PHENOTYPE

FIELD OF THE INVENTION

[0001] This invention relates to an application of marker assisted selection of mammalian subjects for quantitative trait loci (QTL) associated with lactoferrin (Lf) uptake, production, regulation, or secretion, including for example milk or colostrum Lf content, particularly by assaying for the presence of polymorphisms in a gene which is associated with the QTL. BACKGROUND

[0002] 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 relevant to, or prior art or part of the common general knowledge relating to, the presently described or claimed inventions, or that any publication or document that is specifically or implicitly referenced is prior art, or forms part of the common general knowledge as at the priority date of the application. All documents referred to herein are hereby incorporated by reference in their entirety.

[0003] The genetic basis of bovine milk and colostrum production is of great significance to the dairy industry. Accordingly, genetic bases for variations in the composition of milk, for example, the relative amounts of major milk proteins, and the effect of these variations on milk production characteristics and milk processing properties, has been the subject of considerable research, debate, and review. For example, PCT International application PCT/NZ01/00245 (published as WO02/36824) reports that polymorphisms in the bovine Diacylglycerol-o-acyltransferase (DGAT1) gene are associated with increased milk yield and altered milk composition, and in particular that the presence of a K232A mutation in the DGAT1 gene results in a decrease in milk fat percentage, milk fat yield, solid fat content and milk protein percentage, while increasing milk volume and milk protein yield. In another example, PCT International application PCT/NZ02/00157 (published as WO03/104492) _^ reports that polymorphisms in the bovine growth hormone receptor (GHR) gene are associated with an increased milk volume and altered milk composition, and in particular that the presence of the F279Y amino acid variant results in increased milk yield and decreased milk fat and milk protein percentage, as well as a decrease in live weight. For other characteristics of milk composition, the basis for variation is less clear.

[0004] An ability to modulate milk or colostrum composition has the potential to alter farming practices, improve animal and human health, and to produce products which are tailored to meet a range of requirements. In particular, a method of genetically evaluating bovine to select those which express desirable phenotypes, such as desirable milk or colostrum lactoferrin content, would be useful.

[0005] For example, colostrum plays a critical role in the transmission of maternal proteins, such as lactoferrin, to offspring, which is important for the growth and health of newborn mammals. Bovine serum and lacteal secretions (which are representative of these secretions in mammals) contain Lf. Lf is believed to provide protection against certain microbial pathogens and their toxins in newborn mammals. This antimicrobial effect reportedly confers passive immunity until the newborn's own immune system has matured.

[0006] As a result, Lf is ideally suited to use as a nutraceutical or food supplement. Lf may be combined with probiotics to inhibit or reduce adverse effects due to pathogens. Applications include use of Lf as a nutraceutical ingredient to target pathogens which cause infections of mucosal surfaces such as in the nose, eyes, ears, lungs, breast and vagina. Moreover, Lf-containing products are suitable for gut and oral health applications.

[0007] Strategies to increase or decrease milk or colostrum Lf content could provide health benefits and are expected to be economically valuable. Lf is normally present in cows' milk at low levels, and commercial scale purification of Lf typically requires cation exchange chromatography followed by ultrafiltration and diafiltration, or ion-exchange chromatography followed by salt elution regimes to provide a pure or relatively pure Lf-containing protein fraction(s). It will be appreciated, however, that these methods and thus the products prepared using these methods are relatively costly.

[0008] Marker assisted selection, which provides the ability to follow a specific favourable genetic allele, involves the identification of a DNA molecular marker or 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 co-dominant, heterozygous and homozygous animals can be distinctively identified. Once a marker system is established, selection decisions are able to be made very easily as DNA markers can be assayed at any time after a DNA containing sample has been collected from an individual subject, whether embryonic, infant or adult.

[0009] It is an object of the present invention to provide a method for marker assisted selection of a mammalian subject with a desired Lf production, regulation or secretion phenotype, including desired milk or colostrum Lf content, or to provide subjects identified or selected using the method of the invention as well as milk or colostrum produced by the selected subject, uses for such milk or colostrum, or to provide the public with a useful choice.

SUMMARY OF THE INVENTION

[0010] This invention relates to the elucidation of the role of polymorphsims in the gene encoding Lactoferrin (Lf) in the concentration of Lf in mammalian mucosal secretions, such as milk or colostrum, and particularly milk or colostrum Lf content. In particular, the invention relates to the identification for the first time of the 7447 A/G (1145V) polymorphism in the bovine Lf gene. The invention further relates to the association for the first time of the C allele at the 30126 T/C polymorphism, of the G allele at the 7447 A/G polymorphism, and of the C allele at the -7 G/C polymorphism with production of milk and colostrum with decreased Lf content, and of the T allele at the 30126 T/C polymorphism, of the A allele at the 7447 A/G polymorphism, and of the G allele at the -7 G/C _polymorphism with production of milk and colostrum with increased Lf content.

[0011] This gives rise to numerous, and separate, aspects of the invention.

[0012] In one aspect the invention provides a method of determining the genetic status of a mammalian subject comprising determining the Lf allelic profile of the mammal, and determining the genetic status of the mammal with respect to milk or colostrum Lf content, or with respect to capability of producing progeny that will have increased or decreased milk or colostrum Lf content, on the basis of the Lf allelic profile.

[0013] In one embodiment, the mammalian subject is bovine.

[0014] In one embodiment, the genetic status with respect to milk or colostrum Lf content is production of milk or colostrum with increased milk or colostrum Lf content.

[0015] In one embodiment, the genetic status of bovine with respect to milk or colostrum Lf content is suitability for once-daily milking.

[0016] In one embodiment, the genetic status of bovine with respect to milk or colostrum Lf content is production of milk or colostrum with increased Lf content when milked once- daily.

[0017] In one embodiment, the genetic status of bovine with respect to milk or colostrum Lf content is capability of producing progeny that will produce milk or colostrum with increased Lf content when milked once-daily.

[0018] Accordingly, in one embodiment the invention provides a method for identifying or selecting a bovine, the method comprising determining the expression or activity of the Lf gene product, and identifying or selecting bovine that produces milk, colostrum, blood, serum, mucosal secretions, or has mucosal surfaces with increased Lf content, or capable of producing progeny that produce milk, colostrum, blood, serum, mucosal secretions, or having mucosal surfaces with increased Lf content, on the basis of the determination.

[0019] In another embodiment, the genetic status with respect to milk or colostrum Lf content is production of milk or colostrum with decreased milk or colostrum Lf content.

[0020] Accordingly, in one embodiment the invention provides a method for identifying or selecting a bovine, the method comprising determining the expression or activity of the Lf gene product, and identifying or selecting bovine that produces milk, colostrum, blood, serum, mucosal secretions, or has mucosal surfaces with decreased Lf content, or capable of producing progeny that produce milk, colostrum, blood serum, mucosal secretions, or having mucosal surfaces with decreased Lf contenton the basis of the determination.

[0021] In various embodiments, increased Lf content is increased total Lf concentration, such as for example, increased [total LfJmol.L ^{" 1} milk, or increased [total LfJmol.L ^"1 colostrum. Similarly, in various embodiments decreased Lf content is decreased total Lf concentration, such as for example, decreased [total LfJmol.L ^"1 milk, or decreased [total LfJmol.L ^"1 colostrum.

[0022] In various embodiments, increased Lf milk concentration is at least 150mg.L ^" ', at least 160mg.L ^" ', at least 170mg.L ^" ', at least 180mg.L ^" ', at least 190mg.L ^" ', at least 200mg.L ^"1 , at least ^lOmg.L ^"1 , at least 220mg.L ^" ', at least 230mg.L ^" ', at least 240mg.L ^"1 , at least 250mg.L ^" ', at least 260mg.L ^" ', at least 270mg.L ^" ', at least 280mg.L ^" ', at least 290mg.L ^"] , at least 300mg.L ^" ', at least 325mg.L ^" ', at least 350mg.L ^" ', at least 375mg.L ^" ', at least 400mg.L ^"1 , at least 450mg.L ^" ', or at least 500mg.L ^" ' .

[0023] In various embodiments associated with once-daily milking, increased Lf milk concentration is at least 300mg.L ^" ', at least 325mg.L ^"1 , at least 350mg.L ^" ', at least 375mg.L ^" ', at least 400mg.L ^" ', at least 425mg.L ^" ', at least 450mg.L ^" ', at least at least 500mg.L ^" ', at least 525mg.L ^"1 , at least 550mg.L ^" ', at least 575mg.L ^" ', at least eOOmg.L ^"1 , at least 650mg.L ^" ', or at least 700mg.L ^"1 .

[0024] In one embodiment, the genetic status of bovine with respect to milk or colostrum Lf content is production of milk with at least about 600mg.L ^" ' Lf when milked once-daily.

[0025] In various embodiments, increased Lf content is increased Lf concentration, or increased Lf concentration relative to another component, such as for example increased Lf concentration relative to lactoglobulin, for example in milk or colostrum. Similarly, in various embodiments decreased Lf content is decreased Lf concentration, or decreased Lf concentration relative to another component, such as for example, decreased Lf concentration relative to lactoglobulin, for example in milk or colostrum.

[0026] In various embodiments, increased Lf content is increased total Lf yield, such as for example, increased Lf yield in milk, or increased Lf yield in colostrum. Similarly, in various embodiments decreased Lf content is decreased total Lf yield, such as for example, decreased Lf yield in milk, or decreased Lf yield in colostrum.

[0027] In various embodiments, suitability for once-daily milking is capability of producing milk or colostrum with increased Lf concentration relative to milk or colostrum lactoglobulin concentration, more preferably increased Lf concentration relative to milk or colostrum beta-lactoglobulin concentration.

[0028] In one embodiment, increased Lf content when milked once-daily is increased Lf concentration relative to milk or colostrum lactoglobulin concentration, more preferably increased Lf concentration relative to milk or colostrum beta-lactoglobulin concentration.

[0029] It will be appreciated that methods comprising determining the expression or activity of a Lf gene product encompass determining expression from or of a Lf gene.

|0030] In one embodiment, expression or activity of the Lf gene product is determined using Lf mRNA, for example by determining the presence or amount of Lf mRNA. In other embodiments, expression or activity of the Lf gene product is determined using Lf protein, preferably by determining the amount of Lf protein, for example the amount of Lf protein, or by determining the activity of Lf protein, for example the activity of Lf protein present in a sample obtained from the subject, or by determining the amount of a Lf protein variant, derivative or fragment, It will be apparent that the activity of Lf protein, such as that present in a sample obtained from the subject, may be determined by methods well known in the art, for example by determining Lf-mediated antimicrobial activity. In still other embodiments, the expression or activity of the Lf gene product is determined using Lf DNA, preferably by determining the presence or absence of one or more polymorphisms associated with decreased or increased Lf expression or activity, for example one or more or the polymorphisms associated with increased or decreased expression or activity as described herein.

[0031] In another embodiment, the Lf allelic profile of the subject is determined together with the allelic profile of the subject at one or more genetic loci associated with milk or colostrum content, including milk or colostrum protein content. [0032] In one embodiment, the one or more genetic loci is one or more polymorphisms in one or more genes associated with milk or colostrum protein content.

[0033] In one embodiment, the one or more polymorphisms is the AA/BB polymorphism in the bovine beta lactoglobulin gene.

[0034] 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.

[0035] Linkage disequilibrium (LD) 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.)

[0036] It will be apparent that as used herein, the phrase "Lf allelic profile" contemplates data indicative of the presence or absence of one or more alleles at one or more polymorphisms in the Lf gene or which affect expression from the Lf gene or the expression or activity of a Lf gene product or which are associated with variation in the expression from the Lf gene or in the expression or activity of a Lf gene product. In preferred embodiments, the Lf allelic profile comprises data indicative of the presence or absence of one or more alleles at one or more polymorphisms associated with increased or decreased milk or colostrum Lf content.

[0037] For example, in various preferred embodiments the Lf allelic profile of bovine comprises data indicative of

a) the presence or absence of the C allele at the 30126 T/C (rs43706484) polymorphism in the

Lf gene; or

b) the presence or absence of the T allele at the 30126- T/C polymorphism in the Lf gene; or c) the presence or absence of the G allele at the 7447 A/G polymorphism in the Lf gene; or d) the presence or absence of the A allele at the 7447 A/G polymorphism in the Lf gene; or e) the presence or absence of the C allele at the -7 G/C (rs43706485) polymorphism in the Lf gene; or

f) the presence or absence of the G allele at the -7 G/C polymorphism in the Lf gene; or g) the presence or absence of a polymorphism in linkage disequilibrium with any one or more of a) to f) above, and particularly the presence or absence of a polymorphism in 100% linkage disequilibrium (D'= 1.0) with any one or more of a) to f) above, or h) any combination of any two or more of a) to g).

[0038] In other embodiments, the Lf allelic profile comprises data indicative of the presence or absence of one or more alleles at one or more polymorphisms in the promoter of the Lf gene, or in a regulatory region of the Lf gene, or in an intron of the Lf gene, and preferably comprises data indicative of the presence or absence of one or more alleles which affect expression from the Lf gene or the expression or activity of a Lf gene product or which are associated with variation in the expression from the Lf gene or in the expression or activity of a Lf gene product.

[0039] It will further be appreciated that the Lf allelic profile may comprise information correlating the presence or absence of one or more polymorphisms as described above with milk or colostrum Lf content.

[0040] In one embodiment, the allelic profile is determined using nucleic acid obtained from said subject, preferably DNA obtained from said subject, or alternatively, said allelic profile is determined using RNA obtained from said subject.

[0041] In yet a further embodiment, the allelic profile is determined with reference to the amino acid sequence of expressed Lf protein obtained from said subject.

[0042] In another embodiment, the allelic profile is determined with reference to the amount or activity of Lf protein obtained from said subject.

[0043] Conveniently, in said method the presence or absence of DNA encoding wild type Lf in said subject is determined, directly or indirectly, for example using an expressed gene product.

[0044] Alternatively, in said method the presence or absence of at least one nucleotide difference from the nucleotide sequence encoding wild type Lf in said subject is determined, directly or indirectly.

[0045] In various embodiments, the method comprises determining the 30126 T/C Lf allelic profile of the bovine, or determining the 7447 A/G Lf allelic profile of the bovine, or determining the -7 G/C Lf allelic profile of the bovine. More preferably, the method comprises determining the Lf allelic profile at two or more of these polymorphisms, or all three of these polymorphisms.

[0046] More specifically, in said method the presence or absence of one or more of the T allele or C allele at the 30126 T/C polymorphism in the Lf gene is determined, directly or indirectly. For example, the presence of the T allele or C allele at the30126 T/C polymorphism in the Lf gene may be determined using a polymorphism in linkage disequilibrium with the T allele or with the C allele at the 30126 T/C polymorphism.

[0047] Similarly, the presence or absence of a particular allele at the 7447 A/G (1145 V) polymorphism or at the -7 G/C polymorphism in the bovine Lf gene may be determined directly or indirectly, for example by analysis of one or more polymorphisms in LD with one or more of these polymorphisms.

[0048] In one embodiment, the method includes ascertaining, from a sample of material containing DNA obtained from the subject, whether a sequence of the DNA encoding a protein "(A)" having biological activity of wild type Lf 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 low relative milk, colostrum, blood, serum, mucosal secretions, or mucosal surface Lf content, particularly with the production of milk or colostrum with, inter alia, decreased Lf content. 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 high relative milk, colostrum, blood, serum, mucosal secretions, or mucosal surface Lf content, particularly with the production of milk or colostrum with, inter alia, increased Lf content. The presence of both the DNA encoding (A) and the DNA encoding (B) indicates an association with intermediate relative Lf content, particularly with the production of milk or colostrum with, inter alia, intermediate Lf content.

[0049] As used herein, biological activity of wild type Lf protein refers to both expression levels and activity characteristic of Lf protein encoded by the wild type Lf gene.

[0050] In another embodiment, the method includes ascertaining, from a sample of material containing DNA obtained from the subject, whether the wild type Lf gene sequence is present. In still another embodiment, the method includes ascertaining, from a sample of material containing DNA obtained from the subject, the expression of the Lf gene product, preferably by determining the presence or absence of one or more polymorphisms associated with decreased or increased Lf expression, for example one or more promoter polymorphisms associated with increased or decreased expression.

[0051] In another embodiment, the invention includes ascertaining whether mRNA encoding a protein "(A)" having biological activity of a wild type Lf 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 low relative milk, colostrum, blood, serum, mucosal secretions, or mucosal surface Lf content, particularly with the production of milk or colostrum with, inter alia, decreased Lf content. 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 high relative milk, colostrum, blood, serum, mucosal secretions, or mucosal surface Lf content, particularly with the production of milk or colostrum with, inter alia, increased Lf content. Again, the presence of both the mRNA encoding (A) and the mRNA encoding (B) indicates an association with intermediate relative milk, colostrum, blood, serum, mucosal secretions, or mucosal surface Lf content, particularly with the production of milk with, inter alia, intermediate Lf content.

[0052] In another embodiment, the method includes ascertaining the amount of Lf mRNA present in a sample of material containing mRNA obtained from the subject. In another embodiment, the method includes ascertaining the Lf expression profile of the subject, preferably including ascertaining the Lf mRNA expression profile of the subject.

[0053] In another embodiment, the invention includes ascertaining whether a protein "(A)" having biological activity of a wild type Lf is present, or whether a protein "(B)" at least partially 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 low relative milk, colostrum, blood, serum, mucosal secretions, or mucosal surface Lf content, particularly with the production of milk or colostrum with, inter alia, decreased Lf content. The reverse association again holds true, where the presence of (A) and the absence of (B) again indicates an association with high relative milk, colostrum, blood, serum, mucosal secretions, or mucosal surface Lf content, particularly with the production of milk or colostrum with, inter alia, increased Lf content. Again, the presence of both (A) and (B) indicates an association with intermediate relative milk, colostrum, blood, serum, mucosal secretions, or mucosal surface Lf content, particularly with the production of milk with, inter alia, intermediate Lf content.

[0054] In another embodiment, the method includes ascertaining the amount or activity of Lf protein present in a sample of material containing protein obtained from the subject. [0055] In another aspect directed to veterinary or agricultural applications, the invention is a method for determining the Lf genotype of a mammalian subject, as may be desirable to know for breeding purposes.

[0056] In one embodiment, 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 encoding a wild type Lf gene or gene product such as a protein having biological activity of wild type Lf and optionally ascertaining whether the sample contains an (ii) allelic nucleic acid molecule encoding a variant Lf gene or gene product such as a protein lacking biological activity of wild type Lf.

[0057] In another embodiment, 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 of wild type Lf and optionally ascertaining whether the sample contains (iii) a protein lacking biological activity of wild type Lf.

[0058] In a further embodiment, the invention provides a method of determining genetic status of a subject with respect to milk or colostrum Lf content which comprises providing the Lf allelic profile of the subject, together with the allelic profile of the subject at one or more genetic loci associated with milk or colostrum Lf content, and determining the genetic status on the basis of the allelic profiles.

[0059] In one embodiment, the one or more genetic loci is one or more polymorphisms in one or more genes associated with milk or colostrum Lf content.

[0060] In a further aspect, the invention includes a probe containing a nucleic acid molecule sufficiently complementary with a nucleic acid sequence present in NC_007320.3 or a nucleotide sequence encoding a wild type bovine Lf, 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 alleles at one or more of the 30126 T/C polymorphism, the 7447 A/G polymorphism, and the -7 G/C polymorphism in the bovine Lf gene, for example a probe that includes a guanine at the site corresponding to the 7447 A/G polymorphism.

[0061] The invention also includes a primer composition useful for detection of the presence or absence of a wild-type Lf gene, including nucleic acid sequence encoding wild type Lf 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 Lf, or its complement. The nucleic acid sequence can in whole or in part be identified in Genbank references NC_007320.3 or NM_180998. Diagnostic kits including such a composition are also included.

[0062] Particularly contemplated are primers comprising or substantially complementary to a nucleic acid sequence present in NC_007320.3 and within approximately 1 to about 2000 bp of one of the 30126 T/C polymorphism, the 7447 A/G polymorphism, or the -7 G/C polymorphism, more preferably 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 30126 T/C polymorphism, the 7447 A/G polymorphism, or the -7 G/C polymorphism.

[0063] Examples of such primers may be derived from the sequences presented herein as SEQ ID NOs: 1 to 3.

[0064] 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.

[0065] The invention also provides a diagnostic kit including a primer composition useful for determining the presence or absence of a reference Lf gene and/or the presence or absence of nucleic acid encoding reference Lf, the diagnostic kit comprising one or more primers or primer compositions as described herein.

[0066] The invention further includes an antibody composition useful for detection of the presence or absence or amount of wild type Lf 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.

[0067] The invention further provides a diagnostic kit useful in detecting DNA comprising a variant Lf gene, or DNA Or mRNA encoding a variant Lf gene product at least partially lacking wild type activity in a subject which includes first and second primers for amplifying the DNA or mRNA, the primers being complementary to nucleotide sequences of the DNA or mRNA upstream and downstream, respectively, of a polymorphism in the Lf gene which results in increased or decreased Lf levels (particularly increased or decreased Lf content in milk or colostrum). The kit can also include a third or a fourth primer complementary to a naturally occurring mutation of the wild type Lf gene. Preferably the kit includes instructions for use, for example in accordance with a method of the invention.

[0068] In one embodiment at least one of the nucleotide sequences is selected to be from a non-coding region of the reference Lf gene.

[0069] The kit can also include a primer complementary to a naturally occurring mutation of a coding or non-coding portion of the reference Lf gene, for example a mutation in the promoter of the Lf gene. Preferably the kit includes instructions for use, for example in accordance with a method of the invention.

[0070] Thus, in another embodiment the invention provides a method of assessing the genetic status of a bovine with respect to milk or colostrum Lf content which comprises the step of determining the presence or absence of one or more polymorphisms selected from the group comprising:

the 30126 T/C polymorphism in the Lf gene, or

the 7447 A/G (II 45 V) polymorphism in the Lf gene, or

the -7 G/C polymorphism in the Lf gene, or

one or more polymorphisms in linkage disequilibrium with one or more of these polymorphisms.

[0071] In another embodiment the invention provides a method of assessing the genetic status of a bovine with respect to milk or colostrum Lf content which comprises the step of determining the presence or absence of one or more polymorphisms selected from the group comprising:

the C allele or the T allele at the 30126 T/C polymorphism in the Lf gene, or the G allele or the A allele at the 7447 A/G polymorphism in the Lf gene, or the C allele or the G allele at the -7 G/C polymorphism in the Lf gene.

[0072] Again, the one or more polymorphisms can be detected directly or by detection of one or more polymorphisms which are in linkage disequilibrium with the one or more polymorphisms.

[0073] In another aspect, the present invention provides a method for identifying or selecting a mammalian subject with a genotype indicative of one or more desired Lf, production, regulation, or secretion phenotypes. The method comprises determining the Lf allelic profile of said subject, and identifying or selecting the subject on the basis of the determination.

[0074] In one embodiment, the Lf 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 the Lf gene associated with one or more of:

(a) increased or decreased expression or activity of a Lf gene product, or

(b) increased or decreased Lf secretion, or

(c) production of milk, colostrum, blood, serum, mucosal secretions, or having one or more mucosal surfaces with increased or decreased Lf content, or

(d) one or more polymorphisms in linkage disequilibrium with one or more polymorphisms in the Lf gene associated with one or more of (a) to (c) above.

[0075] In one embodiment, the subject is bovine.

[0076] In one embodiment, the phenotype is desired milk or colostrum Lf content.

[0077] In one embodiment, the invention provides a method for selecting a bovine with a Lf allelic profile indicative of increased milk or colostrum Lf content.

[0078] Preferably the method comprises determining the presence of one or more of the T allele at the 30126 T/C polymorphism, the A allele at the 7447 A/G polymorphism, or the G allele at the -7 G/C polymorphism in the Lf gene, and selecting the bovine on the basis of the determination. Alternatively or additionally, the method comprises determining the absence of one or more of the C allele at _. the 30126 T/C polymorphism, ^' the G allele at the 7447 A/G polymorphism, or the C allele at the -7 G/C polymorphism in the Lf gene, and selecting the bovine on the basis of the determination.

[0079] Preferably, the method comprises determining the presence of one or more of the TT genotype at the 30126 T/C polymorphism, the AA genotype at the 7447 A/G polymorphism, or the GG . genotype at the -7 G/C polymorphism in the Lf gene, and selecting the bovine on the basis of the determination.

[0080] In a further embodiment the invention provides a method for selecting a bovine with a Lf allelic profile indicative of decreased milk or colostrum Lf content.

[0081] Preferably the method comprises determining the presence of one or more of the C allele at the 30126 T/C polymorphism, the G allele at the 7447 A/G polymorphism, or the C allele at the -7 G/C polymorphism in the Lf gene, and selecting the bovine on the basis of the determination. Alternatively or additionally, the method comprises determining the absence of one or more of the T allele at the 30126 T/C polymorphism, the A allele at the 7447 A/G polymorphism, or the G allele at the-,-7 G/C polymorphism in the Lf gene, and selecting the bovine on the basis of the determination. .

[0082] Preferably, the method comprises determining the presence of one or more of the CC genotype at the 30126 T/C polymorphism, the GG genotype at the 7447 A/G polymorphism, or the CC genotype at the -7 G/C polymorphism, in the Lf gene, and selecting the bovine on the basis of the determination.

[0083] In a further embodiment the invention provides a method for selecting a bovine with a Lf allelic profile indicative of intermediate milk or colostrum Lf content, preferably of intermediate milk or colostrum Lf content.

[0084] Preferably, the method comprises determining the presence of one or more of the CT genotype at the 30126 T/C polymorphism, the AG genotype at the 7447 A/G polymorphism, or the CG genotype at the -7 G/C polymorphism in the Lf gene, and selecting the bovine on the basis of the determination.

[0085] In one embodiment, the presence of an allele is determined with respect to a Lf polynucleotide (genomic DNA, mRNA or cDNA produced from mRNA) obtained from the bovine.

[0086] In one embodiment, the presence of an allele is determined by sequencing a Lf polynucleotide obtained from the bovine.

[0087] In a further embodiment the determination comprises the step of amplifying a Lf polynucleotide sequence from genomic DNA, mRNA or cDNA produced from mRNA derived from said bovine, for example by PCR.

[0088] Preferably 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 NC_007320.3 or NM_180998 or a naturally occurring flanking sequence.

[0089] In one embodiment at least one of the primers comprises sequence corresponding to at least one of the allele-specific nucleotides described herein.

[0090] In an alternative embodiment, 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.

[0091] In a further embodiment, the presence of an allele is determined by mass spectrometric analysis of a Lf polynucleotide obtained from the bovine. [0092] In an alternative embodiment, 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 NC_007320.3 or NMJ 80998.

[0093] Preferably the probe or probes comprises 12 or more contiguous nucleotides of or complementary to the sequence of NC_007320.3 or NM_180998.

[0094] Preferably the probe or probes comprise sequence corresponding to at least one of the allele-specific nucleotides described herein or complements thereof.

[0095] In an alternative embodiment, the presence of an allele is determined by analysis of a Lf polypeptide obtained from the bovine.

[0096] In a further aspect the invention provides 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.

[0097] In still a further aspect the invention provides 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 provides a herd of bovine so selected, as well as a herd comprising bovine produced by bovine selected by the methods described herein.

[0098] In still a further aspect, the invention provides a method of selecting a herd of bovine by a method of the present invention and then milking said herd once daily instead of the more normal twice daily milking and hence further enriching milk Lf levels

[0099] In a still further aspect, the invention provides a method of determining genetic status of a bovine with respect to one or more milk or colostrum Lf content phenotypes, or with respect to capability of producing progeny predisposed to or with one or more milk or colostrum Lf content phenotypes, the method comprising

providing data about the Lf allelic profile of said bovine, and

determining the genetic status of the bovine on the basis of the data.

[00100] Preferably, the data about the Lf allelic profile comprises data representative of the presence or absence of one or more of the C allele or the T allele at the 30126 T/C polymorphism, the A allele or the G allele at the 7447 A/G polymorphism, or the C allele or the G allele at the -7 G/C polymorphism in the Lf gene.

[00101] Preferably, 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 milk or colostrum Lf content phenotypes, wherein the data is representative of the genetic status of the bovine.

[00102] Preferably, the one or more genetic loci are one or more polymorphisms associated with an increase or decrease in expression or activity of a Lf gene product.

[00103] Preferably the genetic loci is the Lf gene (including all regulatory elements such as the promoter, introns and 3' UTR).

[00104] In one embodiment, the one or more milk or colostrum Lf content phenotypes is selected from the group comprising production of or capability of producing milk with increased Lf content or production of or capability of producing colostrum with increased Lf content. In another embodiment, the one or more milk or colostrum Lf content phenotypes is selected from the group comprising production of or capability of producing milk with decreased Lf content or production of or capability of producing colostrum with decreased Lf content.

[00105] Accordingly, in one embodiment the invention provides a method of determining genetic status of a bovine with respect to milk or colostrum Lf content, or with respect to capability of producing progeny that will have increased or decreased milk or colostrum Lf content, the method comprising

providing data about the Lf allelic profile of the bovine, and

determining the genetic status of the bovine on the basis of the data.

[00106] In a further aspect the invention provides a method for identifying or selecting a mammalian subject with respect to one or more desired Lf production, regulation, or secretion phenotypes, 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:

(a) one or more polymorphisms associated with increased or decreased expression or activity of a Lf gene product, or

(b) one or more polymorphisms in the Lf gene associated with increased or decreased Lf secretion, or

(c) one or more polymorphisms in the Lf gene associated with production of milk, colostrum, blood, serum, mucosal secretions, or having one or more mucosal surfaces with increased or decreased Lf content, or (d) one or more polymorphisms in linkage disequilibrium with one or more polymorphisms in the Lf gene associated with one or more of (a) to (c) above, 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 Lf production, regulation, or secretion phenotypes; and

identifying or selecting the subject on the basis of the result.

[00107] Preferably, the one or more polymorphisms associated with increased or decreased expression or activity of Lf gene product is one or more polymorphisms in the Lf gene.

[00108] In one embodiment, the one or more Lf production, regulation, or secretion phenotypes is one or more milk or colostrum Lf content phenotypes, including increased milk or colostrum Lf content. In a further aspect the invention provides a method for selecting a bovine with one or more desired milk or colostrum Lf content phenotypes, the method comprising

a) providing the result of one or more genetic tests of a sample from the bovine, and b) analysing the result for the presence or absence of one or more polymorphisms selected from the group comprising:

the 30126 T/C polymorphism in the Lf gene, or

the 7447 A/G (1145V) polymorphism in the Lf gene, or

the -7 G/C polymorphism in the Lf gene, or

one or more polymorphisms in linkage disequilibrium with one or more of the 30126

T/C polymorphism in the Lf gene, the 7447 A/G (1145V) polymorphism in the Lf gene, or the -7 G/C polymorphism in the Lf gene,

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 milk or colostrum Lf content phenotypes.

[00109] In other aspects, the invention provides a system for performing one or more of the methods of the invention, said system comprising:

computer processor means for receiving, processing and communicating data;

storage means for storing data including a reference genetic database of the results of genetic analysis of a subject with respect to one or more milk or colostrum Lf content phenotypes and optionally a reference milk or colostrum Lf content trait database of non- genetic factors for one or more subject milk or colostrum Lf content phenotypes; and a computer program embedded within the computer processor which, once data consisting of or including the result of a genetic analysis for which data is included in the reference genetic database is received, processes said data in the context of said reference databases to determine, as an outcome, the genetic status of the subject, said outcome being communicable once known, preferably to a user having input said data.

[00110] Preferably, said system is accessible via the internet or by personal computer.

[00111] Preferably, said reference genetic database comprises or includes the results of one or more analyses of one or more genetic loci associated with one or more milk or colostrum Lf content phenotypes, more preferably the one or more genetic loci are one or more polymorphisms in one or more genes associated with one or more milk or colostrum Lf content phenotypes.

[00112] In yet a further aspect, the invention provides a computer program suitable for use in a system as defined above comprising a computer usable medium having program code embodied in the medium for causing the computer program to process received data consisting of or including the result of at least one genetic analysis of one or more genetic loci associated with one or more milk or colostrum Lf content phenotypes in the context of both a reference genetic database of the results of said at least one genetic analysis and optionally a reference database of non-genetic factors associated with one or more subject milk or colostrum Lf content phenotypes.

[00113] Preferably, the one or more genetic loci are one or more polymorphisms in one or more genes associated with one or more milk or colostrum Lf content phenotypes.

[00114] Preferably the gene is the Lf gene (including all regulatory elements such as the promoter, introns and 3' UTR).

[00115] Preferably, the one or more polymorphisms are one or more polymorphisms associated with an increase or decrease in expression or activity of a Lf gene product.

[00116] In still another aspect, the invention provides a method of determining genetic status of a bovine with respect to milk or colostrum Lf content, or with respect to capability of producing progeny that will have increased or decreased milk or colostrum Lf content, the method comprising

determining milk or colostrum Lf content of the bovine,

determining the Lf allelic profile of the bovine,

comparing the Lf allelic profile of the bovine or the milk or colostrum Lf content of the bovine with that of a bovine having a known Lf allelic profile; determining the genetic status of the bovine on the basis of the comparison.

[00117] It will be appreciated that for the purposes of the comparison, the milk or colostrum Lf content associated with the known Lf allelic profile .is known. It will further be appreciated that the association of milk or colostrum Lf content with a particular Lf allelic profile may be established by the methods described herein.

[00118] In another aspect, the invention relates to an isolated, purified or recombinant nucleic acid molecule comprising nucleotide sequence selected from the group comprising:

(a) at least 12 contiguous nucleotides of NC_007320.3 and comprising a guanine at the 7447 A/G polymorphism; or

(b) at least 12 contiguous nucleotides of NM l 80998 and comprising a guanine at the 7447 A/G polymorphism; or

(c) at least 12 contiguous nucleotides of a variant of (a) or (b); or

(d) at least 12 contiguous nucleotides of any one or more of SEQ ID NOs: 1 to 3 and comprising a guanine at the 7447 A/G polymorphism; or

(e) a complement of any one of (a) to (d); or

(f) a sequence of at least 12 contiguous nucleotides and capable of hybridising to the nucleotide sequence of any one of (a) to (e) under stringent conditions.

[00119] In one embodiment, the Lf nucleic acid molecule is a Lf fragment as defined herein, wherein the Lf fragment comprises one or more of the 30126 T/C polymorphism, the 7447 A/G polymorphism, or the -7 G/C polymorphism, or a combination of any two or more thereof.

[00120] The invention also provides a genetic construct comprising a Lf 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 Lf nucleic acid molecule of the invention, an antibody which selectively binds a polypeptide of the invention, and a method for recombinantly producing a polypeptide of the invention.

[00121] In a further aspect there is provided a method of determining a subject's risk of developing one or more conditions associated with decreased or increased Lf expression or activity comprising providing the results of an analysis of a sample from said subject for the presence or absence of one or more polymorphisms in the Lf gene associated with increased or decreased expression or activity of a Lf gene product, or one or more polymorphisms in linkage disequilibrium with one or more polymorphisms in the Lf gene associated with increased or decreased expression or activity of a Lf gene product, wherein the presence or absence of one or more of said polymorphisms is indicative of the subject's risk of developing one or more conditions associated with decreased or increased Lf expression or activity.

[00122] In one embodiment, the condition associated with decreased or increased Lf expression or activity is mastitis.

[00123] In one embodiment, the presence or absence of one or more of said polymorphisms is indicative of whether a condition suffered by the subject is caused or contributed to by the increased or decreased expression or activity of a Lf gene product.

[00124] In another aspect the invention provides a method of determining a subject's risk of developing one or more conditions associated with Lf production, regulation, or secretion, the method comprising providing the results of an analysis of a sample from said subject for the presence or absence of one or more polymorphisms in the Lf gene associated with increased or decreased expression or activity of a Lf gene product, or one or more polymorphisms in linkage disequilibrium with one or more polymorphisms in the Lf gene associated with increased or decreased expression or activity of a Lf gene product, wherein the presence or absence of one or more of said polymorphisms is indicative of the subject's risk of developing one or more conditions associated with associated with Lf production, regulation, or secretion.

[00125] In one embodiment the one or more conditions is associated with decreased Lf secretion.

[00126] In one embodiment, the one or more conditions is increased susceptibility to one or more infectious diseases.

[00127] In various examples, the infectious disease is an infectious disease of a mucosal surface, or is an infectious disease caused by entry of an infectious agent via a mucosal surface.

[00128] The infectious agent may be a microorganism such as a yeast (including, for example Candida spp.), a virus, including a retrovirus, such as for example HIV, a rotavirus, an adenovirus, a norovirus, a bacteria, or a fungi.

[00129] In one embodiment, the infectious disease is mastitis.

[00130] In one embodiment, the infectious disease is infectious diarrhoea, including infectious diarrhoea caused by viruses, such as Norovirus spp., Rotavirus spp., Adenovirus spp., or Astrovirus spp., bacteria, such as Campylobacter spp., Salmonella spp., Cryptosporidium spp., and Giardia lamblia spp., Shigella spp., Escherichia coli, and Clostridium difficile. For example, in one embodiment where the subject is bovine, cervine, ovine or porcine, the infectious disease is scours.

[00131] 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.

[00132] In one embodiment, the one or more polymorphisms are selected from the group consisting of:

the 30126 T/C polymorphism in the Lf gene, or . ^' . .

the 7447 A/G polymorphism in the Lf gene, or

the -7 G/C polymorphism in the Lf gene, or

one or more polymorphisms in linkage disequilibrium with one or more of these polymorphisms,

wherein the presence or absence of one or more of said polymorphisms is indicative of the subject's risk of developing one or more conditions associated with decreased or increased Lf expression or activity.

[00133] The method can additionally comprise analysing a sample from said subject for the presence of one or more further polymorphisms associated with Lf uptake, production, regulation or secretion, including one or more polymorphisms associated with increased or decreased Lf production or secretion.

[00134] Again, detection of the one or more further polymorphisms may be carried ut directly or by detection of polymorphisms in linkage disequilibrium with the one or more further polymorphisms.

[00135] In another aspect there is provided a method of treating a subject with or at risk of developing one or more conditions associated with Lf production, regulation, or secretion, the method comprising administering to the subject a milk or colostrum product or composition as herein described.

[00136] In one embodiment, the milk or colostrum composition comprises or is derived from milk or colostrum with increased Lf content. In another embodiment, the milk or colostrum composition comprises or is derived from milk or colostrum with decreased Lf content.

[00137] In various embodiments, the subject is a foetal, neonatal, infant or child subject.

[00138] In certain embodiments the composition is a maternal formula, infant formula, follow-on formula, growing-up formula or dietetic product. In other embodiments the composition is a nutraceutical. In still other embodiments, the composition is a pharmaceutical.

[00139] In one embodiment, the method additionally comprises determining the Lf allelic profile of the subject.

[00140] In embodiments relating to maintaining or raising Lf levels in a foetal subject, the composition is suitable for oral administration to a mother during gestation.

[00141] In certain embodiments the composition is a maternal formula, infant formula, follow-on formula, growing-up formula or dietetic product. In other embodiments the composition is a nutraceutical.

[00142] In another aspect the invention relates to an isolated, purified or recombinant protein comprising at least about 10 contiguous amino acids of NP_851341 and comprising a valine at the position corresponding to amino acid 145 of bovine Lf (equivalent to position 145 ofNP_851341).

[00143] In one embodiment the polypeptide is a variant as defined herein.

[00144] In another embodiment the polypeptide is a functional variant of a Lf, or is a functional fragment thereof.

[00145] 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" are to be interpreted in the same manner.

[00146] As described above, in this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 . - shows the effect of lactation stage on the concentration of Lf in bovine milk.

Data shown are average Lf concentrations at each lactation stage, ± standard error of mean. The lowest average Lf concentration was observed at peak lactation (35 days after calving), and the highest average Lf concentration was observed at late lactation. Figure 2 shows the effect of sire on the concentration of Lf in bovine milk. Data shown are average Lf concentrations for the daughters of each sire, ± standard error of mean. Sires are numbered 1 - 6, and these sires formed the basis of the trial pedigree, a Friesian-Jersey Crossbred trial.

Figure 3 shows a graph depicting a QTL for milk Lf concentration on bovine chromosome 22 (solid line) as well as the association analysis of individual markers with the concentration of Lf in bovine milk. The maximum F-value (y-axis) for the QTL was 10.19 and the most likely position for the QTL was estimated at 70 cM. Bootstrap analysis (n = 1000 iterations) showed that the 95% confidence interval for the QTL was 70 - 77 cM. The most highly associated markers were contained within the QTL.

Figure 4 is a schematic of the bovine Lf gene, along with the sequence traces of the polymorphisms identified and subsequently genotyped within the FJXB trial.

Figure 5 is a graph depicting the effect of polymorphism Lf_30126 on the concentration of Lf in milk, at lactation stages peak, mid and late lactation. At each stage of lactation, the TT genotype is associated with higher concentrations of milk Lf. Data shown are means ± standard error.

Figure 6 is a graph depicting the adjusted statistical effect of the Lf gene polymorphisms on the bovine chromosome 22 milk Lf QTL: black diamonds; original QTL, cross; effect of polymorphism Lf_7447, white square; effect of polymorphism Lf_30126, white triangle; effect of polymorphism Lf_-7.

Figure 7 is a graph showing the QTL for milk Lf concentration on bovine chromosome

22 (grey circles) as well as the QTL for Lf mRNA expression (white diamonds). The location for the maximum F-value for both QTLs coincides at approximately 70cM.

Figure 8 - shows the QTL for milk Lf concentration on bovine chromosome 22 (white squares) as well as the effect of fitting the effect of Lf mRNA expression to the milk Lf QTL (black diamonds). This suggests that the QTL for milk Lf concentration and the QTL for Lf mRNA expression are closely related. DETAILED DESCRIPTION OF THE INVENTION

[00147] The present invention recognises for the first time that a polymorphism in the Lf gene in bovine is associated with a QTL for variations in Lf content, and particularly variations in milk or colostrum Lf content.

[00148] For the .sake of clarity, the phrase "milk or colostrum Lf content" is to be read as referring to milk Lf content or colostrum Lf content. It will be apparent to those skilled in the art that milk or colostrum Lf content can readily be determined qualitatively or quantitatively. In some applications, a qualitative comparison of a sample of milk or colostrum relative to another sample (preferably from an individual of known Lf allelic profile) may be sufficient to correlate association with a particular genotype. Methods for quantitative determination of milk or colostrum Lf content are also known in the art, and examples are provided herein.

[00149] The invention provides methods of assessing the genetic status of a bovine with respect to Lf content, more particularly milk or colostrum Lf content. One such method comprises the step of determining the Lf allelic profile of said bovine. Another such method comprises the step of determining the level of the Lf gene product of said bovine.

[00150] The invention also provides methods for identifying or selecting a mammalian subject with a genotype indicative of one or more desired Lf production, regulation, or secretion phenotypes, including desired milk or colostrum Lf content, particularly desired Lf content. One of the major applications of the present invention is in the identification or selection of bovine having one or more of the T allele at the 30126 T/C polymorphism, the A allele at the 7447 A/G polymorphism, or the G allele at the -7 G/C polymorphism in the Lf gene, which are each independently associated with increased milk or colostrum Lf content, or of bovine having one or more of the C allele at the 30126 T/C polymorphism, the G allele at the 7447 A/G polymorphism, or the C allele at the -7 G/C polymorphism in the Lf gene which are each independently associated with decreased milk or colostrum Lf content. Accordingly, one method comprises determining the presence or absence of one or more of the C allele or the T allele at the 30126 T/C polymorphism, the A allele or the G allele at the 7447 A/G polymorphism, or the G allele or the C allele at the -7 G/C polymorphism of the Lf gene, and selecting the bovine on the basis of the determination.

[00151] Additionally, the invention is directed towards the selected non-human subjects, such as bovine and ova or semen from the selected non-human subjects which may be useful in further breeding programs. Bovine so selected will be useful for milk or colostrum production. The invention is also directed towards milk or colostrum produced by the selected bovine or the progeny thereof, as well as dairy products produced from such milk and compositions (including pharmaceutical, nutraceutical, veterinary, and the like) produced from such colostrum.

[00152] The production of a wide variety of dairy products is well known in the art, and dairy products contemplated herein include ice creams, yoghurts and cheeses, dairy based drinks (such as milk drinks including milk shakes, and yogurt drinks), milk powders, dairy based sports supplements, food additives such as protein sprinkles and dietary supplement productsincluding daily supplement tablets.

[00153] Similarly, the production of a wide range of colostrum products, such as but not limited to nutraceutical supplements and the like, is well known in the art. Particularly contemplated herein are colostrum compositions having increased Lf content or produced from colostrum having increased Lf content. Methods to prepare compositions enriched in Lf are also known in the art, as exemplified by those discussed in PCT international patent application PCT/NZ2006/000263 published as WO 2007/043900, incorporated herein by reference in its entirety.

[00154] The present invention recognises that mutations in the gene encoding Lf, as well as Lf levels or activity, may be used as a selection tool to breed animals with higher or lower milk concentrations of Lf. This in turn may allow the production of milk products more suitable to particular markets, such as the production of foods high in Lf.

[00155] The present invention further provides methods to identify subjects at risk of developing conditions associated with one or more Lf intake, production, regulation, or secretion phenotypes, such as decreased Lf uptake or increased or decreased Lf levels, methods to identify subjects who may benefit from a particular treatment regimen (including for example increased Lf intake or supplementation), methods to prevent or treat conditions associated with such phenotypes in subjects in need thereof, as well as compositions and products useful in such methods.

[00156] As used herein, the phrase "once daily milking" and grammatical equivalents means milking once within a twenty-four hour period. The phrase "twice daily milking" and grammatical equivalents means milking twice within a twenty-four hour period.

1 Lactoferrin

[00157] Lactoferrin. is an approximately 80kD iron-binding glycoprotein present in most exocrine fluids, including tears, bile, bronchial mucus, gastrointestinal fluids, cervico-vaginal mucus, seminal fluid and milk. The richest source of lactoferrin is mammalian milk and colostrum. Bovine lactoferrin (UniProtKB/Swiss-Prot P24627) is composed of a single polypeptide chain with 17 disulfide bridges. The three-dimensional structure of bovine lactoferrin comprises two lobes (the N-lobe and C-lobe) of equal size. Each lobe comprises a metal ion-binding pocket; each pocket has the capacity to bind reversibly one Fe ³⁺ ion with high affinity in cooperation with a C0 ₃ ion (Moore SA, Anderson BF, Groom CR, Haridas M, Baker EN. Three-dimensional structure of diferric bovine lactoferrin at 2.8 A resolution. J Mol Biol. (1997) 274(2):222-36). Lactoferrin in bovine milk is naturally about 12 to 18 percent iron saturated, and lactoferrin that is iron saturated to a greater extent has been reported to be useful as an iron supplement or as part of a cancer therapy regime (PCT International Application publication number WO 2006/054908). A reference genomic sequence for the bovine Lf gene (GeneID:280846) is available at Genbank accession number NC 007320.3, while a reference mRNA sequence is available at Genbank accession number NM l 80998, and a reference amino acid sequence for the bovine Lf protein is available at Genbank accession number NP 851341.

[00158] As described herein, the present invention relates to the identification that mutations in the Lf gene lead to variation in Lf content, production, or secretion, particularly variations in milk or colostrum Lf content. The Lf polymorphisms described herein were each closely associated with milk and colostrum Lf concentration phenotype. Animals homozygous for the T allele at the 30126 T/C polymorphism produced milk with approximately 2.5- to 4-fold more Lf than animals homozygous for the C allele at the 30126 T/C polymorphism, depending on the stage of lactation. Similar differences in Lf content phenotype depending on genotype at the 7447 A/G polymorphism and the -7 G/C polymorphism were also observed. See Table 2 herein.

[00159] The CC genotype at the 30126 T/C polymorphism was present in approximately 2% in the Holstein-Friesian x Jersey crossbred trial described herein. The GG genotype at the 7447 A/G polymorphism was present in approximately 6% in the Holstein-Friesian x Jersey crossbred trial, while the CC genotype at the -7 G/C polymorphism was present in approximately 10% in the Holstein-Friesian x Jersey crossbred trial described herein. For example, removal of these latter animals from the herd would increase the herd average Lf concentration by approximately 7%. Removal of both homozygous recessive and heterozygous animals at any polymorphic site would increase the herd average Lf concentration by approximately 25%. Conversely, removal of homozygote wild-type animals would decrease herd average Lf concentration by approximately 21%, and removal of the homozygous wildtype and heterozygous animals would decrease herd average Lf concentration by approximately 61%.

[00160] A reference bovine Lf nucleotide sequence referred to as "wild type Lf ' by the applicants is presented in NC_007320.3, and the corresponding amino acid sequence is presented in NM_180998. Accordingly, as used herein in reference to Lf the term "wild type" recognizes the characteristics of the Lf nucleotide sequences presented in NC_007320.3 and NM l 80998, and of the protein product encoded thereby. For example, when used with reference to activity, the term "wild type" denotes activity associated with the wild type Lf protein. Similarly, when used with reference to expression level, the term "wild type" denotes a level of expression associated with the wild type Lf promoter or of the wild type Lf gene.

[00161] It will be apparent that the term "activity" may refer both to the inherent activity of a single molecule of Lf, 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 Lf molecules 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).

[00162] As used herein, such as when used in reference to an allelic protein lacking the activity of wild type Lf, 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 wild type Lf may be a variant Lf protein of greater or lesser enzymatic activity than that of wild type Lf.

[00163] Methods to assay the expression or activity of Lf are well known in the art. For example, Northern analysis, RT-PCR, or immunostaining for Lf may be used. Another exemplary method utilises reversed-phase HPLC after isoelectric precipitation of milk caseins (Palmano KP, Elgar DF, Detection and quantitation of lactoferrin in bovine whey samples by reversed-phase high-performance liquid chromatography on polystyrene-divinylbenzene. J Chromatogr A. (2002) Feb 22;947(2):307-l 1). Similarly, methods to indirectly measure the activity of Lf are available, and include a determination of the presence, absence or concentration of Fe or an Fe ion, such as Fe ³⁺ , such as that present in a sample obtained from bovine. [00164] In various embodiments the methods of the invention are used in conjunction with other known methods of increasing Lf milk or colostrum concentration or yield, such as those described in NZ patent application NZ550859, published 30 October 2009.

[00165] In various embodiments, including those utilizing milking- withholding periods, and once-daily milking for one to two days followed by twice-daily milking, the methods of the invention are used to produce lactoferrin-enriched milk and milk products comprising at least about 600 mg/1, more preferably at least 800 mg/1, more preferably at least 1000 mg/1, more preferably at least 1500 mg/1, more preferably at least 2000 mg/1, more preferably at least 2500 mg/1, more preferably at least 3000 mg/1, more preferably at least 3500 mg/1, more preferably at least 4000 mg/1, more preferably at least 4500 mg/1, more _. preferably at least 5000 mg/1, most preferably at least 5000 mg/1.

[00166] For example, the invention provides a method of producing a lactoferrin-enriched milk or milk product comprising the steps of:

identifying or selecting bovine as described herein,

providing a milk sample collected from the bovine, and optionally

processing the milk sample to produce a milk product.

[00167] In another example, the invention provides a method of producing a lactoferrin- enriched milk or milk product comprising the steps of:

identifying or selecting bovine as described herein,

providing a milk sample prepared by

a) ceasing to milk the bovine for a period of greater than 24 hours;

b) collecting milk from the bovine within a period of up to 7 days after completion of a); and optionally

processing the milk sample to produce a milk product.

2 Identification and analysis of polymorphisms

[00168] The polymorphisms described herein are numbered according to their position in the genomic nucleotide sequence relative to the +1 translation start site of the bovine Lf gene. Those skilled in the art will recognise that these positions can readily be expressed relative to the coding sequence, or relative to their position in the mature Lf polypeptide. It will be apparent to those skilled in the field that the - convention of identifying polymorphisms effecting an amino acid substitution by their codon position in the gene in which they occur and the amino acid substitution effected thereby is also contemplated herein. Accordingly, the 7447 A/G polymorphism described herein may be referred to by reference to the codon of the Lf gene within which it is located and the amino acid substitution effected, namely the 1145V polymorphism.

[00169] Each of the 30126 T/C polymorphism, the 7447 A/G polymorphism, or the -7 G/C polymorphism can be detected directly or by detection of one or more polymorphisms which are in linkage disequilibrium with one or more of these polymorphisms. 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.)

[00170] Various degrees of linkage disequilibrium are possible. Preferably, 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. Those skilled in the art will appreciate that linkage disequilibrium may also, when expressed with reference to the deviation of the observed frequency of a pair of alleles from the expected, be denoted by a capital D. Accordingly, the phrase "two alleles are in LD" usually means that D does not equal 0. Contrariwise, "linkage equilibrium" denotes the case D = 0. When utilising this nomenclature, the one or more polymorphisms in LD with the one or more polymorphisms specified herein are preferably in LD of greater than about D'=0.6, of about D'=0.7, of about D'=0.75, of about D'=0.8, of about D'=0.85, of about D'=0.9, of about D'=0.91 , of about D'=0.92, of about D'=0.93, of about D'=0.94, of about D'=0.95, of about D'=0.96, of about D'=0.97, of about D'=0.98, of about D'=0.99, or about D'=1.0. (Devlin and Risch 1995; A comparison of linkage disequilibrium measures for fine-scale mapping, Genomics 29: 31 1- 322).

[00171] There are numerous standard methods known in the art for determining whether a particular DNA sequence is present in a sample, many of which include the step of sequencing a DNA sample. Thus in one embodiment of the invention, the step determining whether or not the specified nucleotides are present in a nucleic acid derived from a bovine, includes the step of sequencing the nucleic acid. Methods for nucleotide sequencing are well known to those skilled in the art. [00172] In another aspect, the present invention provides a method for determining the genetic status of a bovine with respect to Lf content, and particularly with respect to milk or colostrum Lf content. 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 Lf is present, and whether a sequence of the DNA encoding (b) an allelic protein lacking the activity of (a) is present.

[00173] 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 Lf, 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 Lf levels, and particularly in milk or colostrum having inter alia a higher Lf content, 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 Lf levels, and particularly in milk or colostrum having inter alia a lower Lf content.

[00174] 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.

[00175] Accordingly, a preferred embodiment of the invention thus includes the step of amplifying a Lf polynucleotide in the presence of at least one primer comprising a nucleotide sequence of or complementary to, the Lf gene (NC_007320.3 and NM_180998) or flanking sequence thereof, and/or in the presence of a such a primer comprising sequence corresponding to or flanking the allele-specific nucleotides described herein. 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).

[00176] Primers suitable for use in PCR based methods of the invention should be sufficiently complementary to the Lf gene sequence, such as NC_007320.3 or NM_180998 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 NC_007320.3 or NM l 80998, or naturally occurring flanking sequences thereof. Examples of such PCR primers may be derived from the sequences presented herein as SEQ ID NOs:l to 3.

[00177] Suitable PCR primers may include a 30126 T/C C allele-specific nucleotide, a 30126 T/C T allele-specific nucleotide, a 7447 A/G G allele-specific nucleotide, a 7447 A/G A allele-specific nucleotide, a -7 G/C C allele-specific nucleotide, or a -7 G/C G allele- specific nucleotide, as described herein. Generation of a corresponding PCR product, or the lack of product, may constitute a test for the presence or absence of the specified nucleotides in the Lf gene of the test bovine.

[00178] 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.

[00179] 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 Lf sequence of NC_007320.3 or NM_180998.

[00180] Such probes should comprise a nucleic acid molecule of sufficient length and sufficiently complementary to the Lf 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.

[00181] With respect to polynucleotide molecules greater than about 100 bases in length, typical stringent hybridization 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+).

[00182] With respect to polynucleotide molecules having a length less than 100 bases, exemplary stringent hybridization 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.

[00183] 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.

[00184] Such probes would typically comprise at least 12 contiguous nucleotides of or complementary to the sequences presented NC_007320.3 or NM l 80998, and may comprise sequence corresponding to the allele-specific nucleotides described herein.

[00185] 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).

[00186] In another aspect, the invention provides a method for determining the genetic status of bovine with respect to Lf content with reference to a sample of material containing mRNA obtained from the bovine. In one embodiment this method includes ascertaining whether a sequence of the mRNA encoding (A) a protein having biological activity of a wild type Lf 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 low relative Lf levels, particularly with the production of milk or colostrum with, inter alia, decreased Lf content. The reverse association again holds true.

[00187] 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 biological activity of a wild type Lf, or in the presence of a pair of primers complementary to a nucleotide sequence encoding a variant Lf protein. It will be appreciated that in embodiments of the invention reliant on assessing the amount of Lf 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. [00188] Other methods to quantitate or otherwise assess the amount of nucleic acid, particularly the amount of m NA are well known in the art. These include Northern analysis using probes able to hybridise to the target Lf mRNA. Such probes should comprise a nucleic acid molecule of sufficient length and sufficiently complementary to the Lf 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 Lf 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.

[00189] In a further aspect, the invention provides a method of determining genetic status of a bovine with respect to milk or colostrum Lf content which comprises determining the Lf allelic profile of said bovine, together with determining the allelic profile of said bovine at one or more genetic loci associated with milk or colostrum Lf content.

[00190] In one embodiment, said genetic loci is a polymorphism in a gene associated with milk or colostrum Lf content, preferably a polymorphism in a gene involved in Lf production or secretion.

[00191] 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. 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 both coding or non-coding regions.

[00192] 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 NCBL SNP database "dbSNP" is incorporated into NCBI's Entrez system and has records for over 17 million refSNPs mapped onto the human genome sequence. Similar databases exist for other mammalian genomes.

[00193] Genotyping approaches to detect SNPs well-known in the art include DNA sequencing, methods that require allele specific hybridization of primers or probes, allele specific incorporation of nucleotides to primers bound close to or adjacent to the polymorphisms (often referred to as "single base extension", or "minisequencing"), 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, 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.

[00194] DNA sequencing allows the direct determination and identification of SNPs. Mini-sequencing involves allowing a primer to hybridize to the DNA sequence adjacent to the SNP site on the test sample under investigation.

[00195] 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 SOLiD ^{1 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).

[00196] 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. [00197] The majority of methods to detect or identify SNPs by site-specific hybridisation 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 hybridization involving a hybridization of a first portion of the target sequence to a capture probe, and hybridization of a second portion of said target sequence to a detection probe.

[00198] US Patent Application Publication No. US 20050042608 (incorporated herein by reference in its entirety) describes a modification of the method of electrochemical detection of nucleic acid hybridization of Thorp et al. (U.S. Pat. No. 5,871 ,918). Briefly, capture probes containing different SNP bases and a sequence of probe bases on each side of the SNP base are immobilized on a different electrode. The extent of hybridization between each capture probe and the nucleic acid target is detected by detecting the oxidation-reduction reaction at each electrode, utilizing a transition metal complex.

[00199] 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.

[00200] 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.

[00201] SNPs can also be determined by ligation-bit analysis. A further discussion of these methods can be found in U.S. Pat. Nos. 5,919,626; 5,945,283; 5,242,794; and 5,952,174.

[00202] 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 fmgerprinting-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).

[00203] 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.

[00204] 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.

[00205] 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.

[00206] 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.

[00207] 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.

[00208] 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.

[00209] 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 protein-encoding gene. Such altered expression can be determined by methods well known in the art, such as quantitative PCR, RT-PCR, quantative 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-synonomous 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.

[00210] The above methods of detecting and identifying SNPs are amenable to use in the methods of the invention.

3 Polynucleotide and polypeptide variants

[00211] 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.

[00212] A "fragment" of a polynucleotide sequence provided herein is a subsequence of contiguous nucleotides that is preferably at least 15 nucleotides in length. The fragments of the invention preferably comprises 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.

[00213] 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.

[00214] Preferably 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.

[00215] The term "primer" refers to a short polynucleotide, usually having a free 3ΌΗ group, that is hybridized 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 1 1 , 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. [00216] The term "probe" refers to a short polynucleotide that is used to detect a polynucleotide sequence that is complementary to the probe, in a hybridization-based assay. The probe may consist of a "fragment" of a polynucleotide as defined herein. Preferably such a probe is at least 5, more preferably at least T O, 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.

[00217] 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 embodiments, 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.

[00218] 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.

3.1 Polynucleotide variants

[00219] 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 %, 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.

[00220] 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.

[00221] The identity of polynucleotide sequences may be examined using the following unix command line parameters:

[00222] bl2seq -i nucleotideseql -j nucleotideseq2 -F F -p blastn

[00223] 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 = ".

[00224] 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,I. 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 provides the facility to perform EMBOSS-needle global alignments between two sequences on line at http:/www.ebi. ac.uk/emboss/align/.

[00225] 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.

[00226] 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/).

[00227] The similarity of polynucleotide sequences may be examined using the following unix command line parameters:

[00228] bl2seq -i nucleotideseql -j nucleotideseq2 -F F -p tblastx

[00229] 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.

[00230] Variant polynucleotide sequences preferably exhibit an E value of less than 1 x 10 ^"10 , more preferably less than 1 x 10 ^"20 , less than 1 x 10 ^~30 , less than 1 x 10 ^"40 , less than 1 x 1Ό ^"50 , less than 1 x 10 ^"60 , less than 1 x 10 ^"70 , less than 1 x 10 ^"80 , less than 1 x l O ^"90 , less than 1 x 10 ^"100 , less than 1 x 10 ^{"1 10} , less than 1 x 10 ^"!2 ° or less than 1 x 10 ^"123 when compared with any one of the specifically identified sequences.

[00231] Alternatively, variant polynucleotides of the present invention hybridize to a specified polynucleotide sequence, or complements thereof under stringent conditions.

[00232] The term "hybridize under stringent conditions", and grammatical equivalents thereof, refers to the ability of a polynucleotide molecule to hybridize 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 hybridize under stringent hybridization conditions can be determined by initially hybridizing under less stringent conditions then increasing the stringency to the desired stringency.

[00233] With respect to polynucleotide molecules greater than about 100 bases in length, typical stringent hybridization 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,). Tm for polynucleotide molecules greater than about 100 bases can be calculated by the formula Tm = 81. 5 + 0. 41% (G + C _. -log' (Na+). (Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Bolton and McCarthy, 1962, PNAS 84: 1390). Typical stringent conditions for polynucleotide of greater than 100 bases in length would be hybridization conditions such as prewashing in a solution of 6X SSC, 0.2% SDS; hybridizing 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.

[00234] With respect to polynucleotide molecules having a length less than 100 bases, exemplary stringent hybridization 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.

[00235] 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 hybridization conditions for a DNA-PNA hybrid having a length less than 100 bases are 5 to 10°C below the Tm.

[00236] Variant polynucleotides of the present invention also encompasses 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.

[00237] 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).

[00238] 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.

3.2 Polypeptide Variants

[00239] The term "variant" with reference to polypeptides encompasses naturally occurring, recombinantly and synthetically produced polypeptides. 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 %, 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 sequences 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.

[00240] 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.

[00241] 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.

[00242] Polypeptide 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 polypeptide sequences may be examined using the following unix command line parameters:

bl2seq -i peptideseql -j peptideseq2 -F F -p blastp

[00243] Variant polypeptide sequences preferably exhibit an E value of less than 1 x 10 ^" '°, more preferably less than 1 x 10 ^"20 , less than 1 x 10 ^"30 , less than 1 x 10 ^"40 , less than 1 x 10 ^"50 , less than 1 x 10 ^"60 , less than 1 x 10 ^"70 , less than 1 x 10 ^"80 , less than 1 x 10 ^"90 , less than 1 10 ^{" ! 0} °, less than 1 x 10 ^" ' ^I0 , less than 1 x 10 ^~120 or less than 1 x 10 ^"123 when compared with any one of the specifically identified sequences.

[00244] 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.

[00245] 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).

[00246] A polypeptide variant of the present invention 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 a variant may be produced by an alternative splicing pattern of the primary RNA transcript to that which produces a wild type polypeptide.

4 Diagnostic kits

[00247] The invention further provides diagnostic kits useful in determining the Lf allelic profile of the mammalian subject, for example for use in the methods of the present invention.

[00248] Accordingly, in one embodiment the invention provides a diagnostic kit which can be used to determine the Lf genotype of the subject's genetic material. One 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 wild type Lf. 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-speeific oligonucleotides in conventional hybridisation, Taqman assays, OLE assays, etc. Alternatively, primers can be designed to permit genotyping by microsequencing.

[00249] 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 Lf 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.

[00250] For example, the kit may include a primer comprising a cytosine at the position corresponding to the 30126 T/C polymorphism in the Lf gene or a nucleotide capable of hybridising to a nucleotide capable of hybridising to a cytosine at the position corresponding to the 30126 T/C polymorphism in the Lf gene. Those skilled in the art will recognise that in such a primer, the cytosine, or the nucleotide capable of hybridising to a nucleotide capable of hybridising to a cytosine, as applicable, may be substituted for a nucleotide analogue having the same discriminatory base-pairing as the substituted nucleotide.

[00251] In another example, the kit may include a primer comprising a thymine at the position corresponding to the 30126 promoter polymorphism in the Lf gene, or comprising a nucleotide capable of hybridising to a nucleotide capable of hybridising to a thymine at the position corresponding to the 30126 promoter polymorphism in the Lf gene. Those skilled in the art will recognise that in such a primer, the thynine, or the nucleotide capable of hybridising to a nucleotide capable of hybridising to a thymine, as applicable, may be substituted for a nucleotide analogue having the same discriminatory base-pairing as the substituted nucleotide. [00252] Those skilled in the art will appreciate that the invention provides kits comprising primers similarly directed to one or more of the 7447 A/G polymorphism, or the -7 G/C polymorphism polymorphism. ·

[00253] In one embodiment, the diagnostic kit is useful in detecting DNA comprising a variant Lf gene or encoding a variant Lf 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 Lf gene which results in increased or decreased Lf levels (particularly increased or decreased Lf content in milk or colostrum). In one embodiment, at least one of the nucleotide sequences is selected to be from a coding region of the Lf gene. In another embodiment, at least one of the nucleotide sequences is selected to be from a non-coding region of the Lf gene. The kit can also include a third primer complementary to a naturally occurring mutation of a coding portion of the wild type Lf gene. Preferably the kit includes instructions for use, for example in accordance with a method of the invention.

[00254] In one embodiment, the diagnostic kit comprises a nucleotide probe complementary to the sequence, or an oligonucleotide fragment thereof, shown in NC_007320.3 or NM_1 80998, 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 NC_007320.3or NM_180998 or complements thereof, so as to bind thereto under stringent conditions. "Stringent hybridisation conditions" takes on its common meaning to a person skilled in the art. Appropriate stringency conditions which promote nucleic acid hybridisation, for example, 6x sodium chloride/sodium citrate (SSC) at about 45°C are known to those skilled in the art, including in Current Protocols in Molecular Biology, John Wiley & Sons, ΝΫ (1989). 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. [00255] In another embodiment, the diagnostic kit comprises an antibody or an antibody composition useful for detection of the presence or absence of wild type Lf and/or the presence or absence of a variant protein at least partially lacking wild type activity, particularly a truncated form of the Lf protein, together with instructions for use, for example in a method of the invention.

5 Sample preparation

[00256] As will be apparent to persons skilled in the art, samples suitable for use in the methods of the present invention may be obtained from tissues or fluids as convenient, and so that the sample contains the moiety or moieties to be tested. For example, where nucleic acid is to be analysed, tissues or fluids containing nucleic acid will be used.

[00257] Conveniently, samples may be taken from milk, colostrum, tissues including blood, serum, and plasma, cerebrospinal fluid, urine, semen or saliva. Tissue samples may be obtained using standard techniques such as cell scrapings or biopsy techniques. For example, the cell or tissue samples may be obtained by using an ear punch to collect ear tissue from bovine. Similarly, blood sampling is routinely performed, for example for pathogen testing, and methods for taking blood samples are well known in the art. 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 analyzed following a fractionation or purification procedure, for example, separation of whole blood into serum or plasma components.

6 Computer-Related Embodiments

[00258] It will also be appreciated that the methods of the invention are amenable to use with and the results analysed by computer systems, software and processes. Computer systems, software and processes to identify and analyse genetic polymorphisms are well known in the art. For example, the results of one or more genetic analyses as described herein may be analysed using a computer system and processed by such a system.

[00259] Both the SNPs and the results of an analysis of the SNPs utilised in the present invention may be "provided" in a variety of mediums to facilitate use thereof. As used in this section, "provided" refers to a manufacture, other than an isolated nucleic acid molecule, that contains SNP information of the present invention. Such a manufacture provides the SNP information in a form that allows a skilled artisan to examine the manufacture using means not directly applicable to examining the SNPs or a subset thereof as they exist in nature or in purified form. The SNP information that may be provided in such a form includes any of the SNP information provided by the present invention such as, for example, polymorphic nucleic acid and/or amino acid sequence information, information about observed SNP alleles, alternative codons, populations, allele frequencies, SNP types, and/or affected proteins, phenotypic effect or association, or any other information provided by the present invention in Tables 1 and 2 and/or the Sequence ID Listing.

[00260] In one application of this embodiment, the SNPs and the results of an analysis of the SNPs utilised in the present invention can be recorded on a computer readable medium. As used herein, "computer readable medium" refers to any medium that can be read and accessed directly by a computer. Such media include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. A skilled artisan can readily appreciate how any of the presently known computer readable media can be used to create a manufacture comprising computer readable medium having recorded thereon SNP information of the present invention. One such medium is provided with the present application, namely, the present application contains computer readable medium (floppy disc) that has nucleic acid sequences used in analysing the SNPs utilised in the present invention, together with derived amino acid sequence, provided/recorded thereon in ASCII text format in a Sequence ID Listing.

[00261] As used herein, "recorded" refers to a process for storing information on computer readable medium. A skilled artisan can readily adopt any of the presently known methods for recording information on computer readable medium to generate manufactures comprising the SNP information of the present invention. -

[00262] A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon SNP information of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the SNP information of the present invention on computer readable medium. For example, sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, represented in the form of an ASCII file, or stored in a database application, such as OB2, Sybase, Oracle, or the like. A skilled artisan can readily adapt any number of data processor structuring formats (e.g., text file or database) in order to obtain computer readable medium having recorded thereon the SNP information of the present invention.

[00263] By providing the SNPs and/or the results of an analysis of the SNPs utilised in the present invention in computer readable form, a skilled artisan can routinely access the SNP information for a variety of purposes. Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium. Examples of publicly available computer software include BLAST (Altschul et at, J. Mol. Biol. 215:403-410 (1990)) and BLAZE (Brutlag et at, Comp. Chem. 17:203-207 (1993)) search algorithms.

[00264] The present invention further provides systems, particularly computer-based systems, which contain the SNP information described herein. Such systems may be designed to store and/or analyze information on, for example, a number of SNP positions, or information on SNP genotypes from a number of subjects. The SNP information of the present invention represents a valuable information source. The SNP information of the present invention stored/analyzed in a computer-based system may be used for such applications as identifying or selecting subjects, in addition to computer-intensive applications as determining or analyzing SNP allele frequencies in a population, mapping disease genes, genotype-phenotype association studies, grouping SNPs into haplotypes, correlating SNP haplotypes with response to particular drugs, or for various other bioinformatic, pharmacogenomic, drug development, or selection or identification applications.

[00265] As used herein, "a computer-based system" refers to the hardware, software, and data storage used to analyze the SNP information of the present invention. The minimum hardware of the computer-based systems of the present invention typically comprises a central processing unit (CPU), an input, an output, and data storage. A skilled artisan can readily appreciate that any one of the currently available computer-based systems are suitable for use in the present invention. Such a system can be changed into a system of the present invention by utilizing the SNP information, such as that provided herewith on the floppy disc, or a subset thereof, without any experimentation.

[00266] As stated above, the computer-based systems of the present invention comprise data storage having stored therein SNP information, such as SNPs and/or the results of an analysis of the SNPs utilised in the present invention, and the necessary hardware and software for supporting and implementing one or more programs or algorithms. As used herein, "data storage" refers to memory which can store SNP information of the present invention, or a memory access facility which can access manufactures having recorded thereon the SNP information of the present invention.

[00267] The one or more programs or algorithms are implemented on the computer-basedsystem to identify or analyze the SNP information stored within the data storage. For example, such programs or algorithms can be used to determine which nucleotide is present at a particular SNP position in a target sequence, or to analyse the results of a genetic analysis of the SNPs described herein. As used herein, a "target sequence" can be any DNA sequence containing the SNP position(s) to be analysed, searched or queried.

[00268] A variety of structural formats for the input and output can be used to input and output the information in the computer-based systems of the present invention. An exemplary format for an output is a display that depicts the SNP information, such as the presence or absence of specified nucleotides (alleles) at particular SNP positions of interest. Such presentation can provide a rapid, binary scoring system for many SNPs Or subjects simultaneously. It will be appreciated that such output may be accessed remotely, for example over a LAN or the internet. Typically, given the nature of SNP. information, such remote accessing of such output or of the computer system itself is available only to verified users so that the security of the SNP information and/or the computer system is maintained. Methods to control access to computer systems and the data residing thereon are well-known in the art, and are amenable to the embodiments of the present invention.

[00269] One exemplary embodiment of a computer-based system comprising SNP information of the present invention that can be used to implement the present invention includes a processor connected to a bus. Also connected to the bus are a main memory (preferably implemented as random access memory, RAM) and a variety of secondary storage devices, such as a hard drive and a removable medium storage device. The removable medium storage device may represent, for example, a floppy disc drive, a CD-ROM drive, a magnetic tape drive, etc. A removable storage medium (such as a floppy disc, a compact disc, a magnetic tape, etc.) containing control logic and/or data recorded therein may be inserted into the removable medium storage device. The computer, system includes appropriate software for reading the control logic and/or the data from the removable storage medium once inserted in the removable medium storage device. The SNP information of the present invention may be stored in a well-known manner in the main memory, any of the secondary storage devices, and/or a removable storage medium. Software for accessing and processing the SNP information (such as SNP scoring tools, search tools, comparing tools, etc.) preferably resides in main memory during execution.

[00270] Accordingly, the present invention provides a system for performing one or more of the methods of the invention, said system comprising:

computer processor means for receiving, processing and communicating data;

storage means for storing data including a reference genetic database of the results of genetic analysis of a mammalian subject with respect to one or more Lf content phenotypes and optionally a reference Lf content phenotypes database of non-genetic factors for mammalian Lf content phenotypes; and ^"

a computer program embedded within the computer processor which, once data consisting of or including the result of a genetic analysis for which data is included in the reference genetic database is received, processes said data in the context of said reference databases to determine, as an outcome, the genetic status of the subject, said outcome being communicable once known, preferably to a user having input said data.

[00271] Preferably, said system is accessible via the internet or by personal computer.

[00272] Preferably, said reference genetic database comprises or includes the results of one or more analyses of one or more genetic loci associated with one or more milk or colostrum Lf content phenotypes, more preferably the one or more genetic loci are one or more polymorphisms in one or more genes associated with one or more milk or colostrum Lf content phenotypes.

[00273] In yet a further aspect, the invention provides a computer program suitable for use in a system as defined above comprising a computer usable medium having program code embodied in the medium for causing the computer program to process received data consisting of or including the result of at least one genetic analysis of one or more genetic loci associated with one or more Lf content phenotypes in the context of both a reference genetic database of the results of said at least one genetic analysis and optionally a reference database of non-genetic factors associated with mammalian Lf content phenotypes.

[00274] Preferably, the one or more genetic loci are one or more polymorphisms in one or more genes associated with one or more milk or colostrum Lf content phenotypes.

7 Compositions useful according to the invention

[00275] A composition useful herein may be formulated as a food, drink, food additive, drink additive, dietary supplement, nutritional product, medical food, enteral or parenteral feeding product, meal replacement, nutraceutical, cosmeceutical or pharmaceutical. Appropriate formulations may be prepared by an art skilled worker with regard to that skill and the teaching of this specification.

[00276] As will be appreciated, the dose of the composition administered, the period of administration, and the general administration regime may differ between subjects depending on such variables as the mode of administration chosen, and the age, sex and/or general health of a subject.

[00277] In one embodiment, compositions useful herein include maternal formulas, infant formulas, follow-on formulas and growing up formulas. Such products are formulated to target nutrients to the foetus, infant and child. It is appreciated that the first life-stages (foetus, infant and growing child) involve significant growth and development. Any support which enhances development can have significant effects on the development of the individual.

[00278] In another embodiment, compositions useful herein include dietetic products.

[00279] The term "maternal formula" as used in this specification means a composition for pregnant woman to take during pregnancy. The term "infant formula" as used in this specification means a composition for infants aged between 0 days and 6 months old. The term "follow-on formula" as used in this specification means a composition for infants aged 6 months to 1 year. The term "growing up formula" as used in this specification means a compositions directed to infants and children aged 1 year upwards. Growing-up formula includes growing-up milk powders as will be understood by those skilled in the art.

[00280] It will further be appreciated by those skilled in the art that the age ranges for the different compositions: "infant formula", "follow-on formula" and "growing-up formula" can vary from child to child depending on the individual's development.

[00281] The term "dietetic product" means a product specially processed or formulated to satisfy particular dietary requirements which exist because of a particular physical or physiological condition and/or specific diseases and disorders and which are presented as such.

[00282] A "subject" is an animal, preferably a mammal, more preferably a mammalian companion animal, a mammalian agricultural animal, or a human. Preferred companion animals include feline, equine, and canine. Preferred agricultural animals include bovine, ovine, cervine, and porcine. , [00283] It will be appreciated that it is not intended to limit the invention to the above example only, many variations, which may readily occur to a person skilled in the art, being possible without departing from the scope thereof as defined in the accompanying claims.

[00284] This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

[00285] The invention consists in the foregoing and also envisages constructions of which the following gives examples only.

EXAMPLE ONE -Analysis of the genetic basis for Lf content of milk and colostrum

[00286] This example describes the investigation of the genetic basis for observed variations in Lf content of colostrum using the results of a Holstein-Friesian X Jersey crossbred trial conducted to facilitate the discovery of QTLs, genes and mutations associated with economically important milk phenotypes.

Materials and methods

1. Trial design

[00287] A Holstein-Friesian x Jersey crossbred trial was conducted using an F2 trial design with a half-sibling family structure. Reciprocal crosses of Holstein-Friesian and Jersey animals were carried out to produce six Fl bulls of high genetic status. 850 F2 female progeny forming the basis of the trial herd were then produced through mating of high genetic status Fl cows with these Fl bulls. The herd was formed over two seasons; animals in cohort one were born in spring 2000, and entered their first lactation in spring 2002, while animals in cohort two were born in spring 2001 and entered their first lactation in spring 2003. A total of 724 F2 cows entered their second lactations and colostrum samples collected from over 600 of these. The animals were farmed under standard New Zealand dairy farming practices using a pasture based management system. All animal work was conducted in accordance with the Ruakura Animal Ethics committee.

2. Lf measurement in milk

[00288] All milk composition measurements were taken during the second lactation. Cows were milked twice daily; milk volume was recorded at each milking. Lf was measured at three time points during lactation: peak lactation (35 days post-calving), mid lactation (mid November) and late lactation (late February). On each collection day, samples were collected from the a.m. and p.m. milkings and combined to make a single composite sample for each animal. For the once-daily milking study undertaken in mid-lactation in lactation 2, all cows were milked once daily for a period of 7 days and a composite am/pm milk sample taken two days prior to once-daily milking and sampled again after days 5 of once-daily milking (am). Lf was measured using ion-exchange chromatography at peak, mid and late lactation (day 50) and by ELISA in mid lactation for the once-daily milking study. Lf was measured using the bovine Lf ELISA quantitation kit (Bethyl laboratories Inc, Montgomery, Texas, USA; catalogue number El 0-126) as per manufacturer's instructions. A reference standard containing lmg/ml of bovine Lf was used to calibrate the assay. The working range of the assay was 0-1000 ug/ml.

3. Genotyping

[00289] Genomic DNA was prepared from whole blood from a total of 1679 animals within the trial pedigree (846 F2 daughters, six Fl sires, 796 Fl dams, and 13 selected F0 sires). An initial whole genome scan was conducted by genotyping each animal for 285 microsatellite markers, obtained primarily from published marker maps. Subsequently, the pedigree was genptyped using the Affymetrix Bovine 10K SNP GeneChip. A total of 6634 informative SNP markers were placed on the map.

4. Candidate gene sequencing

[00290] The coding region of the Lf gene (17 exons) as well as 0.5kb of 5' sequence was sequenced. Intron/exon boundaries were determined. Exons were amplified and sequenced in both directions.

5. Statistical analysis

[00291] The dataset consisted of Lf phenotypes (taken at three time points) collected from two cohorts of F2 animals. Data manipulation was performed using SAS (version 9.1). These data were matched with the following covariates: cohort (cohort 1 or cohort 2), sire (sires 1 - 6), lipid, true protein, casein, lactose, total solids, calving week (0-13), mating week (0-1 1), milk volume, FFA and somatic cell count (<200 or >=200). Mating week was tested at Mid and Late lactations only. The milk composition covariates were matched separately by lactation. Three Lf genotypes thought to be associated with Lf were matched with the data. Animals with missing data points for any of the measurements were excluded from the final datasets. The final matched (including covariates) datasets included 502, 61 1 and 593 animals for Peak, Mid and Late lactations, respectively.

[00292] Statistical analyses were conducted using raw phenotypic data. ANOVA was conducted for Lf phenotypes to determine whether the Lf polymorphisms were significantly associated with milk Lf content. The final ANOVA models for each of the time points were produced using a backward elimination process; all the covariates were included in the model at the first stage of the modeling process and the least significant covariates removed at each subsequent stage until all the remaining covariates were found to be significant (sig. level set at 0.1). The final models were as follows:

L£p = μ+Sire+Lipidp+True protein _P +Lactose _P +Total Solidsp+Calving-weekp+Milk yieldp+residual ₍ p

Lf,M = μ+Sire+Cohort+Lipidivi+True protein _M +Casein average _M +Lactose _M +Mating week _M +Milk yield _M +FFA +residual,w

Lf/L = μ+Sire+Cohort+Lipid _L +True protein _L +Casein average _L +Lactose _L +Milk yield _L +FFAi+residuali

where, i is the individual animal, P=Peak lactation, M=Mid lactation and L=Late lactation

6. QTL detection

[00293] The data used for QTL detection were the residuals from the statistical model. The raw phenotype data (no covariates or modeling) was also used to detect QTLs. QTL detection was conducted using a line of descent model and a half-sib model. Subsequently, the Lf genotypes were included separately as a covariate into the model to determine whether these polymorphisms explained the QTL variation.

Results

1. Milk Lf

[00294] The Lf content of milk differed at peak, mid, and late lactation (Figure 1), with the highest concentration observed at late lactation, and the lowest composition observed at peak lactation. Lf concentration in milk also also differed according to sire (Figure 2).

2. Detection of a QTL for milk Lf on bovine chromosome 22

[00295] Analysis of the Lf within the half-sib models of QTL analysis (Haley et ah, 1994 and Baret et ai, 1998) showed the presence of a significant QTL on bovine chromosome 22 (Figure 1). The maximum F value for the QTL was 10.19, and the most likely position was estimated at 70 cM. Bootstrap analysis (n = 1000) showed that the 95% confidence interval for the QTL was 70-77 cM. There were a total of 205 markers (7 microsatellite markers and 198 single nucleotide polymorphisms) used for this analysis on chromosome 22.

3. Identification of Lf as a candidate gene and detection of polymorphisms affecting Lf content of milk

[00296] Lf binds its ligand, Fe and Fe ions and mediates the transport of same. Lf was identified as a candidate gene for the chromosome 22 Lf QTL effect. To determine whether this gene explained the observed variation in milk Lf content, the Lf coding region in the six Fl sires was sequenced to identify any genetic polymorphisms that could potentially alter the function of this protein. Intron/exon boundaries were determined using the reference sequence NC_007320.3. Primers were designed within introns so that complete sequence was obtained from each exon. An additional 0.5kb of sequence was obtained from the 5' UTR.

[00297] Three polymorphisms which segregated in the same sires as the QTL (sires 3, 4 and 6) were identified and were genotyped in the remainder of the FJXB trial pedigree. The location of these polymorphisms within the gene, and the sequence traces, are shown in Figure 4. The frequency of each Lf genotype is shown in Table 1 below.

Table 1 : Genotype frequencies ofF2 FJXB population

Note: Data based on successfully genotyped animals in F2 population 4. Lf polymorphisms have a significant effect on Lf concentration in milk

[00298] The effect of the Lf 30126 T/C polymorphism on Lf milk concentration is shown in Figure 5. The main effect for genotype on Lf content was significant at P < 0.0000001. Addition of the Lf genotypes as covariates into the ANOVA completely abolished the observed QTL effect on chromosome 22 (Figure 6) for Lf -7 G/C polymorphism, whereas the Lf 30126 T/C polymorphism explained a large proportion, but not all, of the QTL. [00299] Table 2 below shows the mean Lf concentration for the various genotypes at peak, mid, and late lactation.

Table 2: Phenotypic means for F2 FJXB population

5. eQTL for Lf mRNA on chromosome 22

[00300] . In addition to the bovine chromosome 22 QTL for milk Lf concentration, a QTL for the amount of Lf mRNA in liver tissue samples taken in early lactation ("eQTL") was also observed (see Figure 7). Comparison with the QTL for milk Lf concentration shows that the maximum F-value occurs at the same location on chromosome 22 for the milk Lf concentration and the liver expression level (see Figure 7). Further, adjusting the milk Lf phenotype by applying the Lf mRNA as a covariate, abolishes the QTL effect indicating that the genotypic effects on milk lactoferrin will also extend to expression of lactoferrin in other body tissues (see Figure 8).

6. Lf polymorphisms have a significant effect on Lf concentration in colostrum

[00301] A sample of colostrum was taken at the second milking post calving and analysed for lactoferrin by ELISA. Results are shown in Table 3 below.

Table.

173 (14) 352

[00302] SNP Lf_7447 and SNP Lf_-7 showed statistically significant effects (P<0.05 and P<0.01 respectively) by ANOVA.

7. Once daily milking has a synergistic effect with Lf polymorphisms on Lf concentration in milk

[00303] Samples taken during twice daily milking and 5 days after transition to once daily milking were analysed for lactoferrin by ELISA. Results are shown in Table 4 below.

Table 4, Milk lactoferrin concentrations in mid-lactation Friesian-Jersey crossbred cows before and after transition to once-daily milking

[00304] Lactoferrin concentrations in milk were increased approximately two-fold by once-daily milking in mid-lactation. On average the increase was from 273 ± 7 mg/1 to 550 ± 14 mg/1. Polymorphism and milking- effects were all statistically significant (PO.01). As can be seen in Table 4, once-daily milking increased the advantage of the favourable SNP genotypes in terms of increased milk lactoferrin concentrations.

EXAMPLE TWO - Production of lactoferrin-enriched whey powder

[00305] This example describes the production of a lactoferrin-enriched milk product from lactoferrin-enriched milk collected from bovine selected using the methods of the present invention.

[00306] The combination of once-daily milking and one or more of TT genotype at the 30126 T/C polymorphism, the AA genotype at the 7447 A/G polymorphism, or the GG genotype at the -7 G/C polymorphism results in the production of milk containing over 600mg/l of lactoferrin (see Table 4 above).

[00307] In a whey protein fraction this will increase the lactoferrin proportion from 1-2% w/w protein up to 5-6%> w/w protein. Production of a lactoferrin-enriched protein fraction is further enhanced by the application of once-daily milking and selection for favourable Lf alleles in cows where other major proteins, such as the major whey protein β-lactoglobulin, are present at lower concentrations. For example, β-lactoglobulin typically constitutes around 50-60% of whey protein in bulk milks. However, the BB polymorphism of beta lactoglobulin is associated with about a 25% reduction in β-lactoglobulin concentration in milk (Prosser et al., 2000. J. Dairy Research 67, 287-283, Robitaille et al, Quantitative analysis of b- lactoglobulin A and B genetic variants in milk of cows ^-lactoglobulin AB throughout lactation Journal of Dairy Research (2002) 69 651 -654) compared to the AA polymorphism. Selection of cows having the BB polymorphism for β-lactoglobulin in combination with one or more of TT genotype at the 30126 T/C polymorphism, the AA genotype at the 7447 A/G polymorphism, or the GG genotype at the -7 G/C polymorphism in the bovine Lf gene could further increase the lactoferrin proportion up to 7-8% w/w of whey protein.

[00308] Thus, achievement of lactoferrin content of close to 20% w/w in a whey protein fraction can be achieved in milks where β-lactoglobulin was present at around lg/1. Such a concentration would facilitate the enrichment of dairy products with lactoferrin without the need for extensive purification.

Discussion

[00309] The present invention recognises that the Lf polymorphisms described above, together with polymorphisms in linkage disequilibrium, are useful in the identification of subjects with particular Lf production, regulation, or secretion phenotypes, for example as a selection tool to breed agricultural animals with higher or lower production of Lf, such as higher or lower colostrum or milk concentrations of Lf. In particular, the combination of genotype and once-daily milking synergistically enhances the genetic effects on milk lactoferrin content allowing a greater increase in milk lactoferrin concentration to be attained. Publications

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INDUSTRIAL APPLICATION

[00310] The present invention is directed to methods of genotyping mammalian subjects to facilitate the identification or selection of subjects with desired Lf production, regulation, or secretion phenotypes, including desired milk or colostrum Lf content phenotypes. In particular, these phenotypes include increased milk or colostrum Lf content, or decreased milk or colostrum Lf content. It is anticipated that in agricultural applications, herds of bovine selected for such phenotypes will produce milk or colostrum of more desirable Lf content, will allow the production of products with, for example, increased Lf content, and therefore will be of significant socioeconomic benefit.