectroscopy, immunoprecipitation, and peptide fingerprinting are also suitable.

[0267] Those skilled in the art will know that a particular SNP, particularly when it occurs in a regulatory region of a gene such as a promoter, can be associated with altered expression of a gene. Altered expression of a gene can also result when the SNP is located in the coding region of a proteinencoding gene. Such altered expression can be determined by methods well known in the art, such as quantitative PCR, RT-PCR, quantitative Northern analysis, and can thereby be employed to detect such SNPs. Similarly, where a SNP occurs in the coding region of a gene and results in a non-synonymous amino acid substitution, such substitution can result in a change in the function of the gene product. Similarly, in cases where the gene product is an RNA, such SNPs can result in a change of function in the RNA gene product. Any such change in function, for example as assessed in an activity or functionality assay, can be employed to detect such SNPs.

[0268] Any of the above methods of detecting and identifying SNPs are amenable to use in the methods contemplated herein.

[0269] Sample preparation

[0270] In certain examples, the methods contemplated herein will involve taking a sample from an animal (or cell, embryo, nucleus, etc.) to be tested. The sample may be any appropriate tissue or body fluid sample. In one example, the sample is one or more of a cell, blood, muscle, bone, somatic cell(s), saliva, or semen. Such samples can be taken from an animal using standard techniques known in the art, including cell scrapings or biopsy techniques, such as ear punch, or blood sampling. 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 are also amenable to testing using the methods contemplated herein. 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 a biological marker, as will be described further herein after.

[0271] In addition, it should be appreciated that where analysis or observation of a marker in an animal is conducted during gestation, the analysis or observation could be conducted by analysing protein, peptide, nucleic acid or a cell of that animal that may be present in the maternal blood supply, placenta, amniotic fluid or any other maternal tissue or fluid prior to birth of the animal. Accordingly, reference to analysing a nucleic acid of an animal, analysing a protein, a precursor, an isoform and / or fragment thereof of an animal, observing the level or activity of a protein, a precursor, an isoform and / or fragment thereof of an animal, and the like, should be taken to include reference to analysing and / or observing one or more of these from that animal that may be present in a maternal tissue or fluid.

[0272] Likewise, methods for storing and processing biological samples are well known in the art. For example, tissue samples may be frozen until tested if required. In addition, one of skill in the art would realize that some test samples would be more readily analysed following a fractionation or purification procedure, for example, separation of whole blood into serum or plasma components.

[0273] Genetic markers

[0274] A number of the methods contemplated herein involve the determination of the allelic profile of the subject animal, cell, embryo, gamete, or nucleus. As set out herein, various methods for determining the allelic profile are contemplated. A particularly straightforward and informative method involves the analysis of nucleic acid from the animal, cell, embryo, gamete, or nucleus.

[0275] In certain examples, the allelic profile comprises information relating to presence or absence of one or more of the following: a) one or more alleles at a polymorphism associated with decreased expression from a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located; or b) one or more alleles at a polymorphism associated with increased expression from a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located; or c) one or more wild-type alleles at any one of the polymorphisms identified in Table 1, Table 2, or Table 3; or d) one or more variant alleles at any one of the polymorphisms identified in Table 1, Table 2, or Table 3; or e) one or more homozygous wild-type genotypes at any one or more of the polymorphisms identified in Table 1, Table 2, or Table 3; or f) one or more homozygous variant genotypes at any one or more of the polymorphisms identified in Table 1, Table 2, or Table 3; or g) one or more alleles at a polymorphism in linkage disequilibrium with one or more of the polymorphisms recited in any of (a) to (f) above, and particularly a polymorphism in 100% linkage disequilibrium (R2= 1.0) with any one or more of the polymorphisms identified in (a) to (f) above; or h) any combination of any two or more of (a) to (g). It will be appreciated that the precise location of a particular genetic marker contemplated herein may vary slightly from genome to genome; for example, the location of the marker may vary in a different species of animal, or in different breeds of animal. However, persons of skill in the art to which the invention relate will be able to readily identify a particular marker in different genomes through routine sequence alignment and with knowledge that it resides in the equivalent gene. To account for this variation in the location of a genetic marker across genomes reference to a position of a particular marker herein should be taken to mean a position "corresponding to" a particular position of the relevant chromosome of Bos taurus in the ARS-UCD 1.2 genome build. For example, reference to the genetic marker Chr2: 101396615 G>T (rs433745406) on chromosome 2 of Bos taurus should be taken to mean that the genetic marker is at a position corresponding to position 101396615 of the bovine chromosome 2 reference sequence in the ARS-UCD 1.2 genome build.

[0276] In one example, the methods contemplated herein involve the analysis of a nucleic acid to determine whether or not it includes a variation in the nucleotide sequence predicted to result in variation, such as truncation of the protein or variation in the amino acid sequence of the protein. In various examples set out in Table 1, the variation results in a variant protein in which one or more amino acid substitutions are present.

[0277] In one example, the methods contemplated herein involve analysis of a nucleic acid to determine whether or not it includes a variation in the gene at a position corresponding to the position specified in any one of Tables 1 - 3. In one example, the variation is a nonsense variant resulting in truncation of the encoded gene product. In other examples, the variation is a missense variant resulting in an encoded protein having a variant amino acid sequence. In still other examples, the variation is a splice variant, which may result in an encoded protein having a variant amino acid sequence, a truncated protein, or no protein. In various examples, the methods involve analysis of a polymorphism in linkage disequilibrium with said variation.

[0278] The technique(s) used for nucleic acid analysis will depend on the nature of the nucleic acid and / or marker to be detected, as will be appreciated by skilled persons. For example, SNPs will typically be analysed using those techniques capable of resolving single nucleotide differences 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) are suitable to analyse genetic markers which include insertion or deletion of one or more nucleotide.

[0279] 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.

[0280] Accordingly, in a further aspect, the invention relates to a probe containing a nucleic acid molecule sufficiently complementary with a nucleic acid sequence present in any one of SEQ ID NO.s: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21 or a nucleotide sequence encoding a wild-type bovine protein identified in any one of Tables 1 - 3, or its complement, so as to bind thereto under stringent conditions, as well as a diagnostic kit containing such a probe. Particularly contemplated are probes that comprise one or other of the allele-specific nucleotides at one of the polymorphisms identified in Table 1, Table 2, or Table 3.

[0281] The invention also relates to a primer composition useful for detection of the presence or absence of a wild-type gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located, including nucleic acid sequence encoding a wild-type bovine protein identified in any one of Tables 1 - 3 and / or the presence of the nucleic acid sequence encoding a variant protein at least partially lacking wild-type activity. In one form, the composition can include a nucleic acid primer substantially complementary to a nucleic acid sequence encoding wild-type protein, or its complement. The nucleic acid sequence can in whole or in part be identified in one of Genbank references NC_037328.1 to NC_037357.1, NC_006853.1, and NC_082638.1 (the bovine chromosome reference sequences for chromosomes 1 - 29 and the X chromosome, for the mitochondrial chromosome MT, and for the Y chromosome, respectively, in the ARS-UCD 1.2 genome build). Diagnostic kits including such a composition are also included.

[0282] Particularly contemplated are primers comprising or substantially complementary to a nucleic acid sequence present in one of NC_037328.1 to NC_037357.1 and within approximately 1 to about 2000 bp of one of the polymorphisms identified in Table 1, Table 2, or Table 3, for example, within approximately 1 to about 1000 bp, or within approximately 1 to about 500 bp, approximately 1 to about 400 bp, approximately 1 to about 300 bp, approximately 1 to about 200 bp, approximately 1 to about 100 bp, approximately 1 to about 50 bp, or within approximately 1 to about 20 bp of one of the polymorphisms identified in Table 1, Table 2, or Table 3.

[0283] Examples of such primers may be derived from any one of the sequences presented herein as SEQ ID NO.s: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21.

[0284] It will be appreciated by those skilled in the art that a pair of such primers can be used to determine the identity of the nucleotide at a given polymorphism, by, for example the selective generation of an amplicon with one or more sequence-specific primers. Primer compositions comprising a pair of such primers are accordingly contemplated.

[0285] The proteins, isoforms thereof, precursors thereof, and / or fragments thereof as contemplated herein may be analysed using standard techniques known in the art. However, by way of example, peptide sequencing methods, mass spectrometry, Western blotting and ELISA are each suitable for use. It should be appreciated that the analysis of a protein, an isoform or isoforms thereof, precursors thereof, and / or fragments thereof to identify a variation may comprise analysis of the size of the protein, a precursor thereof, an isoform thereof, and / or a fragment thereof.

[0286] The protein (including reference to precursors, fragments and / or isoforms thereof) and nucleic acids encoding same may be detected and the levels thereof compared to a standard using any one or a combination of techniques which are of use in identifying, quantifying and / or highlighting differential levels or expression of one or more proteins. Such techniques will be readily appreciated by persons of ordinary skill in the art to which the invention relates. However, by way of example, the level of protein (including reference to one or more precursors, fragments and / or isoforms thereof) may be measured using protein purification methods, immunological techniques, separation of proteins based on characteristics such as molecular weight and isoelectric point including gel electrophoresis (for example, PAGE, including 2D PAGE) and microfluidics-based technologies as for example in gel-free protein separation techniques, and mass spectrometry (MS) utilizing isobaric label based MS such as iTRAQ or label-free approaches such as multiple reaction monitoring (MRM).

[0287] Appropriate immunological techniques include enzyme linked immunosorbent assay (ELISA) (including sandwich ELISA, double sandwich ELISA, direct ELISA, microparticle ELISA), radioimmunoassay (RIA), immunoprecipitation, Western blotting, immunohistochemical staining, antibody arrays, or agglutination assays. Protocols for carrying out such techniques are readily available; for example, see "Antibodies a Laboratory Manual", Cold Spring Harbor Laboratory Press (1988).

[0288] Antibodies of use in such immunological techniques may be purchased commercially or produced according to standard methodology in the art having regard to the nature of the proteins to be tested. For example, polyclonal antibodies and monoclonal antibodies may be produced in accordance with the procedures described in the text "Antibodies a Laboratory Manual" (Cold Spring Harbor Laboratory Press, 1988) using one or more of the proteins or a fragment thereof as antigen. Preferably monoclonal antibodies are used.

[0289] Methods to determine activity of a protein encoded by a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located are also of use herein, and may be measured using standard methodology as known in the art, having regard to the function of the protein. By way of example, the methods used may involve one or more of the following techniques: Immunoprecipitation, Western blotting, ELISA, mass spectrometry, surface plasmon resonance, isothermal titration calorimetry, luciferase assays and reporter gene assays.

[0290] It should be appreciated that in addition to analysing samples and standards, the methods of the invention may include the testing of one or more positive or negative control samples to ensure the integrity of the results. For example, one could include a sample containing no protein / nucleic acid and one or more samples containing a known level of protein / nucleic acid so that results can be calibrated across different runs of the method.

[0291] Diagnostic kits

[0292] The invention further relates to diagnostic kits useful in determining the allelic profile of the mammalian subject, for example for use in the methods described herein.

[0293] Accordingly, in one example the invention relates to a diagnostic kit which can be used to determine the genotype at one of the polymorphisms identified in Table 1, Table 2, or Table 3 of the subject's genetic material.

[0294] One exemplary kit comprises an array of oligonucleotides wherein at least one oligonucleotide is capable of hybridising with a nucleic acid discriminatory of the allele present at one of the polymorphisms identified in Table 1, Table 2, or Table 3.

[0295] In another example, a kit includes 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 naturally occurring mutations described herein. Such a kit could also include a primer for amplifying the corresponding region of the normal gene that produces a functionally wildtype protein. Usually, such a kit would also include another primer upstream or downstream of the region of the gene. These primers are used to amplify the segment containing the mutation of interest. The actual genotyping is carried out using primers that target specific mutations described herein and that could function as allele-specific oligonucleotides in conventional hybridisation, Taqman assays, OLE assays, etc. Alternatively, primers can be designed to permit genotyping by microsequencing.

[0296] One kit of primers can include first, second and third primers, (a), (b) and (c), respectively. Primer (a) is based on a region containing a mutation such as described above. Primer (b) encodes a region upstream or downstream of the region to be amplified by a primer (a) so that genetic material containing the mutation is amplified, by PCR, for example, in the presence of the two primers. Primer (c) is based on the region corresponding to that on which primer (a) is based, but lacking the mutation. Thus, genetic material containing the non-mutated region will be amplified in the presence of primers (b) and (c). Genetic material homozygous for the wild-type gene will thus provide amplified products in the presence of primers (b) and (c). Genetic material homozygous for the mutated gene will thus provide amplified products in the presence of primers (a) and (b). Heterozygous genetic material will provide amplified products in both cases.

[0297] For example, the kit may include a primer comprising an allele-specific nucleotide at the position corresponding to one of the polymorphisms identified in Table 1, Table 2, or Table 3, or a nucleotide capable of hybridising to a nucleotide capable of hybridising to said allele-specific nucleotide. Those skilled in the art will recognise that in such a primer, the allele-specific nucleotide, or the nucleotide capable of hybridising to a nucleotide capable of hybridising to the allele-specific nucleotide, as applicable, may be substituted for a nucleotide analogue having the same discriminatory base-pairing as the substituted nucleotide. Similarly, nucleotide primers directed to the complementary strand, with appropriate discriminatory nucleotides, are also contemplated.

[0298] In one example, the diagnostic kit is useful in detecting DNA comprising a variant gene or encoding a variant polypeptide at least partially lacking wild-type activity in a subject, such as a bovine, which includes first and second primers for amplifying the DNA, the primers being complementary to nucleotide sequences of the DNA upstream and downstream, respectively, of a polymorphism in the gene which results in one or more increased or decreased productivity or worth traits. In one example, at least one of the nucleotide sequences is selected to be from a coding region of the gene. In another example, at least one of the nucleotide sequences is selected to be from a non-coding region of the gene. The kit can also include a third primer complementary to a naturally occurring mutation of a coding portion of the wild-type gene. In one example the kit includes instructions for use, for example in accordance with a method of the invention.

[0299] In one example, the diagnostic kit comprises a nucleotide probe complementary to the sequence, or an oligonucleotide fragment thereof, shown in any one of NC_037328.1 to NC_037357.1, for example, for hybridisation with mRNA from a sample of cells; means for detecting the nucleotide probe bound to mRNA in the sample with a standard. In a particular aspect, the kit of this aspect of the invention includes a probe having a nucleic acid molecule sufficiently complementary with a sequence presented in any one of NC_037328.1 to NC_037357.1 or complements thereof, so as to bind thereto under stringent conditions. Appropriate wash stringency depends on degree of homology and length of probe. If homology is 100%, a high temperature (65°C to 75°C) may be used. However, if the probe is very short (<100bp), lower temperatures must be used even with 100% homology. In general, one starts washing at low temperatures (37°C to 40°C), and raises the temperature by 3-5°C intervals until background is low enough to be a major factor in autoradiography. The diagnostic kit can also contain an instruction manual for use of the kit.

[0300] In another example, the diagnostic kit comprises an antibody or an antibody composition useful for detection of the presence or absence of wild-type protein and / or the presence or absence of a variant protein at least partially lacking wild-type activity of the protein, together with instructions for use, for example in a method of the invention.

[0301] The invention also relates to a diagnostic kit including a primer composition useful for determining the presence or absence of a reference gene and / or the presence or absence of nucleic acid encoding reference protein, the diagnostic kit comprising one or more primers or primer compositions as described herein.

[0302] The invention further relates to an antibody composition useful for detection of the presence or absence or amount of wild-type protein and / or the presence or absence or amount of a variant protein at least partially lacking wild-type activity, as well as a diagnostic kit containing such an antibody together with instructions for use, for example in a method of the invention.

[0303] Thus, in another example the invention relates to a method of assessing the genetic status of a bovine with respect to one or more productivity and / or worth traits which comprises the step of determining the presence or absence of one or more alleles at any one or more of the polymorphisms identified in Table 1, Table 2, or Table 3, or one or more polymorphisms in linkage disequilibrium with one or more of said polymorphisms.

[0304] In one example, the method of assessing the genetic status of a bovine with respect to one or more productivity and / or worth traits comprises the step of determining one or more of: a) the presence or absence of one or more wild-type alleles at any one or more of the polymorphisms identified in Table 1, Table 2, or Table 3; or b) the presence or absence of one or more variant alleles at any one or more of the polymorphisms identified in Table 1, Table 2, or Table 3; or c) the presence or absence of a polymorphism in linkage disequilibrium with any one of a) or b) above, and particularly the presence or absence of a polymorphism in 100% linkage disequilibrium (R2= 1.0) with any one of a) or b) above, or d) any combination of any two or more of a) to c).

[0305] In another aspect, the present invention relates to a method for identifying, and / or selecting or rejecting a mammalian subject with a genotype indicative of one or more desired productivity and / or worth phenotypes. The method comprises determining the allelic profile of said subject, and identifying, and / or selecting or rejecting the subject on the basis of the determination. In one example, the allelic profile is determined by providing the results of an analysis of a sample from said subject for the presence or absence of one or more alleles at one of the polymorphisms identified in Table 1, Table 2, or Table 3 gene.

[0306] In one example, the one or more alleles is associated with increased or decreased expression or activity of a gene product encoded by a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located.

[0307] In one example, the allelic profile is determined by providing the results of an analysis of a sample from said subject for the presence or absence of one or more polymorphisms in linkage disequilibrium with the one or more alleles associated with increased or decreased expression or activity of a gene product encoded by a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located.

[0308] In one example, the subject is bovine.

[0309] In one example, the phenotype is desired production efficiency and / or robustness, such as increased production efficiency and / or robustness. In one example the phenotype is a capability of producing progeny having or that will have increased production efficiency and / or robustness, such as increase production efficiency.

[0310] In one example, the phenotype is the absence of decreased production efficiency and / or robustness. In one example the phenotype is a capability of producing progeny that do not or will not have decreased production efficiency and / or robustness, such as decreased production efficiency. In one example, the phenotype is a reduced likelihood of having progeny that are less likely to have one or more undesirable lactation traits, or are less likely to be able to confer on their progeny one or more undesirable lactation traits.

[0311] In one example, the invention relates to a method for selecting a bovine with an allelic profile indicative of one or more increased productivity and / or worth traits, such as one or more increased robustness traits, such as increased fertility.

[0312] In one example the method comprises determining the presence of the wild-type allele at one of the polymorphisms identified in Table 1, Table 2, or Table 3, and selecting the bovine on the basis of the determination. Alternatively or additionally, the method comprises determining the absence of the variant allele at one of the polymorphisms identified in Table 1, Table 2, or Table 3, and selecting the bovine on the basis of the determination.

[0313] In one example, the method comprises determining the presence of a homozygous wild-type genotype at one of the polymorphisms identified in Table 1, Table 2, or Table 3, and selecting the bovine on the basis of the determination.

[0314] In a further example the invention relates to a method for identifying and / or rejecting a bovine with an allelic profile indicative of one or more decreased productivity and / or worth traits, such as one or more decreased robustness traits, such as decreased fertility.

[0315] In one example the method comprises determining the presence of the variant allele at one of the polymorphisms identified in Table 1, Table 2, or Table 3, and rejecting the bovine on the basis of the determination. Alternatively or additionally, the method comprises determining the absence of the wild-type allele at one of the polymorphisms identified in Table 1, Table 2, or Table 3, and rejecting the bovine on the basis of the determination.

[0316] In one example, the method comprises determining the presence of a homozygous variant genotype at one of the polymorphisms identified in Table 1, Table 2, or Table 3, and rejecting the bovine on the basis of the determination. In one example, the method comprises determining the absence of a homozygous wild-type genotype at one of the polymorphisms identified in Table 1, Table 2, or Table 3, and rejecting the bovine on the basis of the determination.

[0317] In various examples, the presence of an allele is determined with respect to a polynucleotide (e.g., genomic DNA, mRNA or cDNA produced from mRNA) obtained from the bovine.

[0318] In one example, the presence of an allele is determined by sequencing a polynucleotide obtained from the bovine.

[0319] In a further example the determination comprises the step of amplifying a polynucleotide sequence from genomic DNA, mRNA or cDNA produced from mRNA derived from said bovine, for example by PCR.

[0320] In one example the determination is by use of primers which comprise a nucleotide sequence having at least about 12 contiguous bases of or complementary to the sequence of any one of NC_ 037328.1 to NC_037357.1, and / or to any one of SEQ ID NO.s: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21, or a naturally occurring flanking sequence.

[0321] In one example at least one of the primers comprises sequence corresponding to at least one of the allele-specific nucleotides described herein.

[0322] In an alternative example, the method comprises restriction enzyme digestion of a nucleotide derived from the bovine. Such digestion may also be performed on a product of the PCR amplification described above.

[0323] In a further example, the presence of an allele is determined by mass spectrometric analysis of a polynucleotide obtained from the bovine and comprising a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located.

[0324] In an alternative example, the presence of an allele is determined by hybridisation of a probe or probes comprising a nucleotide sequence of or complementary to the sequence of any one of

[0325] NC_ 037328.1 to NC_037357.1, and / or to any one of SEQ ID NO.s: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21.

[0326] In one example the probe or probes comprises 12 or more contiguous nucleotides of or complementary to the sequence of any one of NC_037328.1 to NC_037357.1, and / or to any one of SEQ ID NO.s: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21.

[0327] In one example the probe or probes comprise sequence corresponding to at least one of the allele-specific nucleotides described herein or complements thereof.

[0328] In an alternative example, the presence of an allele is determined by analysis of a polypeptide obtained from the bovine, wherein said polypeptide is encoded by a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located. In a further aspect the invention relates to a bovine selected by a process of the invention; milk or colostrum produced by the selected bovine or the progeny thereof, as well as compositions and dairy products produced from such milk; compositions produced from such colostrum, and ova or semen produced by or tissue from the selected bovine.

[0329] In still a further aspect the invention relates to a method of selecting a herd of bovine, comprising selecting individuals by a method of the present invention, and segregating and collecting the selected individuals to form the herd. The invention further relates to a herd of bovine so selected, as well as a herd comprising bovine produced by bovine selected by the methods described herein.

[0330] In a still further aspect, the invention relates to a method of determining genetic status of a bovine with respect to one or more one or more productivity and / or worth traits, or with respect to capability of producing progeny predisposed to or with one or more one or more productivity and / or worth traits, the method comprising: providing data about the allelic profile of said bovine, and determining the genetic status of the bovine on the basis of the data.

[0331] In one example, the data about the allelic profile comprises data representative of the presence or absence of one or more of the following: a) one or more alleles at a polymorphism associated with decreased expression from a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located; or b) one or more alleles at a polymorphism associated with increased expression from a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located; or c) one or more wild-type alleles at any one of the polymorphisms identified in Table 1,

[0332] Table 2, or Table 3; or d) one or more variant alleles at any one of the polymorphisms identified in Table 1, Table 2, or Table 3; or e) one or more homozygous wild-type genotypes at any one or more of the polymorphisms identified in Table 1, Table 2, or Table 3; or f) one or more homozygous variant genotypes at any one or more of the polymorphisms identified in Table 1, Table 2, or Table 3; or g) one or more alleles at a polymorphism in linkage disequilibrium with one or more of the polymorphisms recited in any of (a) to (f) above, and particularly a polymorphism in 100% linkage disequilibrium (R2= 1.0) with any one or more of the polymorphisms identified in (a) to (f) above; or h) any combination of any two or more of (a) to (g).

[0333] In one example, the method additionally comprises providing data comprising the result of at least one analysis of one or more genetic loci associated with one or more one or more productivity and / or worth traits, wherein the data is representative of the genetic status of the bovine.

[0334] In one example, the one or more genetic loci are one or more polymorphisms associated with an increase or decrease in expression or activity of a gene product encoded by a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located. In one example the genetic locus is a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located (including all regulatory elements such as the promoter, introns and 3' UTR).

[0335] In a further aspect the invention relates to a method for identifying, and / or selecting or rejecting a non-human mammalian subject with respect to one or more desired productivity and / or worth traits, the method comprising: providing the result of one or more genetic tests of a sample from the subject, and analysing the result for the presence or absence of one or more polymorphisms selected from the group comprising:

[0336] (a) one or more polymorphisms associated with increased or decreased expression or activity of a gene product encoded by a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located, or

[0337] (b) one or more polymorphisms in linkage disequilibrium with one or more polymorphisms of (a), wherein a result indicative of the presence or absence of one or more of said polymorphisms is indicative of a subject with one or more desired productivity and / or worth traits; and identifying, and / or selecting or rejecting the subject on the basis of the result.

[0338] In one example, the one or more polymorphisms associated with increased or decreased expression or activity of the gene product is one or more polymorphisms in a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located.

[0339] In a further aspect the invention relates to a method for selecting a bovine with one or more desired productivity and / or worth traits, the method comprising: providing the result of one or more genetic tests of a sample from the bovine, and analysing the result for the presence or absence of any one or more of the following: a) one or more alleles at a polymorphism associated with decreased expression from a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located; or b) one or more alleles at a polymorphism associated with increased expression from a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located; or c) one or more wild-type alleles at any one of the polymorphisms identified in Table 1, Table 2, or Table 3; or d) one or more variant alleles at any one of the polymorphisms identified in Table 1, Table

[0340] 2, or Table 3; or e) one or more homozygous wild-type genotypes at any one or more of the polymorphisms identified in Table 1, Table 2, or Table 3; or f) one or more homozygous variant genotypes at any one or more of the polymorphisms identified in Table 1, Table 2, or Table 3; or g) one or more alleles at a polymorphism in linkage disequilibrium with one or more of the polymorphisms recited in any of (a) to (f) above, and particularly a polymorphism in 100% linkage disequilibrium (R2 = 1.0) with any one of (a) to (f) above; or h) any combination of any two or more of (a) to (g); wherein a result indicative of the presence or absence of one or more of said polymorphisms is indicative of a bovine with one or more desired productivity and / or worth traits.

[0341] In another aspect, the invention relates to an isolated, purified, synthesised, or recombinant nucleic acid molecule comprising nucleotide sequence selected from the group comprising any one of: a) at least 12 contiguous nucleotides of a nucleotide sequence corresponding to that of one of SEQ ID NO.s: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21 and comprising the nucleotides corresponding to the variant allele as set out in Table 1 at the polymorphic locus present in the sequence; b) at least 12 contiguous nucleotides of any one of NC_037328.1 to NC_037357.1 and comprising a variant allele-specific nucleotide or nucleotides at one of the polymorphisms identified in Table 1, Table 2, or Table 3; c) at least 12 contiguous nucleotides of a variant of a) or b); or d) a complement of any one of a) to c); or e) a sequence of at least 12 contiguous nucleotides and capable of hybridising to the nucleotide sequence of any one of a) to d) under stringent conditions.

[0342] The invention also relates to a genetic construct comprising a nucleic acid molecule of the invention, a vector comprising the genetic construct or a nucleic acid sequence as described above, a host cell comprising the genetic construct or vector, a polypeptide encoded by a variant gene as described herein, a nucleic acid molecule as described herein, an antibody which selectively binds a polypeptide as described herein, and a method for recombinantly producing a polypeptide as described herein.

[0343] In a further aspect the invention relates to a method of determining a subject's risk of developing one or more diseases or conditions associated with decreased or increased expression or activity of a gene product encoded by a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located, the method comprising providing the results of an analysis of a sample from said subject for the presence or absence of one or more alleles at any one or more of the polymorphisms identified in Table 1, Table 2, or Table 3, or one or more polymorphisms associated with increased or decreased expression or activity of a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located, or one or more polymorphisms in linkage disequilibrium with one or more of the polymorphisms identified in Table 1, Table 2, or Table 3, wherein the presence or absence of one or more of said polymorphisms is indicative of the subject's risk of developing one or more diseases or conditions associated with decreased or increased expression or activity.

[0344] The one or more polymorphisms can be detected directly or by detection of one or more polymorphisms which are in linkage disequilibrium with said one or more polymorphisms.

[0345] In one example, the one or more polymorphisms are selected from the group consisting of: a) one or more alleles at a polymorphism associated with decreased expression from a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located; or b) one or more alleles at a polymorphism associated with increased expression from a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located; or c) one or more wild-type alleles at any one of the polymorphisms identified in Table 1, Table 2, or Table 3; or d) one or more variant alleles at any one of the polymorphisms identified in Table 1, Table

[0346] 2, or Table 3; or e) one or more homozygous wild-type genotypes at any one or more of the polymorphisms identified in Table 1, Table 2, or Table 3; or f) one or more homozygous variant genotypes at any one or more of the polymorphisms identified in Table 1, Table 2, or Table 3; or g) one or more alleles at a polymorphism in linkage disequilibrium with one or more of the polymorphisms recited in any of (a) to (f) above, and particularly a polymorphism in 100% linkage disequilibrium (R2= 1.0) with any one of (a) to (f) above; or h) any combination of any two or more of (a) to (g); wherein the presence or absence of one or more of said polymorphisms is indicative of the subject's risk of developing one or more diseases or conditions, such as one or more diseases or conditions associated with decreased or increased expression or activity of a gene product encoded by a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located.

[0347] In various examples, the presence of one or more of the following is indicative of an increased risk of developing one or more diseases or conditions, such as one or more diseases or conditions associated with decreased expression or activity of a gene product encoded by a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located: a) one or more variant alleles at any one of the polymorphisms identified in Table 1, Table

[0348] 2, or Table 3; or b) one or more alleles at a polymorphism in linkage disequilibrium with one or more of the polymorphisms recited in (a) above, and particularly a polymorphism in 100% linkage disequilibrium (R2= 1.0) with one or more of the polymorphisms recited in (a) above; or c) any combination of any two or more of (a) to (b).

[0349] In various examples, the absence of one or more of the following is indicative of a decreased risk of developing one or more diseases or conditions, such as one or more diseases or conditions associated with increased expression or activity of a gene product encoded by a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located: a) one or more variant alleles at any one of the polymorphisms identified in Table 1, Table

[0350] 2, or Table 3; or b) one or more alleles at a polymorphism in linkage disequilibrium with one or more of the polymorphisms recited in (a) above, and particularly a polymorphism in 100% linkage disequilibrium (R2= 1.0) with one or more of the polymorphisms recited in (a) above; or c) any combination of any two or more of (a) to (b).

[0351] In various examples, the presence of one or more of the following is indicative of a decreased risk of developing one or more diseases or conditions, such as one or more diseases or conditions associated with increased expression or activity of a gene product encoded by a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located: a) one or more variant alleles at any one of the polymorphisms identified in Table 1, Table 2, or Table 3; or b) one or more alleles at a polymorphism in linkage disequilibrium with one or more of the polymorphisms recited in (a) above, and particularly a polymorphism in 100% linkage disequilibrium (R2= 1.0) with one or more of the polymorphisms recited in (a) above; or c) any combination of any two or more of (a) to (b).

[0352] In various examples, the absence of one or more of the following is indicative of an increased risk of developing one or more diseases or conditions associated with increased expression or activity of a gene product encoded by a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located: a) one or more variant alleles at any one of the polymorphisms identified in Table 1, Table 2, or Table 3; or b) one or more alleles at a polymorphism in linkage disequilibrium with one or more of the polymorphisms recited in (a) above, and particularly a polymorphism in 100% linkage disequilibrium (R2= 1.0) with one or more of the polymorphisms recited in (a) above; or c) any combination of any two or more of (a) to (b).

[0353] In another aspect the invention relates to an isolated, purified, synthesised, or recombinant protein encoded by a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located, wherein said gene comprises the variant allele at said polymorphism.

[0354] In one example the polypeptide is a variant as defined herein.

[0355] In another example the polypeptide is a functional variant of a protein encoded by a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located, or is a functional fragment thereof.

[0356] Polynucleotides and variants thereof

[0357] The term "polynucleotide(s)," as used herein, means a single or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length but preferably at least 15 nucleotides, and include as non-limiting examples, coding and non-coding sequences of a gene, sense and antisense sequences complements, exons, introns, genomic DNA, cDNA, pre- mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polypeptides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers and fragments. A number of nucleic acid analogues are well known in the art and are also contemplated.

[0358] A "fragment" of a polynucleotide sequence provided herein is a subsequence of contiguous nucleotides that is usually at least 15 nucleotides in length. The fragments of the invention preferably comprise at least 20 nucleotides, more preferably at least 30 nucleotides, more preferably at least 40 nucleotides, more preferably at least 50 nucleotides and most preferably at least 60 contiguous nucleotides of a polynucleotide of the invention. A fragment of a polynucleotide sequence can be used in antisense, gene silencing, triple helix or ribozyme technology, or as a primer, a probe, included in a microarray, or used in polynucleotide-based selection methods. The term "fragment" in relation to promoter polynucleotide sequences is intended to include sequences comprising cis-elements and regions of the promoter polynucleotide sequence capable of regulating expression of a polynucleotide sequence to which the fragment is operably linked.

[0359] In one example fragments of polynucleotide sequences of the invention comprise at least 20, more preferably at least 30, more preferably at least 40, more preferably at least 50, more preferably at least 100, more preferably at least 200, more preferably at least 300, more preferably at least 400, more preferably at least 500, more preferably at least 600, more preferably at least 700, more preferably at least 800, more preferably at least 900 and most preferably at least 1000 contiguous nucleotides of a polynucleotide of the invention.

[0360] The term "primer" refers to a short polynucleotide, usually having a free 3'OH group, that is hybridised to a template and used for priming polymerization of a polynucleotide complementary to the template. Such a primer is preferably at least 5, more preferably at least 6, more preferably at least 7, more preferably at least 9, more preferably at least 10, more preferably at least 11, more preferably at least 12, more preferably at least 13, more preferably at least 14, more preferably at least 15, more preferably at least 16, more preferably at least 17, more preferably at least 18, more preferably at least 19, more preferably at least 20 nucleotides in length.

[0361] The term "probe" refers to a short polynucleotide that is used to detect a polynucleotide sequence that is complementary to the probe, in a hybridisation-based assay. The probe may consist of a "fragment" of a polynucleotide as defined herein. In one example such a probe is at least 5, more preferably at least 10, more preferably at least 20, more preferably at least 30, more preferably at least 40, more preferably at least 50, more preferably at least 100, more preferably at least 200, more preferably at least 300, more preferably at least 400 and most preferably at least 500 nucleotides in length.

[0362] The term "variant" as used herein refers to polynucleotide or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain examples, variants of the polynucleotides and polypeptides possess biological activities that are the same or similar to those of the wild-type polynucleotides or polypeptides. The term "variant" with reference to polynucleotides and polypeptides encompasses all forms of polynucleotides and polypeptides as defined herein.

[0363] The phrase "functional variant" recognises that it is possible to vary the amino acid sequence of a protein while retaining substantially equivalent functionality. For example, a protein can be considered a functional variant of another protein for a specific function if the variant peptide is immunologically cross-reactive with the original protein, and preferably has at least substantially the same function as the original protein.

[0364] Polynucleotide variants

[0365] Variant polynucleotide sequences preferably exhibit at least 50%, more preferably at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%), at least 57%, at least 58%, at least 59%, at least 60%, at least 61 %, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a specified polynucleotide sequence. Identity is found over a comparison window of at least 20 nucleotide positions, preferably at least 50 nucleotide positions, at least 100 nucleotide positions, or over the entire length of the specified polynucleotide sequence.

[0366] Polynucleotide sequence identity can be determined in the following manner. The subject polynucleotide sequence is compared to a candidate polynucleotide sequence using BLASTN (from the BLAST suite of programs, version 2.2.10 [Oct 2004]) in bl2seq (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250), which is publicly available from NCBI (ftp: / / ftp.ncbi.nih.gov / blast / ). The default parameters of bl2seq are utilized except that filtering of low complexity parts should be turned off.

[0367] The identity of polynucleotide sequences may be examined using the following UNIX command line parameters: bl2seq -I nucleotideseql -j nucleotideseq2 -F F -p blastn

[0368] The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. The bl2seq program reports sequence identity as both the number and percentage of identical nucleotides in a line "Identities =

[0369] Polynucleotide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs (e.g., Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443- 453). A full implementation of the Needleman-Wunsch global alignment algorithm is found in the needle program in the EMBOSS package (Rice,P. Longden,!. and Bleasby,A. EMBOSS: The European Molecular Biology Open Software Suite, Trends in Genetics June 2000, vol 16, No 6. pp.276-277) which can be obtained from http: / / www.hgmp.mrc.ac.uk / Software / EMBOSS / . The European Bioinformatics Institute server also relates to the facility to perform EMBOSS-needle global alignments between two sequences online at http: / www.ebi. ac.uk / emboss / align / .

[0370] Alternatively the GAP program may be used which computes an optimal global alignment of two sequences without penalizing terminal gaps. GAP is described in the following paper: Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.

[0371] Polynucleotide variants of the present invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polypeptides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.10 [Oct 2004]) from NCBI (ftp: / / ftp.ncbi.nih.gov / blast / ). The similarity of polynucleotide sequences may be examined using the following unix command line parameters: bl2seq -I nucleotideseql -j nucleotideseq2 -F F -p tblastx

[0372] The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. The size of this database is set by default in the bl2seq program. For small E values, much less than one, the E value is approximately the probability of such a random match.

[0373] Variant polynucleotide sequences preferably exhibit an E value of less than 1 x 10-10, more preferably less than 1 x IO-20, less than 1 x IO-30, less than 1 x IO-40, less than 1 x IO-50, less than 1 x IO-60, less than 1 x IO-70, less than 1 x IO-80, less than 1 x IO-90, less than 1 xlO-100, less than 1 x 10_110, less than 1 x IO-120or less than 1 x IO-123when compared with any one of the specifically identified sequences.

[0374] Alternatively, variant polynucleotides of the present invention hybridise to a specified polynucleotide sequence, or complements thereof under stringent conditions.

[0375] The term "hybridise under stringent conditions", and grammatical equivalents thereof, refers to the ability of a polynucleotide molecule to hybridise to a target polynucleotide molecule (such as a target polynucleotide molecule immobilized on a DNA or RNA blot, such as a Southern blot or Northern blot) under defined conditions of temperature and salt concentration. The ability to hybridise under stringent hybridisation conditions can be determined by initially hybridising under less stringent conditions then increasing the stringency to the desired stringency.

[0376] With respect to polynucleotide molecules greater than about 100 bases in length, typical stringent hybridisation conditions are no more than 25 to 30°C (for example, 10°C) below the melting temperature (Tm) of the native duplex (see generally, Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Ausubel et al., 1987, Current Protocols in Molecular Biology, Greene Publishing,). Typical stringent conditions for polynucleotide of greater than 100 bases in length would be hybridisation conditions such as prewashing in a solution of 6X SSC, 0.2% SDS; hybridising at 65°C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in IX SSC, 0.1% SDS at 65°C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65°C.

[0377] With respect to polynucleotide molecules having a length less than 100 bases, exemplary stringent hybridisation conditions are 5 to 1.0°C below Tm. On average, the Tm of a polynucleotide molecule of length less than 100 bp is reduced by approximately (500 / oligonucleotide length)°C.

[0378] With respect to the DNA mimics known as peptide nucleic acids (PN As)<‘> (Nielsen et al., Science. 1991 Dec 6;254(5037): 1497-500) Tm values are higher than those for DNA- DNA or DNA- RNA hybrids, and can be calculated using the formula described in Giesen et al., Nucleic Acids Res. 1998 Nov 1 ;26(21): 5004-6. Exemplary stringent hybridisation conditions for a DNA-PNA hybrid having a length less than 100 bases are 5 to 10°C below the Tm. Variant polynucleotides of the present invention also encompass polynucleotides that differ from the sequences of the invention but that, as a consequence of the degeneracy of the genetic code, encode a polypeptide having similar activity to a polypeptide encoded by a polynucleotide of the present invention. A sequence alteration that does not change the amino acid sequence of the polypeptide is a "silent variation". Except for ATG (methionine) and TGG (tryptophan), other codons for the same amino acid may be changed by art recognized techniques, e.g., to optimize codon expression in a particular host organism.

[0379] Polynucleotide sequence alterations resulting in conservative substitutions of one or several amino acids in the encoded polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).

[0380] Variant polynucleotides due to silent variations and conservative substitutions in the encoded polypeptide sequence may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.10 [Oct 2004]) from NCBI (ftp: / / ftp.ncbi.nih.gov / blast / ) via the tblastx algorithm as previously described.

[0381] Polypeptides and variants thereof

[0382] As will be appreciated from this disclosure, polypeptides useful in the determination of genetic status and / or allelic profile are provided herein. These include full length polypeptides, and fragments of or domains present in those polypeptides, including for example polypeptide variants and fusion polypeptides. Isolated, purified, synthesised, or recombinant polypeptides (including polypeptide variants and fusion polypeptides) are particularly contemplated.

[0383] In one example, the polypepeptide, polypeptide variant, or fusion polypeptide comprises, consists essentially of, or consists of 10 or more contiguous amino acids encoded by a variant gene as contemplated herein.

[0384] Proteins suitable for use herein include naturally-occurring proteins and peptides, and derivatives thereof including proteins and peptides having one or more amino acid variations from a naturally-occurring protein or peptide.

[0385] The term "amino acid" refers to natural amino acids, non-natural amino acids, and amino acid analogues. Unless otherwise indicated, the term "amino acid" includes both D and L stereoisomers if the respective structure allows such stereoisomeric forms.

[0386] Natural amino acids include alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gin or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (lie or I), leucine (Leu or L), Lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y) and valine (Vai or V).

[0387] Non-natural amino acids include, but are not limited to, azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, naphthylalanine ("naph"), aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6- aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3- aminoisbutyric acid, 2- aminopimelic acid, tertiary-butylglycine ("tBuG"), 2,4-diaminoisobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N-ethyl glycine, N-ethylasparagine, homoproline ("hPro" or "homoP"), hydroxylysine, allo-hydroxylysine, 3-hydroxyproline ("3Hyp"), 4- hydroxyproline ("4Hyp"), isodesmosine, allo-isoleucine, N-methylalanine ("MeAla" or "Nime"), Nalkylglycine ("NAG") including N-methylglycine, N- methylisoleucine, N-alkylpentylglycine ("NAPG") including N-methylpentylglycine. N- methylvaline, naphthylalanine, norvaline ("Norval"), norleucine ("Norleu"), octylglycine ("OctG"), ornithine ("Orn"), pentylglycine ("pG" or "PGIy"), pipecolic acid, thioproline ("ThioP" or "tPro"), homoLysine ("hLys"), and homoArginine ("hArg").

[0388] The term "amino acid analogue" refers to a natural or non-natural amino acid where one or more of the C-terminal carboxy group, the N-terminal amino group and side-chain functional group has been chemically blocked, reversibly or irreversibly, or otherwise modified to another functional group. For example, aspartic acid-(beta-methyl ester) is an amino acid analogue of aspartic acid; N- ethylglycine is an amino acid analogue of glycine; or alanine carboxamide is an amino acid analogue of alanine. Other amino acid analogues include methionine sulfoxide, methionine sulfone, S- (carboxymethyl)-cysteine, S-(carboxymethyl) cysteine sulfoxide and S-(carboxymethyl)-cysteine sulfone.

[0389] The term "expression construct" refers to a genetic construct that includes elements that permit transcribing the polynucleotide molecule of interest, and, optionally, translating the transcript into a polypeptide. An expression construct typically comprises in a 5' to 3' direction:

[0390] (1) a promoter, functional in the host cell into which the construct will be introduced,

[0391] (2) the polynucleotide to be expressed, and

[0392] (3) a terminator functional in the host cell into which the construct will be introduced.

[0393] Expression constructs of the invention are inserted into a replicable vector for cloning or for expression, or are incorporated into the host genome. Specifically contemplated herein are expression constructs comprising the polynucleotide to be expressed, such as one of the nucleic acids specified herein, operatively linked to a heterologous promoter, or operatively linked to a heterologous terminator, or operatively linked to both a heterologous promoter and a heterologous terminator.

[0394] The term "vector" refers to a polynucleotide molecule, usually double stranded DNA, which is used to transport the genetic construct into a host cell. In certain examples the vector is capable of replication in at least one additional host system, such as E. coli.

[0395] A "fragment" of a polypeptide is a subsequence of the polypeptide, typically one that performs a function that is required for activity, such as enzymatic or binding activity, and / or provides a three- dimensional structure of the polypeptide or a part thereof, such as an epitope. It will be appreciated that a fragment of a polypeptide may possess or elicit a different function or functions from that possessed or exhibited by the full-length polypeptide from which it is derived.

[0396] Reference is made herein to "isoforms" and "precursors" of polypeptides, including of a protein encoded by a gene identified in Table 1, Table 2, or Table 3. Persons skilled in the art to which the inventions relate will readily understand these terms. However, for the avoidance of any doubt, protein isoforms are different forms of a protein coded from the same gene. For example, different forms of a protein can be produced by alternative splicing of RNA transcripts to form different mRNA sequences or by different glycosylation or other posttranslational modification. For the avoidance of any doubt, protein precursors (including pre-pro-proteins or pre-pro-peptides and pro-proteins or propeptides) are usually inactive or differently active forms of a protein or peptide that can be activated by post- and / or co-translation modification or processing.

[0397] As used herein, the term "peptide" refers a short polymer of amino acids linked together by peptide bonds. While it will be recognised that the names associated with various classes of amino acid polymers (e.g., peptides, proteins, polypeptides, etc.) are somewhat arbitrary, peptides are generally of about 50 amino acids or less in length. A peptide can comprise natural amino acids, nonnatural amino acids, amino acid analogues, and / or modified amino acids. A peptide can be a subsequence of naturally occurring protein or a non-natural, including a synthetic, sequence.

[0398] As used herein, the term "synthetic peptide" encompasses a peptide having a distinct amino acid sequence from those found in natural peptides and / or proteins. A "synthetic peptide," as used herein, can be produced or synthesized by any suitable method (e.g., recombinant expression, chemical synthesis, enzymatic synthesis, etc.), and can include any chemical modification to a parent peptide, and may include, but is not limited to such methods as truncations, deletions, cyclization or non-peptidic synthetic or semi-synthetic derivatives that retain the same biological function(s) as the starting peptide. Methods of protein synthesis, such as solid state synthesis, are well known in the art.

[0399] The terms "peptide mimetic" or "peptidomimetic" refer to a peptide-like molecule that emulates a sequence derived from a protein or peptide. A peptide mimetic or peptidomimetic can contain amino acids and / or non-amino acid components. Examples of peptidomimetics include chemically modified peptides, peptoids (side groups are appended to the nitrogen atom of the peptide backbone, rather than to the a-carbons), [3-peptides (amino group bonded to the 3 carbon rather than the a-carbon), etc. Chemical modification includes one or more modifications at amino acid side groups, a-carbon atoms, terminal amine group, or terminal carboxy group. A chemical modification can be adding chemical moieties, creating new bonds, or removing chemical moieties. Modifications at amino acid side groups include, without limitation, acylation of lysine E-amino groups, N-alkylation of arginine, histidine, or lysine, alkylation of glutamic or aspartic carboxylic acid groups, lactam formation via cyclization of lysine e-amino groups with glutamic or aspartic acid side group carboxyl groups, hydrocarbon "stapling" (e.g., to stabilize alpha-helix conformations), and deamidation of glutamine or asparagine. Modifications of the terminal amine group include, without limitation, the desamino, N- lower alkyl, N-di-lower alkyl, constrained alkyls (e.g., branched, cyclic, fused, adamantyl) and N-acyl modifications. Modifications of the terminal carboxy group include, without limitation, the amide, lower alkyl amide, constrained alkyls (e.g., branched, cyclic, fused, adamantyl) alkyl, dialkyl amide, and lower alkyl ester modifications. Lower alkyl is C1-C4 alkyl. Furthermore, one or more side groups, or terminal groups, can be protected by protective groups known to the ordinarily skilled peptide chemist. The a-carbon of an amino acid can be mono- or dimethylated.

[0400] It will be appreciated that any one of the proteins or peptides described herein in certain examples comprises one or more non-naturally occurring amino acids, one or more amino acid analogues, or is or comprises a synthetic peptide or polypeptide or a peptide mimetic. Similarly, it will be appreciated that any one of the proteins or peptides described herein will in certain examples be the starting point for one or more modifications, synthetic methods, or protein engineering methods to develop a peptide analogue having a desired biological activity - for example, a qualitatively similar bioactivity as the parent protein or peptide, but an effect of a quantitatively different magnitude, or indeed a different bioactivity from that elicited by the parent protein or peptide.

[0401] In various examples, one or more of the polypeptides described herein comprises a fusion polypeptide. For example, a fusion polypeptide as contemplated herein will in certain examples comprise one or more fragments or domains, such as one or more functional domains, derived from, comprising or consisting of one of the sequences presented herein fused to another amino acid sequence to provide a fusion polypeptide.

[0402] The term "fusion polypeptide", as used herein, refers to a polypeptide comprising two or more amino acid sequences, for example two or more polypeptide domains, fused through respective amino and carboxyl residues by a peptide linkage to form a single continuous polypeptide. It should be understood that the two or more amino acid sequences can either be directly fused or indirectly fused through their respective amino and carboxyl termini through a linker or spacer or an additional polypeptide.

[0403] The term "polypeptide", as used herein, encompasses amino acid chains of any length but preferably at least 10 amino acids, including full-length proteins, in which amino acid residues are linked by covalent peptide bonds. Polypeptides described herein are purified natural products, or are produced partially or wholly using recombinant or synthetic techniques. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide variant, or derivative thereof.

[0404] It will be understood that, for the particular polypeptides and proteins contemplated herein, natural variations can exist between individuals of a species. These variations may be demonstrated by (an) amino acid difference(s) in the overall sequence or by deletions, substitutions, insertions, inversions or additions of (an) amino acid(s) in said sequence. Amino acid substitutions which do not essentially alter biological and / or immunological activities, are well known. Amino acid replacements between related amino acids or replacements which have occurred frequently in evolution are, inter alia, Ser / Ala, Ser / Gly, Asp / Gly, Asp / Asn, Ile / Val. Other amino add substitutions include Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Thr / Phe, Ala / Pro, Lys / Arg, Leu / Ile, Leu / Val and Ala / Glu. Based on this information, methods for rapid and sensitive protein comparison and determining the functional similarity between homologous proteins were developed. Such amino acid substitutions of the exemplary examples described herein, as well as variations having deletions and / or insertions are within the scope of the invention as long as the resulting proteins retain useful biological activity and / or immune reactivity. This explains why one or more proteins described herein, when isolated from different individuals, strains, field isolates, or species, may have identity levels below 100%, while still representing the same protein with the same immunological characteristics and / or biological function. Those variations in the amino acid sequence of a certain protein described herein that still provide a protein having useful biological activity related to that of a protein specifically identified herein are considered functional equivalents. For example, those variations in the amino acid sequence of a certain protein described herein that still provide a protein capable of reacting with an antibody specific to a protein specifically identified herein are considered as immunologically functional equivalents of the proteins identified herein, and as such do not essentially influence the immunogenicity of the protein.

[0405] When a protein is used for example for diagnostic or therapeutic purposes, for example for reacting with antibodies, or for mediating a biological effect, for example one or more of the biological functions associated with the native protein in vivo, while it can be expedient to do so it is not necessary to use the whole protein. It is also possible to use a polypeptide fragment of that protein (as such or coupled to a carrier or as a component in a fusion polypeptide, for example) or a polypeptide fragment derived from that protein or a related amino acid sequence that is capable of eliciting a desired biological effect, such as an immune response against that protein or of being recognised by an antibody specific to that protein, of mediating a cell-signalling effect, or the like. Such a polypeptide fragment may be referred to with reference to the function it possesses, such as the function it shares with the full-length protein from which it was derived. For example, a polypeptide fragment having an immunological effect may be referred to as an immunogenic fragment, where an "immunogenic fragment" is understood to be a fragment of the full-length protein that retains its capability to induce an immune response in a vertebrate host or be recognised by an antibody specific to the parent protein. Similarly, a polypeptide fragment retaining or possessing one or more biological effects elicited by the full-length protein from which it was derived, or possessing a related or different biological effect, is referred to herein as a "bioactive fragment" or a "bioactive polypeptide fragment". Likewise, a polypeptide having a biological effect, such as a polypeptide capable of stimulating a biological response in a cell or eliciting a therapeutic effect, may be referred to herein as a "bioactive fragment" or a "bioactive polypeptide fragment", or grammatical equivalents thereof.

[0406] A variety of techniques is available to identify such polypeptide fragments, as well as DNA fragments encoding such fragments. For example, in the case of immunogenic fragments, such fragments may comprise one or more determinants or epitopes. Well-established empirical and in silico methods for the detection of epitopes exist and are well known to those skilled in the art. For example, computer algorithms are able to designate specific protein fragments as the immunologically important epitopes on the basis of their sequential and / or structural agreement with epitopes that are known. The determination of these regions is typically based on a combination of the hydrophilicity criteria and secondary structural features. An immunogenic fragment (or epitope) usually has a minimal length of 6, more commonly 8 amino acids, preferably more then 8, such as 9, 10, 12, 15 or even 20 or more amino acids. The nucleic acid sequences encoding such a fragment therefore have a length of at least 18, more commonly 24 and preferably 27, 30, 36, 45 or even 60 nucleic acids.

[0407] Similarly, those skilled in the art will be aware of methods to identify bioactive fragments using various assays targeted at identifying or detecting a particular biological response. Representative methods suitable for use in the identification or detection of bioactive fragments contemplated herein are presented below, including in the Examples.

[0408] The term "variant" with reference to polypeptides encompasses naturally occurring, recombinantly, and synthetically produced polypeptides, including those comprising one or more nonnatural amino acids, one or more amino acid analogues, and peptide mimetics. Variant polypeptide sequences preferably exhibit at least 50%, more preferably at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a sequence of the present invention. Identity is found over a comparison window of at least 20 amino acid positions, preferably at least 50 amino acid positions, at least 100 amino acid positions, or over the entire length of a polypeptide of the invention.

[0409] Polypeptide sequence identity can be determined in the following manner. The subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (from the BLAST suite of programs, version 2.2.10 [Oct 2004]) in bl2seq, which is publicly available from NCBI (ftp: / / ftp.ncbi.nih.gov / blast / ). The default parameters of bl2seq are utilized except that filtering of low complexity regions should be turned off.

[0410] Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs. EMBOSS-needle (available at http: / www. ebi.ac.uk / emboss / align / ) and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity.

[0411] Polypeptide variants contemplated herein also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polypeptides can be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.10 [Oct 2004]) from NCBI (ftp: / / ftp.ncbi.nih.gov / blast / ). The similarity of polypeptide sequences can be examined using the following unix command line parameters: bl2seq -I peptideseql -j peptideseq2 -F F -p blastp

[0412] Variant polypeptide sequences preferably exhibit an E value of less than 1 x IO-10, more preferably less than 1 x IO-20, less than 1 x IO-30, less than 1 x IO-40, less than 1 x IO-50, less than 1 x IO-60, less than 1 x IO-70, less than 1 x IO-80, less than 1 x IO-90, less than 1 xlO-100, less than 1 x 10_110, less than 1 x IO-120or less than 1 x IO-123when compared with any one of the specifically identified sequences.

[0413] The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. For small E values, much less than one, this is approximately the probability of such a random match. Conservative substitutions of one or several amino acids of a described polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).

[0414] A polypeptide variant contemplated herein also encompasses that which is produced from the nucleic acid encoding a polypeptide, but differs from the wild-type polypeptide in that it is processed differently such that it has an altered amino acid sequence. For example, in one example a variant is produced by an alternative splicing pattern of the primary RIMA transcript to that which produces a wild-type polypeptide.

[0415] Methods of Breeding and cloning

[0416] Various examples of methods described herein relate to breeding one or more animals, including one or more animals identified and / or selected by a method as contemplated herein. It should be appreciated that any appropriate breeding method 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.

[0417] 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.

[0418] As noted previously herein, the methods of the invention may be used to identify animals suitable for cloning. They may also be used during cloning processes, to determine the allelic profile of, for example, a cell, nucleus, embryo, gamete, or cloned animal. Any appropriate cloning method could be used. However, by way of example, specifically contemplated 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 PJ and Cibelli, JB 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 J, Borowicz P, Wang Z, Nowak RA. Anim Reprod Sci. 2013 Jan 30;136(4):231-44. doi: 10.1016 / j.anireprosci.2012.10.030. Epub 2012 Nov 8.

[0419] As used herein, the term "embryo" should be taken broadly to include an organism from the first division of the zygote. In certain examples, 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.

[0420] As set out herein, examples of the methods contemplated are for the selection or rejection of one or more cells. In certain examples, such "cells" may include a gamete (for example, sperm or ovum) or zygote. The selection of such cells will enable a number of subsequent methods to be pursued, for example, the use of such selected cells in an IVF program, for example. In other examples, such cells may be somatic cells, embryonic cells, embryonic stem cells, cells in a cell line, and cells of use in cloning, for example. Selection of these cells may be of use in cloning procedures, or preparing cell lines for use in cloning and other procedures, for example.

[0421] Certain methods described herein refer to "fusing a first and a second gamete" to form a zygote. This and like phrases 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.

[0422] Reference is made herein to certain methods used to identify whether or not an animal, cell, embryo, gamete, or nucleus is "suitable" (or at least "more or less suitable") for, for example, production purposes or breeding purposes, or to identify whether or not an animal is "suitable" (or at least "more or less suitable") for inclusion in a herd. In one example, the production purpose is milking. In one example, the production purpose is meat production.

[0423] In one example, a cell, animal or embryo is identified as unsuitable or less suitable on the basis of an allelic profile comprising data indicative of the presence of one or more of: a) one or more alleles at a polymorphism associated with decreased expression from a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located; or b) one or more variant alleles at any one of the polymorphisms identified in Table 1, Table

[0424] 2, or Table 3; or c) one or more homozygous variant genotypes at any one or more of the polymorphisms identified in Table 1, Table 2, or Table 3; or d) one or more alleles at a polymorphism in linkage disequilibrium with one or more of the polymorphisms recited in any of (a) to (c) above, and particularly a polymorphism in 100% linkage disequilibrium (R2= 1.0) with any one or more of the polymorphisms identified in (a) to (c) above; or e) any combination of any two or more of (a) to (d).

[0425] In one example, a cell, animal or embryo is identified as suitable or more suitable on the basis of an allelic profile comprising data indicative of the absence of one or more of: a) one or more alleles at a polymorphism associated with decreased expression from a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located; or b) one or more variant alleles at any one of the polymorphisms identified in Table 1, Table

[0426] 2, or Table 3; or c) one or more homozygous variant genotypes at any one or more of the polymorphisms identified in Table 1, Table 2, or Table 3; or d) one or more alleles at a polymorphism in linkage disequilibrium with one or more of the polymorphisms recited in any of (a) to (c) above, and particularly a polymorphism in 100% linkage disequilibrium (R2= 1.0) with any one or more of the polymorphisms identified in (a) to (c) above; or e) any combination of any two or more of (a) to (d).

[0427] "More or less" suitable refers to a comparison between the animal, cell, embryo, gamete, or nucleus and an animal, cell, embryo, gamete, or nucleus that has (or in some examples does not have) a biological marker linked to a deleterious effect on productivity and / or worth of an animal, or a comparison between the same animal, cell, embryo, gamete, or nucleus if it had (or in some examples did not have) a biological marker linked to a deleterious effect on productivity and / or worth of an animal. Specifically contemplated are comparisons based on the allelic profile of the subject animal, cell, embryo, gamete, or nucleus.

[0428] As mentioned herein before, methods for breeding and cloning animals are contemplated. In certain examples, methods described herein are used for selecting animals (including selecting their gametes, for example) for such purposes.

[0429] Such methods may comprise identifying at least one first animal on the basis of its genetic status with respect to productivity and / or worth (in one example, using one or more methods as described herein) and mating said animal with a second animal. In one example, the method may comprise further identifying at least one second animal on the basis of its genetic status with respect to productivity and / or worth (in one example, using one or more methods as described herein). In a preferred example, the mating will produce one or more progeny.

[0430] Breeding methods are also contemplated which comprise selecting a first and / or a second gamete where it / they have been identified on the basis of genetic status, for example with reference to allelic profile, and fusing the first gamete and second gamete to form a zygote. In one example, the method may comprise: selecting a first gamete and / or a second gamete using a method as described herein and fusing said first gamete with said second gamete to form a zygote. The method may further comprise selecting any embryo that results from this process using one or more methods contemplated herein.

[0431] Also encompassed are breeding methods which comprise selecting an embryo that has been identified on the basis of its genetic status with respect to productivity and / or worth, for example, with reference to its allelic profile. In specifically contemplated examples, a selected embryo is used to breed an animal.

[0432] Methods of cloning an animal are also contemplated, the methods comprising selecting a cell for cloning where it has been identified on the basis of its genetic status with respect to productivity and / or worth, for example, with reference to its allelic profile. In specifically contemplated examples, a selected cell is used to clone an animal.

[0433] In certain examples, one or more methods described herein (such as those for selecting or rejecting one or more cell or embryo) is used to identify and select appropriate gametes and embryos for these breeding methods. For example, in one example, the method comprises selecting a gamete, embryo, gamete, or cell identified on the basis of its genetic status with respect to productivity and / or worth, for example, with reference to its allelic profile.

[0434] In various examples, methods for determining whether or not one or more cell or embryo is suitable for being used to produce an animal (for example, using breeding or cloning methods) with an allelic profile associated with one or more desirable productivity and / or worth traits are contemplated.

[0435] In one example, the breeding and / or cloning methods described herein further comprise transferring one or more embryo to a gestational carrier, in accordance with any number of techniques known in the art. In certain examples of the breeding methods contemplated herein, animals are mated using any appropriate methods including naturally, artificial insemination, or IVF. In one example, individual gametes are selected for use in the process. In one example, gametes are selected using a method as described herein; for example, a method comprising the identification of animals on the basis of genetic status with respect to productivity and / or worth, for example, with reference to allelic profile, and gametes from those animals selected for use in a breeding program or process or gametes may be tested in accordance with this disclosure and then selected for use in a breeding program or process.

[0436] In one particular example, a method of selecting or rejecting one or more animal is used to select the first and / or second animal and their gametes used in IVF. In another example, a method of selecting or rejecting one or more cells is used to select a first and / or second gamete and selected gametes used in IVF. Following selection of male and female gametes, the female gamete is fertilised in vitro. At the relevant time, one or more embryo is transferred to a gestational carrier.

[0437] In one example, in vitro fertilisation of a female gamete is performed and then a method as described herein is used to determine whether or not an embryo has a desired genotype / phenotype and should be selected or rejected for further use in a breeding programme. In various examples, this occurs where individual gametes, or animals from which they have been obtained or derived, have not been tested to determine their genetic status with respect to productivity and / or worth prior to fertilisation. Accordingly, in certain examples the methods of breeding include those 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 progeny is tested and selected). Alternatively, in other examples the method is used where the individual gametes or animals from which they have been obtained or derived have been tested and selected on the basis of having a desirable genotype / phenotype, for quality control purposes or to double check that the resulting embryos have the same desirable genotype / phenotype.

[0438] Optionally, following mating of the animals, the genetic status of one or more progeny is determined to determine whether or not any progeny has or may be inferred to have the desired characteristics. Such testing may occur at any time during the life of the progeny, including before birth; by way of example only, testing an embryo, a foetus, amniotic fluid, placenta, maternal blood, at birth.

[0439] In certain examples, gametes are fused to form a zygote. Methods for fusing gametes are known in the art.

[0440] In certain cases, cloning is used to generate an animal. In such cases, the method comprises selecting at least one first animal that has been identified on the basis of its genetic status with respect to productivity and / or worth, for example, with reference to its allelic profile, 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 Ci bell I, JB 2010. Methods in Molecular Biology 636: 155-177. In certain examples, at the relevant time during the cloning procedure, one or more embryo will be transferred to a gestational carrier. In certain examples, a cloning procedure utilises a cell derived from a cell line, optionally a cell selected in a method as contemplated herein, or a cell derived from such a cell. In certain examples, a method as contemplated herein is used to select such a cell which is, or cell line whose cells are, capable of being used to generate an animal having one or more desired productivity and / or worth traits. In one example, the cell line may be an embryonic cell line.

[0441] In certain examples, one or more cells of use in cloning is selected using a method as presented herein. In specifically contemplated examples, following selection of one or more cells a cloning procedure is conducted. In one example, an animal is identified on the basis of its genetic status with respect to productivity and / or worth, for example, with reference to its allelic profile, and cells from those animals are selected for use in a cloning process. Similarly, cells from a cell line identified on the basis of their genetic status with respect to productivity and / or worth, for example, with reference to their allelic profile will be of use in generating an animal that has one or more desired productivity and / or worth traits. Similar methods will in certain examples be useful to identify animals whose cells could be used to generate cell lines for cloning purposes.

[0442] In one particular example, a method of selecting or rejecting one or more animal on the basis of its genetic status with respect to productivity and / or worth, for example, with reference to its allelic profile, is used to select an animal for cloning. In another example, a method of selecting or rejecting one or more cells on the basis of their genetic status with respect to productivity and / or worth, for example, with reference to their allelic profile, is used to select one or more cells of use in cloning.

[0443] Optionally, at various stages during the cloning procedure, one or more tests can be carried out to identify whether or not any cloned animal carries a marker linked to a deleterious effect on productivity and / or worth. Such testing may occur at any time during the life of the cloned animal. By way of example only, testing of a blastocyst, an embryo, a foetus, amniotic fluid, placenta, maternal blood, at birth.

[0444] The breeding and cloning methods contemplated herein will in certain examples involve subjecting one or more cell, zygote, embryo and / or foetus, for example, to any one of a number of standard growth and / or gestation methods.

[0445] In one example, if an animal, cell, embryo, gamete, or nucleus is identified to be heterozygous for a marker linked to a deleterious effect on productivity and / or worth of an animal, it is not selected for the purposes of breeding or cloning. This will help avoid the possible mating of carriers of the deleterious effect on productivity and / or worth during current or future breeding programme, for example.

[0446] Forming a Herd

[0447] The invention also relates to methods for forming a herd of animals. Such methods comprise selecting or rejecting an animal on the basis of its genetic status with respect to productivity and / or worth, for example, with reference to its allelic profile. In one example, the method comprises selecting or rejecting an animal on the basis of its genetic status with respect to productivity and / or worth, for example, with reference to its allelic profile, and forming a herd of selected animals. In certain examples, such methods will be performed on one or more cells, such as one or more cells obtained from an animal, or one or more cells, nuclei, embryos to be used to generate an animal for subsequent inclusion in a herd. In certain examples, where an animal is identified to have for example an allelic profile associated with one or more undesirable productivity and / or worth traits, it will be rejected and not selected for inclusion in the herd.

[0448] In one example, an animal that is or has been identified as being heterozygous for a marker linked to an undesired or deleterious effect on productivity and / or worth, for example carrying both a variant allele and a wild-type allele at any one of the polymorphisms identified in Table 1, Table 2, or Table 3, is selected for inclusion in a herd. More typically, and particularly where the marker is associated with a substantial undesired or deleterious effect on productivity and / or worth (whether in the homozygous state or heterozygous state), a heterozygous animal is not chosen for inclusion in a herd.

[0449] The invention should also be taken to relate a herd formed by the methods described herein, including a herd comprising only those animals for which the genetic status with respect to productivity and / or worth has been determined, for example using a method as described herein, and / or progeny therefrom, such as for example a herd free of animals for which genetic status with respect to productivity and / or worth, for example allelic profile, has not been determined, or free of animals rejected for inclusion in the herd.

[0450] The herd of animals may be formed for any desirable reason. However, by way of example only, in certain examples particularly applicable to agricultural animals it will be desirable to form a herd for beef farming or milk production.

[0451] Gene editing methods

[0452] As noted herein before, the identification that an alteration in any one of the genes in which one of the polymorphisms identified in Table 1, Table 2, or Table 3, is located is associated with a deleterious effect on productivity and / or worth of an animal allows for the targeted correction of an alteration in the gene having this effect using cloning and / or gene editing processes in which one or more genetic alteration is introduced into the gene. For example, a specific alteration may be introduced into a cell, nucleus, nucleic acid or embryo that may be used to generate an animal. Accordingly, the invention relates to one or more nucleic acids (including expression constructs, vectors) used in the generation of an animal, cell, nucleus, or embryo, as well as any animal, cell, nucleus, or embryo into which an alteration has been introduced in accordance with this disclosure.

[0453] The correction of an alteration in a gene linked to an undesired or deleterious effect on productivity and / or worth of an animal will in certain examples allow for the production of animals and more broadly the formation of herds which have desirable productivity and / or worth or an increased level of productivity and / or worth compared to an animal / animals which have an alteration linked to a deleterious effect on productivity and / or worth of an animal.

[0454] The genetic alteration introduced, for example introduced into the gene or any sequence associated with the function of the gene or gene product, may be of any nature, including insertion of one or more nucleotide, deletion of one or more nucleotide, and / or substitution of one or more nucleotide. In one example, the genetic alteration corrects a variation in the gene which is associated with an undesired or deleterious effect on productivity and / or worth of an animal. In one example, the genetic alteration is one which corrects a variation in the gene which disrupts the gene. In one example, the genetic alteration is one which increases the level and / or activity of the protein encoded by the gene compared to the level and / or activity of the protein if it did not include the alteration.

[0455] In one example, the genetic alteration includes a genetic alteration located at one of the polymorphisms identified in Table 1, Table 2, or Table 3. In one example, the one or more genetic alteration is a substitution of the variant nucleotide(s) at the polymorphism with the wild-type nucleotide(s).

[0456] In one example, one or more cell used to generate an animal includes individual gametes, zygotes, embryos, somatic cells, cells from a cell line, for example. In one example, where IVF is used, a genetic alteration is introduced into a gamete or zygote, for example. In one example, where cloning is used, a genetic alteration is introduced into a somatic cell or cell from a cell line, for example.

[0457] Such methods may further comprise testing or screening one or more cell, embryo, or animal after a gene editing step to ensure they include the desired genetic alteration.

[0458] Any one of a number of standard methods may be used to introduce one or more genetic alterations, for example to one or more of the genes in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located.

[0459] Endonuclease-based systems, also referred to as Sequence-specific nuclease (SSN) systems, have rapidly become the principal gene editing tools used in molecular biology, amenable for use in gene disruption and gene targeting, in addition to targeted incorporation of new genetic material into host genomes. Endonuclease-based systems for gene editing allow the modification of genomes with high precision, efficiency, and flexibility. Examples of endonuclease-based approaches for gene editing include systems comprising, without limitations, zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), meganucleases (such as MegaTALs), and CRISPR / Cas9 and related systems for example alternate enzyme combinations such as Cas9 nickase combined with a reverse transcriptase for prime editing, or attenuated Cas9 guide RNA targeted enzymes to catalyse targeted nucleotide transitions or transversions.

[0460] In one example, the generation of a modified cell as contemplated herein is by targeted genome modification comprising the use of SSNs. In one example, the SSNs are selected from ZFNs, TALENs, or CRISPR / Cas. In one example, the SSN is selected from a TALEN. In another example, the SSN is selected from a CRISPR / Cas system, such as a CRISPR / Cas9 or a CRISPR / Casl2a system. Particularly contemplated gene editing systems for the production of a modified cell are CRISPR / Cas9 and its various derivatives, including CRISPR systems utilising modified Cas9 proteins, functional fragments thereof, and / or homologues or variants thereof including CRISPR / Cas9 targeted synthetic protein complexes to mediate specific transitions or transversions or in example of prime editing a synthetic RNA template inclusive of the CRISPR guide and template for targeted repair after reverse transcription mediated by a CAS9-reverse transcriptase fusion protein.

[0461] In various examples contemplated herein, the introduction of selected specific DNA variations into a target genome is through the use of the CRISPR / Cas9 editing system or one of its many variations. Briefly, the basis of the CRISPR / Cas9 editing system is Clustered Regularly Interspaced Short Palindromic Repeats sequences or guide RNAs (gRNAs) that are specifically designed to bind to selected locations in the genome in close proximity of the desired change. The gRNA forms a complex with CRISPR associated protein 9 (Cas9), an endonuclease. Targeted binding of the gRNA-Cas9 complex induces a modification at the guide binding site that in its simplest form is a double strand break. As described herein, other forms of modification are possible through the use of Cas9 or Casl2a (a related endonuclease) that have been themselves modified to alter their activity.

[0462] Those skilled in the art will understand that the gene editing reagents can be delivered to cells in many forms, including by DNA expression vectors, or simply as synthesised nucleic acids and purified Cas9 protein. In the context of the present invention, where a specific change is desired at a specific target locus, in one example a template DNA carrying the desired mutation is added to the target cell, and this sequence is incorporated into the cell's genome by homologous recombination (HR). The efficiency of HR is greatly increased due to the double strand break induced by Cas9 at the target site.

[0463] Other examples rely on another potential consequence of Cas9 cleavage, which is a phenomenon called nonhomologous end joining (NHEJ) where one or more nucleotides are added to or removed from the Cas9 cleaved ends. This results in insertions or, more typically, deletions, usually in an uncontrolled manner. This will in certain examples be useful if one is aiming to cause a frameshift mutation or other mutation to for example knock a gene function out, but is less commonly used where a specific point mutation is desired.

[0464] There are many available modifications to the basic CRISPR / Cas9 editing system. These share (and rely upon) the fundamental characteristic of specificity in genome binding site. Many of these methods aim to both increase the efficiency of homologous recombination whilst reducing the possibly of undesired or off-target edits, such as undesired NHEJ events. One such method utilises a modified Cas9 protein that cleaves DNA through a single strand nick (a so-called Cas9 nickase) that can be targeted to a site with a single gRNA, or with a pair of guides catalysing two nicks in close proximity on opposite DNA strands, such as the RuvC mutant / HNH mutant combination described below. The nick or nicks catalyse an HR event with the donor nucleic acid. This method, like the prime editing methods described below, typically has a lower frequency of undesired NHEJ events.

[0465] Other methods contemplated herein utilise the specificity of genome location targeting afforded by the CRISPR / Cas9 editing system. Particularly contemplated methods suitable for use in accordance with this disclosure include those utilising nickases, such as Cas9 variants comprising a RuvC mutant or an HNH mutant which typically are targeted using separate, sequence-specific guide RNAs; prime editing methods, which generally utilise chimeric fusion proteins comprising an inactivated Cas9 (such as an HNH mutant as described above) fused to a reverse transcriptase (such as Moloney Murine Leukemia Virus reverse transcriptase or variants thereof) in combination with prime editing guide RNA (pegRNA), an RNA consisting of the guide sequence and a template to direct the fusion protein to the target site and that when reverse transcribed results in a single strand DNA donor template carrying the desired mutation that is then incorporated into the genome through DNA repair; base editors, which modify nucleotides without introducing double strand breaks, either by base transition (pyrimidine-to-pyrimidine or purine-to-purine), such as transitions catalysed by cytidine deaminases which convert cytosine to thymine via hydrolytic deamination or adenine base editors (such as E. coli TadA) which convert adenosine to guanosine, and by base transversion (pyrimidine-to-purine or vice versa), such as cytosine to guanosine transversions by base editors such as CGBE1 (comprising Cas9 nickase, a uracil DNA N-glycosylase and a APOBEC1 cytidine deaminase); gene editing via direct delivery of ribonucleoprotein (RNP) complexes comprising Cas9 protein combined with gRNA (as opposed to, for example, delivery of Cas9-expressing polynucleotides or plasmids via transfection or viral transduction); and editing methods using protospacer-adjacent motif (PAM)-less Cas9 variants, in which the sequence specificity of, for example, S. pyogenes Cas9 for the PAM sequence NGG is altered.

[0466] These methods, along with methods to detect and verify the desired gene edits and identify off- target edits are helpfully summarised in, for example, Naeem et al., Latest Developed Strategies to Minimize the Off-Target Effects in CRISPR-Cas-Mediated Genome Editing, Cells 2020 Jul; 9(7): 1608, incorporated in its entirety by reference herein.

[0467] Another method for modifying the genome to obtain a desired mutation relies on cell mediated homologous recombination events undertaken in cell culture. These events occur at a variable rate depending on the targeted loci and rely on the introduction to the cell of a single or double stranded template construct carrying the desired mutation sequence with homologous regions extending either side of the mutation site. This genome modification process is often performed in embryonal stem cells or somatic cells obtained from the targeted species. In many examples, one or more genes for positive and negative antibiotic selection are included in the construct and are contiguous with the donor sequence. The one or more antibiotic selection genes enable the rejection of cells in which random integration (non homologous) events have occurred and the selection of cells which have undergone the desired homologous recombination event. Typically, the antibiotic selection gene(s) are flanked in the construct by recombinase recognition sites, whereby the gene or genes can be removed in the presence of the recombinase to provide a largely unmodified genome except for the targeted variation. Animals are then able to be produced carrying the cellular genome modification through the formation of chimeric blastocysts, for example by ES cell injection of the modified cells and subsequent selective breeding.

[0468] In one example, the methods of generating an animal, including a modified animal as contemplated herein comprises mating a first non-human animal with a second non-human animal.

[0469] In one example, the method comprises fusing said first gamete with a second non-human gamete to form a zygote. In one example, the zygote is maintained under conditions conducive to cellular development and / or differentiation, for example, as are typically employed in IVF.

[0470] In various examples, the second non-human animal or second gamete has been modified as contemplated herein.

[0471] In one example, following modification of one or more or both of the 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.

[0472] In one example, the embryo is maintained under conditions conducive to development and / or differentiation. In one example, the method comprises transferring the embryo to a gestational carrier.

[0473] In one example, the method comprises nuclear transfer of said one or more nuclei. In one example, the one or more selected cells or nuclei are used to clone an animal.

[0474] Accordingly, the invention also relates to a method of cloning a non-human animal, the method comprising at least the step of modifying one or more non-human cells as described herein, and maintaining the one or more cells under conditions conducive to cellular development, replication, and / or differentiation or subsequent cellular development, replication, and / or differentiation.

[0475] As set out herein, examples of the methods contemplated are for the modification of one or more cells. In certain examples, such cells will include a gamete (for example, sperm or ovum) or a zygote. The modification of such cells will enable a number of subsequent methods to be pursued, for example, the use of such selected cells in an IVF program or for cloning, for example. In other examples, such cells may be somatic cells, embryonic cells, embryonic stem cells, cells in a cell line, for example. Modified cells will in certain examples contemplated herein be of use in cloning procedures, or for preparing cell lines for use in cloning and other procedures.

[0476] In one example, gene editing is combined with cloning. The nucleus from a modified cell is then used for cloning, for example in known cloning processes, such as chromatin transfer, somatic cell nuclear transfer and embryo splitting. It should be appreciated that in certain examples one or more alteration other than the modifications specifically described herein in relation to the gene is also introduced into the cell or cell's genome if desired. Again, at the relevant time, one or more embryos may be implanted into a carrier female animal for gestation according to methods known in the art.

[0477] In certain examples, the methods of these examples optionally comprise determining the genetic status of the cell or animal, including a cloned cell or cloned animal, for example to determine whether or not any cell or animal has the desired genetic modification. Such testing may occur at any time during the process of generating or during the life of said animal or a relation thereof. By way of example, testing of a blastocyst, an embryo, a foetus, amniotic fluid, placenta, maternal blood, or the animal at birth are each contemplated. In one example, an embryo is tested prior to transferring to the gestational carrier. In another example, the animal is tested at birth.

[0478] In one example, the gene editing methods contemplated herein are carried out for the purpose of generating (or at least increasing the likelihood of generating) an animal which has a desired genetic modification, for example a genotype associated with one or more desired productivity and / or worth traits, such as increased production efficiency and / or robustness. For example, the gene editing methods are carried out for the purpose of generating (or at least increasing the likelihood of generating) an animal with one or more desired lactation traits, and which is suitable for production and / or breeding purposes.

[0479] In one example, the methods are carried out for the purpose of generating (or at least increasing the likelihood of generating) a cell, nucleus or embryo used to generate an animal which has a desired genetic modification, for example a genotype associated with one or more desired productivity and / or worth traits, such as increased production efficiency and / or robustness. For example, in one example the method is for generating a cell, nucleus or embryo which is of use in a method for generating an animal, the method comprising at least the step of introducing a genetic alteration to a gene of the cell, nucleus or embryo, wherein the gene is a gene in which any one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located. The invention also relates to cells, nuclei or embryos generated using a gene editing method as contemplated herein, including those capable of use in a method of generating a non-human animal.

[0480] Embryonic stem cells are frequently used in conjunction with genomic modification methods such as those described herein to generate chimeric blastocysts which are in turn used to generate modified embryos and animals. Selective breeding methods are often employed thereafter to provide a population of desired, genetically modified, animals.

[0481] In various alternative examples, somatic cell cloning is used, in which nuclei from genetically modified cells are injected into single cell embryos which are then implanted into recipients.

[0482] A particularly contemplated method for producing a modified animal comprises the direct injection of the gene editing reagents or their derivatives described above into one or more early- stage embryos. Typically, the embryos are screened preimplantation, for example via sequencing, for the desired edit before implantation, but methods where embryos are directly implanted and the animals selected once born are also contemplated.

[0483] As will be appreciated by those skilled in the art, the target species will frequently influence the choice of which of the various methods of producing modified cells, embryos, and animals is employed.

[0484] Particular examples of animals contemplated herein, including animals derived from cells, gametes, embryos and animals generated using a gene editing or breeding method described herein include an animal selected from one of the group consisting of bovine, including Bos taurus and Bos indicus, such as cattle or buffalo breeds used in agriculture, whether beef or dairy cattle including crossbred dairy cattle or and crossbred beef cattle, including Aberdeen Angus (Angus), Ayrshire, Braunvieh, Brown Swiss, Danish Red, Friesian, Hereford, Holstein, Holstein-Friesian, Jersey, Simmental, or buffalo such as water buffalo Bubalus bubalis); ovine, including sheep widely used in agriculture (whether for meat, dairy, or wool production, or production of other products), such as Meatlinc, Dorset, Romney, Dorsetx Rambouillet, Finnsheep cross, Merino; and porcine, including pigs and including for example pig breeds such as Large White, Duroc, Yorkshire, Landrace Large WhitexLandrace cross).

[0485] In one example, the breeding method involves IVF. In this example, one typically will first choose individual male and female gametes based on their genetic status, allelic profile, or otherwise, conduct gene editing methods on one or both gametes to introduce at least one desired alteration, such as a desired alteration into one or more of the gene identified in Table 1, Table 2, or Table 3, and fertilise the female gamete in vitro. Alternatively, individual male and female gametes will be chosen, the female gamete fertilised in vitro, and then a gene editing method conducted on the fertilised zygote to introduce at least one desired alteration, such as the desired alteration in the gene. It should be appreciated that one or more other alteration could also be introduced into the genome of a gamete or zygote. At the relevant time, one or more embryos may be transferred to a gestational carrier according to methods known in the art. In one example, gene editing is combined with cloning. In this example, one typically will first choose an animal for use in cloning based on its genetic status or otherwise. A cell from the animal will in certain examples be subject to gene editing methods to introduce at least one desired alteration, for example a desired alteration into the gene. The nucleus from such cell will then be used for cloning, for example in known cloning processes, such as chromatin transfer, somatic cell nuclear transfer and embryo splitting. It should be appreciated that one or more other alteration could also be introduced into the genome if desired. Again, at the relevant time, one or more embryos may be implanted into a carrier female animal for gestation according to methods known in the art.

[0486] In certain examples, the methods of these examples optionally comprise conducting a method described herein to determine the genetic status of the cell or animal, including a cloned cell or cloned animal, for example to determine whether or not any cell or animal has a desired genetic status, for example a desired allelic profile, or a desired alteration. Such testing may occur at any time during the process and life of any animal. By way of example testing of a blastocyst, an embryo, a foetus, amniotic fluid, placenta, maternal blood, at birth are each contemplated. In one example, an embryo is tested prior to transferring to the gestational carrier. In another example, the animal is tested at birth.

[0487] In one example, the gene editing methods contemplated herein are conducted for the purpose of generating (or at least increasing the likelihood of generating) an animal which has a desired genetic status, for example an allelic profile associated with one or more desired productivity and / or worth traits. For example, the gene editing methods are conducted for the purpose of generating (or at least increasing the likelihood of generating) an animal which is suitable for production and / or breeding purposes.

[0488] In one example, the methods are conducted for the purpose of generating (or at least increasing the likelihood of generating) a cell, nucleus or embryo used to generate an animal which has a desired genetic status, for example an allelic profile associated with one or more desired productivity and / or worth traits. For example, in one example the method is for generating a cell, nucleus or embryo which is of use in a method for generating an animal, the method comprising at least the step of introducing a genetic alteration to the gene of the cell, nucleus or embryo. The invention also relates to cells, nuclei or embryos generated using a gene editing method as contemplated herein, including those capable of use in a method of generating a non-human animal.

[0489] The invention is further described with reference to the following examples. It will be appreciated that the invention as claimed is not intended to be limited in any way by these examples.

[0490] EXAMPLES

[0491] These examples describe the determination of genetic variations in Bos taurus that are associated with variation in one or more robustness traits, such as fertility. Accordingly, the presence of one or more of the variations (i.e., the presence of a variant allele) identified herein is indicative of one or more deleterious effects on productivity and / or worth in bovine.

[0492] Methods

[0493] The variants described here have been identified through genome-wide association analyses powered to detect non-additive effects. These analyses were conducted on bulls that were either physically genotyped on, or imputed to the GeneMark COMvl genotyping platform. All variants of interest influence one or more fertility traits associated with sperm quality and / or quantity, such as sperm production, sperm motility, and / or sperm morphology.

[0494] Linear mixed-model association analyses powered to detect non-additive genetic effects were conducted in BOLT-LMM using a leave-one-segment-out approach for production efficiency and robustness traits including the traits identified in Table A below - traits routinely collected for animal (bull) evaluation. Average, low and high scores for each trait are shown in Table A.

[0495] Table A. Units and scores for fertility traits

[0496] Results Allele frequencies in the Holstein-Friesian (HF), Jersey (J), and at the population level in New

[0497] Zealand for each of the variant alleles described herein are presented in Table 2 below.

[0498] Table 2. Genotyping results for the 6 variants of interest - NZ incidence

[0499] The statistically significant increased culling (culling %, see below) of animals that are homozygous at any one of these polymorphisms presented in Table 2 strongly supports the inventors' position that these variant alleles have a deleterious effect on an animal, such as a deleterious effect on one or more productivity and / or worth traits including, for example, a deleterious effect on one or more fertility traits, such as one or more sperm quality or quantity traits, such as those set out in the Examples below. Allele frequency in the 1000 Bull Genomes dataset for each variant was then determined to assess incidence in international populations, as shown in Table 2A below.

[0500] Table 2A. Genotyping results for the 6 variants of interest - International incidence

[0501] Accordingly, the determination of the allelic profile of animals at one or more of the polymorphisms presented in Table 2 (for example, the identification and rejection of animals carrying the T allele at the Chr2: 101396615G>T polymorphism (rs433745406) is desirable for, amongst other motivations, herd improvement and to optimise production and / or worth, for example of the animals themselves or their progeny.

[0502] Further information for each of these polymorphisms is provided below in Table 3.

[0503] able 3. Details of the polymorphisms identified herein. RefSeq mRNA transcript ID for the first transcript of each gene. * SEQ ID NO. : in the accompanying sequence ID listing of the gene carrying the wild type allele

[0504] The observed deleterious effect of the variant allele at each of these polymorphisms strongly supports the inventors' view that the presence of one r more of these variants is undesirable and may negatively impact an animal's productivity and / or worth, in particular, its fertility. Accordingly, the etermination of the allelic profile of an animal (for example, the identification and rejection of animals carrying the variant allele) at any one or more of he polymorphisms depicted in Tables 1 - 3 is desirable for, amongst other motivations, herd improvement and to optimise production.

[0505] Example 1: Assessment of the impact of the Chr2:101396615G>T (rs433745406) polymorphism in the bovine SPAG16 gene.

[0506] This example describes the determination of a genetic variation on Bos taurus chromosome 2 in the gene encoding the protein Sperm-associated antigen 16 (SPAG16), and an assessment of its impact on productivity and / or worth.

[0507] The SPAG16 gene is denoted in the Ensembl database with the identifier ENSBTAG00000018549.6. Three SPAG16 mRNA transcripts and the encoded SPAG16 proteins are identified in the Ensembl database as follows:

[0508] • ENSBTAT00000024688.6 (used herein), protein ENSBTAP00000024688.6);

[0509] • ENSBTAT00000093116.1, protein ENSBTAP00000078412.1;

[0510] • ENSBTAT00000105070.1, protein ENSBTAP00000083778.1.

[0511] The variation is a nucleotide substitution at position 101,396,615 on chromosome 2 (Chr2: 101396615 G>T (rs433745406)). The T allele results in the introduction in transcripts ENSBTAT00000024688.6 and ENSBTAP00000024688.6 of a missense mutation leading to the substitution of the glutamic acid at amino acid position 257 with a premature stop codon (SPAG16 p.Glu257Ter, SPAG16 p.E257*) and a truncated protein. The inventors believe without wishing to be bound by any theory, that the variation will result in a decrease or loss of function of the protein.

[0512] Research and Results

[0513] This variation was identified in a study of semen quality phenotypes, comprising records for 5,562 past and present recorded bulls, and imputed sequence genotypes for 14,887,157 variants. Significant statistical associations were observed for a recessive genetic effect with the variant at chr2: 101396615, which maps within the gene SPAG16, for the sperm motility phenotypes Forward Progress Motility (p=4.07x l0-17) and Live Sperm Percentage Motility (p=4.33xl0-11).

[0514] Cull data was examined to determine the effect of the variant on culling rate amongst bulls. The majority of bulls homozygous for the variant had been culled, with "semen quality" recorded as the reason for culling in 100% of cases (compared to 8.2% across all culled bulls, p=1.9xl0-7).

[0515] The effects of the variant on fertility were then assessed, as measured using the non-return rate in inseminated cows. Bulls homozygous for the variant gave non-return rates 17.3 percentage points lower than other bulls (p=2.03x l0-7), and a drop of 23.6 percentage points when considering only inseminations using frozen straws (p=9.86x l0-3). For liquid straws, the drop was of 14.9 percentage points.

[0516] Accordingly, the variant is considered to have a substantial negative effect on fertility, and particularly on sperm quality and / or quantity.

[0517] The variant is observed in the Holstein-Friesian subpopulation (n=2,470 bulls), with an allele frequency of 3.8%. It was not observed in Jerseys (n= 1,438 bulls). The allele frequency across all bulls (including cross breeds) was 3.3%. As shown in Table 4, 54 other variants are in LD with the variant with R2>0.7, 50 with R2>0.8, and 42 with R2>0.9.

[0518] Table 4. SNPs in LD (R2> 0.7, R2> 0.8, R2> 0.9) with Chr2:101396615

[0519] The observed substantial reduction in fertility, for example the observed reductions in the sperm quality phenotype sperm motility, such as Forward Progress Motility and Live Sperm Percentage Motility, the reduction in semen quality leading to increased culling of bulls and / or increased nonreturn rates in cows inseminated with semen from bulls homozygous for the variant allele, is undesirable. This in turn negatively impacts the animal's productivity and / or worth. Accordingly, the determination of the SPAG16 allelic profile of an animal (for example, the identification and rejection of animals carrying the T allele at the Chr2: 101396615 G>T (rs433745406) polymorphism, and in certain examples the identification and rejection of animals homozygous for the T allele at this polymorphism) is desirable for, amongst other motivations, herd improvement and to optimise production and / or worth.

[0520] Sequence information for SPAG16 and the Chr2: 101396615 G>T (rs433745406) polymorphism is provided below:

[0521] SEQ ID NO.: 1 > bosTau9_ensGene_ENSBTAT00000024688.5 range=chr2: 101338464- 101409333. The genomic sequence (plus strand) presenting the reference form of the SPAG16 gene carrying the Chr2: 101396615G wild type allele at the Chr2: 101396615 G>T (rs433745406) polymorphism. The polymorphic locus of interest in the SPAG126 gene, Chr2: 101396615, is nucleotide position 58152 in SEQ ID NO. : 1.

[0522] SEQ ID No.: 2 > Amino acid sequence of the reference form of the SPAG16 protein encoded by the wild-type gene, with glutamic acid at amino acid position 257.

[0523] SEQ ID NO.: 3 > bosTau9_ensGene_ENSBTAT00000024688.5 range=chr2: 101338464- 101409333. The genomic sequence (plus strand) presenting the reference form of the SPAG16 gene carrying the Chr2: 101396615T variant allele at the Chr2: 101396615 G>T (rs433745406) polymorphism. The polymorphic locus of interest in the SPAG16 gene, Chr2: 101396615, is nucleotide position 58152 in SEQ ID NO. : 2.

[0524] SEQ ID No.: 4 > Amino acid sequence of the truncated SPAG16 polypeptide encoded by the variant gene carrying the variant Chr2: 101396615T allele.

[0525] Example 2: Assessment of the impact of the Chr4:30657607 G>A polymorphism in the bovine DNAH11 gene.

[0526] This example describes the determination of genetic variations on Bos taurus chromosome 4 in the gene encoding the protein Dynein axonemal heavy chain 11 (DNAH11), and an assessment of their impact on productivity and / or worth.

[0527] The DNAH11 gene is denoted in the Ensembl database with the identifier ENSBTAG00000013078.8, with a single transcript recorded and denoted with the identifier ENSBTAT00000061103.4. The encoded DNAH11 protein is denoted in the Ensembl database with the identifier ENSBTAP00000053699.4.

[0528] One variation is a nucleotide substitution at position 30657607 on chromosome 4 (Chr4:30657607 G>A). The A allele results in the substitution of valine at amino acid position 4118 with methionine (e.g., DNAH11 p.Val4118Met, DNAH11 p.V4118M, ENSBTAP00000053699.4: p.Val4118Met, ENSBTAP00000053699.4: p.V4118M).

[0529] A second variation is a nucleotide substitution at position 30411361 on chromosome 4 (Chr4:30411361 CA>C (rs465963699)0. The C allele results in a frameshift (denoted herein as Aspl791Thr (fsTer62)) that reduces the protein length to 1680 amino acids, replacing the sequence starting from amino acid position 1620 with the altered amino acid sequence TYLTFSQRELNLNRLHATFPNSSTALQICSLKMIRMSLHTGRLECIAKTRSTSHSQLRVNA [amino acids 1620 - 1680 of SEQ ID NO. : 10 herein, based on the UCD 1.2 build using the ENSEMBL .3 dataset], followed by premature termination (e.g., DNAH11 p.Aspl791ThrfsTer62, DNAH11 p.D1791TfsTer62, ENSBTAP00000053699.4:p.Aspl791ThrfsTer62, ENSBTAP00000053699.4:p.D1791TfsTer62.

[0530] The inventors believe without wishing to be bound by any theory, that these variations will result in a decrease or loss of function of the protein.

[0531] Research and Results

[0532] The Chr4:30657607 G>A variation was identified in a study of semen quality phenotypes, comprising records for 5,562 past and present recorded bulls, and imputed sequence genotypes for 14,887,157 variants. Significant statistical associations were observed for a recessive genetic effect with the variant at Chr4:30657607, which maps within the gene DNAH11, for the phenotypes Abnormal Tail Percentage (p=l.llx lO-27), Distal Reflex without Protoplasmic Droplet (p=1.73x l0-25) Live Sperm Percentage Motility (p=2.11x l0-25), Distal Reflex with Protoplasmic Droplet (1.54x l0-13), Normal Percentage (p=4.34xl0-13), and Forward Progressive Motility (7.45xl0-11).

[0533] Cull data was examined to determine the effect of the variant on culling rate amongst bulls. Compared to the rate across all bulls (8.2%), the culling rate in bulls homozygous for the variant was approximately four times higher (31.9%; p=3.2xl0-7).

[0534] Accordingly, the variant is considered to have a negative effect on fertility, and particularly on sperm quality and / or quantity.

[0535] The variant is most commonly found in the Jersey breed, with a minor allele frequency of 25% (n=l,438 bulls). The frequency in Holstein-Friesian bulls was 1.5% (n=2,470 bulls). Across all bulls, the allele frequency was 11%.

[0536] As shown in Table 5, 95 other variants are in LD with the variant with R2>0.7, 60 with R2>0.8, and 30 with R2>0.9.

[0537] Table 5. SNPs in LD (R2> 0.7, R2> 0.8, R2> 0.9) with Chr4:30657607

[0538]

[0539] The observed reduction in fertility, for example the observed reductions in the sperm quality phenotypes Abnormal Tail Percentage, Distal Reflex without Protoplasmic Droplet, Live Sperm Percentage Motility, Distal Reflex with Protoplasmic Droplet, Normal Percentage, and Forward Progressive Motility, the reduction in semen quality leading to increased culling of bulls, associated with the variant allele, is undesirable. This in turn negatively impacts the animal's productivity and / or worth.

[0540] Accordingly, the determination of the DNAH11 allelic profile of animals (for example, the identification and rejection of animals carrying the A allele at the Chr4:30657607 G>A (rs457253516) polymorphism, and in certain examples the identification and rejection of animals homozygous for the A allele at this polymorphism) is desirable for, amongst other motivations, herd improvement and to optimise production.

[0541] Sequence information for DNAH11 and the Chr4:30657607 G>A (rs457253516) polymorphism is provided below:

[0542] SEQ ID NO.: 5 > bosTau9_ensGene_ ENSBTAT00000061103.3. The coding sequence (plus strand) of the reference form of the DNAH11 gene carrying the Chr4:30657607G wild type allele at the Chr4:30657607 G>A (rs457253516) polymorphism. The polymorphic locus of interest in the DNAH11 gene, Chr4:30657607, is nucleotide position 12352 in SEQ ID NO.: 5.

[0543] SEQ ID NO.: 6 > Amino acid sequence of the reference form of the DNAH11 protein encoded by the wild-type gene.

[0544] SEQ ID NO.: 7 > bosTau9_ensGene_ ENSBTAT00000061103.3. The coding sequence (plus strand) of the reference form of the DNAH11 gene carrying the Chr4:30657607A variant allele at the Chr4:30657607 G>A (rs457253516) polymorphism. The polymorphic locus of interest in the DNAH11 gene, Chr4:30657607, is nucleotide position 12352 in SEQ ID NO.: 7. SEQ ID NO.: 8 > Amino acid sequence of the DNAH11 protein encoded by the variant gene carrying the variant Chr4:30657607A allele. The variant amino acid is present at amino acid position 4118 in SEQ ID NO. : 8.

[0545] Chr4:30411361 CA>C (rs465963699)

[0546] The Chr4:30411361 CA>C (rs465963699) variation in the gene DNAHllwas observed in a study of semen quality phenotypes, comprising records for 5,562 past and present recorded bulls, and imputed sequence genotypes for 14,887,157 variants. Significant statistical associations were observed for a recessive genetic effect with the variant at Chr4:30411361, causing decreases in the phenotypes Normal Percentage (-14.4; p=3.54x l0-8), Live Sperm Percentage Motility (-9.42; p=4.06xl0-8), and Forward Progress Motility (-0.62; p=1.39x l0-5), as well as an increase in Distal Reflex with Protoplasmic Drop Percentage (+2.08; p=1.29xl0-7).

[0547] Cull data was examined to determine the effect of the variant on culling rate amongst bulls. Again, this is expected to elucidate evidence of reduced semen quality. Compared to the rate across all bulls (8.2%), the culling rate in bulls homozygous for the invention was approximately five times higher (40%; p=1.5xl0-3). Accordingly, the Chr4:30411361 CA>C (rs465963699) variant is considered to have a negative effect on fertility, and particularly on sperm quality and / or quantity.

[0548] The variant was most commonly found in the Jersey breed, with a minor allele frequency of 8.1% (n=l,438 bulls). The frequency in Holstein-Friesian bulls was 0.2% (n=2,470 bulls). Across all bulls, the allele frequency was 3.3%.

[0549] The Chr4:30411361 CA>C (rs465963699) variant was also observed in genome-wide association studies of genomic evaluation traits testing for non-additive effects, where these analyses comprised records of past and present cows and imputed to sequence genotypes (16,453,913 variants). The production efficiency and robustness traits including the traits identified in Table B below - traits routinely collected for animal (cow) evaluation. Average, low and high scores for each trait are shown in Table B.

[0550] Table B. Units and scores for production efficiency and robustness traits in cows

[0551] *CSDHeifer = days after planned calving date in heifers

[0552] The number of cows included in each analysis differed based on available genotypic and phenotypic data and is indicated by N below. Statistically significant associations were observed for the Chr4:30411361 CA>C (rs465963699) variant for the following traits:

[0553] • capacity - p=5.7xl0-25; N=139,033; effect size= -0.45.

[0554] • days after planned calving date in heifers - p=4.6xl0-11; N=208,459; effect size= 6.62.

[0555] • dairy conformation - p=5.7xl0-21; N = 139,033; effect size= -0.45.

[0556] • fat yield during first lactation - p= 4.6xl0-11; N=203,767; effect size= -8.29. • liveweight - p= 1.6xl0-13; N = 138,420; effect size= -13.44.

[0557] As seen in Table 6 below, two other variants are in LD with the variant with R2>0.7, two with R2>0.8, and 0 with R2>0.9.

[0558] Table 6. SNPs in LD (R2> 0.7) with Chr4:30411361

[0559] The observed reduction in fertility, for example the observed reductions in the sperm quality phenotypes Normal Percentage, Live Sperm Percentage Motility, and Forward Progress Motility, and the observed increase in Distal Reflex with Protoplasmic Drop Percentage, and the reduction in semen quality leading to increased culling of bulls, associated with the variant C allele at the Chr4:30411361 CA>C (rs465963699) polymorphism, is undesirable. This reduction in fertility in turn negatively impacts the animal's productivity and / or worth.

[0560] Furthermore, the observed reductions in production and / or worth phenotypes capacity, days after planned calving date, dairy confirmation, fat yield, and liveweight, are likewise undesirable. These again negatively affect the animal's productivity and / or worth.

[0561] Accordingly, the determination of the DNAH11 allelic profile of animals (for example, the identification and rejection of animals carrying the C allele at the Chr4:30411361 CA>C (rs465963699) polymorphism, and in certain examples the identification and rejection of animals homozygous for the C allele at this polymorphism) is desirable for, amongst other motivations, herd improvement and to optimise production.

[0562] As described above, SEQ ID NO.: 5 presents the coding sequence (plus strand) of the reference form of the DNAH11 gene carrying the Chr4:30411361CA wild type allele at the Chr4:30411361 CA>C (rs465963699) polymorphism. The polymorphic locus of interest in the DNAH11 gene, Chr4:30411361, is nucleotide position X* in SEQ ID NO. : 5. Similarly, SEQ ID NO. : 6 presents the amino acid sequence of the wild type DNAH11 protein.

[0563] Sequence information for the Chr4:30411361 CA>C (rs465963699) polymorphism is provided below:

[0564] SEQ ID NO.: 9 > bosTau9_ensGene_ ENSBTAT00000061103.3. The coding sequence (plus strand) of the reference form of the DNAH11 gene carrying the Chr4:30411361C variant allele at the Chr4:30411361 CA>C (rs465963699) polymorphism. The polymorphic locus of interest in the DNAH11 gene, Chr4:30411361, is nucleotide position 4856 in SEQ ID NO. : 9.

[0565] SEQ ID NO.: 10 > Amino acid sequence of the DNAH11 protein encoded by the variant gene carrying the variant Chr4:30411361 C allele. The variant amino acid sequence begins at amino acid position 1620 in SEQ ID NO. : 10, with the resulting premature termination and truncated 1680 amino acid polypeptide shown. Example 3: Assessment of the impact of the Chr8:98463916 G>A (rs459888392) polymorphism in the bovine ACTL7B gene.

[0566] This example describes the determination of a genetic variation on Bos taurus chromosome 8 in the gene encoding the protein Actin-like 7B (ACTL7B), and an assessment of its impact on productivity and / or worth.

[0567] The ACTL7B gene is denoted in the Ensembl database with the identifier ENSBTAG00000025932.7, with a single transcript recorded and denoted with the identifier ENSBTAT00000025932.7. The encoded ACTL7B protein is denoted in the Ensembl database with the identifier ENSBTAP00000054716.1. SEQ ID NO.s: 11 and 13 present the sequence from an earlier version of the transcript, ENSBTAT00000025932.6, which differs from ENSBTAT00000025932.7 in the UTR. The amino acid coding sequence is unchanged between these versions.

[0568] The variation is a nucleotide substitution at position 98463916 on chromosome 8 (Chr8:98463916 G>A). The A allele results in the introduction of a missense mutation leading to the substitution of the alanine amino acid at amino acid position 53 with valine (ACTL7B p. Ala53Val, ACTL7B p.A53V). The inventors believe without wishing to be bound by any theory, that the variation will result in a decrease or loss of function of the protein.

[0569] Research and Results

[0570] The variation was identified in a study of semen quality phenotypes, comprising records for 5,562 past and present recorded bulls, and imputed sequence genotypes for 14,887,157 variants. Significant statistical associations were observed for a recessive genetic effect with the variant at Chr8:98463916, which maps within the gene ACTL7B, for increases in the phenotypes abnormal acrosome percentage (p=1.90x l0-16) and abnormal head percentage (p=5.16x l0-41), and a decrease in the phenotype percentage normal (p=2.72xl0-13).

[0571] Cull data was examined to determine the effect of the variant on culling rate amongst bulls. Compared to the rate across all bulls (8.2%), the culling rate in bulls homozygous for the variant was approximately 3 times higher (24%; p=0.014).

[0572] The variant is observed in the Holstein-Friesian subpopulation (n=2,470 bulls) with an allele frequency of 18.5%. It is much rarer in Jerseys (n=l,438 bulls) with an allele frequency of only 0.1%. Across all bulls (n=5,562), the frequency was 12.6%.

[0573] As shown in Table 7, 304 other variants are in LD with the variant with R2>0.7, 293 with R2>0.8, and 291 with R2>0.9.

[0574] Table 7. SNPs in LD (R2> 0.7, R2> 0.8, R2> 0.9) with Chr8:98463916.

[0575]

[0576] The observed substantial reduction in fertility, for example the observed increases in the deleterious sperm quality phenotypes abnormal acrosome percentage and abnormal head percentage, and a decrease in percentage normal sperm, is undesirable. This in turn negatively impacts the animal's productivity and / or worth.

[0577] Accordingly, the determination of the ACTL7B allelic profile of animals (for example, the identification and rejection of animals carrying the A allele at the Chr8:98463916 G>A (rs459888392) polymorphism, and in certain examples the identification and rejection of animals homozygous for the A allele at this polymorphism) is desirable for, amongst other motivations, herd improvement and to optimise production.

[0578] Sequence information for ACTL7B and for the Chr8:98463916 G>A (rs459888392) polymorphism is provided below:

[0579] SEQ ID NO.: 11 > bosTau9_ensGene_ENSBTAT00000025932.6 range=chr8:98462724- 98464130. The genomic sequence (minus strand) presenting the reference form of the ACTL7B gene carrying the Chr8:98463916G wild type allele at the Chr8:98463916 G>A (rs459888392) polymorphism. The polymorphic locus of interest in the ACTL7B gene, Chr8:98463916, is nucleotide position 215 in SEQ ID NO. : 11.

[0580] SEQ ID No.: 12 > Amino acid sequence of the reference form of the ACTL7B protein encoded by the wild-type gene.

[0581] SEQ ID NO.: 13 > bosTau9_ensGene_ENSBTAT00000025932.6 range= chr8:98462724- 98464130. The genomic sequence (minus strand) presenting the reference form of the ACTL7B gene carrying the Chr8:98463916A variant allele at the Chr8:98463916 G>A (rs459888392) polymorphism. The polymorphic locus of interest in the ACTL7B gene, Chr8:98463916, is nucleotide position 215 in SEQ ID NO.: 13. SEQ ID No.: 14 > Amino acid sequence of the variant ACTL7B protein encoded by the variant gene carrying the variant Chr8:98463916A allele.

[0582] Example 4: Assessment of the impact of the Chrl7:63363268 TOT (rs458604940) polymorphism in the bovine TCHP gene.

[0583] This example describes the determination of a genetic variation on Bos taurus chromosome 17 in the gene encoding the protein Trichoplein keratin filament binding (TCHP), and an assessment of its impact on productivity and / or worth.

[0584] The TCHP gene is denoted in the Ensembl database with the identifier ENSBTAG00000006504.7 Three TCHP mRNA transcripts and the encoded TCHP proteins are identified in the Ensembl database as follows:

[0585] • ENSBTAT00000129259.1 (canonical), protein ENSBTAP00000087137.1;

[0586] • ENSBTAT00000045400.5, protein ENSBTAP00000042791.4;

[0587] • ENSBTAT00000118119.1, protein ENSBTAP00000101620.1.

[0588] The TCHP mRNA transcript sequences used herein, SEQ ID NO.s: 15 and 17, however, are based on the canonical NCBI version of the TCHP transcript identified as NM_001164026, with the encoded protein identified as NP_001157498.1.

[0589] The variation is a nucleotide substitution leading to a frameshift mutation at position 63363268 on chromosome 17 (Chrl7:63363268 TG>T (rs434709552)). The T allele results in the introduction of a frameshift mutation. The frameshift causes a substantial change (Gln44ArgfsTer31) in sequence then early termination, with the amino acid sequence from Glutamine 44 (inclusive) replaced by 30 amino acids (RCPTSAAPNRQSGAPGPPTSGACMPTSGRR, being the C-terminal 30 amino acids in SEQ ID NO. : 18), followed by a stop codon (TGA). The inventors believe without wishing to be bound by any theory, that the variation will result in a decrease or loss of function of the protein.

[0590] Research and Results

[0591] The variant was identified in a study of semen quality phenotypes, comprising records for 5,562 past and present recorded bulls, and imputed sequence genotypes for 14,887,157 variants. Significant statistical associations were observed for a recessive genetic effect with the variant at Chrl7:63363268, which maps within the gene TCHP, for the phenotypes Forward Progress Motility (p= 1.77x l0-8), Initial Concentration (p=5.36x l0-6), Live Sperm Percentage Motility (p=1.19x l0-8), and Percentage Normal (p=4.28x l0-11).

[0592] Cull data was examined to determine the effect of the variant on culling rate amongst bulls. Among all culled bulls, 75% of homozygous affected bulls for this variant were culled due to semen quality issues, compared to a rate of 8.2% across all bulls (p=5.5x l0-4). The variant was also associated with a moderate (9.3 percentage points) recessive decrease in non-return rate, a measure of fertility (p=0.00165) in liquid straw inseminations, and a decrease of 9.0 percentage points across all inseminations (p=0.00667).

[0593] Accordingly, the variant is considered to have a substantial negative effect on fertility, and particularly on sperm quality and / or quantity. The variant is observed in the Holstein-Friesian subpopulation (n=2,470 bulls) with an allele frequency of 7.8%. It was not observed in Jerseys (n=l,438 bulls). Across all bulls (n=5,562), the frequency was 4.4%.

[0594] As seen in Table 8 below, 169 other variants are in LD with the variant with R2>0.7, 120 have R2>0.8, and 85 have R2>0.9.

[0595] Table 8. SNPs in LD (R2> 0.7, R2> 0.8, R2> 0.9) with Chrl7:63363268.

[0596] The observed substantial reduction in fertility, for example the observed reductions in the sperm quality and / or quantity phenotypes Forward Progress Motility, Live Sperm Percentage Motility, Live Sperm Percentage Motility, and Percentage Normal, the reduction in semen quality leading to increased culling of bulls and / or increased non-return rates in cows inseminated with semen from bulls homozygous for the variant allele, is undesirable. This in turn negatively impacts the animal's productivity and / or worth. Accordingly, the determination of the TCHP allelic profile of animals (for example, the identification and rejection of animals carrying the T allele at the Chrl7:63363268T G>T (rs434709552) polymorphism) is desirable for, amongst other motivations, herd improvement and to optimise production.

[0597] Sequence information for TCHP and the Chrl7:63363268 TG>T (rs434709552) polymorphism is provided below:

[0598] SEQ ID NO.: 15 > bosTau9_refseq_NM001164026.2 range=Chrl7:63350533-63365001. The genomic sequence (minus strand) presenting the reference form of the TCHP gene carrying the Chrl7:63363268TG wild type allele at the Chrl7:63363268 TG>T (rs434709552) polymorphism. The polymorphic locus of interest in the TCHP gene, Chrl7:63363268, is nucleotide position 1586 in SEQ ID NO.: 15.

[0599] SEQ ID NO.: 16 Amino acid sequence of the reference form of the TCHP protein encoded by the wild-type gene.

[0600] SEQ ID NO.: 17 > bosTau9_refseq_NM001164026.2 range=Chrl7:63350533-63365001. The genomic sequence (minus strand) presenting the reference form of the TCHP gene carrying the Chrl7:63363268T variant allele at the Chrl7:63363268 TG>T (rs434709552) polymorphism. The polymorphic locus of interest in the TCHP gene, Chrl7:63363268, is nucleotide position 1586 in SEQ ID NO. : 17.

[0601] SEQ ID NO. 18: > Amino acid sequence of the variant truncated TCHP polypeptide encoded by the variant gene carrying the Chrl7:63363268T allele.

[0602] Example 5: Assessment of the impact of the Chr25:1194914 G>C (rs433354487) polymorphism in the bovine IFT140 gene.

[0603] This example describes the determination of a genetic variation on Bos taurus chromosome 25 in the gene encoding the protein Intraflagellar transport 140 (IFT140), and an assessment of its impact on productivity and / or worth. The IFT140 gene is denoted in the Ensembl database with the identifier ENSBTAG00000007245.7. Two mRNA transcripts and the encoded IFT140 proteins are identified in the Ensembl database as follows:

[0604] • ENSBTAT00000047406.5 (canonical), protein ENSBTAP00000044615.4;

[0605] • ENSBTAT00000124675.1, protein ENSBTAP00000074908.1.

[0606] SEQ ID NO.s: 19 and 21 present the sequence from an earlier version of the transcript, ENSBTAT00000047406.4, which differs from ENSBTAT00000047406.5 in the UTR. The amino acid coding sequence is unchanged between these versions.

[0607] The variation is a nucleotide substitution at position 1194914 on chromosome 25 (Chr25: 1194914 G>C). The C allele results in the substitution of the glutamine amino acid at amino acid position 810 in the wild-type protein with glutamic acid (IFT140 p. Gln810Glu, IFT140 p.Q810E). The inventors believe without wishing to be bound by any theory, that the variation will result in a decrease or loss of function of the protein.

[0608] Research and Results

[0609] The variation was identified in a study of semen quality phenotypes, comprising records for 5,562 past and present recorded bulls, and imputed sequence genotypes for 14,887,157 variants. Significant statistical associations were observed for a recessive genetic effect with the variant at Chr25: 1194914, which maps to the IFT140 gene (a component of intraflagellar transport complex A (IFTA)), with an increase in abnormal acrosome percentage (p=2.08xl0-112) and a decrease in percentage normal (p=7.99xl0-17).

[0610] Cull data was examined to determine the effect of the variant on culling rate amongst bulls. Among all culled bulls, 40% of homozygous affected bulls for this variant were culled due to semen quality issues, compared to a rate of 8.2% across all bulls (p=6.6x l0-7). The variant also had a small (1.9 percentage points) but significant (p=0.022) deleterious recessive effect on non-return rate (NRR), a measure of fertility. Interestingly, the variant also had an additive effect on NRR (0.5 percentage points per allele, p=0.007).

[0611] Accordingly, the variant is considered to have a negative effect on fertility, and particularly on sperm quality and / or quantity.

[0612] The variant is observed in the Holstein-Friesian subpopulation (n=2,470 bulls) with an allele frequency of 12.4%. It is much rarer in Jerseys (n=l,438 bulls) with an allele frequency of only 0.1%. Across all bulls (n=5,562), the frequency was 8.6%.

[0613] As shown in Table 9, 31 other variants are in LD with the variant with R2>0.7. All of these also have R2>0.8, and eight have R2>0.9.

[0614] Table 9. SNPs in LD (R2> 0.7, R2> 0.8, R2> 0.9) with Chr25:1194914.

[0615]

[0616] The observed substantial reduction in fertility, for example the observed reductions in the sperm quality and / or quantity phenotypes abnormal acrosome percentage and percentage normal, the reduction in semen quality leading to increased culling of bulls and / or increased non-return rates in cows inseminated with semen from bulls homozygous for the variant allele, is undesirable. This in turn negatively impacts the animal's productivity and / or worth.

[0617] Accordingly, the determination of the IFT140 allelic profile of animals (for example, the identification and rejection of animals carrying the C allele at the Chr25: 1194914 G>C (rs433354487) polymorphism, and in certain examples the identification and rejection of animals homozygous for the C allele at this polymorphism) is desirable for, amongst other motivations, herd improvement and to optimise production.

[0618] Sequence information for IFT140 and the Chr25: 1194914 G>C (rs433354487) polymorphism is provided below:

[0619] SEQ ID NO.: 19 > bosTau9_ensGene_ ENSBTAT00000047406.4 range= chr25: 1184871- 1241910. The genomic sequence (minus strand) presenting the reference form of the IFT140 gene carrying the Chr25: 1194914G wild type allele at the Chr25: 1194914 G>C (rs433354487) polymorphism. The polymorphic locus of interest in the IFT140 gene, Chr25: 1194914, is nucleotide position 43451 in SEQ ID NO.: 19.

[0620] SEQ ID NO.: 20 > Amino acid sequence of the reference form of the IFT140 protein encoded by the wild-type gene.

[0621] SEQ ID NO.: 21 > bosTau9_ensGene_ ENSBTAT00000047406.4 range= chr25: 1184871- 1241910. The genomic sequence (minus strand) presenting the reference form of the IFT140 gene carrying the Chr25: 11949140 variant allele at the Chr25: 1194914 G>C (rs433354487) polymorphism. The polymorphic locus of interest in the IFT140 gene, Chr25: 11...

Claims

1. CLAIMS1. A method for identifying, and / or selecting or rejecting a non-human animal subject, or a subject comprising an isolated cell, embryo, gamete, or nucleus, with one or more desired or undesired productivity or worth phenotypes, the method comprising determining an allelic profile of the animal, isolated cell, embryo, gamete, or nucleus, and identifying, and / or selecting or rejecting the animal, isolated cell, embryo, gamete, or nucleus on the basis of the determination, wherein the allelic profile comprises data indicative of the presence or absence of one or more alleles at one or more of the polymorphisms identified in Table 1, Table 2, or Table 3 herein.

2. The method as claimed in claim 1, wherein the allelic profile is determined by providing the results of an analysis of a sample from said subject for the presence or absence of one or more alleles at a polymorphism in a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is present, wherein: a) said one or more alleles is associated with increased or decreased expression or activity of a gene product encoded by a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is present; or b) said one or more alleles is associated with increased or decreased expression of or from a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is present; or c) said one or more alleles is associated with one or more polymorphisms in linkage disequilibrium with one of the polymorphisms identified in Table 1, Table 2, or Table 3.

3. The method of claim 1 or 2 wherein the non-human animal subject is bovine.

4. The method as claimed in any one of claims 1 to 3, wherein the allelic profile is determined by determining the presence or absence of the wild-type allele at any one or more of the polymorphisms identified in Table 1, Table 2, or Table 3.

5. The method as claimed in any one of claims 1 to 4, wherein the allelic profile is determined by determining the presence or absence of the variant allele at any one or more of the polymorphisms identified in Table 1, Table 2, or Table 3.

6. A method for identifying, and / or selecting or rejecting a bovine with respect to one or more productivity or worth traits, or with respect to capability of producing progeny that will have one or more desired productivity or worth traits, the method comprising providing data about an allelic profile of the bovine, and identifying, and / or selecting or rejecting the bovine on the basis of the data, wherein the data about an allelic profile of the bovine comprises data indicative of the presence or absence of one or more alleles at one or more of the polymorphisms identified in Table 1, Table 2, or Table 3 herein.

7. The method as claimed in claim 6, wherein the data about an allelic profile comprises a) data indicative of the presence or absence of one or more alleles at one or more polymorphisms which affect expression of or from a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is present;b) data indicative of the presence or absence of one or more alleles at one or more polymorphisms which affect expression or activity of a gene product encoded by a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is present; or c) data indicative of the presence or absence of one or more alleles at one or more polymorphisms which are associated with increased or decreased expression from a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is present; d) data indicative of the presence or absence of one or more alleles at one or more polymorphisms which are associated with increased or decreased expression or activity of a gene product encoded by a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is present, or e) data indicative of the presence or absence of one or more alleles at one or more polymorphisms in linkage disequilibrium with one or more of the alleles identified in any one or more of (a) to (d) above.

8. The method as claimed in claim 6 or 7, wherein the one or more polymorphisms is selected from the group consisting of the polymorphisms identified in Table 1, Table 2, or Table 3.

9. The method as claimed in any one of claims 6 to 8, wherein the data about an allelic profile comprises data indicative of the presence or absence of one or more alleles at one or more polymorphisms selected from the group comprising: a) the polymorphisms identified in Table 1; or b) a polymorphism in linkage disequilibrium with one or more of the polymorphisms recited in (a) above, and particularly a polymorphism in 100% linkage disequilibrium (R2 = 1.0) with one or more of the polymorphisms recited in (a) above, or c) any combination of any two or more of (a) to (b).

10. A method of determining genetic status of a bovine with respect to one or more productivity and / or worth traits, or with respect to capability of producing progeny that will have one or more desired productivity and / or worth traits, the method comprising determining the allelic profile of the bovine, and determining the genetic status of the bovine on the basis of the allelic profile, wherein the allelic profile comprises data indicative of the presence or absence of one or more alleles at one or more of the polymorphisms identified in Table 1, Table 2, or Table 3 herein.

11. The method according to claim 10, wherein the allelic profile comprises information relating to the level or activity of a gene product encoded by a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is present.

12. The method according to claim 10 or 11, wherein the method additionally comprises a comparison to the allelic profile of a bovine with known genetic status with respect to one or more productivity and / or worth traits.

13. A method for identifying, and / or selecting or rejecting a non-human animal subject with respect to one or more productivity and / or worth traits, the method comprising: providing the result of one or more genetic tests of a sample from the subject;analysing the result for information relating to the presence or absence of one or more of the following: a) one or more alleles at a polymorphism associated with decreased expression from a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located; or b) one or more alleles at a polymorphism associated with increased expression from a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located; or c) one or more wild-type alleles at any one of the polymorphisms identified in Table 1,Table 2, or Table 3; or d) one or more variant alleles at any one of the polymorphisms identified in Table 1, Table2, or Table 3; or e) one or more homozygous wild-type genotypes at any one or more of the polymorphisms identified in Table 1, Table 2, or Table 3; or f) one or more homozygous variant genotypes at any one or more of the polymorphisms identified in Table 1, Table 2, or Table 3; or g) one or more alleles at a polymorphism in linkage disequilibrium with one or more of the polymorphisms recited in any of (a) to (f) above, and particularly a polymorphism in 100% linkage disequilibrium (R2 = 1.0) with any one or more of the polymorphisms recited in (a) to (f) above; or h) any combination of any two or more of (a) to (g); wherein a result indicative of the presence or absence of one or more of said alleles is indicative of a subject with one or more desired or undesired productivity and / or worth traits; and identifying, and / or selecting or rejecting the subject on the basis of the result.

14. The method according to claim 13 comprising: analysing the result for information relating to the presence or absence of one or more of the following: a) one or more alleles at a polymorphism associated with decreased expression from a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located; or b) one or more variant alleles at any one of the polymorphisms identified in Table 1, Table2, or Table 3; or c) one or more homozygous variant genotypes at any one or more of the polymorphisms identified in Table 1, Table 2, or Table 3; or d) one or more alleles at a polymorphism in linkage disequilibrium with one or more of the polymorphisms recited in any of (a) to (c) above, and particularly a polymorphism in 100% linkage disequilibrium (R2 = 1.0) with any one or more of the polymorphisms recited in (a) to (c) above; or e) any combination of any two or more of (a) to (d); wherein a result indicative of the presence of one or more of said alleles, genotypes, or polymorphisms is indicative of a subject with one or more undesired productivity and / or worth traits; andwherein a result indicative of the absence of one or more of said alleles, genotypes, or polymorphisms is indicative of a subject with one or more desired productivity and / or worth traits; and identifying, and / or selecting or rejecting the subject on the basis of the result.

15. The method according to claim 13 comprising: analysing the result for information relating to the presence or absence of one or more of the following: a) one or more alleles at a polymorphism associated with increased expression from a gene in which one of the polymorphisms identified in Table 1, Table 2, or Table 3 is located; or b) one or more wild-type alleles at any one of the polymorphisms identified in Table 1, Table 2, or Table 3; or c) one or more homozygous wild-type genotypes at any one or more of the polymorphisms identified in Table 1, Table 2, or Table 3; or d) one or more alleles at a polymorphism in linkage disequilibrium with one or more of the polymorphisms recited in any of (a) to (c) above, and particularly a polymorphism in 100% linkage disequilibrium (R2 = 1.0) with any one or more of the polymorphisms recited in (a) to (c) above; or e) any combination of any two or more of (a) to (d); wherein a result indicative of the presence of one or more of said alleles, genotypes, or polymorphisms is indicative of a subject with one or more desired productivity and / or worth traits; and wherein a result indicative of the absence of one or more of said alleles, genotypes, or polymorphisms is indicative of a subject with one or more undesired productivity and / or worth traits; and identifying, and / or selecting or rejecting the subject on the basis of the result.

16. The method as claimed in any one of claims 1 to 15, wherein the allelic profile and / or the identity of one or both alleles at one or more of said polymorphisms is determined in a genotyping method.

17. The method as claimed in claim 16, wherein said genotyping method comprising the hybridisation of nucleic acid obtained from the subject with one or more immobilised nucleic acid molecules.

18. The method as claimed in any one of claims 1 to 17, wherein the allelic profile and / or the identity of one or both alleles at one or more of said polymorphisms is determined on the basis of data obtained in a genotyping method, said genotyping method comprising the hybridisation of nucleic acid obtained from the subject with one or more immobilised nucleic acid molecules.

19. The method according to any one of claims 1 to 18, wherein the productivity and / or worth trait is a fertility trait.

20. The method according to any one of claims 1 to 19, wherein the undesired productivity and / or worth trait is decreased fertility.

21. The method according to any one of claims 1 to 19, wherein the desired productivity and / or worth trait is increased fertility.

22. The method according to claim 19, wherein the fertility trait is selected from the group consisting of sperm motility, sperm production, and sperm morphology.

23. The method according to claim 20, wherein the decreased fertility trait is selected from the group consisting of decreased % total motile sperm, decreased % progressive motile sperm, increased % non-motile sperm, decreased sperm speed, decreased ejaculate volume, decreased sperm concentration, decreased total sperm number, decreased total live sperm, abnormal head morphology including increased incidence of abnormal sperm head morphology, and abnormal tail morphology including increased incidence of abnormal tail morphology..

24. The method according to claim 21, wherein the increased fertility trait is selected from the group consisting of increased % total motile sperm, increased % progressive motile sperm, increased % non-motile sperm, increased sperm speed, increased ejaculate volume, increased sperm concentration, increased total sperm number, increased total live sperm, normal head morphology including increased incidence of normal sperm head morphology, and normal tail morphology including increased incidence of normal tail morphology..

25. A bovine identified or selected by the method of any one of claims 3 to 24.

26. The bovine as claimed in claim 25, wherein the bovine is a bull.

27. Collected semen produced by a bovine as claimed in claim 26.

28. The bovine as claimed in claim 25, wherein the bovine is a cow.

29. A method of selecting a herd of bovine, comprising selecting individuals by the method of any one of claims 1 to 24, and segregating and collecting the selected individuals to form the herd.

30. A herd of bovine selected by the method of claim 29.

31. A herd of bovine comprising two or more bovine, wherein the bovine are the progeny of one or more bovine selected by the method of any one of claims 3 to 24.

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