GB2453254A

GB2453254A - Genotyping bovines for SCARB1 polymorphisms

Info

Publication number: GB2453254A
Application number: GB0817719A
Authority: GB
Inventors: Natalie Louise Thomas; Hamish Evan Ward; Stephen Richard Davis; Klaus Lehnert; Julia Leigh Barnett; Sarah Dianne Berry; Russell Grant Snell; Alastair Kenneth Hugh Macgibbon; Richard Spelman; John Frederick Pearson; Penny Elizabeth Beattie
Original assignee: ViaLactia Biosciences NZ Ltd
Current assignee: ViaLactia Biosciences NZ Ltd
Priority date: 2007-09-26
Filing date: 2008-09-26
Publication date: 2009-04-01
Also published as: GB2453254A8; AU2008227070A1; NZ561999A; GB0817719D0; IE20080786A1

Abstract

The present invention provides methods of genotyping bovine for desired milk or tissue colour or b -carotene content traits by determining the SCARB1 genotype of said bovine, particularly as the presence or absence of the C or G allele at the C-321G promoter polymorphism in the SCARB1 gene is associated with variation in milk or tissue colour or b -carotene content. The methods include determining the SCARB1 allelic profile of the bovine, determining the genetic merit of the bovine and selecting or non-selecting it accordingly. Detection of the SCARB1 polymorphism may utilise linkage disequilibrium, expression or activity of the gene or gene product. A kit for genotyping a bovine, suitable primer sequences and the SCARB1 gene sequence are also provided.

Description

MARKER ASSISTED SELECTION OF BOVINE FOR DESIRED MILK OR TISSUE

CONTENT

FIELD OF THE INVENTION

I0001J This invention relates to an application of marker assisted selection of bovine for a quantitative trait loj (QTL) associated with milk and tissue colour and [3-carotene content, particularly by assaying for the presence of polymorphisms in a gene which is associated with the QTL.

BACKGROUND

[0002) The genetic basis of bovine milk production is of immense significance to the dairy industry. An ability to modulate milk volumes and composition has the potential to alter farming practices 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 traits, such as desirable milk fat colour or composition, would be useful.

Similarly, a method of genetically evaluating bovine with desirable tissue colour or composition would be useful.

[00031 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, PCI International application PCT/NZO1/00245 (published as W002/36824) reports that polymorphisms in the bovine Diacylglycerol..oacyltransferase (DGAT I) 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/NZO2JOO1 57 (published as W003/104492) 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] The yellow colour of milk and milk fat, caused primarily by the presence of (3-carotene, is considered a negative characteristic in some consumer markets. Conversely, other markets prize the yellow colour, while foods enriched in f3carotene have been associated with health benefits. Consequently, strategies to modulate milk colour could be economically valuable. Although environmental factors, such as diet, lactation stage and milk volume, influence milk colour, previous research suggests that some of the variation in milk colour may be attributable to genetics (Winkelman el a!., 1999). Similarly, different consumer markets variously perceive tissue colour.

100051 Strategies to modulate milk colour or 13-carotene content or tissue colour or 13-carotene content could provide health benefits and are expected to be economically valuable.

(3-carotene and vitamin A deficiencies are still major health problems (particularly in developing countries) leading to blindness and childhood mortality. Milk with increased (3-carotene content would be of benefit, for example in markets where other dietary sources of (3- carotene are scarce or not commonly consumed.

[00061 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 animal, whether embryonic, infant or adult.

100071 It is an object of the present invention to provide a method for marker assisted selection of bovine with desired milk colour or milk (3-carotene content or tissue colour or tissue (3-carotene content; and/or to provide animals selected using the method of the invention as well as milk produced by or tissue(s) derived from the selected animals; and/or to provide the public with a useful choice.

SUMMARY OF THE INVENTION

[00081 This invention relates to the elucidation of the role of the gene encoding Scavenger receptor class B, member I (SCARB 1, also known as CD36 antigen, collagen type I receptor, and thrombospondin receptor-like 1) in milk or tissue colour or (3-carotene content, particularly milk fat colour and (3-carotene content. In particular, the invention relates to the identification of the C-321G promoter polymorphism in the SCARB1 gene, and to the association of the C allele with production of milk and particularly milk fat with increased [3-carotene content for the first time.

[0009] This gives rise to numerous, and separate, aspects of the invention.

100101 In one aspect the invention provides a method of determining the genetic merit of a bovine with respect to milk or tissue colour or 13-carotene content or with respect to capability of producing progeny that will have increased or decreased milk or tissue colour or 13-carotene content, which comprises determining the SCARBI allelic profile of the bovine, and determining the genetic merit of the bovine on the basis of the SCARB I allelic profile.

[0O1IJ In one embodiment, milk 13-carotene content is milk fat 13-carotene content.

[00121 In another embodiment, milk content is milk colour, more preferably milk fat colour.

(0013J In still further embodiments, the tissue is selected from liver or adipose tissue.

[0014] In one embodiment, the genetic merit with respect to milk or tissue colour or [3-carotene content is production of milk or tissue with increased colour or 13-carotene content.

[0015] Accordingly, in various embodiments the invention provides a method for identifying or selecting a bovine that produces milk, tissue, milk fat, or tissue fat with increased 13-carotene content, or that is capable of producing progeny that produce milk, tissue, milk fat, or tissue fat with increased 13-carotene content, comprising determining the expression or activity of the SCARBI gene product, and identifying or selecting the bovine on the basis of the determination.

10016] In another embodiment, the genetic merit with respect to milk or tissue colour or 13-carotene content is production of milk or tissue with decreased colour or 13-carotene content.

Accordingly, in various embodiments the invention provides a method for identifying or selecting a bovine that produces milk, tissue, milk fat, or tissue fat with decreased 13-carotene content, or capable of producing progeny that produce milk, tissue, milk fat, or tissue fat with decreased 13-carotene content, comprising determining the expression or activity of the SCARB1 gene product, and identifying or selecting the bovine on the basis of the determination.

[00171 In one embodiment, the method comprises determining the expression or activity of a SCARBI gene or gene product, and preferably identifying or selecting the bovine on the basis of the determination.

[0018] In one embodiment, expression or activity of the SCARBI gene product is determined using SCARB1 mRNA, for example by determining the presence or amount of SCARBI mRNA. In other embodiments, expression or activity of the SCARBI gene product is determined using SCARB1 protein, preferably by determining the amount of SCARB1 protein, for example the amount of SCARB 1 protein, or by determining the activity of SCARBI protein, for example the enzymatic activity of SCARB1 protein present in a sample obtained from the bovine. It will be apparent that the activity of SCARE 1 protein (such as that present in a sample obtained from the bovine) may be determined by determining the localisation of SCARBI or of SCARBI ligand, for example by determining the ratio of intracellular SCARBI to extracellular SCARBI, or by determining the the ratio of intracellular SCARE I ligand to extracellular SCARBI ligand, or by determining the transport or uptake of SCARB 1 or of SCARB I ligand across, out of, or into a cell or membrane. In still other embodiments, the expression or activity of the SCARE 1 gene product is determined using SCARBI DNA, preferably by determining the presence or absence of one or more polymorphisms associated with decreased or increased SCARBI expression or activity, for example one or more promoter polymorphisms associated with increased or decreased expression, or one or more coding sequence polymorphisms associated with increased or decreased expression or activity.

10019] In another embodiment, the SCARBI allelic profile of the bovine is determined together with the allelic profile of the bovine at one or more genetic loci associated with milk or tissue colour or fl-carotene content.

[00201 In one embodiment, the one or more genetic loci is one or more polymorphisms in one or more genes associated with milk or tissue colour or fl-carotene content.

[0021] 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.

[00221 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 infers the presence of the other. (Reich DE et al; Linkage disequilibrium in the human genome, Nature 2001, 411:199-204.) [0023] It will be apparent that as used herein, the phrase "SCARBI allelic profile" contemplates data indicative of the presence or absence of one or more alleles at one or more polymorphisms in the SCARBI gene or which affect expression from the SCARB1 gene or the expression or activity of a SCARB1 gene product or which are associated with variation in the expression from the SCARBI gene or in the expression or activity of a SCARBI gene product. In preferred embodiments, the SCARB I ailelic 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 tissue colour or -carotenc content. For example, in preferred embodiments the SCARB1 allelic profile comprises data indicative of the presence or absence of the C allele, or of the presence or absence of the G allele, at the C-321G promoter polymorphism in the SCARE! gene. In other embodiments, the SCARB1 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 SCARBI gene, or in a regulatory region of the SCARB 1 gene, or in an intron of the SCARE 1 gene, or in a coding region of the SCARE 1 gene, and preferably comprises data indicative of the presence or absence of one or more alleles which affect expression from the SCARB1 gene or the expression or activity of a SCARBI gene product or which are associated with variation in the expression from the SCARB I gene or in the expression or activity of a SCARE I gene product.

100241 It will further be appreciated that the SCARBI allelic profile may comprise information correlating the presence or absence of one or more polymorphisms as described above with milk or tissue colour or f3-carotene content.

[00251 In one embodiment, the allelic profile is determined using nucleic acid obtained from said bovine, preferably DNA obtained from said bovine, or alternatively, said allelic profile is determined using RNA obtained from said bovine.

[0026] In yet a further embodiment, the allelic profile is determined with reference to the amino acid sequence of expressed SCARE! protein obtained from said bovine.

[0027] In another embodiment, the allelic profile is determined with reference to the amount or activity of SCARE I protein obtained from said bovine.

[0028] Conveniently, in said method the presence or absence of DNA encoding a reference SCARE I gene product, or of nucleotide sequence comprising a reference SCARB I gene, in said bovine is determined, directly or indirectly, for example using an expressed SCARB1 gene product.

10029] Alternatively, in said method the presence or absence of at least one nucleotide difference from the nucleotide sequence of a reference SCARB1 gene, for example, at least one nucleotide difference from the nucleotide sequence encoding reference SCARE 1, in said bovine is determined, directly or indirectly.

[00301 In one embodiment, the method comprises determining the SCARBI C-32lG promoter allelic profile of the bovine.

100311 More specifically, in said method the presence or absence of one or more of the C allele or G allele at the C-32lG promoter polymorphism in the SCARBI gene is determined, directly or indirectly. For example, the presence of the C allele or of the G allele at the C- 321(3 promoter polymorphism in the SCARB1 gene may be determined using a polymorphism in linkage disequilibrium with the C allele or with the G allele at the C-321G promoter polymorphism.

[0032J In one embodiment, the method includes ascertaining, from a sample of material containing DNA obtained from the bovine, whether a sequence of the DNA encoding a protein "(A)" having biological activity of reference SCARB1 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.

[00331 As used herein, biological activity of reference SCARB 1 protein refers to both expression levels and activity characteristic of SCARB 1 protein expressed from the reference SCARB1 gene.

[0034J In another embodiment, the method includes ascertaining, from a sample of material containing DNA obtained from the bovine, whether a reference SCARB1 gene sequence is present. In still another embodiment, the method includes ascertaining, from a sample of material containing DNA obtained from the bovine, the expression of the SCARB1 gene product, preferably by determining the presence or absence of one or more polymorphisms associated with decreased or increased SCARB I expression, for example one or more promoter polymorphisms associated with increased or decreased expression.

100351 In one embodiment, this method includes ascertaining whether a sequence of the mRNA encoding a protein "(A)" having biological activity of a reference SCARB 1 is present, or whether a sequence of the mRNA encoding a protein "(B)" at least partially lacking the activity of (A) is present, or whether a sequence of the mRNA encoding (A) and a sequence of the mRNA encoding (B) are both present.

[00361 In another embodiment, the method includes ascertaining the amount of SCARB I mRNA present in a sample of material containing mRNA obtained from the bovine.

[00371 In another embodiment, the method includes ascertaining whether a protein "(A)" having biological activity of a reference SCARBI 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.

[0038] In another embodiment, the method includes ascertaining the amount or activity of SCARB I protein present in a sample of material containing protein obtained from the bovine.

(0039] In another aspect, the invention is a method for determining the SCARB1 genotype of a bovine, as may be desirable to know for breeding purposes.

[0040] hi one embodiment, the method includes ascertaining, with reference to a sample of material containing nucleic acid obtained from the bovine and uncontaminated by heterologous nucleic acid, whether the sample contains (i) nucleic acid molecule encoding a protein having biological activity of reference SCARB I and optionally ascertaining whether the sample contains an (ii) allelic nucleic acid molecule encoding a protein lacking biological activity of reference S CARB 1.

[0041J In another embodiment, the method includes ascertaining, with reference to a sample of material containing protein obtained from the bovine and uncontaminated by heterologous protein, whether the sample contains (i) a protein having biological activity of reference SCARB1 and optionally ascertaining whether the sample contains a protein lacking biological activity of reference SCARBI.

100421 In a further embodiment, the invention provides a method of determining genetic merit of a bovine with respect to milk or tissue 13-carotene content which comprises determining the SCARB I allelic profile of the bovine, together with determining the allelic profile of the bovine at one or more genetic loci associated with milk or tissue 13-carotene content.

10043] In one embodiment, the one or more genetic loci is one or more polymorphisms in one or more genes associated with milk or tissue 13-carotene content, preferably one or more polymorphisms in one or more genes involved in 13-carotene uptake or metabolism.

[0044J In a further aspect, the invention includes a probe comprising a nucleic acid molecule sufficiently complementary with a nucleic acid sequence comprising a bovine SCARB I gene or encoding a bovine SCARBI gene product, or its complement, so as to bind thereto under stringent conditions, as well as a diagnostic kit containing such a probe.

(0045J The invention also includes a primer composition useful for detection of the presence or absence of a reference SCARB I gene andior the presence or absence of nucleic acid encoding reference SCARBI. In one form, the composition can include a nucleic acid primer substantially complementary to a nucleic acid sequence comprising a reference SCARB1 gene or encoding reference SCARB1, or its complement. The nucleic acid sequence can in whole or in part be identified in SEQ ID No. 1 or SEQ ID No.3. The invention also includes a primer composition useful for detection of the presence or absence of a variant SCARBI gene and/or the presence of the DNA encoding a variant SCARBI protein at least partially lacking reference SCARB I activity. In one form, the composition can include a nucleic acid primer substantially complementary to a nucleic acid sequence comprising a variant SCARBI gene or encoding a variant SCARBI protein, or its complement. The nucleic acid sequence can in whole or in part be identified in SEQ ID NO:! or SEQ ID NO:3. Diagnostic kits including such a composition are also included.

[0046] Particularly contemplated are primers comprising or substantially complementary to a nucleic acid sequence present in SEQ ID NO: I and within approximately 1 to about 2000 bp of the C-321G promoter polymorphism, more preferably within approximately I to about 1000 bp, or within approximately I to about 500 bp, approximately I to about 400 bp, approximately 1 to about 300 bp, approximately I to about 200 bp, approximately I to about bp, approximately I to about 50 bp, or approximately 1 to about 20 bp of the C-32lG promoter polymorphism.

[00471 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 the C-32l (3 promoter 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.

[0048] The invention also provides a diagnostic kit including a primer composition useful for determining the presence or absence of a reference SCARB1 gene and/or the presence or absence of nucleic acid encoding reference SCARB 1, the diagnostic kit comprising one or more primers or primer compositions as described herein.

[0049] The invention further includes an antibody composition useful for determining the presence or absence of reference SCARBI protein, or for determining the presence or absence of a variant protein at least partially lacking reference SCARB I activity, or for determining the expression of SCARB 1 protein, as well as a diagnostic kit containing such an antibody together with instructions for use, for example in a method of the invention.

[00501 The invention further provides a diagnostic kit useful in detecting DNA comprising a variant SCARBI gene, or DNA or mRNA encoding a variant SCARBI gene at least partially lacking reference activity, in a bovine which includes first and second primers for amplifying the DNA or mRNA, the primers being complementary to nucleotide sequences of the DNA upstream and downstream, respectively, of a polymorphism in the SCARBI gene which results in or is associated with increased or decreased milk or tissue colour or 13-Carotene content.

[00511 In one embodiment at least one of the nucleotide sequences is selected to be from a non-coding region of the reference SCARB1 gene.

100521 The kit can also include a primer complementary to a naturally occurring mutation of a coding or non-coding portion of the reference SCARB I gene, for example a mutation in the promoter of the SCARB I gene. Preferably the kit includes instructions for use, for example in accordance with a method of the invention.

[0053] Thus, in another embodiment the invention provides a method of assessing the genetic merit of a bovine with respect to milk or tissue 13-carotene content which comprises the step of determining the presence or absence of one or more polymorphisms selected from the group comprising: the C-321G promoter polymorphism in the SCARBI gene, or one or more polymorphisms in linkage disequilibrium with the C-321G promoter polymorphism in the SCARB1 gene [0054] In another embodiment the invention provides a method of assessing the genetic merit of a bovine with respect to milk or tissue colour or 13-carotene content which comprises the step of determining the presence or absence of one or more polymorphisms selected from the group comprising: the C allele at the C-321G promoter polymorphism, or the G allele at the C-32 I G promoter polymorphism.

[0055] 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.

10056] Thus, in another embodiment the invention provides a method of assessing the genetic merit of a bovine with respect to milk or tissue 13-carotene content which comprises the step of determining the presence or absence of the G allele at the C-32 1 G promoter polymorphism in the SCARB1 gene. The invention further provides a method of assessing the genetic merit of a bovine with respect to milk or tissue 13-carotene content which comprises the step of determining the presence or absence of the C allele at the C-32 10 promoter polymorphism in the SCARBI gene.

[0057] In another aspect, the present invention provides a method for selecting a bovine with a genotype indicative of desired milk colour or of desired milk 13-carotene content or of desired tissue colour or of desired tissue 13-carotene content. The method comprises determining the bovine SCARB I allelic profile of said bovine, and selecting the bovine on the basis of the determination.

[0058J In one embodiment, the invention provides a method for identifying or selecting a bovine with increased milk colour or increased milk 13-carotene content, preferably increased milk fat colour or increased milk fat 13-carotene content.

[0059] In another embodiment, the invention provides a method for identifying or selecting a bovine with increased tissue colour or increased tissue 13-carotene content, preferably increased tissue fat colour or increased tissue fat 13-carotene content.

[0060] In one embodiment, the invention provides a method for identifying or selecting a bovine with a SCARB1 allelic profile indicative of increased milk colour or increased milk [3-carotene content, preferably of increased milk fat colour or increased milk fat 13-carotene content.

[0061J In another embodiment, the invention provides a method for identifying or selecting a bovine with a SCARB1 allelic prolile indicative of increased tissue colour or increased tissue 13-carotene content.

[0062J Preferably the method comprises determining the presence of the C allele at the C-32 10 promoter polymorphism in the SCARB I gene, and selecting the bovine on the basis of the determination. Alternatively or additionally, the method comprises determining the absence of the G allele at the C-321G promoter polymorphism in the SCARBI gene, and selecting the bovine on the basis of the determination.

[00631 Preferably, the method comprises determining the presence of the CC genotype at the C-32 10 promoter polymorphism in the SCARB 1 gene, and selecting the bovine on the basis of the determination.

[0064] In another embodiment, the invention provides a method for identifying or selecting a bovine with decreased milk colour or decreased milk 13-carotene content, preferably decreased milk fat colour or decreased milk fat 13-carotene content.

100651 In another embodiment, the invention provides a method for identifying or selecting a bovine with decreased tissue colour or decreased tissue 13-carotene content, preferably decreased tissue fat colour or decreased tissue fat f3-carotene content.

100661 In a further embodiment the invention provides a method for selecting a bovine with a SCARBI allelic profile indicative of decreased milk colour or decreased milk 13-carotene content, preferably of decreased milk fat colour or decreased milk fat 13-carotene content.

100671 In another embodiment, the invention provides a method for selecting a bovine with a SCARBI allelic profile indicative of decreased tissue colour or decreased tissue 13-carotene content.

100681 Preferably the method comprises determining the absence of the C allele at the C- 321G promoter polymorphism in the SCARBI gene, and selecting the bovine on the basis of the determination. Alternatively or additionally, the method comprises determining the presence of the G allele at the C-32lG promoter polymorphism in the SCARBI gene, and selecting the bovine on the basis of the determination.

[00691 Preferably, the method comprises determining the presence of the GO genotype at the C-32 1 G promoter polymorphism in the SCARB 1 gene, and selecting the bovine on the basis of the determination.

10070] In one embodiment, the invention provides a method for identifying or selecting a bovine with intermediate milk colour or intermediate milk 13-carotene content, preferably intermediate milk fat colour or intermediate milk fat 13-carotene content.

[0071] In another embodiment, the invention provides a method for identifying or selecting a bovine with intermediate tissue colour or intermediate tissue 13-carotene content, preferably intermediate tissue fat colour or intermediate tissue fat 13-carotene content.

100721 In a further embodiment the invention provides a method for selecting a bovine with a SCARB I allelic profile indicative of intermediate milk colour or intermediate milk 13- carotene content, preferably of intermediate milk fat colour or intermediate milk fat 13-carotene content.

[0073] In another embodiment, the invention provides a method for selecting a bovine with a SCARB I allelic profile indicative of intermediate tissue c olour or intermediate tissue 13-carotene content.

100741 Preferably, the method comprises determining the presence of the CG genotype at the C-321G promoter polymorphism in the SCARBI gene, and selecting the bovine on the basis of the determination.

[0075] In one embodiment, the presence of the C allele or of the G allele is determined with respect to a SCARBI polynucleotide (genomic DNA, rnRNA or eDNA produced from mRNA) obtained from the bovine.

[0076] In one embodiment, the presence of the C allele or of the G allele is determined by sequencing a SCARB 1 polynucleotide obtained from the bovine.

(0077] In a further embodiment the determination comprises the step of amplifying a SCARB I polynucleotide sequence from genomic DNA, mRNA or cDNA produced from mRNA derived from said bovine, for example by PCR.

[0078] 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 SEQ ID NO:l or SEQ ID NO:3 or a naturally occurring flanking sequence.

100791 In one embodiment at least one of the primers comprises sequence corresponding to at least one of the allele-specific nucleotides described herein.

J0080J 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.

100811 In a further embodiment, the presence of the C allele or of the G allele is determined by mass spectrometric analysis of a SCARBI polynucleotide, such as that obtained from the bovine or from a method as described herein.

(0082] In an alternative embodiment, the presence of the C allele or of the G allele is determined by hybridisation of a probe or probes comprising a nucleotide sequence of or complementary to the sequence of SEQ ID NO:l or SEQ ID NO:3.

100831 Preferably the probe or probes comprises 12 or more contiguous nucleotides of or complementary to the sequence of SEQ ID NO: I or SEQ ID NO:3.

(00841 Preferably the probe or probes comprise sequence corresponding to the C allele-specific or the G allele-specific nucleotides described herein or complements thereof.

[0085] In an alternative embodiment, the presence of the C allele or of the G allele is determined by analysis of a SCARB I polypeptide obtained from the bovine.

10086] In a further aspect the invention provides a bovine selected by a process of the invention; milk produced by the selected bovine or the progeny thereof as well as dairy products produced from such milk; and ova or semen produced by or tissue from the selected bovine.

[0087] 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.

[00881 In a still further aspect, the invention provides a method of determining genetic merit of a bovine with respect to one or more milk or tissue colour or t3-carotene content traits, or with respect to capability of producing progeny predisposed to or with one or more milk or tissue colour or 3-carotene content traits, the method comprising providing data about the SCARBI allelic profile of said bovine, and determining the genetic merit of the bovine on the basis of the data.

[0089] Preferably, the data about the SCARB1 allelic profile comprises data representative of the presence or absence of the C allele or the G allele at the C-321G promoter polymorphism in the SCARB1 gene.

100901 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 tissue colour or 3-carotene content traits, wherein the data is representative of the genetic merit of the bovine.

100911 Preferably, the one or more genetic loci are one or more polymorphisms associated with an increase or decrease in expression or activity of a SCARB1 gene product.

10092] Preferably the genetic loci is the SCALRBI gene (including all regulatory elements such as the promoter, introns and 3'UTR).

100931 In one embodiment, the one or more milk or tissue colour or 3-carotene content traits is selected from the group comprising production of or capability of producing milk with increased milk colour, production of or capability of producing milk with increased milk 13-carotene content, increased tissue colour, or increased tissue 13-carotene content.

[0094] In another embodiment, the one or more milk or tissue colour or 13-carotene content traits is selected from the group comprising production of or capability of producing milk with decreased milk colour, production of or capability of producing milk with decreased milk 13-carotene content, decreased tissue colour, or decreased tissue 13-carotene content.

(0095J Accordingly, in one embodiment the invention provides a method of determining genetic merit of a bovine with respect to milk or tissue colour or 13-carotene content, or with respect to capability of producing progeny that will have increased or decreased milk or tissue colour or 13-carotene content, the method comprising providing data about the SCARBI allelic profile of the bovine, and determining the genetic merit of the bovine on the basis of the data.

[0096J Preferably, the data about the SCARB1 allelic profile comprises data representative of the presence or absence of the C allele or the G allele at the C-32 I G promoter polymorphism in the SCARB 1 gene.

100971 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 tissue colour or f3-c&otene content traits, wherein the data is representative of the genetic merit of the bovine.

[0098] Preferably, the one or more genetic loci are one or more polymorphisms associated with an increase or decrease in expression or activity of a SCARBI gene product.

10099] Preferably the genetic loci is the SCARBI gene (including all regulatory elements such as the promoter, introns and 3'UTR).

100100] In a further aspect the invention provides a method for identifying or selecting a bovine with respect to one or more milk or tissue colour or 13-carotene content traits, the method comprising providing the result of one or more genetic tests of a sample from the bovine, and analysing the result for the presence or absence of one or more polymorphisms associated with increased or decreased expression or activity of SCARB1 gene product, or one or more polymorphisms in linkage disequilibrium with one or more polymorphisms associated with increased or decreased expression or activity of SCARB 1 gene product, 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 tissue colour or 13-carotene content traits; and identifying or selecting the bovine on the basis of the result.

100101] Preferably, the one or more polymorphisms associated with increased or decreased expression or activity of SCARB 1 gene product is one or more polymorphisms in the SCARBI gene.

1001021 In a further aspect the invention provides a method for selecting a bovine with one or more desired milk or tissue colour or a-carotene content traits, 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 C-321G promoter polymorphism in the SCARBI gene, or one or more polymorphisms in linkage disequilibrium with the C-321G promoter polymorphism in the SCARBI 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 tissue colour or 3-carotene content traits.

[00103] 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 bovine with respect to one or more milk or tissue colour or 13-carotene content traits and optionally a reference milk or tissue colour or 13-carotene content trait database of non-genetic factors for one or more bovine milk or tissue colour or 13-carotene content traits; 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 merit of the bovine, said outcome being communicable once known, preferably to a user having input said data.

[00104] Preferably, said system is accessible via the internet or by personal computer.

100105] 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 tissue colour or 13-carotene content traits, 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 tissue colour or 13-carotene content traits.

100106] 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 tissue colour or J3-carotene content traits 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 bovine milk or tissue colour or 13-carotene content traits.

1001071 Preferably, the one or more genetic loci are one or more polymorphisms in one or more genes associated with one or more milk or tissue colour or 13-carotene content traits.

[00108] Preferably the gene is the SCARBI gene (including all regulatory elements such as the promoter, introns and 3'UTR).

100109] Preferably, the one or more polymorphisms are one or more polymorphisms associated with an increase or decrease in expression or activity of a SCARBI gene product.

[00110] In still another aspect, the invention provides a method of determining genetic merit of a bovine with respect to milk or tissue colour or 13-carotene content, or with respect to capability of producing progeny that will have increased or decreased milk or tissue colour or 13-carotene content, the method comprising determining milk or tissue colour or 13-carotene content of the bovine, determining the SCARBI allelic profile of the bovine, comparing the SCARBI allelic profile of the bovine or the milk or tissue colour or 13-carotene content of the bovine with that of a bovine having a known SCARB1 allelic profile; determining the genetic merit of the bovine on the basis of the comparison.

100111] It will be appreciated that for the purposes of the comparison, the milk or tissue colour or 13-carotene content associated with the known SCARB1 allelic profile is known. It will further be appreciated that the association of milk or tissue colour or 13-carotene content with a particular SCARB1 allelic profile may be established by the methods described herein.

1001121 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 SEQ ID NO:I and comprising the C-321G promoter polymorphism; or (b) any one or more of SEQ ID NOs:5 -44; or (c) a complement of(a) or (b); or (d) a sequence of at least 12 contiguous nucleotides and capable of hybridising to the nucleotide sequence of any one of (a) to (c) under stringent conditions.

[00113] In one embodiment, the SCARBI nucleic acid molecule is a SCARB1 fragment as defined herein, wherein the SCARB1 fragment comprises the C-321G promoter polymorphism.

1001141 The invention also provides a genetic construct comprising a SCARBI 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 SCARB I 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.

1001151 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.

[001161 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 a graph showing the range and seasonal effect of 13-carotene concentration determined as described in the Example. Data for peak (35 days post calving), mid (late November) and late (late February) lactation is shown.

Figure 2 shows a graph depicting a QTL for milk fat colour on bovine chromosome 17. The maximum F-value of 10.6 occurred at 71 centimorgans (cM). The approximate map location of the SCARBI gene was between two inicrosatellite markers BMS1879 at 50.7cM and BM1862 at 67.5 cM.

Figure 3 is a schematic showing a graphical representation of the C-321G promoter polymorphism in bovine SCARBI. A: Graphical representation of the predicted gene structure of bovine SCARBI where vertical bars represent exons, as we]l as 5' UTR and 3' UTR. B. Identification of the C/G polymorphism at position -321 relative to the + 1 translation start site (promoter). The polymorphism (highlighted) was heterozygous in five Fl sires, and one sire was homozygous for the G allele.

Figure 4 is a graph showing the effect ofSCARBI genotype on milk fat colour. Data shown are means � s.e.m. Data shown are for peak lactation but are representative of the additional two time points that were measured. The main effect of SCARB1 genotype was significant at P <0.01.

Figure 5 is a graph showing the adjusted statistical effect of SCARB1 genotype on the bovine chromosome 17 milk fat colour QTL. Solid line: milk fat colour QTL (residual of modeled data excluding SCARB I genotype). Dotted line: milk fat colour QTL after SCARB I genotype included in statistical model (residual of modeled data including SCARB 1 genotype as a fixed effect). The decrease in QTL significance (F-value) suggests a significant association of the SCARBI genotype with the milk fat colour QTL variation.

Figure 6 is a graph showing the n-carotene content of bovine adipose tissue. The main effect of the SCARBI genotype was significant at p < 0.01. Data shown are means � s.e.m. Significance between specific genotype groups is indicated by asterix (** P <0.01). For the CC, CG and GO genotype groups there were n=5, n=12 and n=7 animals, respectively.

Figure 7 is a graph showing the effect SCARBI genotype on liver SCARBI mRNA levels as measured by quantitative real time PCR. Data shown are the fold change means relative to a reference sample � s.e.m. Significance between genotype groups is indicated by asterix (** P <0.01). For the CC, CG and GG genotype groups there were n=7, n13 and n8 animals, respectively.

DETAILED DESCRIPTION OF THE INVENTION

100117] The present invention recognises for the first time that a polymorphism in the SCARB 1 gene in bovine is associated with a QTL for variations in milk colour and milk 3-carotene content, and for variations in tissue colour and tissue j3-carotene content.

[001181 For the sake of clarity, the phrase "milk or tissue colour or (3-carotene content" is to be read as referring to milk colour or milk (3-carotene content or tissue colour or tissue (3-carotene content. Grammatical equivalents or components thereof are to be read likewise, such that the phrase "milk or tissue colour" is to be read as "milk colour or tissue colour".

[001191 It will be apparent to those skilled in the art that milk or tissue colour can readily be determined qualitatively or quantitatively. For example, a visual comparison may in many cases be sufficient to qualitatively determine a sample of milk or tissue having increased colour, or decreased colour, relative to another sample. Methods for quantitative determination of milk or tissue colour or (3-carotene content are also known in the art, and examples are provided herein.

(001201 The invention provides methods of assessing the genetic merit of a bovine with respect to milk 13-carotene content, more particularly milk fat 13-carotene content. One such method comprises the step of determining the SCARB I allelic profile of said bovine. Another such method comprises the step of determining the level of the SCARB1 gene product of said bovine.

[001211 The invention also provides a method for selecting a bovine with a genotype indicative of desired milk (3-carotene content, particularly desired milk fat (3-carotene content.

One of the major applications of the present invention is in the selection of bovine having the C allele or the 0 allele of the C-321G promoter polymorphism in the SCARBI gene, which are associated with increased milk fat (3-carotene content and milk fat colour, and decreased milk fat 13-carotene content and milk fat colour, respectively. Accordingly, one method comprises determining the presence or absence of the C allele or of the 0 allele at the C-32lG promoter polymorphism of the SCARB I gene, and selecting the bovine on the basis of the determination.

[00122 Additionally, the invention is directed towards the selected bovine and semen from the selected bovine which may be useful in further breeding programs. Bovine so selected will be useful for milk production. The invention is also directed towards milk produced by the selected bovine or the progeny thereof, as well as dairy products produced from such milk, 1001231 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 products including daily supplement tablets.

[00124] Similarly, the production of a wide range of tissue products, such as but not limited to meat, organs, pelts, fluids and the like, is well known in the art. Particularly contemplated herein are meat, organs, blood and serum having increased or decreased colour or (3-carotene content.

[00125] The present invention recognises that polymorphisms in the gene encoding SCARBI, as well as SCARBI levels or activity, may be used as a selection tool to breed animals with higher or lower milk concentrations of 13-carotene (and thus milk fat colour).

This in turn may allow the production of milk products more suitable to markets favouring white milk and milk products, or the production of milk products more suitable to markets favouring yellow milk and milk products, or the production of milk and milk products, such as foods, high in 13-carotene.

1 SCARB1 [00126] SCARB1 is believed to be involved in the uptake of carotenoids into cells. A Drosophila knockout model is reported to be blind, with no carotenoids present, and vitamin A deficient (Kiefer C., Ct al., 2002).

1001271 The present invention relates to the identification that a polymorphism in the SCARB 1 gene leads to variation in milk or tissue colour or 13-carotene content, particularly variation in milk fat colour and milk fat 13-carotene content, and tissue fat colour and tissue fat 13-carotene content.

1001281 The genomic sequence comprising the bovine SCARBI gene is presented herein as SEQ ID NO:l. The predicted exon structure is shown in SEQ ID NO:l, and the derived amino acid sequence is presented herein as SEQ ID NO:2. The predicted coding sequence of bovine SCARBI is presented as SEQ ID NO:3, and a provisional sequence is available as NCBI accession number NM_174597.2 (01:31341575). This coding sequence is derived from a cDNA clone, the sequence of which is available as NCBI accession number AFOI 9384 (GI:2429347). The amino acid sequence encoded by the reference coding sequence of SEQ ID NO:3 is presented herein as SEQ ID NO:4, and is itself available as NCBI accession number NP 777022.1 (GI:27807079).

[001291 As described herein, the SCARB1 C-321G polymorphism was closely associated with milk colour phenotype. Animals homozygous for the C allele (CC genotype) produced milk with more 13-carotene than heterozygous animals (CO genotype), who in turn produce milk with more 13-carotene than animals homozygous for the 0 allele (GO genotype). This effect was observed at three stages of lactation.

[00130] The CC genotype for SCARBI was present in 23.52% of animals in the Holstein-Friesian x Jersey crossbred trial, while the CG genotype was present in 48.85% of animals, and the remaining 27.64% of animals were of GO genotype.

1001311 A reference bovine SCARBI nucleotide sequence is presented as SEQ ID NO:1, and the compiled reference coding sequence is presented as SEQ ID NO:3, with the corresponding amino acid sequences presented as SEQ ID NO:2, and SEQ ID NO:4, respectively. Accordingly, as used herein with respect to SCARB I, such as use with respect to a SCARBI gene or a SCARB1 gene product, the term "reference" recognizes the characteristics of the SCARBI nucleotide sequences presented as SEQ ID NOs: I and 3, arid of the protein product encoded thereby. For example, when used with respect to activity, the term "reference" denotes activity associated with the reference SCARB I protein. Similarly, when used with respect to expression amount, the term "reference" denotes a level of expression associated with the reference SCARB 1 gene or promoter.

1001321 It will be apparent that the term"activit-y" may refer both to the inherent activity of a single molecule of SCARB 1, which may be reference activity or may be less or greater than reference 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 SCARB1 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).

[00133] As used herein, such as when used in reference to an allelic protein lacking the activity of reference SCARB 1, 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 reference SCARBI may be a variant SCARB I protein of greater or lesser activity than that of reference SCARB 1.

(00134] Methods to assay the activity of SCARBI are well known in the art. For example, one such method utilises quantification of SCARE I mRNA, such as that described herein.

Another exemplary method utilises an assay of the uptake of 3-caroterie as described in During and Harrison (2007).

1001351 The genetic polymorphism identified in the bovine SCARB1 gene is identified as a variant in SEQ ID NO:l and 3, and is reported in Figure 3. The nucleic acid and proteins sequences of the SCARBI G and C alleles at the C-321G promoter polymorphic site are shown in Figure 3B.

2 identification and analysis of polymorphisms (00136) It will be apparent to those skilled in the field that the convention of identifying promoter polymorphisms by their position relative to the +1 translation start site of the gene in which they occur is followed herein. Accordingly, the C-321G polymorphism in the SCARBI gene described herein lies 321 nucleotides upstream of the +1 translation start site of the SCARBI gene.

1001371 The C-321G promoter polymorphism can be detected directly or by detection of one or more polymorphisms which are in linkage disequilibrium with the C-321G promoter polymorphism. 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 meais 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, 411:199-204.) [00138] 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 the C- 321G promoter polymorphism of the SCARB1 gene. (Devlin and Risch 1995; A comparison of linkage disequilibrium measures for fine-scale mapping, Genomics 29: 311-322).

100139] There are numerous standard methods known in the art for detennining 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.

[00140] In one aspect, the present invention provides a method for determining the genetic merit of a bovine with respect to milk or tissue p3-carotene content, and particularly with milk fat colour or n-carotene content. In one embodiment 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 reference SCARB1 is present, and whether a sequence of the DNA encoding "(B)" an allelic protein lacking the activity of (A) is present. In another embodiment, the method includes ascertaining, from a sample of material containing DNA obtained from the bovine, whether the reference SCARB I gene sequence is present. In still another embodiment, the method includes ascertaining, from a sample of material containing DNA ob tained from the bovine, the expression of the SCARB I gene product, preferably by determining the presence or absence of one or more polymorphisms associated with decreased or increased SCARB I expression, for example one or more promoter polymorphisms associated with increased or decreased expression.

[00141J 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).

One embodiment of the invention thus includes a step in which ascertaining whether a sequence of the DNA encoding (A) is present, or 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 reference SCARB 1, 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 f-carotene levels, and particularly in milk or tissue having inter alia a higher fl-carotene 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 fl-carotene levels, and particularly in milk or tissue having inter alia a lower fl-carotene content.

[001421 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 protein 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.

[00143J Accordingly, a preferred embodiment of the invention thus includes the step of amplifying a SCARB I polynucleotide in the presence of at least one primer comprising a nucleotide sequence of, or complementary to, the SCARB 1 gene (SEQ ID NO: 1 and SEQ ID NO:3) or flanking sequence thereof, and/or in the presence of a such a primer comprising sequence corresponding to or flanking the C-321G C allele-specific or C-32l0 G 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).

[00144J Primers suitable for use in PCR based methods of the invention should be sufficiently complementary to the SCARB1 gene sequence, such as SEQ ID NO: I or SEQ ID NO:3 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 SEQ ID NO: I or SEQ ID NO:3, or naturally occurring flanking sequences thereof. Examples of such PCR primers are presented herein as SEQ ID NOS:5-44.

[00145J Suitable PCR primers may include sequence corresponding to the C-321G C allele-specific or C-32 1 G G allele-specific SCARB 1 nucleotides 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 SCARB1 gene of the test bovine.

1001461 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 he a PCR product amplified as described above.

[001471 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 the C-321G C allele-specific or C-321G 0 allele-specific nucleotides may comprise the additional steps of hybridisation of a probe derived from the SCARBI sequence of SEQ ID NO:! or SEQ ID NO:3.

[00148J Such probes should comprise a nucleic acid molecule of sufficient length and sufficiently complementary to the SCARB I 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.

[001491 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, 100 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+).

[001501 With respect to polynucleotide molecules having a length less than 100 bases, exemplary stringent hybridization conditions are 5 to 100 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. 1001511 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.

100152] Such probes would typically comprise at least 12 contiguous nucleotides of or complementary to the sequences presented SEQ ID NO:l or SEQ ID NO:3, and may comprise sequence corresponding to the allele-specific nucleotides described herein.

[00153] 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).

[00154] As will be apparent to a person skilled in the art, promoter function may be determined by various well-known methods, for example use in reporter systems. For example, one approach of determining the allelic state of a promoter would be via a reporter construct, where the promoter of interest is fused upstream of a reporter gene (e.g. luciferase), and the activity of the reporter is determined and correlates with promoter function.

[00155] Accordingly, in one embodiment the method for determining the genetic merit of a bovine with respect to milk or tissue colour or f3-carotene content includes ascertaining, from a sample of material containing DNA obtained from the subject, whether a sequence of the DNA encoding the promoter of the SCARBI gene is associated with higher or lower relative levels of expression.

1001561 In another aspect, the invention provides a method for determining the genetic merit of bovine with respect to milk or tissue colour or 13-carotene 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 reference SCARBI 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.

100157] Again, if an amplification method such as PCR is used in ascertaining whether a sequence of the mRNA encoding (B) is present, and whether a sequence of the mRNA encoding (A) 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 reference SCARB 1, or in the presence of a pair of primers complementary to a nucleotide sequence encoding a variant SCARB I protein. It will be appreciated that in embodiments of the invention reliant on assessing the amount of SCARBI mRNA present in a sample, quantitative amplification methods well known in the art may be employed, for example quantitative RT-PCR, microarray analysis, arid other methods described herein.

[00158] Other methods to quantitate or otherwise assess the amount of nucleic acid, particularly the amount of mRNA are well known in the art. These include Northern analysis using probes able to hybridise to the target SCARBI mRNA. Such probes should comprise a nucleic acid molecule of sufficient length and sufficiently complementary to the SCARBI 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 SCARB1 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.

[00159] In a further aspect, the invention provides a method of determining genetic merit of a bovine with respect to milk or tissue n-carotene content which comprises determining the SCARBI allelic profile of said bovine, together with determining the allelic profile of said bovine at one or more genetic loci associated with milk or tissue 3-carotene content.

[00160] In one embodiment, said genetic loci is a polymorphism in a gene associated with milk or tissue 3-carotene content, preferably a polymorphism in a gene involved in J3-carotene uptake or metabolism.

[001611 The 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.

1001621 A SNP is a single base change or point mutation resulting in genetic variation between individuals. SNPs are believed to occur in mammalian genomes approximately once every 100 to 300 bases, and can occur in coding or non-coding regions. Due to the redundancy of the genetic code, a SNP in the coding region may or may not change the amino acid sequence of a protein product. A SNP in a non-coding region can, for example, alter gene expression by, for example, modifying control regions such as promoters, transcription factor binding sites, processing sites, ribosomal binding sites, mRNA stability, and affect gene transcription, processing, and translation.

100163] SNPs can facilitate large-scale association genetics studies, and there has recently been great interest in SNP discovery and detection. SNPs show great promise as markers for a number of phenotypic traits (including latent traits), such as for example, disease propensity and severity, weliness propensity, drug responsiveness including, for example, susceptibility to adverse drug reactions, and as described herein association with desirable phenotypic traits.

Knowledge of the association of a particular SNP with a phenotypic trait, coupled with the knowledge of whether a subject has said particular SNP, can enable the targeting of diagnostic, preventative and therapeutic applications to allow better disease management, to enhance understanding of disease states, to develop selective breeding regimes, and to identify subjects of desirable genetic merit.

100164] Indeed, a number of databases have been constructed of known SNPs, and for some such SNPs, the biological effect associated with a SNP. Understandably, there has been a focus on human genetics. For example, the NCBI SNP database "dbSNP" is incorporated into NCBJ's Entrez system and can be queried using the same approach as the other Entrez databases such as PubMed and GenBank. This database has records for over 1.5 million SNPs mapped onto the human genome sequence. Each dbSNP entry includes the sequence context of the polymorphism (i.e., the surrounding sequence), the occurrence frequency of the polymorphism (by population or individual), and the experimental method(s), protocols, and conditions used to assay the variation, and can include information associating a SNP with a particular phenotypic trait. Similar databases are available for a number of species of commercial and scientific interest.

[00165] There has been and continues to be a great deal of effort to develop methods that reliably and rapidly identify new SNPs associated with a phenotypic trait. This is no trivial task, at least in part because of the complexity of mammalian genomic DNA (e.g., the haploid human genome of 3 x I ü base pairs, while current estimates of the size of the haploid bovine genome arc in the range of 2.6 -2.7 x i� base pairs), and the associated sensitivity and discriminatory requirements.

[00166) 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 (oflen 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 SN? lies in a coding region and results in an amino acid change.

[00167] DNA sequencing allows the direct determination arid identification of SNPs. The benefits in specificity and accuracy are generally outweighed for screening purposes by the diflicuities inherent in whole genome, or even targeted subgenome, sequencing.

[001681 Mini-sequencing involves allowing a primer to hybridize to the DNA sequence adjacent to the SN? site on the test sample under investigation. The primer is extended by one nucleotide using all four differentially tagged fluorescent dideoxynucleotides (A,C,G, or T), and a DNA polymerase. Only one of the fbur nucleotides (homozygous case) or two of the four nucleotides (heterozygous case) is incorporated. The base that is incorporated is complementary to the nucleotide at the SN? position.

1001691 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 GS FLX pyrosequencing platform available from 454 Life Sciences (Branford, CT) which can generate 100 million nucleotide data in a 7.5 hour run with a single machine, and solid-state sequencing methods, such as that utilised in the SOLiD sequencing platform (Applied Biosystems, Foster City, CA).

[00170J 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.) arc 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.

1001711 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, PCI publication WO 95/ 12607). US Application 20050059030 (incorporated herein in its entirety) describes a method for detecting a single nucleotide polymorphism 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 two hybridization events: 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. Both hybridization events happen in the same reaction, and the order in which hybridisation occurs is not critical.

[00172] US Application 20050042608 (incorporated herein in its entirety) describes a modification of the method of electrochemicai detection of nucleic acid hybridization of Thorp et al. (U.S. Pat. No. 5,871,918). Briefly, capture probes are designed, each of which has a different SNP base and a sequence of probe bases on eaeh side of the SNP base. The probe bases are complementary to the corresponding target sequence adjacent to the SNP site. Each capture probe is immobilized on a different electrode having a non-conductive outer layer on a conductive working surface of a substrate. 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. These differences in the oxidation rates at the different electrodes are used to determine whether the selected nucleic acid target has a single nucleotide polymorphism at the selected SNP site.

1001731 The technique of Lynx Therapeutics (Hayward, Calif.) using MEGATYPE technology can genotype very large numbers of SNPs simultaneously from small or large pools of genomic material. This technology uses fluorescently labeled probes and compares the collected genomes of two populations, enabling detection and recovery of DNA fragments spanning SNPs that distinguish the two populations, without requiring prior SNP mapping or knowledge.

100174] A number of other methods for detecting and identifying SNPs exist. These include the use of mass spectrometry, for example, to measure probes that hybridize to the SNP. This technique varies in how rapidly it can be performed, from a few samples per day to a high throughput of many thousands of SNPs per day, using mass code tags. A preferred example is the use of mass spectrometric determination of a nucleic acid sequence which comprises the polymorphisms of the invention, for example, which includes the C-32 I G promoter polymorphism in the SCARB1 gene (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.

[001751 SNPs can also be determined by ligation-bit analysis. This analysis requires two primers that hybridize to a target with a one nucleotide gap between the primers. Each of the four nucleotides is added to a separate reaction mixture containing DNA polymerase, ligase, target DNA and the primers. The polymerase adds a nucleotide to the 3'end of the first primer that is complementary to the SNP, and the ligase then ligates the two adjacent primers together. Upon heating of the sample, if ligation has occurred, the now larger primer will remain hybridized and a signal, for example, fluorescence, can be detected. 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.

1001761 US Patent 6,821,733 (incorporated herein in its entirety) describes methods to detect differences in the sequence of two nucleic acid molecules that includes the steps of: contacting two nucleic acids under conditions that allow the formation of a four-way complex and branch migration; contacting the four-way complex with a tracer molecule and a detection molecule under conditions in which the detection molecule is capable of binding the tracer molecule or the four-way complex; and determining binding of the tracer molecule to the detection molecule before and after exposure to the four-way complex. Competition of the four-way complex with the tracer molecule for binding to the detection molecule indicates a difference between the two nucleic acids.

[00177] Protein-and proteomics-based approaches are also suitable for polymorphism detection and analysis. Polymorphisms which result in or are associated with variation in expressed proteins can be detected directly by analysing said proteins. This typically requires 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.

Proteornic methodologies are well known in the art, and have great potential for automation.

For example, integrated systems, such as the Proteom1Q 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.

[001781 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 ES! 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.

1001791 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.

[001801 It will be apparent that the presence or absence of the C allele or of the 0 allele at the C-321G promoter polymorphism in the SCARBI gene may also be determined by analysis of a polypeptide sample, derived from a bovine.

[00181] 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.

[00182] A large number of methods reliant on the conformational variability of nucleic acids have been developed to detect SNPs.

[001831 For example, Single Strand Conformational Polymorphism (SSCP, Orita et aL, PNAS 1989 86:2766-2770) is a method reliant on the ability of single-stranded nucleic acids to form secondary structure in solution under certain conditions. The secondary structure depends on the base composition and can be altered by a single nucleotide substitution, causing differences in electrophoretic mobility under nondenaturing conditions. The various polymorphs are typically detected by autoradiography when radioactively labelled, by silver staining of bands, by hybridisation with detectably labelled probe fragments or the use of fluorescent PCR primers which are subsequently detected, for example by an automated DNA sequencer.

[00184] Modifications of SSCP are well known in the art, and include the use of differing gel running conditions, such as for example differing temperature, or the addition of additives, and different gel matrices. Other variations on SSCP are well known to the skilled artisan, including,RNA-SSCp, restriction endonuclease fingerprinting-SSCP, dideoxy fingerprinting (a hybrid between dideoxy sequencing and SSCP), bi-directional dideoxy fingerprinting (in which the dideoxy termination reaction is performed simultaneously with two opposing primers), and Fluorescent PCR-SSCP (in which PCR products are internally labelled with multiple fluorescent dyes, may be digested with restriction enzymes, followed by SSCP, and analysed on an automated DNA sequencer able to detect the fluorescent dyes).

[00185) Other methods which utilise the varying mobility of different nucleic acid structures include Denaturing Gradient Gel Electrophoresis (DGGE), Temperature Gradient Gel Eleetrophoresis (TGGE), and Hetcroduplex Analysis (HEY). Here, variation in the dissociation of double stranded DNA (for example, due to base-pair mismatches) results in a change in electrophoretic mobility. These mobility shifts are used to detect nucleotide variations.

[00186) Denaturing High Pressure Liquid Chromatography (HPLC) is yet a further method utilised to detect SNPs, using HPLC methods well-known in the art as an alternative to the separation methods described above (such as gel electophoresis) 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, [00187) 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. For example, cleavage of mismatches within RNA:DNA heteroduplexes by RNase A, of heteroduplexes by, for example bacteriophage T4 endonuclease Yll or 17 endonuclease I, of the 5' end of the hairpin loops at the junction between single stranded and double stranded DNA by cleavase I, and the modification of mispaired nucleotides within heteroduplexes by chemical agents commonly used in Maxam-Gilbert sequencing chemistry, are all well known in the art.

[00188] Further examples include the Protein Translation Test (PIT), 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. Variations are detected by binding of, for example, the MutS protein, a component of Escherichia coli DNA mismatch repair system, or the human hMSH2 and GTBP proteins, to double stranded DNA heteroduplexes containing mismatched bases. DNA duplexes are then incubated with the mismatch binding protein, and variations are detected by mobility shift assay. For example, a simple assay is based on the fact that the binding of the mismatch binding protein to the heteroduplex protects the heteroduplex from exonuclease degradation.

[00189] 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 a ltered 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, for example where the SNP is associated with codons of varying usage and thus with tRNAs of differing abundance. Such altered expression can be determined by methods well known in the art, 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.

[00190] The above methods of detecting and identifying SNPs are amenable to use in the methods of the invention.

3 Polynucleotide and polypeptide variants [001911 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.

1001921 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.

[00193J 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.

[00194] 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.

[001951 The term "primer" refers to a short polynucleotide, usually having a free 3'OH 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 11, more preferably at least 12, more preferably at least 13, more preferably at least 14, more preferably at least 15, more preferably at least 16, more preferably at least 17, more preferably at least 18, more preferably at least 19, more preferably at least 20 nucleotides in length.

[00196] 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 10, more preferably at least 20, more preferably at least 30, more preferably at least 40, more preferably at least 50, more preferably at least 100, more preferably at least 200, more preferably at least 300, more preferably at least 400 and most preferably at least 500 nucleotides in length.

[00197] 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 reference polynucleotides or polypeptides. The term "variant" with reference to polynucleotides and polypeptides encompasses all forms of polynucleotides and polypeptides as defined herein.

3.1 Polynucleotide variants [00198] 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.

109199] 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:I/flp.ncbi.nih.govlblastl'). The default parameters of bl2seq are utilized except that filtering of low complexity parts should be turned off.

(00200] The identity of polynucleotide sequences may be examined using the following unix command line parameters: 1002011 bl2seq -i nucleotideseqi -j nucleotideseq2 -F F -p blastn [002021 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 [00203] 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/SoftwarelEMB OSS/. 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/alignl.

[002041 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.

[00205] 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://flp.ncbi.nih.govfblastl).

[002061 The similarity of polynucleotide sequences may be examined using the following unix command line parameters: [00207) bl2seq -i nucleotideseq 1 -j nucleotideseq2 -F F -p tblastx t002081 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.

[00209J Variant polynucleotide sequences preferably exhibit an E value of less than I x 10.10, more preferably less than I x 10.20, less than 1 x 10.30, less than I x 1040, less than I x 1 0°, less than I x 1 0°, less than I x 10.70, less than I x 10.80, less than I x 1 00, less than x 10.100, less than I x 101b0, less than 1 x 10.120 or less than I x lO when compared with any one of the specifically identified sequences.

1002101 Alternatively, variant polynucleotides of the present invention hybridize to a specified polynucleotide sequence, or complements thereof under stringent conditions.

[002111 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.

[00212J 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 a!., 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 lx ssc, 0.1% SDS at 65°C and two washes of 30 minutes each n 0.2X SSC, 0.1% SDS at 65°C.

1002131 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)°C.

100214] With respect to the DNA mimics known as peptide nucleic acids (PNAs) (Nielsen et al., Science. 1991 Dec 6;254(5037):1497-500) Tm values are higher than those for DNA-DNA or DNA-RNA hybrids, arid 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.

[00215] 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 TOG (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.

1002161 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).

1002171 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.

1002181 Polypeptide Variants [002191 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.

[002201 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/blastl). The default parameters of bl2seq are utilized except that filtering of low complexity regions should be turned off.

[002211 Polypeptide sequence identity may also be caJculated 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.uklemboss/align/) and GAP (Fluang, 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.

100222] Polypeptidc 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.govlblast/). The similarity of polypeptide sequences may be examined using the following unix command line parameters: bl2seq -i peptideseq 1 -j peptideseq2 -F F -p blastp 1002231 Variant polypeptide sequences preferably exhibit an E value of less than I x I 00, more preferably less than 1 x 1020, less than I x 10.30, less than 1 x 100, less than 1 X 1050, less than I x l0°, less than 1 x 10.70, less than 1 x 1080, less than 1 x less than 1 x10 100, less than 1 x less than I x 10120 or less than 1 x l0.123 when compared with any one of the specifically identified sequences.

1002241 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.

100225] Conservative substitutions of one or several amino acids of a described polypeptide sequence without significantly altering its biological activity arc 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).

[002261 A polypeptide variant of the present invention also encompasses that which is produced from the nucleic acid encoding a polypeptide, but differs from the reference 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 reference polypeptide.

4 Diagnostic kits 1002271 The invention further provides diagnostic kits useful in determining the bovine SCARBI allelic profile of bovine, for example for use in the methods of the present invention.

1002281 Accordingly, in one embodiment the invention provides a diagnostic kit which can be used to determine the SCARB I genotype of bovine 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 a non-reference allele at a polymorphism. Such a kit could also include a primer for amplifying the corresponding region of the reference gene, for example one that produces a reference SCARB1. 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 polymorphism of interest. The actual genotyping is carried out using primers that target specific alleles such as those described herein, and that could function as allele-specific oligonucleotides in conventional hybridisation, Taqman assays, OLE assays, etc. Alternatively, primers can be designed to permit genotyping by microsequencing.

1002291 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 SCARBI mutation. 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.

100230] For example, the kit may include a primer comprising a cytosine at the position corresponding to the C-321G promoter polymorphism in the SCARB1 gene or comprising a nucleotide capable of hybridising to a cytosine at the position corresponding to the C-32 10 promoter polymorphism in the SCARB I gene. Those skilled in the art will recognise that in such a primer, the cytosine, or the nucleofide 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.

1002311 In another example, the kit may include a primer comprising a guanine at the position corresponding to the C-32 1 G promoter polymorphism in the SCARB I gene, or comprising a nucleotide capable of hybridising to a guanine at the position corresponding to the C-32]G promoter polymorphism in the SCARBI gene. Those skilled in the art will recognise that in such a primer, the guanine, or the nucleotide capable of hybridising to a guanine, as applicable, may be substituted for a nucleotide analogue having the same discriminatory base-pairing as the substituted nucleotide.

1002321 In one embodiment, the diagnostic kit is useful in detecting DNA comprising a variant SCARBI gene or DNA or mRNA encoding a variant SCARBI polypeptide at least partially lacking reference activity in a bovine 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 portion of the DNA encoding SCARBI which results in increased 13-carotene levels (particularly increased J3-carotene content in milk fat), preferably wherein at least one of the nucleotide sequences is selected to be from a non-coding region of the reference SCARB 1 gene. The kit can also include a third primer complementary to a naturally occurring mutation of a coding portion of the reference SCARBI gene. Preferably the kit includes instructions for use, for example in accordance with a method of the invention.

100233] In one embodiment, the diagnostic kit comprises a nucleotide probe complementary to the sequence, or an oligonucleotide fragment thereof, shown in SEQ ID NO:l or SEQ ID NO:3, for example, for hybridisation with mRNA from a sample of cells; means for detecting the nucleotide probe bound to rnRNA 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 SEQ ID NO: br SEQ ID NO:3 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, NY (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 (<lOObp), lower temperatures must be used even with 100% homology. In general, one starts washing at low temperatures (3 7°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.

[002341 In another embodiment, the diagnostic kit comprises an antibody or an antibody composition useful for detection of the presence or absence of reference SCARB I, or determining the presence or absence of a variant protein at least partially lacking reference activity, or determining the level of expression of the SCARB1 protein, together with instructions for use, for example in a method of the invention.

Sample preparation 100235] 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.

1002361 Conveniently, samples may be taken from milk, 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 100237] It will also be appreciated that the methods of the invention are amenable to use with and the results analysed by computer systems, software arid processes. Computer systems, software and processes to identif' 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.

1002381 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 thcy 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 I -3 and/or the Sequence H) Listing.

1002391 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 flo ppy discs, hard disc storage medium, arid 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 SN? 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.

1002401 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.

1002411 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 0B2, 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.

100242J 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 (Aitsehul et at, J. Mol. Biol. 215:403-410 (1990)) and BLAZE (Brutlag et at, Comp. Chem. 17:203-207 (1993)) search algorithms.

[00243j 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 SN? 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 storedlanalyzed 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 SN? haplotypes with response to particular drugs, or for various other bioinformatic, pharmacogenomic, drug development, or selection or identification applications.

[002441 As used herein, "a computer-based system" refers to the hardware, software, and data storage used to analyze the SN? 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.

[002451 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.

[002461 The one or more programs or algorithms are implemented on the computer-based system 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.

100247] 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 andior 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.

1002481 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.

1002491 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 bovine with respect to one or more milk or tissue colour or 13-carotene content traits and optionally a reference milk or tissue colour or 13-carotene content traits database of non-genetic factors for bovine milk or tissue colour or 13-carotene content traits; 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 merit of the bovine, said outcome being communicable once known, preferably to a user having input said data.

[00250] Preferably, said system is accessible via the internet or by personal computer.

[00251] 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 tissue colour or 13-carotene content traits, 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 tissue colour or 13-carotene content traits.

[002521 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 tissue colour or 13-carotene content traits 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 bovine milk or tissue colour or 13-carotene content traits.

[002531 Preferably, the one or more genetic loci are one or more polymorphisms in one or more genes associated with one or more milk or tissue colour or 13-carotene content traits.

100254] 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 skiUed in the art, being possible without departing from the scope thereof as defined in the accompanying claims.

1002551 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.

1002561 The invention consists in the foregoing and also envisages constructions of which the following gives examples only.

EXAMPLE -Analysis of the genetic basis for milk fat colour 1002571 This example describes the investigation of the genetic basis for observed variations in milk fat colour using the results of a Hoistein-Friesian X Jersey cross-bred trial conducted to facilitate the discovery of QTLs, genes and mutations associated with economically important milk traits.

Materials and methods 1. Trial design 1002581 A Hoistein-Friesian x Jersey crossbred trial was conducted using an F2 trial design with a half-sibling family structure. Reciprocal crosses of Hoistein-Friesian and Jersey animals were carried out to produce six Fl bulls of high genetic merit. 850 F2 female progeny forming the basis of the trial herd were then produced through mating of high genetic merit 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 (during which milk fat colour was measured).

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. Milk fat colour measurement 100259] All milk measurements were taken during the animals' second lactation. Cows were milked twice daily; milk volume was recorded at each milking. Milk fat colour 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. Milk fat colour was measured as previously described (Winkelman et a!., 1999). Briefly, nonsaponifiable material (including carotenoids) was extracted from fresh milk samples and the absorbance at 450nm was measured. Fat colour (p.ig 3-carotene/mg milk fat) was calculated (Winkelman et al., 1999).

3. Genotyping [002601 Genomic DNA was prepared from whole blood from a total of 1665 animals within the trial pedigree (850 F2 daughters, six Fl sires, 796 Fl dams, and 13 selected FO 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 genotyped using the Affymetrix Bovine 10K SNP GeneChip. A total of 6634 informative SNP markers were placed on the map.

4. Candidate gene sequencing [002611 SCARBI was identified as a candidate gene for the milk fat colour QTL on chromosome 17. Intron/exon boundaries were determined using the UniGene bovine Bt.4520.

The promoter sequence was amplified using the primers presented as SEQ ID NOS: 5 and 6.

Exons were amplified using the primers presented as SEQ ID NOS: 7-44 and sequenced in both directions. The reference gene sequence for the SCARBI gene is shown in SEQ ID NO.

1. In addition to coding regions, an additional 1.2 kb of 5' untranslated region was also sequenced. The predicted structure of the bovine gene is shown in Figure 3. Primers were designed within introns so that complete sequence was obtained from each exon.

5. Statistical analysis [00262J The dataset consisted of milk fat colour phenotypes collected during the animals' second lactation for two cohorts of F2 animals. Data manipulation was performed using SAS (version 9.1). Phenotype data for milk fat colour was recorded at three time points during lactation (peak, mid and late lactation). These data were matched with the following covariates: cohort (cohort I or cohort 2), sire (sires I -6), milk fat%, milk protein%, lactose%, milk solids%, milk yield, condition score, live weight (average taken for � seven days around each of the milk fat colour time points at peak, mid and late lactation), somatic cell count (threshold of 200,000 cells during � seven days around sampling times), free fatty acids (as an indicator of milk fat quality, measured in the same sample as milk fat colour at peak, mid and late lactation), calving week, and estrus week. Animals with missing data points for any of the measurements were excluded and the final datasets included 597, 648 and 632 observations at peak, mid and late lactation, respectively.

[002631 Analyses were conducted using both raw and log-transformed data. ANOVA was conducted for the milk fat colour phenotype at peak, mid and late lactation. The final ANOVA models for each of the lactations 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). Thus, the final models were as follows: peak lactation (sire, cohort, protein%, calving week), mid lactation (sire, cohort) and late lactation (sire, cohort, protein%, milk solid%, and somatic cell count).

6. QTL detection [00264] The data used for QTL detection were the residuals from each model for both non-transformed and log- transformed data. The raw phenotype data (no covariates or modeling) was also used to detect QTLs. Since the same results were detected with each kind of data, results presented below used non-transformed, modeled data. QTL detection was conducted using a line of descent model (Haley et al. 1994) and a half-sib model (Baret et al. 1998). Subsequently, the SCARB I polymorphism was included as a covariate into the models for peak, mid and late lactation, and ANOVA was performed to test for an association between the SCARB1 mutation arid [3-carotene concentration of milk.

7. Tissue SCARBI mRNAmeasurements 1002651 Subcutaneous adipose tissue biopsies (-500mg tissue) and liver biopsies (-100 mg tissue) were taken from a subset of 40 cows (8 CC animals, 21 CO animals and 11 GG animals). Total RNA was isolated using the Qiagen RNeasy method, according to manufacturer's instructions. RNA yields were quantified by absorbance at 260nm and RNA integrity was determined using the Agilent Bioanalyser. SCARB I mRNA was determined by quantitative real time PCR using the Roche Universal Probe library system. An Intron spanning assay was used to prevent amplification of any contaminating genomic DNA. The assay was designed using SEQ ID NO: 3 as a reference sequence. The details of the primers (SEQ ID NOs: 42 and 43) and amplicon sequence (SEQ ID NO:44) are presented in Table I below. Universal probe number 62 was used for the assay (sequence cagcaggl).

Table 1: Details of SCARB1 quantitative real time PCR assay Primer Length Tm %GC Sequence Left 18 59 50 tggtgccctcaatcatca [SEQ ID NO:42] Right 22 60 45 catgttgaaagacaggctgttg [SEQ ID NO:43] Amplicon 74 tggtgccctcaatcatcaagcagcaggtcctcaagaatgtgcgcatcgac _________ _______ ______ cccaacagcctgtctttcaacatg [SEQ ID NO:44J 1002661 Results are expressed as fold-change relative to animal single reference sample (this animal was not included in the subsequent analysis). Genotype effects were determined by ANOVA, using genotype as a fixed effect, and the significance level was set at p = 0.05.

(002671 p-carotene adipose tissue measurements were determined using IIPLC with commercial standards, based on a published method (Huishof et al., 2006). The inter-assay variation was on average 5%.

Resu1t 1. Detection of a major QTL for milk fat colour on bovine chromosome 17 [00268] Analysis of the n-carotene data within the line of descent model of QTL analysis showed the presence of a significant QTL on bovine chromosome 17 (Figure 2). The maximum F value for the QTL was 10.6, and the most likely position was estimated at 71 cM.

2. Identification of SCARB1 as a candidate gene and detection of a polymorphism [00269] The SCARBI gene was thought to be approximately located between markers BMS1879 at 50.7 cM and BM1862 at 67.5 cM on bovine chromosome 17. To determine whether the SCARBI gene was involved in the observed variation in milk colour and 1-carotene content, the SCARB 1 promoter and coding regions were sequenced in the six Fl sires to identif' any genetic polymorphisms that could potentially alter the fimction, activity, or expression of this protein. The predicted structure of the bovine gene is shown in Figure 3, andinSEQIDNOs:l and3.

[002701 One polymorphism in the bovine SCARB 1 gene was identified. A substitution of C to 0 at genomic nucleotide position -321 (see SEQ ID NO:!) in the promoter of the SCARB 1 gene was heterozygous in five Fl sires. The remaining sire was homozygous for the 0 allele. To determine whether this polymorphism was associated with the milk colour and 13-carotene content phenotypes, the remainder of the FJXB trial pedigree was genotyped.

The frequency of each SCARB1 genotype in the F2 population is shown in Table 2 below.

Table 2: Genotype frequencies of F2 population Genotype Total CC 194 23.52 CG 403 48.84 OG 228 27.64 Total 825 100.00 [00271] The SCARB 1 genotype was subsequently introduced into the statistical model as a fixed effect. As shown in Figure 5, this reduced the QTL significance (F-value), showing a close association of the SCARB1 genotype with the milk fat colour QTL variation.

3. SCARB1 polymorphism has a significant effect on n-carotene concentration in milk (00272] The effect of the SCARB 1 polymorphism on milk fat colour is shown in Table 3 below and in Figure 4.

Table 3: Genotype effect on milk fat colour Milk fat colour genotype N Peak Lactation N Mid Lactation N Late Lactation 116 160 9.52 152 7.55 293 10.87 320 8.91 298 7.19 178 10.22 186 8.81 174 6.73 [00273] Animals homozygous for the C allele produce milk with greater milk fat colour and greater concentrations of 13-carotene than heterozygous (CG) animals. Furthermore, heterozygous animals produce milk with greater milk fat colour and greater concentrations of 13-carotene than animals homozygous for the G allele. The genotype effect was similar at each stage of lactation.

4. SCARB1 polymorphism has a significant effect on SCARB1 mRNA levels in liver tissue [00274] The effect of the SCARBI polymorphism on SCARB1 mRNA levels in liver tissue is shown in Figure 7. SCARBI mRNA levels were measured in liver samples from a subset of 40 F2 cows. The relative expression level of SCARB I mRNA was significantly lower in liver tissue from animals homozygous for the C allele. Heterozygous animals had intermediate expression of SCARB I mRNA, and animals homozygous for the G allele had the highest relative expression of SCARB 1 mRNA.

5. SCARB1 polymorphism has a significant effect on adipose tissue 13-carotene content.

[00275] The effect of the SCARB I polymorphism on adipose tissue 13-carotene content is shown in Figure 6. Animals homozygous for the C allele had higher concentrations of b-carotene in adipose tissue than heterozygous animals or animals homozygous for the G allele.

Discussion [00276] The present invention recognises that the SCARB1 polymorphism described above is useful as a selection tool to breed animals with higher or lower milk concentrations of 13-carotene, with increased or decreased milk fat colour, or with increased or decreased tissue colour or 13-carotene content. Such a strategy may allow the production of, for example, milk products more suitable to particular markets, depending on the preference for white or yellow milk and milk products, or the dietary or health requirements prevalent in a market.

Publications Ausubel et al., 1987, Current Protocols in Molecular Biology, Greene Publishing.

Baret P., S. Knott and P. Visscher, 1998 On the use of linear regression and maximum likelihood for qtl mapping in half-sib designs. Genet. Res. 72: 149-158.

Bolton and McCarthy, PNAS 1962, 84:1390.

Bowie et al., Science 1990, 247, 1306.

Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989).

Devlin and Risch 1995; A comparison of linkage disequilibrium measures for fine-scale mapping, Genomics 29: 311-322.

During and Harrison, J Lipid Research 2007 Oct.48(l0) 2283-94.

Giesen et a)., Nucleic Acids Res. 1998, Nov l;26(21):5004-6.

Haley C.S., S. Knott, J. Elsen, 1994 Mapping quantitative trait loci in crosses between outbred lines using least squares. Genetics 136: 1195-1207.

Huang, X., On Global Sequence Alignment. Computer Applications in the Biosciences 1994, 10, 227-235.

Huishof P., 1. van Roeke-Jansen, P. van de Bovenkamp, C. West, 2006 Variation in retinol and carotenoid content of milk and milk products in the Netherlands. J Food Comp. Anal.

19: 67-75.

Kiefer C, Sumser E, Wemet M1F, Von Lintig J.,A class B scavenger receptor mediates the cellular uptake of carotenoids in Drosophila.Proc Natl Acad Sci USA. 2002 Aug 6;99(16):1058l-6.

Mullis et a!., Eds. 1994 The Polymera.se Chain Reaction, Birkhauser.

Needleman, S. B. and Wunsch, C. D., J. Mol. Biol. 1970, 48, 443-453.

Nielsen et al., Science 1991, Dec 6;254(5037):1497-500.

Onta et a!., PNAS 1989, 86:2766-2770.

Reich DE et al; Linkage disequilibrium in the human genome, Nature 2001, 411:199-204.

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.

Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press.

Tatiana A. Tatusova, Thomas L. Madden, Blast 2 sequences -a new tool for comparing protein and nucleotide sequences, FEMS Microbiol Lett. 1999, 174:247-250.

Winkelman et al., Estimation of Heritabilities and Correlations Associated with Milk Color Traits, J Dairy Sci. 1999, 82:215-224.

INDUSTRIAL APPLICATION

[00277J The present invention is directed to methods of genotyping bovine to facilitate the selection of animals with altered milk or tissue colour or 13-carotene content traits. In particular, such traits include desired milk fat colour or 13-carotene content, or desired tissue colour or 13-carotene content. It is anticipated that herds of bovine selected for such traits will produce milk fat and tissue of more desirable colour (whether more or less yellow), or more desirable 13-carotene content, and therefore be of significant economical benefit to farmers.

SEQUENCE LISTING

<110> Vialactia Biosciences (NZ) Limited <120> arker Assisted Selection of Bovine for Desired t'411k or Tissue Content <130> 566948 JBM <160> 44 <170> Patentln version 3.2 <210> 1 <211> 92944 <212> DNA <213> Bos taurus <221> variant <222> (1188).. (1188) <223> -321 C/G polymorphism <221> CDS <222> (1509).. (1634) <221> misc feature <222> (27335).. (27384)

Claims

1. A method of determining genetic merit of a bovine with respect to milk or tissue colour or 13-carotene content or with respect to capability of producing progeny that will have increased or decreased milk or tissue colour or 13-carotene content, which comprises the step of determining the SCARBI allelic profile of said bovine, and determining the genetic merit of the bovine on the basis of the SCARB 1 allelic profile.

2. A method as claimed in claim I, wherein the milk 13-carotene content is milk fat 13-carotene content.

3. A method as claimed in claim 1, wherein the allelic profile is determined with respect to DNA, mRNA andlor protein obtained from said bovine.

4. A method as claimed in claim 1, wherein the allelic profile is determined by determining the presence or absence of the C allele at the C-32 1 G promoter polymorphism of the bovine SCARBI gene.

5. A method as claimed in claim I, wherein the allelic profile is determined by determining the presence or absence of the G allele at the C-32 1 G promoter polymorphism of the bovine SCARB1 gene.

6. A method as claimed in claim 4 or claim 5 wherein the allelic profile of the C-321G promoter polymorphism in the SCARB1 gene is determined by the use of one or more polymorphisms in linkage disequilibrium with this polymorphism.

7. A method as claimed in claim I wherein the allelic profile is determined by determining the expression or activity of a SCARB I gene or gene product.

8. A method of identifying or selecting a bovine having a desired SCARBI allelic profile comprising determining the allelic profile according to the method of any one of claims I to 7 and identifying or selecting said bovine on the basis of the determination.

9. A bovine selected by the method of claim 8.

10. A method of determining genetic merit of a bovine with respect to milk or tissue colour or 13-carotene content, or with respect to capability of producing progeny that will have increased or decreased milk or tissue colour or 13-carotene content, the method comprising providing data about the SCARBI allelie profile of said bovine, and determining the genetic merit of the bovine on the basis of the data. c

11. A method for identifying or selecting a bovine with respect to milk or tissue colour or 3-carotene content, or with respect to capability of producing progeny that will have one or more desired milk or tissue colour or n-carotene content traits, the method comprising providing data about the SCARB1 allelic profile of said bovine, and identifying or selecting the bovine on the basis of the data.

12. A method as claimed in claim 10 or 11, wherein the the SCARBI allelic profile comprises data indicative of the presence or absence of one or more alleles at one or more polymorphisms which affect expression from the SCARB I gene or the expression or activity of a SCARB 1 gene product or which are associated with increased or decreased expression from the SCARB I gene or with increased or decreased expression or activity of a SCARB 1 gene product, or one or more polymorphisms in linkage disequilibrium with one or more of said polymorphisms.

13. A method as claimed in claim 12, wherein the one or more polymorphisms is in the SCARB1 gene.

14. A method as claimed in claim 12 or claim 13, wherein the one or more polymorphisms is one or more polymorphisms selected from the group comprising the C-32 10 promoter polymorphism in the SCARB 1 gene, or one or more polymorphisms which are in linkage disequilibrium with the C-321G promoter polymorphism in the SCARB I gene.

15. A method as claimed in any one of claims 10 to 14, wherein the milk or tissue colour or 13-carotene content is increased milk colour or increased milk 13-carotene content.

16. A method as claimed in any one of claims 10 to 14, wherein the milk or tissue colour or 13-carotene content is increased tissue colour or increased tissue 13-carotene content.

17. A method as claimed in claim 15 or claim 16 comprising determining (a) the presence of the C allele at the C-321G promoter polymorphism in the SCARBI gene, or (b) the absence of the G allele at the C-321G promoter polymorphism in the SCARB I gene, or (c) both (a) and (b), and identifying or selecting the bovine on the basis of the determination.

18. A method as claimed in any one of claims 10 to 14, wherein the milk or tissue colour or 13-carotene content is decreased milk colour or decreased milk 13-carotene content.

19. A method as claimed in any one of claims 10 to 14, wherein the milk or tissue colour or 13-carotene content is decreased tissue colour or decreased tissue (3-carotene content.

20. A method as claimed in claim 18 or claim 19 comprising determining (a) the absence of the C allele at the C-321G promoter polymorphism in the SCARBI gene, or (b) the presence of the G allele at the C-32 10 promoter polymorphism in the SCARB 1 gene, or (c) both (a) and (b), and identifying or selecting the bovine on the basis of the determination.

21. A method as claimed in claim 14, further comprising the step of amplifying at least a fragment of the bovine SCARB 1 gene sequence to determine the presence or absence of one or more polymorphisms associated with increased or decreased expression or activity of a SCARBI gene product.

22. A method as claimed in claim 21, wherein the primers used in the amplification are selected from the group consisting of SEQ ID NOs: 5 to 44.

23. A method as claimed in claim 12 or claim 13 wherein the presence or absence of one or more polymorphisms associated with increased or decreased expression or activity of SCARB I gene product is determined by determining the expression or activity of a SCARB I gene or gene product.

24. A probe or primer comprising a nucleotide sequence having about at least 12 contiguous bases of SEQ ID NO: I or SEQ ID NO: 3.

25. The probe or primer of claim 24 comprising a cytosine at the position corresponding to the C-32 10 promoter polymorphism in the SCARB I gene, or comprising a nucleotide capable of hybridising to a cytosine at the position corresponding to the C-32 10 promoter polymorphism in the SCARB 1 gene.

26. The probe or primer of claim 24 comprising a guanine at the position corresponding to the C-3210 promoter polymorphism in the SCARBI gene, or comprising a nucleotide capable of hybridising to a guanine at the position corresponding to the C- 321 G promoter polymorphism in the SCARB 1 gene.

27. A probe or primer having about at least 2 contiguous bases of one of SEQ 1D NOs: 5-44.

28. A probe or primer as claimed in claim 24 comprising a nucleotide sequence having at least about 12 contiguous bases of SEQ ID NO: 1 or SEQ ID NO: 3 wherein the about 12 contiguous bases comprise or are within about I to about 2000 nucleotides of the C-321G promoter polymorphism in the SCARBI gene.

29. A pair of primers comprising two primers as claimed in claim 28.

30. A bovine identified by the method of any one of claims II to 23.

31. A bovine as claimed in claim 9 or 30, wherein the bovine is a bull.

32. Collected semen produced by a bovine as claimed in claim 31.

33. A bovine as claimed in claim 9 or 30, wherein the bovine is a cow.

34. A method of selecting a herd of bovine, comprising selecting individuals by the method of arty one of claims 8 or 11 to 23, and segregating and collecting the selected individuals to form the herd.

35. A herd of bovine selected by the method of claim 34.

36. A herd of bovine comprising two or more bovine, wherein the bovine are the progeny of one or more bovine selected by the method of any one of claims 8 or 11 to 23.

37. Collected or pooled milk produced by bovine as claimed in claim 33.

38. Collected or pooled milk produced by bovine as claimed in claim 35.

39. Collected or pooled milk as claimed in claim 37 or 38 having increased or decreased milk colour or increased or decreased 1-carotene content when compared to milk produced by a bovine having a SCARBI gene comprising the nucleotide sequence of SEQ ID NO: 1.

40. A dairy product made from the milk as claimed in any one of claims 37 to 39.

41. A kit for genotyping a bovine with respect to one or more milk or tissue colour or carotene content traits, comprising a probe or primer as defined in any one of claims 24 to 28 or a pair of primers as defined in claim 29.

42. An isolated, purified or recombinant nucleic acid molecule comprising nucleotide sequence selected from (a) at least 12 contiguous nucleotides of SEQ ID NO:l and comprising the C-321G promoter polymorphism; or (b) any one or more of SEQ ID NOs:5 -44; or (c) a complement of(a) or (b); or (d) a sequence of at least 12 contiguous nucleotides and capable of hybridising to the nucleotide sequence of any one of (a) to (c) under stringent conditions

43. A vector comprising the nucleic acid of claim 42.

44. A method of determining genetic merit of a bovine with respect to milk or tissue colour or 13-carotene content, or with respect to capability of producing progeny that will have increased or decreased milk or tissue colour or 13-carotene content, the method comprising determining milk or tissue colour or 13-carotene content of the bovine, determining the SCARBI allelic profile of the bovine, comparing the SCARBI allelic profile of the bovine or the milk or tissue colour or 13-carotene content of the bovine with that of a bovine having a known SCARB1 allelic profile; determining the genetic merit of the bovine on the basis of the comparison.