GB2455657A

GB2455657A - b-carotene 15,15-monooxygenase for genotyping bovine milk for colour or b-carotene content

Info

Publication number: GB2455657A
Application number: GB0823253A
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 Oxley; Elizabeth Marie Beattie
Original assignee: ViaLactia Biosciences NZ Ltd
Current assignee: ViaLactia Biosciences NZ Ltd
Priority date: 2007-12-21
Filing date: 2008-12-19
Publication date: 2009-06-24
Also published as: AU2008261149A1; GB0823253D0

Abstract

The present invention provides methods of genotyping bovine for desired milk production phenotypes, such as milk colour or milk b -carotene content phenotypes, by determining the b -carotene 15,15'-monooxygenase (BCMO1) genotype of said bovine. In particular, the methods are directed to determining the genotype at one or more of the C-1054T promoter polymorphism in the BCMO1 gene, the G15929A (G278R) polymorphism in the BCMO1 gene, or the A18068G (N341D) polymorphism in the BCMO1 gene, wherein each is associated with variation in milk colour or milk b -carotene content. Isolated, purified or recombinant polynucleotides and polypeptides are also provided, as are milk and milk products from bovine selected by the methods described herein.

Description

I

MARKER ASSISTED SELECTION OF BOVINE FOR DESiRED MILK CONTENT

FIELD OF THE INVENTION

[00011 This invention relates to aji application of marker assisted selection of bovine for a quantitative trait loci (QTL) associated with milk colour and 13-carotene content, particularly by assaying for the presence of polymorphisms in a gene which is associated with the QTL.

BACKGROUND

(00021 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 fanning 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.

(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, PCT International application PCTINZOI/00245 (published as W002/36824) reports that polymorphisms in the bovine Diacylglycerol-o-acyltransferaSe 1 (DGATI) 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 lnternational application PCTINZO2/00i57 (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.

(00041 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 (3-carotene 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).

100051 Strategies to modulate milk colour or content (for example 13-carotene content) could provide health benefits and are expected to be economically valuable. 13-carotene and vitamin A deficiencies are still major health problems (jarticularly in developing countries) leading to blindness and childhood mortality. Milk with increased 13-carotene content would be of benefit, for example in markets where other dietary sources of 13-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 arc 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 content, particularly milk 13-carotene content; and/or to provide animals selected using the method of the invention as well as milk produced by the selected animals; and/or to provide the public with a useful choice.

SUMMARY OF THE INVENTION

f0008J This invention relates to the elucidation of the role of the gene encoding 13-carotene 15', 15'-monooxygenase I (BCMOI) [EC:1.14.99.36] in milk colour or 13-carotene content, particularly milk fat colour and 13-carotene content. In particular, the invention relates to the identification of the C-1054T polymorphism in the promoter of the BCMO1 gene, of the G15929A (G278R) polymorphism in exon 6 of the BCMO1 gene, and of the A18068G (N341D) polymorphism in exon 7 of the BCMO1 gene, and their association with variations in milk colour or content, for the first time. This includes the association of each of the T allele at the C-1054T polymorphism, the G allele at the G15929A (G278R) polymorphism, and the G allele at the A18068G (N341D) polymorphism with production of milk fat with increased 13-carotene content, for the first time. Furthermore, this includes the association of each of the C allele at the C-1054T polymorphism, the A allele at the G15929A (G278R) polymorphism, and the A allele at the A18068G (N341D) polymorphism with production of milk fat with decreased f3-carotene content, for the first time.

100091 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 colour or 13-carotene content or with respect to capability of producing progeny that will have increased or decreased milk colour or 13-carotene content, which comprises determining the BCMOI allelic profile of the bovine, and determining the genetic merit of the bovine on the basis of the BCMOI allelic profile.

100111 In one embodiment, milk content is milk fat content, more preferably milk fat 13-carotene content.

100121 In one embodiment, milk colour is milk fat colour.

100131 In one embodiment, the genetic merit with respect to milk colour or 13-carotene content is production of milk with increased colour or 13-carotene content, preferably production of milk with increased yellow colour.

100141 In one embodiment, the genetic merit with respect to milk colour or 13-carotene content is capability of producing progeny that will have increased milk colour or milk 13-carotene content.

100151 In various embodiments, the BCMOI allelic profile is determined by determining the expression or activity of a BCMO I gene product. It will be appreciated that methods comprising determining the expression or activity of a BCMOI gene product encompass determining expression from or of a BCMO1 gene.

100161 Accordingly, in various embodiments the invention provides a method for identifying or selecting a bovine that produces milk with increased 13-carotene content, or that is capable of producing progeny that produce milk with increased 13-carotene content, comprising determining the expression or activity of a BCMO 1 gene product, and identifying or selecting the bovine on the basis of the determination.

100171 In another embodiment, the genetic merit with respect to milk colour or 13-carotene content is production of milk with decreased colour or 13-carotene content, preferably production of milk with decreased yellow colour.

100181 In a further embodiment, the genetic merit with respect to milk colour or 13-carotene content is capability of producing progeny that will have decreased milk colour or milk 13-carotene content.

100191 Accordingly, in various embodiments the invention provides a method for identifying or selecting a bovine that produces milk with decreased f'-carotene content, or capable of producing progeny that produce milk with decreased 3-carotene content, comprising determining the expression or activity of a BCMOI gene product, and identifying or selecting the bovine on the basis of the determination.

(00201 In one embodiment, expression or activity of the BCMOI gene product is determined using BCMOI mRNA, for example by determining the presence or amount of BCMOI mRNA. In other embodiments, expression or activity of the BCMO1 gene product is determined using BCMO1 protein, preferably by determining the presence or amount of BCMOI protein, for example the amount of BCMO1 protein, or by determining the activity of BCMOI protein, for example the enzymatic activity of BCMOI protein present in a sample obtained from the bovine. In still other embodiments, the expression or activity of a BCMOI gene product is determined using BCMOI DNA, preferably by determining the presence or absence of one or more polymorphisms associated with decreased or increased BCMOI 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.

10021] In another embodiment, the BCMOI allelic profile of the bovine is determined together with the allelic profile of the bovine at one or more genetic loci associted with milk colour or p3-carotene content.

100221 In one embodiment, the one or more genetic loci is one or more polymorphisms in one or more genes associated with milk colour or 3-carotene content.

100231 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.

10024] 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.) (0025J It will be apparent that as used herein, the phrase "BCMO1 allelic profile" contemplates data indicative of the presence or absence of one or more alleles at one or more polymorphisms in the BCMOI gene or which affect expression from the BCMOI gene or the expression or activity of a BCMOI gene product or which are associated with variation in the expression from the BCMOI gene or in the expression or activity of a BCMOI gene product.

In preferred embodiments, the BCMOI allelic profile comprises data indicative of the presence or absence of one or more alleles at one or more polymorphisms associated with increased or decreased milk colour or J3-carotene content. For example, in preferred embodiments the BCMOI allelic profile comprises data indicative of the presence or absence of the C allele or of the presence or absence of the T allele at the C-10541 polymorphism, or data indicative of the presence or absence of the A allele or of the presence or absence of the O allele at the 015929A (G278R) polymorphism, or data indicative of the presence or absence of the A allele or of the presence or absence of the 0 allele at the A18068G (N341D) polymorphism in the BCMO1 gene. In other embodiments, the BCMOI 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 BCMOI gene, or in a regulatory region of the BCMO1 gene, or in an intron of the BCMO1 gene, or in a coding region of the BCMOI gene, and preferably comprises data indicative of the presence or absence of one or more alleles which affect expression from the BCMOI gene or the expression or activity of a BCMO1 gene product or which are associated with variation in the expression from the BCMOI gene or in the expression or activity of a BCMOI gene product.

100261 In one embodiment, the BCMO1 allelic profile consists of data indicative of the presence or absence of the C allele or of the presence or absence of the T allele at the C- 10541 polymorphism, or data indicative of the presence or absence of the A allele or of the presence or absence of the G allele at the G 1 5929A (G278R) polymorphism, or data indicative of the presence or absence of the A allele or of the presence or absence of the 0 allele at the A18068G (N341D) polymorphism in the BCMO1 gene, or of any combination of such data.

100271 It will further be appreciated that the BCMOI allelic profile may comprise information correlating the presence or absence of one or more polymorphisms as described above with milk colour or J-carotene content.

100281 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.

100291 In yet a further embodiment, the allelic profile is determined with reference to the amino acid sequence of BCMO I protein obtained from said bovine.

100301 In another embodiment, the allelic profile is determined with reference to the amount or activity ofBCMOI protein obtained from said bovine.

100311 Conveniently, in said method the presence or absence of DNA encoding a wild type BCMOI gene product, or of nucleotide sequence comprising a wild type BCMO1 gene, in said bovine is determined, directly or indirectly, for example using an expressed BCMOI gene product.

100321 Alternatively, in said method the presence or absence of at least one nucleotide difference from the nucleotide sequence of a wild type BCMOI gene, for example, at least one nucleotide difference from the nucleotide sequence encoding wild type BCMO1, in said bovine is determined, directly or indirectly.

100331 More specifically, in said method the presence or absence of one or more of the group comprising the C allele at the C-1054T promoter polymorphism in the bovine BCMOI gene, the T allele at the C-l054T promoter polymorphism in the bovine BCMOI gene, the G allele at the G15929A (G278R) polymorphism in the bovine BCMOI gene, the A allele at the G15929A (G278R) polymorphism in the bovine BCMOI gene, the A allele at the A18068G (N341D) polymorphism in the bovine BCMOI gene, and the G allele at the A18068G (N341D) polymorphism in the bovine BCMO1 gene, is determined, directly or indirectly.

100341 For example, the presence of the C allele or the T allele at the C-1054T promoter polymorphism may be determined using a polymorphism in linkage disequilibrium with the C allele or with the T allele at the C-I 054T promoter polymorphism. Similarly, the presence of the A allele or the 0 allele at the G15929A (G278R) polymorphism may be determined using a polymorphism in linkage disequilibrium with the A allele or with the G allele at the G 1 5929A (G278R) polymorphism. Likewise, the presence of the A allele or the G allele at the A18068G (N341D) polymorphism may be determined using a polymorphism in linkage disequilibrium with the A allele or with the G allele at the A18068G (N341D) polymorphism.

100351 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 wild type BCMOI is present, or whether a sequence of the DNA encoding an allelic protein "(B)" at least partially lacking the activity of (A) is present, or whether a sequence of the DNA encoding (A) and a sequence of the DNA encoding (B) are both present. The absence of the DNA encoding (A) and the presence of the DNA encoding (B) indicates an association with high relative 13-carotene levels, particularly with the production of milk with, infer alia, increased fat colour. The reverse association holds true, where the presence of the DNA encoding (A) and the absence of the DNA encoding (B) indicates an association with low relative 13-carotene levels, particularly with the production of milk with, inter alia, decreased fat colour. The presence of both the DNA encoding (A) and the DNA encoding (B) indicates an association with intermediate relative 13-carotene levels, particularly with the production of milk with, inter a/ia, intermediate fat colour.

100361 As used herein, biological activity of wild type BCMO1 protein refers to both expression levels and activity characteristic of BCMOI protein expressed from the wild type BCMOI gene.

100371 In another embodiment, the method includes ascertaining, from a sample of material containing DNA obtained from the bovine, whether a wild type BCMOI 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 BCMO1 gene product, preferably by determining the presence or absence of one or more polymorphisms associated with decreased or increased BCMOI expression, for example one or more promoter polymorphisms associated with increased or decreased expression.

100381 In one embodiment, this method includes ascertaining, from a sample of material containing mRNA obtained from the bovine, whether inRNA encoding a protein "(A)" having biological activity of a wild type BCMO1 is present, or whether mRNA encoding a variant protein "(B)" at least partially lacking the activity of (A) is present, or whether mRNA encoding (A) and mRNA encoding (B) are both present. The absence of the mRNA encoding (A) and the presence of the mRNA encoding (B) again indicates an association with high relative 13-carotene levels, particularly with the production of milk with, inter alia, increased fat colour. The reverse association again holds true. Again, the presence of both the mRNA encoding (A) and the mRNA encoding (B) indicates an association with intermediate relative 13-carotene levels, particularly with the production of milk with, inter alia, intermediate fat colour.

100391 In another embodiment, the method includes ascertaining the amount of BCMOI mRNA present in a sample of material containing mRNA obtained from the bovine.

100401 In another embodiment, the method includes ascertaining, from a sample of material containing protein obtained from the bovine, whether a protein "(A)" having biological activity of a wild type BCMOI is present, or whether a variant protein "(B)" at least partially lacking the activity of (A) is present, or whether (A) and (B) are both present.

The absence of(A) and the presence of(B) again indicates an association with high relative 13-carotene levels, particularly with the production of milk with, inter a/ia, increased fat colour andlor high relative 13-carotene content. The reverse association again holds true. Further, the presence of both (A) and (B) indicates an association with intermediate relative 13-carotene levels, particularly with the production of milk with, inter a/ia, intermediate fat colour.

100411 In another embodiment, the method includes ascertaining the amount or activity of BCMO1 protein present in a sample of material containing protein obtained from the bovine.

[0042J In a further embodiment, the invention provides a method of determining genetic merit of a bovine with respect to milk 13-carotene content which comprises determining the BCMOI allelic profile of the bovine, together with determining the allelic profile of the bovine at one or more genetic loci associated with milk 13-carotene content.

100431 In one embodiment, the one or more genetic loci is one or more polymorphisms in one or more genes associated with milk 13-carotene content, preferably one or more polymorphisms in one or more genes involved in 13-carotene uptake or metabolism.

100441 In one embodiment the gene involved in 13-carotene uptake is the SCARB I gene (the sequence of which is available at NCBI accession number NM_I 74597.2, GI:3 1341575).

(0045J In another embodiment the gene involved 13-carotene metabolism is selected from BCO2 (the sequence of which is available at NCBI accession number NM_00 11 01987, GI:156120622)orBCMO1.

(00461 For example, the one or more polymorphisms in a gene associated with 13-carotene uptake is the C-32 1 G promoter polymorphism in the SCARB I gene. The association of the C allele at this polymorphism with production of milk and particularly milk fat with increased f3-carotene content, and the association of the G allele at this polymorphism with production of milk and particularly milk fat with decreased 13-carotene content, is discussed in the applicant's co-pending New Zealand Patent application NZ 561999, United Kingdom Patent application GB 0817719.8, Australian Patent application AU 2008227070, and Irish Patent application IE 2008/0786.

100471 For example, the one or more polymorphisms in a gene associated with 13-carotene metabolism the W8OStop G/A polymorphism in the BCO2 gene. The association of the A allele with production of milk and particularly milk fat with increased 13-carotene content, and the association of the G allele at this polymorphism with production of milk and particularly milk fat with decreased 13-carotene content, is discussed in the applicant's co-pending New Zealand Patent application NZ 561998, United Kingdom Patent application GB 0815964.2, Australian Patent application AU 2008207705, and Irish Patent application IE 2008/0710.

100481 Accordingly, in one embodiment the presence or absence of one or more of the group comprising the C allele at the C-1054T promoter polymorphism in the bovine BCMO1 gene, the T allele at the C-1054T promoter polymorphism in the bovine BCMOI gene, the G allele at the G15929A (G278R) polymorphism in the bovine BCMO1 gene, the A allele at the G15929A (G278R) polymorphism in the bovine BCMOI gene, the A allele at the A18068G (N341D) polymorphism in the bovine BCMOI gene, and the G allele at the A18068G (N341D) polymorphism in the bovine BCMOI gene, is determined, directly or indirectly, together with the presence or absence of one or more of the group comprising the C allele at the C-32 I G promoter polymorphism in the SCARB 1 gene, the 0 allele at the C-32 1 G promoter polymorphism in the SCARB I gene, the G allele at the W8OStop G/A polymorphism in the BCO2 gene, the A allele at the W8OStop 0/A polymorphism in the BCO2 gene.

[0049] In a further aspect, the invention includes a probe comprising a nucleic acid molecule sufficiently complementary with a nucleic acid sequence comprising a bovine BCMO I gene or encoding a bovine BCMO I gene product, or its complement, so as to bind thereto under stringent conditions, as well as a diagnostic kit containing such a probe.

[0050] The invention also includes a primer composition useful for detection of the presence or absence of a wild type BCMOI gene andlor the presence or absence of nucleic acid encoding a wild type BCMOI gene product, such as wild type BCMO1 protein. In one form, the composition can include a nucleic acid primer substantially complementary to a nucleic acid sequence comprising a wild type BCMO1 gene or encoding a wild type BCMOI gene product, or its complement. The nucleic acid sequence can in whole or in part be identified in SEQ ID No.1 or SEQ ID No. 2. The invention also includes a primer composition useful for detection of the presence or absence of a variant BCMO1 gene and/or the presence of the DNA encoding a variant BCMOI gene product, such as a variant BCMO1 * protein at least partially lacking wild type BCMOI activity. In one form, the composition can include a nucleic acid primer substantially complementary to a nucleic acid sequence comprising a variant BCMO1 gene or encoding a variant BCMOI gene product, or its complement. Again, the nucleic acid sequence can in whole or in part be identified in SEQ ID NO:l or SEQ ID NO:2. Diagnostic kits including such a composition are also included.

100511 Particularly contemplated are primers comprising or substantially complementary to a nucleic acid sequence present in SEQ ID NO: 1 and within approximately I to about 2000 bp of the C-I 054T polymorphism, more preferably within approximately 1 to about 1000 bp, or within approximately I to about 500 bp, approximately 1 to about 400 bp, approximately I to about 300 bp, approximately I to about 200 bp, approximately I to about 100 bp, approximately I to about 50 bp, or approximately I to about 20 bp of the C-1054T polymorphism.

100521 Also contemplated are primers comprising or substantially complementary to a nucleic acid sequence present in SEQ ID NO: I and within approximately I to about 2000 bp of the G15929A (G278R) 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 I to about 300 bp, approximately I to about 200 bp, approximately I to about bp, approximately 1 to about 50 bp, or approximately I to about 20 bp of the G15929A (G278R) polymorphism.

100531 Further 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 Al 8068G (N34 1 D) polymorphism, more preferably within approximately Ito about 1000 bp, or within approximately 1 to about 500 bp, approximately I to about 400 bp, approximately I to about 300 bp, approximately I to about 200 bp, approximately 1 to about bp, approximately I to about 50 bp, or approximately I to about 20 bp of the Al 8068G (N34 1 D) polymorphism.

100541 Examples of such primers are presented herein as SEQ ID NOs: 4 -17.

100551 It will be appreciated by those skilled in the art that a pair of such primers can be used to determine the identity of the nucleotide at a given polymorphism, by, for example the selective generation of an amplicon with one or more sequence-specific primers. Primer compositions comprising a pair of such primers are accordingly contemplated.

100561 The invention also provides a diagnostic kit including a primer composition useful for determining the presence or absence of a wild type BCMO1 gene and/or the presence or absence of nucleic acid encoding wild type BCMOI, the diagnostic kit comprising one or more primers or primer compositions or probes or probe compositions as described herein.

100571 The invention further includes an antibody composition useful for determining the presence or absence of wild type BCMO1 protein, or for determining the presence or absence of a variant BCMOI protein such as a variant BCMO1 protein at least partially lacking wild type BCMOI activity, or for determining the expression of BCMOI protein, as well as a diagnostic kit containing such an antibody together with instructions for use, for example in a method of the invention.

100581 The invention further provides a diagnostic kit useful in detecting DNA comprising a variant BCMOI gene, or DNA or mRNA encoding a variant BCMOI gene product at least partially lacking wild type 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 RNA upstream and downstream, respectively, of a polymorphism in the BCMOI gene which results in or is associated with increased or decreased 13-carotene levels (particularly increased or decreased milk colour or 13-carotene content.

100591 In one embodiment at least one of the nucleotide sequences is selected to be from a non-coding region of the wild type BCMOI gene. The kit can also include a primer complementary to a naturally occurring mutation of a coding or non-coding portion of the wild type BCMO 1 gene, for example a mutation in the promoter of the BCMO 1 gene.

Preferably the kit includes instructions for use, for example in accordance with a method of the invention.

100601 In another embodiment the invention provides a method of assessing the genetic merit of a bovine with respect to milk content, the method comprising determining the presence or absence of the C-lO54T promoter polymorphism in the bovine BCMOI gene.

100611 Thus, in another embodiment the invention provides a method of assessing the genetic merit of a bovine with respect to milk content which comprises the step of determining the presence or absence of one or more polymorphisms selected from the group comprising: the C-1054T promoter polymorphism in the bovine BCMOI gene, the GI 5929A (G278R) polymorphism in the bovine BCMO1 gene, or the Al 8068G (N34 1 D) polymorphism in the bovine BCMO 1 gene.

[0062J In another embodiment the invention provides a method of assessing the genetic merit of a bovine with respect to milk 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-1054T promoter polymorphism in the bovine BCMOI gene, the T allele at the C-1054T promoter polymorphism in the bovine BCMOI gene, the G allele at the G15929A (G278R) polymorphism in the bovine BCMOI gene, the A allele at the G15929A (G278R) polymorphism in the bovine BCMOI gene, the A allele at the A18068G (N341D) polymorphism of the bovine BCMOI gene, or the G allele at the A18O68G (N341D) polymorphism of the bovine BCMO1 gene.

100631 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.

100641 In another aspect, the present invention provides a method for identifying or selecting a bovine with a genotype indicative of desired milk colour or of desired milk J3-carotene content. The method comprises determining the bovine BCMOI allelic profile of said bovine, and selecting the bovine on the basis of the determination.

100651 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.

100661 in one embodiment, the invention provides a method for identifying or selecting a bovine with a BCMOI allelic profile indicative of increased milk colour or increased milk 13-carotene content, preferably of increased milk fat colour or increased milk fat 13-carotene content.

100671 In one example, the method comprises determining the absence of one or more of the C allele at the C-1054T promoter polymorphism in the BCMO1 gene, the A allele at the G15929A (G278R) polymorphism in the BCMO1 gene, or the A allele at the A18068G (N341D) polymorphism in the BCMO1 gene, and identifying or selecting the bovine on the basis of the determination. Alternatively or additionally, the method comprises determining the presence of one or more of the T allele at the C-i 054T promoter polymorphism in the BCMOI gene, the G allele at the G15929A (G278R) polymorphism in the BCMO1 gene, or the G allele at the A18068G (N34ID) polymorphism in the BCMOI gene, and identifying or selecting the bovine on the basis of the determination.

10068J In one example, the method comprises determining the presence of one or more of the IT genotype at the C-1054T promoter polymorphism in the BCMOI gene, the GG genotype at the GI 5929A (G278R) polymorphism in the BCMOI gene, or the GG genotype at the A18068G (N341D) polymorphism in the BCMOI gene, and selecting the bovine on the basis of the determination. In a further example, the method comprises determining the presence of at least two of said genotypes, for example the method comprises determining the genotype at each polymorphism of the group comprising the C-1054T promoter polymorphism in the BCMOI gene, the G15929A (G278R) polymorphism in the BCMOI gene, and the A18068G (N34lD) polymorphism in the BCMOI gene.

100691 In one embodiment, the invention provides a method for identifying or selecting a bovine with a BCMOI allelic profile indicative of intermediate milk fat colour or intermediate milk fat 13-carotene content.

(00701 In one embodiment, the method comprises determining the presence of the C allele and of the T allele at the C-10541' promoter polymorphism in the BCMO1 gene, and selecting --... -- 100711 ln a further embodiment, the metho1 comprises ucuermining the presence of the CT genotype at the C-I 054T promoter polymorphism in the BCMO I gene, and selecting the bovine on the basis of the determination.

100721 In a further embodiment the invention provides a method for selecting a bovine with a BCMOI allelic profile indicative of decreased milk fat colour or decreased milk fat 13-carotene content.

100731 In one example, the method comprises determining the presence of one or more of the C allele at the C-1054T promoter polymorphism in the BCMO1 gene, the A allele at the G15929A (G278R) polymorphism in the BCMOI gene, or the A allele at the A18068G (N341D) polymorphism in the BCMO1 gene, and selecting the bovine on the basis of the determination. Alternatively or additionally, the method comprises determining the absence of one or more of the T allele at the C-1054T promoter polymorphism in the BCMOI gene, the G allele at the G15929A (G278R) polymorphism in the BCMO1 gene, or the G allele at the A18068G (N341 D) polymorphism in the BCMOI gene, and selecting the bovine on the basis of the determination.

[0074J In one example, the method comprises determining the presence of one or more of the CC genotype at the C-1054T promoter polymorphism in the BCMOI gene, the GO genotype at the 01 5929A (G278R) polymorphism in the BCMOI gene, or the GO genotype at the Al 80680 (N341D) polymorphism in the BCMO1 gene, and selecting the bovine on the basis of the determination. In a further example, the method comprises determining the presence of at least two of said genotypes, for example the method comprises determining the genotype at each polymorphism of the group comprising the C-1054T promoter polymorphism in the BCMOI gene, the G15929A (G278R) polymorphism in the BCMO1 gene, and the A18068G (N341D) polymorphism in the BCMO1 gene.

100751 In one embodiment, the presence or absence of any one or more of the above alleles is determined with respect to a BCMOI polynucleotide (such as genomic DNA, mRNA or cDNA produced from niRNA) obtained from the bovine.

100761 For example, the presence or absence of any one or more of the above alleles is determined by sequencing a BCMOI polynucleotide obtained from the bovine.

100771 In a further embodiment the determination comprises the step of amplifying a BCMO1 polynucleotide sequence from genomic DNA, mR. NA or cDNA produced from mRNA derived from said bovine, for example by PCR.

100781 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 present in SEQ ID NO:! or SEQ ID NO:2 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.

100801 In an alternative embodiment, the method comprises restriction enzyme digestion of a nuc!eotide 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 or absence of any of the above alleles is determined by mass spectrometric analysis of a BCMOI polynucleotide, such as that obtained from the bovine or from a method as described herein.

[0082] In an alternative embodiment, the presence or absence of any of the above alleles is determined by hybridisation of a probe or probes comprising a nucleotide sequence of or complementary to the sequence of SEQ ID NO: 1 or SEQ ID NO:2.

[00831 Preferably the probe or probes comprises 12 or more contiguous nucleotides of or complementary to the sequence in SEQ IDNO:1 or SEQ ID NO:2.

100841 Preferably the probe or probes comprise sequence corresponding to one of the allele-specific nucleotides described herein or complements thereof.

100851 In an alternative embodiment, the presence or absence of any of the C allele or of the G allele above alleles is determined by analysis of a BCMO1 polypeptide obtained from the bovine.

100861 In one embodiment, the presence of one or more of the C allele at the C-1054T promoter polymorphism, the A allele at the G15929A (G278R) polymorphism, or the A allele at the Al 8068G (N341D) polymorphism is determined by detecting a reduction in the amount of or an absence of BCMOI gene product, or a reduction in the activity of BCMO1 polypeptide.

100871 In one embodiment, the presence of one or more of the T allele at the C-I 054T promoter polymorphism, the G allele at the G15929A (G278R) polymorphism, or the G allele at the A18068G (N341D) polymorphism is determined by detecting an increase in the amount oCBCMO1 gene product, or an increase in the activity of BCMOI polypeptide.

100881 In a further aspect the invention provides a bovine selectcd 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 the selected bovine.

100891 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.

100901 In a still further aspect, the invention provides a method of determining genetic merit of a bovine with respect to one or more milk colour or 3-carotene content phenotypes, or with respect to capability of producing progeny predisposed to or with one or more milk colour or 13-carotene content phenotypes, the method comprising providing data about the BCMO1 allelic profile of said bovine, and determining the genetic merit of the bovine on the basis of the data.

100911 In one embodiment, the data about the BCMOI allelic profile comprises data representative of the presence or absence of one or more of the group comprising the C allele at the C-1054T promoter polymorphism in the bovine BCMO1 gene, the T allele at the C-1 054T promoter polymorphism in the bovine BCMO 1 gene, the G allele at the G15929A (G278R) polymorphism in the bovine BCMOI gene, the A allele at the G15929A (G278R) polymorphism in the bovine BCMOI gene, the A allele at the A18O68G (N34ID) polymorphism of the bovine BCMOI gene, or the G allele at the A18068G (N341D) polymorphism of the bovine BCMOI gene.

100921 In one example, the method additionally comprises providing data comprising the result of at least one analysis of one or more genetic loci associated with one or more milk colour or 13-carotene content phenotypes, wherein the data is representative of the genetic merit of the bovine.

100931 In one example, the one or more genetic loci are one or more polymorphisms associated with an increase or decrease in expression or activity of a BCMOI gene product.

100941 For example, the genetic loci is the BCMOI gene (including all regulatory elements such as the promoter, introns and 3'UTR).

100951 In one embodiment, the one or more milk colour or 13-carotene content phenotypes is selected from the group comprising production of or capability of producing milk with increased milk colour or production of or capability of producing milk with increased milk J3-carotene content.

100961 In another embodiment, the one or more milk colour or 13-carotene content phenotypes is selected from the group comprising production of or capability of producing milk with decreased milk colour or production of or capability of producing milk with decreased milk 13-carotene content.

100971 Accordingly, in one embodiment the invention provides a method of determining genetic merit of a bovine with respect to milk colour or 13-carotene content, or with respect to capability of producing progeny that will have increased or decreased milk colour or J3-carotene content, the method comprising providing data about the BCMOI allelic profile of the bovine, and determining the genetic merit of the bovine on the basis of the data.

100981 In one embodiment, 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 13-carotene content phenotypes, wherein the data is representative of the genetic merit of the bovine.

100991 In one example, the one or more genetic loci are one or more polymorphisms associated with an increase or decrease in expression or activity of a BCMO1 gene product.

1001001 For example, the genetic loci is the BCMOI gene (including all regulatory elements such as the promoter, introns and 3'UTR).

1001011 In one embodiment, the data comprises the result of one or more genetic tests of a sample from the bovine, and the determination comprises analysing the result for the presence or absence of one or more polymorphisms associated with increased or decreased expression or activity of BCMO1 gene product, or one or more polymorphisms in linkage disequilibrium with one or more polymorphisms associated with increased or decreased expression or activity of BCMO I 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 phenotypes; and identifying or selecting the bovine on the basis of the result.

1001021 In one example, the one or more polymorphisms associated with increased or decreased expression or activity of BCMO1 gene product is one or more polymorphisms in the BCMO1 gene.

1001031 In a further aspect the invention provides a method for selecting a bovine that produces milk fat with increased or decreased 13-carotene content, or capable of producing progeny that produce milk fat with increased or decreased 13-carotene content, the method compnsing 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 in the BCMO1 gene associated with increased or decreased expression or activity of BCMOI gene product, or one or more polymorphisms in linkage disequilibrium with one or more polymorphisms in the BCMOI gene associated with increased or decreased expression or activity ofBCMO1 gene product, wherein a result indicative of the presence or absence of one or more of said polymorphisms is indicative of a bovine that produces milk fat with increased or decreased 13-carotene content, or that is capable of producing progeny that produce milk fat with increased or decreased 13-carotene content.

1001041 In one embodiment, the one or more polymorphisms is selected from the group comprising: the C-I 054T promoter polymorphism in the bovine BCMO I gene, the G15929A (G278R) polymorphism in the bovine BCMOI gene, the A18068G (N341D) polymorphism of the bovine BCMO1 gene, or one or more polymorphisms in linkage disequilibrium with one or more of these polymorphisms.

1001051 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 colour or 13-carotene content phenotypes and optionally a reference milk colour or 13-carotene content phenotype database of non-genetic factors for one or more bovine milk colour or 13-carotene content phenotypes; and a computer program embedded within the computer processor which, once data consisting of or including the result of a genetic analysis for which data is included in the reference genetic database is received, processes said data in the context of said reference databases to determine, as an outcome, the genetic merit of the bovine, said outcome being communicable once known, preferably to a user having input said data.

(001061 In one example, said system is accessible via the internet or by personal computer.

(001071 In one embodiment, 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 colour or 13-carotene content phenotypes, more preferably the one or more genetic loci are one or more polymorphisms in one or more genes associated with one or more milk colour or 13-carotene content phenotypes.

[001081 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 colour or 13-carotene content phenotypes in the context of both a reference genetic database of the results of said at least one genetic analysis and optionally a reference database of non-genetic factors associated with one or more bovine milk colour or 13-carotene content phenotypes.

[001091 In one embodiment, the one or more genetic loci are one or more polymorphisms in one or more genes associated with one or more milk colour or 13-carotene content phenotypes.

1001101 In one example, the one or more polymorphisms are one or more polymorphisms associated with an increase or decrease in expression or activity of a BCMO I gene product.

1001111 In still another aspect, the invention provides a method of determining the genetic merit of a bovine with respect to milk colour or 13-carotene content, or with respect to capability of producing progeny that will have increased or decreased milk colour or 13-carotene content, the method comprising determining milk or tissue colour or 13-carotene content of the bovine, determining the BCMOI allelic profile of the bovine, comparing the BCMOI aHelic profile of the bovine or the milk or tissue colour or [3-carotene content of the bovine with that of a bovine having a known BCMOI allelic profile; determining the genetic merit of the bovine on the basis of the comparison.

1001121 It will be appreciated that for the purposes of the comparison, the milk colour or [3-carotene content associated with the known BCMOI allelic profile is known. It will further be appreciated that the association of milk colour or [3-carotene content with a particular BCMO1 allelic profile may be established by the methods described herein.

1001131 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:! and comprising one or more of the C-1054T promoter polymorphism, the G15929A (G278R) polymorphism, or the A18068G (N341D) polymorphism; or (b) from 12 to 1791 contiguous nucleotides of SEQ ID NO:2 and comprising one or more of the C-1054T promoter polymorphism, the G15929A (G278R) polymorphism, or the Al 8068G (N341D) polymorphism; or (c) at least 12 contiguous nucleotides of a functional variant of SEQ ID NO: 1; or (d) at least 12 contiguous nucleotides of a functional variant of SEQ ID NO:2; or (e) at least 12 contiguous nucleotides of any one of SEQ ID NOs:4 -17; or (f) a complement of any one of (a) to (e); or (g) a sequence of at least 12 contiguous nucleotides and capable of hybridising to the nucleotide sequence of any one of (a) to (1) under stringent conditions.

1001141 In one embodiment, the nucleic acid molecule comprises nucleotide sequence selected from (a) at least 12 contiguous nucleotides of SEQ ID NO: 1 and comprising a cytosine at the C-i 054T promoter polymorphism; or (b) at least 12 contiguous nucleotides of SEQ ID NO:2 and comprising a cytosine at the C-1054T polymorphism; or (c) at least 12 contiguous nucleotides of SEQ ID NO: I and comprising a thymine at the C- I 054T polymorphism; or (d) at least 12 contiguous nucleotides of SEQ ID NO:2 and comprising a thymine at the C-i 054T polymorphism; or (e) at least 12 contiguous nucleotides of SEQ ID NO: 1 and comprising a guanine at the the G I 5929A (0278R) polymorphism; or (f) at least 12 contiguous nucleotides of SEQ ID NO:2 and comprising a guanine at the G15929A (G278R)polymorphism; or (g) at least 12 contiguous nucleotides of SEQ ID NO:1 and comprising an adenine at the G15929A (G278R)polymorphism; or (h) at least 12 contiguous nucleotides of SEQ ID NO:2 and comprising an adenine at the G15929A (0278R)polymorphism; or (i) at least 12 contiguous nucleotides of SEQ ID NO:1 and comprising a guanine at the the A18068G (N341D) polymorphism; or ) at least 2 contiguous nucleotides of SEQ ID NO:2 and comprising a guanine at the A180680 (N341D)polymorphism; or (k) at least 12 contiguous nucleotides of SEQ ID NO: I and comprising an adenine at the Al 80680 (N341D)polymorphism; or (I) at least 12 contiguous nucleotides of SEQ ID NO:2 and comprising an adenine at the A18068G (N341D)polymorphism; or (m) a complement of any one of(a) to (1); or (n) a sequence of at least 12 contiguous nucicotides and capable of hybridising to the nucleotide sequence of any one of(a) to (m) under stringent conditions.

(001151 In one embodiment, the BCMOI nucleic acid molecule consists of a nucleic acid sequence as defined in any one of (a) to (n) above.

1001161 In one embodiment, the BCMO1 nucleic acid molecule is a BCMOI fragment as defined herein, for example a BCMO 1 fragment comprising one or more of the C-I 054T promoter polymorphism, the G15929A (G278R) polymorphism, the A18068G (N341D) polymorphism, or a combination of any two or more thereof.

1001171 In one embodiment, the nucleic acid molecule comprises nucleotide sequence selected from the group comprising: (a) from 12 to at least about 20000, or at least about 10000, contiguous nucleotides of SEQ ID NO:I and comprising one or more of the C-1054T promoter polymorphism, the GI 5929A (G278R) polymorphism, or the Al 8068G (N341 D) polymorphism; or (b) from 12 to 1791 contiguous nucleotides of SEQ ID NO:2 and comprising one or more of the C-1054T promoter polymorphism, the G15929A (G278R) polymorphism, or the Al 80680 (N341 D) polymorphism; or (c) from 12 to at least about 20000, or at least about 10000, contiguous nucleotides of a functional variant of SEQ ID NO: 1; or (d) from 12 to 1791 contiguous nucleotides of a functional variant of SEQ ID NO:2; or (e) a complement of any one of (a) to (d); or (f) a sequence of at least 12 contiguous nucleotides and capable of hybridising to the nucleotidc sequence of any one of (a) to (c) under stringent conditions.

1001181 In one embodiment, the BCMO nucleic acid molecule consists of nucleotide sequence selected from the group comprising: (a) from 12 to about 20000 contiguous nucleotides of SEQ ID NO: 1 and comprising one or more of the C-1054T promoter polymorphism, the G15929A (G278R) polymorphism, or the Al 8068G (N341D) polymorphism; or (b) from 12 to about 1791 contiguous nucleotides of SEQ 1D NO:2 and comprising one or more of the C-l054T promoter polymorphism, the G15929A (G278R) polymorphism, or the A18068G (N341D) polymorphism; or (c) from 12 to about 20000 contiguous nucleotides of a functional variant of SEQ ID NO:1; or (d) from 12 to about 1791 contiguous nucleotides of a functional variant of SEQ ID NO:2; or (e) any one of SEQ ID NOs:4 -17; or (f) a complement of any one of(a) to (e); or (g) a sequence of from 12 to about 20000 contiguous nucleotides and capable of hybridising to the nucleotide sequence of any one of (a) to (f) under stringent conditions.

1001 19J In another aspect the invention relates to an isolated, purified or recombinant polypeptide comprising at least about 10 contiguous amino acids of SEQ ID NO:3.

100120] In one embodiment, the polypeptide comprises one or more of the following: (a) arginine at the position corresponding to amino acid 278 of SEQ ID NO:3; or (b) an amino acid other than glycine at the position corresponding to amino acid 278 of SEQ ID NO:3; or (c) aspartate at the position corresponding to amino acid 341 of SEQ ID NO:3; or (d) an amino acid other than asparagine at the position corresponding to amino acid 341 of SEQ ID NO:3; or (e) any combination of(a) or (b) and (c) or (d).

1001211 In one embodiment, the polypeptide consists of at least about 10 contiguous amino acids of SEQ ID NO:3, at least about 20, at least about 30, at least about 40, at least about 50, at least about 100, at least about 150, at least about 200, or at least about 250 contiguous amino acids of SEQ ID NO:3, or consists of an amino acid sequence as defined in any one of (a) to (e) above.

1001221 In one embodiment the polypeptide is a variant as defined herein.

1001231 In one example, the polypeptide has at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO:3 or with an amino acid sequence present in SEQ ID NO:3.

1001241 In another embodiment the polypeptide is a functional variant of a BCMO1, or is a functional fragment thereof.

1001251 In one example, the polypeptide (for example, the functional variant or functional fragment) has at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 110%, at least about 120%, at least about 130%, at least about 140%, at least about 150%, or at least about 200% of the ezymatic activity of a BCMOI polypeptide having the amino acid sequence of SEQ ID NO:3.

1001261 For example, in one embodiment the isolated, purified or recombinant polypeptide comprises an amino acid sequence having at least 95% sequence identity with a sequence present in SEQ ID NO:3, wherein the polypeptide comprises at least about 100 amino acids.

1001271 In a further example, the isolated, purified or recombinant polypeptide comprises an amino acid sequence having at least 95% sequence identity with a sequence present in SEQ ID NO:3, wherein the polypeptide comprises one or more of the following: (a) argimne at the position corresponding to amino acid 278 of SEQ ID NO:3; or (b) an amino acid other than glycine at the position corresponding to amino acid 278 of SEQ ID NO:3; or (c) aspartate at the position corresponding to amino acid 341 of SEQ ID NO:3; or (d) an amino acid other than asparagine at the position corresponding to amino acid 341 of SEQ ID NO:3; or (e) any combination of (a) or (b) and (c) or (d).

1001281 The invention also provides a genetic construct comprising a BCMO1 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 BCMOI 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.

1001291 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.

1001301 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 milk 13-carotene content determined as described in the Example. Data for peak (35 days post calving), mid (late November) and late (late February) lactation is shown.

These data were obtained from a Friesian-Jersey crossbred trial, as described in the materials and methods.

Figure 2 shows a graph depicting a QTL for milk 13-carotene contenton bovine chromosome 18. The maximum F-value of 7.8 occurred at 15 centimorgans (cM). The grey bar shows the 95% confidence interval for the QTL.

Figure 3 shows a schematic showing a graphical representation of polymorphism in the bovine BCMOI gene. A: Graphical representation of the predicted exonic structure of bovine BCMOI gene. B. Identification (indicated by arrows) of the Cu' promoter polymorphism at position -1054 relative to the + 1 translation start site (i.e., in the promoter), a G/A polymorphism at position 15929 (G278R, exon 6), and an A/G polymorphism at position 18068 (N341D, exon 7). The -1054 and 15929 polymorphisms were heterozygous in 2 of the six Fl sires (sires 405 and 740) and the 18068 polymorphism was heterozygous in one sire (405).

Figure 4 shows the statistical effect of each genotypic state for the following BCMO1 polymorphisms: G15929A (G278R), A18068G (N341D) and C-1054T polymorphisms on milk 13-carotene content at peak lactation. A) BCMOI_G15929A. B) BCMOI_A18068G. C) BCMO1_C-1054T. Similar effects were seen at the other two time points during lactation (mid, late lactation).

Figure 5 shows the adjusted statistical effect of BCMOI_G15929A (G278R) on bovine chromosome 18 milk 13-carotene QTL (mid lactation data shown). Solid line: milk 13-carotene QTL (residual of log-transformed, modeled data). Dotted line: milk 13-carotene QTL after BCMOI_GI 5929A accounted for (residual of log-transformed, modeled data including BCMO1_G1 5929A as fixed effect). The decrease in QTL significance (F-value) suggests a significant association of the BCMO 1 genotype with the milk 13-carotene content QTL variation.

Figure 6 shows the adjusted statistical effect of BCMOI_A18068G (N341D) on bovine chromosome 18 milk 13-carotene QTL (mid lactation data shown). Solid line: milk 13-carotene QTL (residual of log-transformed, modeled data). Dotted line: milk 13-carotene QTL after BCMO 1_Al 8068G accounted for (residual of log-transformed, modeled data including BCMO1_A18068G as fixed effect). The decrease in QTL significance (F-value) suggests a significant association of the BCMOI genotype with the milk 13-carotene content QTL variation.

Figure 7 shows the adjusted statistical effect of BCMO1_C-1054T polymorphism on bovine chromosome 18 milk 13-carotene QTL (mid lactation data shown).

Solid line: milk 13-carotene QTL (residual of log-transformed, modeled data).

Dotted line: milk 13-carotene QTL after BCMOI_C-1054T accounted for (residual of log-transformed, modeled data including BCMO 1_C-I 054T as fixed effect).

Figure 8 shows the enzyme activities for the following recombinant BCMOI proteins: 278G/341N (wildtype), 278G/34lD, 278R1341N, 278R1341D expressed in CHO-KI cells, with enzyme activity assayed in cell lysates, as described in the example. Data presented are means � standard error for n = 2 independent experiments.

DETAILED DESCRIPTION OF THE INVENTION

1001311 The present invention recognises for the first time that polymorphisms in the BCMO1 gene in bovine is associated with a QTL for variations in milk colour and variations in milk 13-carotene content.

1001321 For the sake of clarity, the phrase "milk colour or 13-carotene content" is to be read as refen-ing to milk colour or milk 13-carotene content. Grammatical equivalents or components thereof are to be read likewise.

1001331 It will be apparent to those skilled in the art that milk 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 having increased colour, or decreased colour, for example increased yellow colour, relative to another sample, for example relative to milk produced by bovine having wild type BCMO1. It will similarly be appreciated by those skilled in the art that reference herein to milk having increased colour may be considered as a reference to milk that is less white, and vice versa. Methods for quantitative determination of milk colour or 13-carotene content are also known in the art, and examples are provided herein.

1001341 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 BCMOI allelic profile of said bovine. Another such method comprises the step of determining the level of a BCMOI gene product of said bovine.

1001351 The invention also provides a method for selecting a bovine with a genotype indicative of desired milk 13-carotene content, particularly desired milk fat 13-carotene content.

One of the major applications of the present invention is in the selection of bovine having the T allele or the C allele at the C-1054T promoter polymorphism in the BCMOI gene, which are associated with increased milk 13-carotene content and milk colour, and decreased milk 13-carotene content and milk colour, respectively. Accordingly, one method comprises determining the presence or absence of the C allele or of the T allele at the C-I 054T promoter polymorphism of the BCMOI gene, and selecting the bovine on the basis of the determination.

1001361 Another of the applications of the present invention is in the selection of bovine having the G allele or the A allele at the G15929A (G278R) polymorphism in the BCMO1 gene, which are associated with increased milk fat 13-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 G allele or the A allele at the Gi 5929A (G278R) polymorphism of the bovine BCMO1 gene, and selecting the bovine on the basis of the determination.

1001371 Another of the applications of the present invention is in the selection of bovine having the G allele or the A allele at the A18068G (N341D) polymorphism in the BCMOI gene, which are associated with increased milk fat 3-carotene content and milk fat colour, and decreased milk fat 3-carotene content and milk fat colour, respectively. Accordingly, one method comprises determining the presence or absence of the G allele or the A allele at the A18068G (N341D) polymorphism of the bovine BCMOI gene, and selecting the bovine on the basis of the determination.

1001381 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.

1001391 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.

1001401 The present invention recognises that polymorphisms in the gene encoding BCMO1, as well as I3CMOI levels or activity, may be used as a selection tool to breed animals with higher or lower milk concentrations of n-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 p3-carotene.

1 BCMO1 1001411 BCMOI is a key regulatory enzyme for the metabolism of 3-carotene to vitamin A. BCMOI catalyses the symmetrical cleavage of f3-carotene, resulting in the formation of two molecules of retinal. Retinal (also known as retinaldehyde) is the aldehyde isomer of vitamin A, and can be reversibly reduced to produce retinol or irreversibly oxidized to produce retinoic acid. In animals, the major isoform of vitamin A is retinol. The conversion of 3-carotene to vitamin A in animals is usefully reviewed in von Lintig, J. and Vogt, K., 2004.

1001421 The genomic sequence comprising the bovine BCMO1 gene is presented herein as SEQ ID No.1. The predicted amino acid coding sequence of bovine BCMO1 is presented herein as SEQ ID No. 2, and a provisional reference sequence is available as NCBI accession number NM_001024559.1 (01:66792909). This coding sequence is derived from a cDNA clone, the sequence of which is available as NCBI accession number DQ008469.1 (GL62999033). The amino acid sequence encoded by this coding sequence is presented herein as SEQ ID No. 3, and is itself available as NCBI accession number NP 001019730.1 (01:66792910).

1001431 The present invention relates to the identification that one or more mutations in the BCMO 1 gene leads to variation in milk content and composition, particularly variation in milk colour and milk n-carotene content, and are associated with one or more milk content or milk colour phenotypes.

1001441 As described herein, the BCMO1 mutations were closely associated with milk content phenotype. For example (and as described herein), animals homozygous for the C allele (CC genotype) at the C-1054T promoter polymorphism (wild type promoter) produced milk with less f3-carotene than animals homozygous for the T allele (TT genotype). This effect was observed at three stages of lactation, and within two sire families that carried the mutation. Animals homozygous for the C allele (wild type promoter) also produced milk with less 13-carotene than heterozygous animals. Likewise, animals homozygous for the A allele of the G15929A polymorphism and animals homozygous for the A allele of the Al 80680 polymorphism produced milk with less 13-carotene than animals homozygous for either of the respective 0 alleles.

1001451 The C-i 054T CC genotype for BCMOI was present in 74.43% of F2 animals in the Holstein-Friesian x Jersey crossbred (FJXB) trial, while the CT genotype was present in 24.49% of F2 animals, and the TT genotype in 1.08% (see Table 2 herein). The 01 5929A GO genotype for BCMOI was present in 74.64% of F2 animals in the Hoistein-Friesian x Jersey crossbred (FJXB) trial, while the GA genotype was present in 24.05% of F2 animals, and the AA genotype in 1.31% (see Table 2 herein). The A18068G AA genotype for BCMOI was present in 84.64% of F2 animals in the Hoistein-Friesian x Jersey crossbred (FJXJ3) trial, while the AG genotype was present in 14.76% of F2 animals, and the GO genotype in 0.60% (see Table 2 herein). The details of the FJXB trial are detailed in the materials and methods.

(00146] A reference bovine BCMOI nucleotide sequence referred to as "wild type BCMOI" determined by the applicants is presented as SEQ ID NO:1, and the compiled coding sequence is presented as SEQ ID NO:2, with the corresponding amino acid sequence is presented as SEQ ID NO:3. It will be appreciated that the polymorphisms described herein are noted as variants in SEQ ID NO:!, where the wild-type allele is depicted in SEQ ID NO:l, and the non-wild-type allele is noted as the variant nucleotide. Accordingly, as used herein with respect to BCMOI, such as use with respect to a BCMOI gene or a BCMOI gene product, the term "wild type" recognizes the *characteristics of the BCMO1 nucleotide sequences presented as SEQ ID NOs: I and 2, and of the protein product encoded thereby.

For example, when used with respect to enzymatic activity, the term "wild type" denotes activity associated with the wild type BCMOI enzyme. Similarly, when used with respect to expression level, the term "wild type" denotes a level of expression associated with the wild type BCMOI gene or with the wild type BCMOI promoter.

1001471 It will be apparent that the term "activity" may refer both to the inherent enzymatic activity of a single molecule of BCMO1, which may be wild type activity or may be less or greater than wild type activity as may depend, for example on the amino acid sequence, the presence of any amino acid substitutions, the availability of co-factors, and the like, as well as to the total enzymatic activity of the population of BCMOI molecules present (for example, in a bovine or in a sample taken from a bovine), as may depend on both the enzymatic activity of each molecule present and the level of expression (for example, how many such molecules are present).

1001481 As used herein, such as when used in reference to an allelic protein lacking the activity of wild type BCMO 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 wild type BCMO1 may be a variant BCMOI protein of greater or lesser enzymatic activity than that of wild type BCMO1.

(001491 Methods to assay the activity of BCMO1 are well known in the art. For example, one such method quantifies BCMOI mRNA, for example using a method well known in the art including quantitative RT-PCR, TaqManTM assays, or the like. Another exemplary method utilises the assay of the BCMOI substrate fl-carotene described herein, where the disappearance of fl-carotene (and the associated reduction in absorbance at 450nm) correlates with BCMO1 enzymatic activity.

[00150] The C-1054T promoter polymorphism, the G15929A (G278R) polymorphism in exon 6 of the BCMOI gene, and the Al 80680 (N341 D) polymorphism in exon 7 of the BCMOI gene are each identified in the bovine BCMO1 gene sequence presented as SEQ ID NO: I in the sequence ID listing as variants.

2 Identification and analysis of polymorphisms 1001511 It will be apparent to those skilled in the field that the convention of identifying polymorphisms by their position in the genomic sequence relative to the +1 translation start site of the gene in which they occur is followed herein. Accordingly, the C-i 054T polymorphism in the BCMOI gene described herein lies 1054 nucleotides upstream of the +1 translation start site of the BCMOI gene. Those skilled in the art will also recognise that these positions can readily be expressed relative to the coding sequence (for example for non-intronic polymorphisms).

1001521 It will similarly be apparent to those skilled in the field that the convention of identifying polymorphisms effecting an amino acid substitution by their codon position in the gene in which they occur and the amino acid substitution effected thereby is also contemplated herein. Accordingly, the G15929A polymorphism and the A18068G polymorphism described herein may be referred to by reference to the codon of the BCMOI gene within which they are located and the amino acid substitution effected, namely the G278R polymorphism, and the N341D polymorphism, respectively.

j00153J The polymorphisms described herein can be detected directly or by detection of one or more polymorphisms which are in linkage disequilibrium with these polymorphisms.

Linkage disequilibrium is a phenomenon in genetics whereby two or more mutations or polymorphisms are in such close genetic proximity that they are co-inherited. This means that in genotyping, detection of one polymorphism as present implies the presence of the other.

(Reich DE et al; Linkage disequilibrium in the human genome, Nature 2001,411:199-204.) 1001541 Various degrees of linkage disequilibrium are possible. Preferably, the one or more polymorphisms in linkage disequilibrium with one or more of the polymorphisms specified herein are in greater than about 60% linkage disequilibrium, are in about 70% linkage disequilibrium, about 75%, about 80%, about 85%, about 90%, about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% linkage disequilibrium with one or more of the polymorphisms selected from the group comprising the C-i 054T promoter polymorphism of the BCMO1 gene, the G15929A (G278R) polymorphism of the BCMOI gene, or the A18068G (N34ID) polymorphism of the BCMO1 gene. (Devlin and Risch 1995; A comparison of linkage disequilibrium measures for fine-scale mapping, Genomics 29: 311-322).

1001551 There are numerous standard methods known in the art for determining whether a particular DNA sequence is present in a sample, many of which include the step of sequencing a DNA sample. Thus in one embodiment of the invention, the step determining whether or not the specified nucleotides are present in a nucleic acid derived from a bovine, includes the step of sequencing the nucleic acid. Methods for nucleotide sequencing are well known to those skilled in the art.

1001561 In one aspect, the present invention provides a method for determining the genetic merit of a bovine with respect to milk content, and particularly with milk colour or 13-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 wild type BCMOI 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 wild type BCMO1 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 BCMO1 gene product, preferably by determining the presence or absence of one or more polymorphisms associated with decreased or increased BCMO1 expression, for example one or more promoter polymorphisms associated with increased or decreased expression.

1001571 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 wild type BCMO 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 13-carotene levels, and particularly in milk having inter alia a higher 13-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 13-carotene levels, and particularly in milk having inter alia a lower 13-carotene content.

1001581 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.

1001591 Accordingly, a preferred embodiment of the invention thus includes the step of amplifying a BCMOI polynucleotide in the presence of at least one primer comprising a nucleotide sequence of, or complementary to, the BCMOI gene (SEQ ID NO:l and SEQ ID NO:2) or flanking sequence thereof, and/or in the presence of a such a primer comprising sequence corresponding to or flanking the C-1054T polymorphism, the G15929A (G278R) polymorphism, or the Al 8068G (N34 I D) polymorphism, or comprising sequence including one or other of the allele-specific polymorphic nucleotides at the C-i 054T polymorphism, the G15929A (G278R) polymorphism, or the A18O68G (N341D) polymorphism as 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., 1987).

1601 Primers suitable for use in PCR based methods of the invention should be sufficiently complementary to the BCMOI gene sequence, such as SEQ ID NO: 1 or SEQ ID NO:2 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:2, or naturally occurring flanking sequences thereof. Examples of such PCR primers are presented herein as SEQ ID NOs: 4-17.

100161] Suitable PCR primers may include sequence corresponding to the C-1054T C allele-specific or C-1054T T allele-specific BCMO1 nucleotides described herein. Similarly, PCR primers may include sequence corresponding to the GI 5929A (G278R) G allele-specific or G 1 5929A (G278R) A allele-specific BCMO I nucleotides, or sequence corresponding to the A18068G (N341D) A allele-specific or A18068G (N341D) G allele- specific BCMO1 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 BCMOI gene of the test bovine.

1001621 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 nucicotide sequence digested may be a PCR product amplified as described above.

100163] 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 for example the C-1054T C allele-specific or C-1054T T allele-specific nucleotides may comprise the additional steps of hybridisation of a probe derived from the BCMOI sequence of SEQ ID NO:1 or SEQ ID NO:2.

1001641 Such prdbes should comprise a nucleic acid molecule of sufficient length and sufficiently complementary to the BCMO 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.

1001651 With respect to polynucleotide molecules greater than about 100 bases in length, typical stringent hybridization conditions are no more than 25 to 300 C (for example, 10° C) below the melting temperature (Tm) of the native duplex (see generally, Sambrook et al., 1987; Ausubel et al., 1987). i'm for polynucleotide molecules greater than about 100 bases can be calculated by the formula Tm = 81. 5 + 0. 41% (G + C-log (Na+).

1001661 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. 1001671 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.

1001681 Such probes would typically comprise at least 12 contiguous nucleotides of or complementary to the sequences presented SEQ ID NO:1 or SEQ ID NO:2, and may comprise sequence corresponding to the allele-specific nucleotides described herein.

1001691 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., 1987).

1001701 As will he apparent to a person skilled in the art, promoter function may bc 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.

1001711 Accordingly, in one embodiment the method for determining the genetic merit of a bovine with respect to milk colour or f'-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 BCMOI gene associated with higher or lower relative levels of expression is present.

1001721 In another aspect, the invention provides a method for determining the genetic merit of bovine with respect to milk colour or a-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 wild type BCMO 1 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.

1001731 In another aspect, the invention provides a method for determining the genetic merit of bovine with respect to milk content with reference to a sample of material containing mRNA obtained from the bovine. In one embodiment this method includes ascertaining whether a sequence of the mRNA encoding (A) a protein having biological activity of a wild type BCMOI is present, and whether a sequence of the mRNA encoding (B) a protein at least partially lacking the activity of (A) is present, and may include determining the amount of mRNA. The absence of the mRNA encoding (A) and the presence of the mRNA encoding (B), or a decrease in the amount of the mRNA encoding (A) compared to wild type levels, again indicates an association with high relative 3-carotene levels, particularly with the production of milk with, inter alia, increased fat colour. The reverse association again holds true.

1001741 Again, if an amplification method such as PCR is used in ascertaining whether a sequence of the niRNA encoding (A) is present, or whether a sequence of the mRNA encoding (B) is present, the method includes amplifying the mRNA, for example in the presence of a pair of primers complementary to a nucleotide sequence encoding a protein having biological activity of a wild type BCMO1, or in the presence of a pair of primers complementary to a nucleotide sequence encoding a variant BCMOI protein. It will be appreciated that in embodiments of the invention reliant on assessing the amount of BCMO 1 mRNA present in a sample, quantitative amplification methods well known in the art may be employed, for example quantitative RT-PCR, microarray analysis, and other methods described herein.

1001 75J Other methods to quantitate or otherwise assess the amount of nucleic acid, particularly the amount of mRl\IA are well known in the art. These include Northern analysis using probes able to hybridise to the target BCMO1 mRNA. Such probes should comprise a nucleic acid molecule of sufficient length and sufficiently complementary to the BCMO1 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 BCMO1 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.

[001761 In a further aspect, the invention provides a method of determining genetic merit of a bovine with respect to milk content which comprises determining the BCMOI allelic profile of said bovine, together with determining the allelic profile of said bovine at one or more genetic loci associated with milk content, including milk 13-carotene content.

1001771 In one embodiment, said genetic loci is a polymorphism in a gene associated with milk 13-carotene content, preferably a polymorphism in a gene involved in 13-carotene uptake or metabolism. Preferably the gene involved in 13-carotene uptake is the SCARBI gene (the sequence of which is available at NCBI accession number NM_174597.2, Gl:31341575).

Preferably the gene involved 13-carotene metabolism is selected from BCO2 (the sequence of which is available at NCBI accession number NMOO 1101987, GI: 156120622) or BCMO 1.

1001781 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.

1001791 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.

1001801 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 SN P 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.

1001811 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 NCBI'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.

1001821 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 9 base pairs, while current estimates of the size of the haploid bovine genome are in the range of 2.6 -2.7 x i� base pairs), and the associated sensitivity and discriminatory requirements.

1001831 Genotyping approaches to detect SNPs well-known in the art include DNA sequencing, methods that require allele specific hybridization of primers or probes, allele specific incorporation of nucleotides to primers bound close to or adjacent to the polymorphisms (often referred to as "single base extension", or "minisequencing"), allele-specific ligation (joining) of oligonucleotides (ligation chain reaction or ligation padlock probes), allele-specific cleavage of oligonucleotides or PCR products by restriction enzymes (restriction fragment length polymorphisms analysis or RFLP) or chemical or other agents, resolution of allele-dependent differences in electrophoretic or chromatographic mobilities, by structure specific enzymes including invasive structure specific enzymes, or mass spectrometry. Analysis of amino acid variation is also possible where the SNP lies in a coding region and results in an amino acid change.

[001841 DNA sequencing allows the direct determination and identification of SNPs. The benefits in specificity and accuracy are generally outweighed for screening purposes by the difficulties inherent in whole genome, or even targeted subgenome, sequencing.

1001851 Mini-sequencing involves allowing a primer to hybridize to the DNA sequence adjacent to the SNP 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 four 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 SNP position.

[001861 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).

1001871 A number of methods currently used for SNP detection involve site-specific and/or allele-specific hybridisation. These methods are largely reliant on the discriminatory binding of oligonucleotides to target sequences containing the SNP of interest. The techniques of Illumina (San Diego, CA), Affymetrix (Santa Clara, CA.) and Nanogen Inc. (San Diego, Calif.) are particularly well-known, and utilize the fact that DNA duplexes containing single base mismatches are much less stable than duplexes that are perfectly base-paired. The presence of a matched duplex is usually detected by fluorescence. A number of whole-genome genotyping products and solutions amenable or adaptable for use in the present invention are now available, including those available from the above companies.

f00188] The majority of methods to detect or identify SNPs by site-specific hybridisation require target amplification by methods such as PCR to increase sensitivity and specificity (see, for example U.S. Pat. No. 5,679,524, PCT publication WO 98/59066, PCT publication WO 95/12607). US 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.

1001891 US Application 20050042608 (incorporated herein in its entirety) describes a modification of the method of electrochemical detection of nucleic acid hybridization of Thorp et al. (U.S. Pat. No. 5,871,918). Briefly, capture probes are designed, each of which has a different SNP base and a sequence of probe bases on each 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.

(001901 The technique of Lynx Therapeutics (Hayward, Calif.) using MEGATYPETM 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.

1001911 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-1054T promoter polymorphism, the G15929A (G278R) and the A18068G (N34ID) exonic polymorphisms, in the BCMO1 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.

1001921 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.

(00193] 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.

1001941 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.

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

For example, integrated systems, such as the ProteomlQTM 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.

1001951 The majority of proteomic methods of protein identification utilise mass spectrometry, including ion trap mass spectrometry, liquid chromatography (LC) and LCIMSn 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 a phosphorylation or glycosylation, and thus have utility in determining polymorphisms that result in or are associated with variation in post-translational modifications of proteins.

1001961 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.

1001971 It will be apparent that the presence or absence of the C allele or of the I allele at the C-1054T promoter polymorphism, or the presence or absence of the G or of the A allele at the G15929A exonic polymorphism, or the presence or absence of the G or of the A allele at the Al 8068G exonic polymorphism, in the BCMOI gene may also be determined by analysis of a polypeptide sample, derived from a bovine.

1001981 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.

1001991 A large number of methods reliant on the conformational variability of nucleic acids have been developed to detect SNPs.

1002001 For example, Single Strand Conformational Polymorphism (SSCP, Orita et a!., 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.

1002011 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).

1002021 Other methods which utilise the varying mobility of different nucleic acid structures include Denaturing Gradient Gel Electrophoresis (DGGE), Temperature Gradient Gel Electrophoresis (TGGE), and Heteroduplex Analysis (HET). 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.

100203] 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.

L00204] 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 heteroiluplexes by RNase A, of heteroduplexes by, for example bacteriophage T4 endonuclease Yll or T7 endonuclease 1, 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.

[002051 Further examples include the Protein Translation Test (PTT), used to resolve stop codons generated by variations which lead t 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.

1002061 Those skilled in the art will know that a particular SNP, particularly when it occurs in a regulatory region of a gene such as a promoter, can be associated with altered expression of a gene. Altered expression of a gene can also result when the SNP is located in the coding region of a protein-encoding gene, 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.

1002071 The above methods of detecting and identifying SNPs are amenable to use in the methods of the invention.

3 Polynucleotides, polypeptides, and variants 100208] 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.

1002091 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.

1002101 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.

1002111 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.

1002121 A "fragment" of a polypeptide or an amino acid sequence provided herein is a subsequence of contiguous amino acids that is preferably at least 10 amino acids in length.

The fragments of the invention preferably comprise or consist of at least about 15 amino acids, at least 20 amino acids, more preferably at least 30 amino acids, more preferably at least 40 amino acids, more preferably at least 50 amino acids and most preferably at least 60 contiguous amino acids of a polypeptide of the invention. A fragment of a polypeptide sequence can be used in the methods described herein, for example in polypeptide-based selection methods as described herein, and polypeptide fragments retaining functional activity may be used in methods of the invention that are reliant on such activity.

1002131 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.

1002141 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.

1002151 The term "variant" as used herein refers to polynucleotide or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the polynucleotides and polypeptides possess biological activities that are the same or similar to those of the wild type polynucleotides or polypeptides. The term "variant" with reference to polynucleotides and polypeptides encompasses all forms of polynucleotides and polypeptides as defined herein.

3.1 PoLynucleotide variants 1002161 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.

1002171 Polynucleotide sequence identity can be determined in the following manner. The subject polynucleotide sequence is compared to a candidate polynucleotide sequence using BLASTN (from the BLAST suite of programs, version 2.2.10 [Oct 2004]) in bl2seq (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences -a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250), which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blastl). The default parameters of bl2seq are utilized except that filtering of low complexity parts should be turned off.

1002181 The identity of polynucleotide sequences may be examined using the following Unix command line parameters: 1002 191 bl2seq -i nucleotideseqi -j nucleotideseq2 -F F-p blastn 1002201 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 = ".

1002211 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,1. 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.nirc.ac.uk/SoftwareIEMBOSS/. 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.uklemboss/align/.

[00222J 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 Biosciences 10, 227-235.

1002231 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 20041) from NCBI (ftp://ftp.ncbi.nih.govfblastl).

1002241 The similarity of polynucleotide sequences may be examined using the following unix command line parameters: 1002251 bl2seq -i nucleotideseqi -j nucleotideseq2 -F F -p tblastx 1002261 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.

100227J Variant polynucleotide sequences preferably exhibit an E value of less than I x more preferably less than 1 x 10.20, less than I x j30, less than I x 100, less than I x i050, less than 1 x l00, less than I x 1070, less than I x 1080, less than I x 10.90, less than I x 10 100, less than 1 x l0b0, less than 1 x lO20 or less than I x 10.123 when compared with any one of the specifically identified sequences.

1002281 Alternatively, variant polynucleotides of the present invention hybridize to a specified polynucleotide sequence, or complements thereof under stringent conditions.

1002291 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.

1002301 With respect to polynucleotide molecules greater than about 100 bases in length, typical stringent hybridization conditions are no more than 25 to 30°C (for example, 10°C) below the melting temperature (Tm) of the native duplex (see generally, Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Ausubel et al., 1987, Current Protocols in Molecular Biology, Greene Publishing,). Tm for polynucleotide molecules greater than about 100 bases can be calculated by the formula Tm 81. 5 + 0. 41% (G + C-log (Na+). (Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Bolton and McCarthy, 1962, PNAS 84:1390). Typical stringent conditions for polynucleotide of greater than 100 bases in length would be hybridization conditions such as prewashing in a solution of 6X SSC, 0.2% SDS; hybridizing at 65°C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in 1X SSC, 0.1% SDS at 65°C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65°C.

1002311 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 Iength)°C.

J00232J With respect to the DNA mimics known as peptide nucleic acids (PNAs) (Nielsen Ct al., Science. 1991 Dec 6;254(5037):1497-500) Tm values are higher than those for DNA-DNA or DNA-RNA hybrids, and can be calculated using the formula described in Giesen et al., Nucleic Acids Res. 1998 Nov l;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.

1002331 Variant polynucleotides of the present invention also encompasses polynucleotides that differ from the sequences of the invention but that, as a consequence of the degeneracy of the genetic code, encode a polypeptide having similar activity to a polypeptide encoded by a polynucleotide of the present invention. A sequence alteration that does not change the amino acid sequence of the polypeptide is a "silent variation". Except for ATG (methionine) and TGG (tryptophan), other codons for the same amino acid may be changed by art recognized techniques, e.g., to optimize codon expression in a particular host organism.

1002341 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., Bowic et al., 1990, Science 247, 1306).

1002351 Variant polynucleotides due to silent variations and conservative substitutions in the encoded polypeptide sequence may be detennined 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/blastl) via the tblastx algorithm as previously described.

3.2 Polypeptide Variants 1002361 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.

1002371 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/blastJ). The default parameters of bl2seq are utilized except that filtering of low complexity regions should be turned off.

1002381 Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs. EMBOSS-needle (available at http:/www.ebi.ac.uk/emboss/alignl) and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity.

1002391 Polypeptide variants of the present invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polypeptides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.10 [Oct 2004]) from NCBI (ftp://ftp.ncbi.nih.govfblastl). The similarity of polypeptide sequences may be examined using the following unix command line parameters: bl2seq -i peptideseq I -j peptideseq2 -F F -p blastp 1002401 Variant polypeptide sequences preferably exhibit an E value of less than 1 x more preferably less than I x 1020, less than I x 1030, less than 1 x lO°, less than I x 1050, less than I x I0°, less than I x less than I x 10.80, less than 1 x less than I x10 oo less than I x 10h10, less than I x I020 or less than I x l0.123 when compared with any one of the specifically identi fled sequences.

1002411 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.

1002421 Conservative substitutions of one or several amino acids of a described polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).

1002431 A polypeptide variant of the present invention also encompasses that which is produced from the nucleic acid encoding a polypeptide, but differs from the wild type polypeptide in that it is processed differently such that it has an altered amino acid sequence.

For example a variant may be produced by an alternative splicing pattern of the primary RNA transcript to that which produces a wild type polypeptide.

4 Diagnostic kits 1002441 The invention further provides diagnostic kits useful in determining the bovine BCMO1 allelic profile of bovine, for example for use in the methods of the present invention.

1002451 Accordingly, in one embodiment the invention provides a diagnostic kit which can be used to determine the BCMOI 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-wild type 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 wild type BCMOI or a functionally wild type BCMOI. 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.

1002461 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 BCMO1 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.

1002471 For example, the kit may include a primer comprising a cytosine at the position corresponding to the C-1054T promoter polymorphism in the BCMO1 gene or comprising a nucleotide capable of hybridising to a cytosine at the position corresponding to the C-I 054T promoter polymorphism in the BCMOI gene. Those skilled in the art will recognise that in such a primer, the cytosine, or the nucleotide capable of hybridising to a cytosine, as applicable, may be substituted for a nucleotide analogue having the same discriminatory base-pairing as the substituted nucleotide.

100248J In another example, the kit may include a primer comprising a thymine at the position corresponding to the C-i 054T promoter polymorphism in the BCMO 1 gene, or comprising a nucleotide capable of hybridising to a thymine at the position corresponding to the C-1054T promoter polymorphism in the BCMOI gene. Those skilled in the art will recognise that in such a primer, the thymine, or the nucleotide capable of hybridising to a thymine, as applicable, may be substituted for a nucicotide analogue having the same discriminatory base-pairing as the substituted nucleotide.

1002491 Those skilled in the art will appreciate that the invention provides kits comprising primers similarly directed to the G15929A (G278R) polymorphism, or to the A18068G (N34 ID) polymorphism.

1002501 In one embodiment, the diagnostic kit is useful in detecting DNA comprising a variant BCMOI gene or DNA or mRNA encoding a variant BCMOI 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 BCMOI which results in increased a-carotene levels (particularly increased 3-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 wild type BCMOI gene. The kit can also include a third primer complementary to a naturally occurring mutation of a coding portion of the wild e BCMOI gene. Preferably the kit includes instructions for use, for example in accordance with a method of the invention.

1002511 In one embodiment, the diagnostic kit comprises a nucleotide probe complementary to the sequence, or an oligonucleotide fragment thereof, shown in SEQ ID NO:! or SEQ ID NO:2, for example, for hybridisation with mRNA from a sample of cells; means for detecting the nucleotide probe bound to mRNA in the sample with a standard. In a particular aspect, the kit of this aspect of the invention includes a probe having a nucleic acid molecule sufficiently complementary with a sequence presented in SEQ ID NO: I or SEQ ID NO:2 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 (<1 OObp), lower temperatures must be used even with 100% homology. In general, one starts washing at low temperatures (37°C to 40°C), and raises the temperature by 3-5°C intervals until background is low enough to be a major factor in autoradiography. The diagnostic kit can also contain an instruction manual for use of the kit.

[002521 The invention also includes kits for detecting the presence of BCMO1 protein in a biological sample. For example, the kit can include a compound or agent capable of detecting BCMOI protein in a biological sample; and a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect BCMOI protein.

1002531 In one embodiment, the diagnostic kit comprises an antibody or an antibody composition useful for detection of the presence or absence of wild type BCMOI, or determining wild type BCMOI andlor the presence or absence of a variant protein at least partially lacking wild type activity, or determining the level of expression of the BCMOI protein, together with instructions for use, for example in a method of the invention.

1002541 For antibody-based kits, the kit can include: (1) a first antibody (e.g., attached to a solid support) which binds to a polypeptide corresponding to a marker; and, optionally, (2) a second, different antibody which binds to either the polypeptide or the first antibody and is conjugated to a detectable agent.

1002551 The kit can also include a buffering agent, a preservative, or a protein stabilizing agent. The kit can also include components necessary for detecting the detectable agent (e.g., an enzyme or a substrate). The kit can also contain a control sample or a series of control samples which can be assayed and compared to the test sample contained. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit.

Sample preparation 1002561 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.

1002571 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 1002581 It will also be appreciated that the methods of the invention are amenable to use with and the results analysed by computer systems, software and processes. Computer systems, software and processes to identify and analyse genetic polymorphisms are well known in the art. For example, the results of one or more genetic analyses as described herein may be analysed using a computer system and processed by such a system.

1002591 Both the SNPs and the results of an analysis of the SNPs utilised in the present invention may be "provided" in a variety of mediums to facilitate use thereof. As used in this section, "provided" refers to a manufacture, other than an isolated nucleic acid molecule, that contains SNP information of the present invention. Such a manufacture provides the SNP information in a form that allows a skilled artisan to examine the manufacture using means not directly applicable to examining the SNPs or a subset thereof as they exist in nature or in purified form. The SNP information that may be provided in such a form includes any of the SNP information provided by the present invention such as, for example, polymorphic nucleic acid andIor 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 2 and 3 and/or the Sequence ID Listing.

1002601 In one application of this embodiment, the SNPs and the results of an analysis of the SNPs utilised in the present invention can be recorded on a computer readable medium.

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

j00261 J 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.

1002621 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 082, 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.

1002631 By providing the SNPs andlor the results of an analysis of the SNPs utilised in the present invention in computer readable form, a skilled artisan can routinely access the SNP information for a variety of purposes. Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium.

Examples of publicly available computer software include BLAST (Altschul Ct at, J. Mol. Biol. 215:403-410 (1990)) and BLAZE (Brutlag et at, Comp. Chem. 17:203-207 (1993)) search algorithms.

1002641 The present invention further provides systems, particularly computer-based systems, which contain the SNP information described herein. Such systems may be designed to store andlor analyze information on, for example, a number of SNP positions, or information on SNP genotypes from a number of subjects. The SNP information of the present invention represents a valuable information source. The SNP information of the present invention stored/analyzed in a computer-based system may be used for such applications as identifying or selecting subjects, in addition to computer-intensive applications as determining or analyzing SNP allele frequencies in a population, mapping disease genes, genotype-phenotype association studies, grouping SNPs into haplotypes, correlating SNP haplotypes with response to particular drugs, or for various other bioinformatic, pharmacogenomic, drug development, or selection or identification applications.

1002651 As used herein, "a computer-based system" refers to the hardware, software, and data storage used to analyze the SNP information of the present invention. The minimum hardware of the computer-based systems of the present invention typically comprises a central processing unit (CPU), an input, an output, and data storage. A skilled artisan can readily appreciate that any one of the currently available computer-based systems are suitable for use in the present invention. Such a system can be changed into a system of the present invention by utilizing the SNP information, such as that provided herewith on the floppy disc, or a subset thereof, without any experimentation.

1002661 As stated above, the computer-based systems of the present invention comprise data storage having stored therein SNP information, such as SNPs andlor 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 prcsent invention, or a memory access facility which can access manufactures having recorded thereon the SNP information of the present invention.

1002671 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.

1002681 A variety of structural formats for the input and output can be used to input and output the information in the computer-based systems of the present invention. An exemplary format for an output is a display that depicts the SNP information, such as the presence or absence of specified nucleotides (alleles) at particular SNP positions of interest. Such presentation can provide a rapid, binary scoring system for many SNPs or subjects simultaneously. It will be appreciated that such output may be accessed remotely, for example over a LAN or the internet. Typically, given the nature of SNP information, such remote accessing of such output or of the computer system itself is available only to verified users so that the security of the SNP information and/or the computer system is maintained. Methods to control access to computer systems and the data residing thereon are well-known in the art, and are amenable to the embodiments of the present invention.

1002691 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.

[002701 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 colour or f'-carotene content phenotypes and optionally a reference milk colour or f3-carotene content phenotypes database of non-genetic factors for bovine milk colour or f3-carotene content phenotypes; and a computer program embedded within the computer processor which, once data consisting of or including the result of a genetic analysis for which data is included in the reference genetic database is received, processes said data in the context of said reference databases to determine, as an outcome, the genetic merit of the bovine, said outcome being communicable once known, preferably to a user having input said data.

1002711 Preferably, said system is accessible via the Internet or by personal computer.

1002721 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 colour or 3-carotene content phenotypes, more preferably the one or more genetic loci are one or more polymorphisms in one or more genes associated with one or more milk colour or 13-carotene content phenotypes, preferably one or more polymorphisms in one or more genes involved in 13-carotene uptake or metabolism.

1002731 Preferably the one or more genes involved in 13-carotene uptake is the SCARBI gene. Preferably the one or more genes involved 13-carotene metabolism is selected from BCO2 or BCMOI.

1002741 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 colour or 13-carotene content phenotypes in the context of both a reference genetic database of the results of said at least one genetic analysis and optionally a reference database of non-genetic factors associated with bovine milk colour or 13-carotene content phenotypes.

1002751 Preferably, the one or more genetic loci are one or more polymorphisms in one or more genes associated with one or more milk colour or 13-carotene content phenotypes, preferably one or more polymorphisms in one or more genes involved in 13-carotene uptake or metabolism.

1002761 Preferably the one or more genes involved in 13-carotene uptake is the SCARBI gene. Preferably the one or more genes involved 13-carotene metabolism is selected from BCO2 or BCMOI.

1002771 It will be appreciated that it is not intended to limit the invention to the above example only, many variations, which may readily occur to a person skilled in the art, being possible without departing from the scope thereof as defined in the accompanying claims.

1002781 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.

1002791 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 1002801 This example describes the investigation of the genetic basis for observed variations in milk fat colour using the results of a Holstein-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 1002811 A Holstein-Friesian x Jersey crossbred trial was conducted using an F2 trial design with a half-sibling family structure. Reciprocal crosses of Holstein-Friesian and Jersey animals were carried out to produce six Fl bulls of high genetic 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 1002821 Cows were milked twice daily; milk volume was recorded at each milking. Milk fat colour was measured at three time points during the second 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 al., 1999). Briefly, nonsaponifiable material (including carotenoids) was extracted from fresh milk samples and the absorbance at 450nm was measured. Fat colour (gig 3-carotene/g milk fat) was calculated (Winkelman etal., 1999).

3. Genotyping [002831 Genomic DNA was prepared from whole blood from a total of 1679 animals within the trial pedigree (846 F2 daughters, six Fl sires, 796 Fl dams, and 13 selected 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 used for QTL analysis.

4. Candidate gene sequencing 1002841 BCMO1 was identified as a candidate gene for the milk fat colour QTL on chromosome 18, based on its mapped location to bovine chromosome 18, within the 95% confidence interval of the QTL. Intron/exon boundaries were determined by homology with the human gene sequence. The promoter was amplified using the primers presented as SEQ ID NOS:4-7 and sequenced in both directions. Exons were also sequenced in both directions.

The determined wild-type gene sequence for the BCMOI gene is shown in SEQ ID NO: I. 5. Statistical analysis 1002851 Data analysis was performed using SAS (version 9.1). Phenotype data for milk fat colour was recorded at three stages of 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 I 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.

1002861 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 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 (significance level set at 0.1). Thus, the final models were as follows: peak lactation (sire, cohort, milk protein%, calving week), mid lactation (sire, cohort) and late lactation (sire, cohort, milk protein%, milk solid%, and somatic cell count).

6. QTL detection 1002871 The data used for QTL analysis 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 analysis was conducted using a line of descent model and a half-sib model. Subsequently, the BCMOI mutation was included as a covariate into the models for peak, mid and late lactation, and QTL analysis was performed to test for an association between the BCMOI mutation and QTL significance.

7. Cloning and expression of recombinant BCMO1 1002881 Bovine cDNA was obtained by reverse-transcriptase PCR from mRNA extracted from small intestine tissue of a Friesian cow. The full length BCMOI coding sequence was obtained by PCR using the Advantage GC polymerase (Clontech, California, USA). The forward primer (TOTGCGGCCGCCATGGAAATAATATFTGG [SEQ ID NO:12]) contained a No/I site (underlined) and the reverse primer (GATAGTCCTCACGGCCAAAA [SEQ ID NO:13]) was placed in the 3' UTR. The resultant PCR product was T/A cloned, using the T/A PCR-produced 3'-adcnosinc overhang, into pGEMT-easy (Promega, Wisconsin, USA), and the inserted DNA from selected clones were sequenced to verify BCMO1 sequence integrity. The BCMO1 gene was subsequently digested out of pGEMT-easy with Not!, and ligated into the pFLAG-CMV-2 vector (Sigma-Aldrich), also linearised with Noti, to create a FLAG-BCMOI fusion protein.

[002891 Resulting clones were digested with NotI to determine the presence of full-legth BCMOI insert, and with BamHI, to determine the orientation of the insert. The insert from selected clones were sequenced to verify the full length sequence of the BCMOI coding sequence, and to verify the reading frame. Site directed mutagenesis was then performed to generate clones containing each of the following amino acids 278G/341D, 278R/341N and 278R/341D, (with the wildtype protein being 278G/341N) using the QuikChange Mutagenesis Kit, (Stratagene, La Jolla, California), following manufacturer's instructions.

The primers used for the site-directed mutagenesis are presented in Table I below.

Table 1: primer sequences used for Site-directed mutagenesis Primer name Primer sequence G I 5929A (G278R) -sense strand cctggccttccacgggaggacaagac [SEQ ID NO: 14] G I 5929A (G278R) -anti-sense strand gtcttgtcctcccgtggaaggccagg [SEQ ID NO: 15] Al 8068G (N34 1 D) -sense strand tctacttggccaacctggacgaggactttaaggag [SEQ ID NO: 16] Al 8068G (N34 ID) -anti-sense strand ctccttaaagtcctcgtcaggttggccaagtaga [SEQ ID NO: 17] 1002901 Sequencing verified the site-directed mutagenesis and the plasmid DNA was purified by midi-prep (Qiagen, following manufacturer's instructions). For transfection experiments, CHO-Ki cells (ATCC, Manasass, VA) were seeded into 75 mm2 flasks (1.5 million cells per flask, with 2OmL DMEM). After 24 hours, at approximately 50% confluency, the cells were transfected with 10 tg plasmid DNA, with each of the clones separately transfected, using Fugene 6 as a transfection agent as per manufacturer's instructions (Roche Applied Science). One flask was transfected per plasmid, per experiment.

Cells were harvested 48 hours following transfection for measurement of BCMOI activity.

Expression of a -64 kDa FLAG-BCMOI protein in cells was verified by western blotting, for each of the protein variants, using the FLAG M2 monoclonal antibody (Sigma-Aldrich) at a concentration of 1: 1000, and detected using the ECL detection kit (GE Healthcare), according to manufacturer's instructions.

[002911 BCMOI activity was measured as followed. Cells were harvested 48 hours following transfection using the protocol of During et al., 1996. Briefly, cells were rinsed twice with Solution A (50 mM HEPES, 1.15% KCI, 1 mM EDTA, 0.1 mM DTT) and scraped into two mL Solution A. The cell suspension was centrifuged at 2000 x g, for 10 minutes at 4°C, and the cell pellet was resuspended into 350 tl Solution A. The cells were then disrupted using the FastPrep instrument (MP Biomedicals, Solon, Ohio) and using lysis matrix D (2mL capped tube containing 1.4mm ceramic spheres; QBiogene). The lysate was then centrifuged at 9000 xg, for 30 minutes at 4°C. The resulting supernatant was pippeted off the pellet, snap frozen in liquid nitrogen and stored at -80°C for subsequent enzyme assay. Enzyme activity was determined by measuring the conversion of [i-carotene to retinal, as described in During et al., 1996, but using 25 iL cell lysate in a total reaction volume of 50 1.tL. Relative enzyme activity was calculated as fmol retinal formed per mm (frnol retinal/mm). Protein assays were conducted using a modified Lowry assay (Bio-rad protein assay).

Results 1. il-Carotene concentration in milk is influenced by sire and by stage of lactation.

1002921 The variation in 3-carotene concentration in milk throughout lactation is shown in Figure 1.

2. Detection of a major QTL for milk fat colour on bovine chromosome 15 1002931 Analysis of the 13-carotene data within the half- sib model of QTL analysis showed the presence of a significant QTL on bovine chromosome 18 (Figure 2). The maximum F value for the QTL was 7.8, and the most likely position was estimated at 15 cM. Bootstrap analysis (n 1000) showed that the 95% confidence interval for the QTL was 5 -30 cM (shown as a grey bar in Figure 1). The information content for the markers used in mapping on chromosome 18 ranged between 0.625 and 0.847 and averaged 0.774. There were a total of 190 markers (6 microsatellite markers and 184 single nucleotide polymorphisms) that comprised the chromosome 18 genetic map.

3. Identification of BCMOI as a candidate gene and detection of a polymorphism 1002941 13-carotene 15', 15' -monooxygenase (BMO 1) catalyses the symmetrical cleavage of fl-carotene to vitamin A (see von Lintig, J. and Vogt, K., 2004) and is located within the milk 13-carotene QTL confidence interval bovine chromosome 18 (Figure 2).

Therefore, this gene was identified as a strong candidate for the chromosome 18 milk fat colour QTL. To determine whether this gene explained the observed variation, the BCMOI region in the six Fl sires was sequenced to identify any genetic polymorphisms that could potentially alter the function, activity, or expression of this enzyme. Intron/exon boundaries were determined using the UniGene bovine gene prediction Bt.15605 and by sequence comparison with the human BCMOI gene sequence (UniGene Hs.212l72). 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.

1002951 Three polymorphisms in the bovine BCMOI gene were identified. The first was a 5' C to T substitution at genomic nucleotide position -1054 (relative to the +1 translation start site), and was heterozygous in two of the six Fl sires. Two single nucleotide polymorphisms causing amino acid substitutions were discovered; one in exon 6: G15929A (G278R) and one in exon 7: A18068G (N341D). G15929A was heterozygous in two Fl sires, with the remaining four Fl sires homozygous for the G allele, while A18068G was heterozygous in one Fl sire, with the remaining five sires homozygous for the A allele. To determine whether these polymorphisms were associated with the QTL effect, the remainder of the FJXB trial pedigree was genotyped. The frequency for each BCMO1 genotype is shown in

Table 2 below.

Table 2: Genotype frequencies of F2 population Polymorphism Genotype Total % C-1054T CC 623 74.43 CT 205 24.49 ___________ Ti' 9 1.08 G15929A GG 627 74.64 GA 202 24.05 _____________ AA 11 1.31 A18068G AA 711 84.64 AG 124 14.76 _____________ GG 5 0.60 4. BCMOI polymorphisms have an effect on n-carotene concentration in milk 1002961 The association of the BCMOI polymorphisms with variation in milk fat colour (3-carotene concentration) within offspring of the two heterozygous sires is shown in Table 3 below and in Figure 4.

Table 3: Genotype effect on milk fat colour.

Milk fat colour Qtg 3-carotene/g milk fat) Polymorphism Genotype Peak Lactation Mid Lactation Late Lactation C-1054T CC 9.22 (0.27) 7.78 (0.27) 6.56 (1.15) CT 10.37 (0.20) 8.64 (0.21) 6.72 (0.76) 11' 10.90 (0.18) 9.37 (0.13) -7.45 (0.39) G15929A GG 11.34(0.17) 9.32 (0.13) 7.32 (0.09) GA 9.67 (0.24) 8.15 (0.20) 6.71 (0.16) AA 8.34 (0.60) 7.94 (0.75) 5.73 (0.58) A18068G AA 10.84 (0.15) 9.0 (0.12) 7.12 (0.09) AG 11.15 (0.39) 9.11 (0.29) 7.35 (0.19) GG 15.95 (0.9) 11.45 (0.38) 8.70 (0.72) Data shown are means with standard errors in brackets.

1002971 Inclusion of the BCMOI C-1054T, or G15929A, or A18068G genotypes as fixed effects in the statistical model reduced the QTL effect for milk n-carotene to below a statistically significant threshold. The QTLs resulting from the adjusted statistical effects are shown in Figures 5, 6 and 7.

100298] For the BCMOI C-1054T polymorphism, animals homozygous for the T allele produce milk with greater concentrations of f3-carotene than homozygous C animals. For the GI 5929A polymorphism, animals homozygous for the G allele produce milk with greater concentrations of 13-carotene than homozygous A animals. For the Al 8068G polymorphism, animals homozygous for the G allele produce milk with greater concentrations of J3-carotene than homozygous A animals. For each polymorphism, the effects were similar at each of the three lactation time points that were measured.

1002991 For the C-1054T polymorphism, animals homozygous for the C allele produced milk of approximately 15.4%, 17% and 12% less 13-carotene than animals homozygous for the T allele, at peak, mid and late lactation, respectively. For the G15929A polymorphism, animals homozygous for the A allele produced milk of approximately 26%, 14.8%, and 21.7% less 13-carotene than animals homozygous for the G allele, at peak, mid and late lactation, respectively. For the Al 8068G polymorphism, animals homozygous for the A allele produced milk of approximately 32%, 21%, and 18% less 13-carotene than animals homozygous for the G allele, at peak, mid and late lactation, respectively.

5. Effect of BCMO1 polypolymorphisms on enzymatic activity of recombinant BCMO1 1003001 The effect of the BCMO1 polymorphisms on the enzymatic activity of recombinant BCMO1 was determined as described above.

[003011 The enzymatic activity of recombinant BCMO1 carrying the 341D and 278G residues, the 341N and 278R residue, or the 341D and the 278R residues (compared to wild type of 278G/341N), is shown in Figure 8. Data are representative of two independent experiments, and the protein content was equal for each lysate within an experiment.

1003021 The above data show that the presence of the 341 D residue (instead of wild type 341 N) results in markedly decreased enzyme activity. Notably, the 278R version has similar enzymatic activity to wild-type-BCMO1, as defined by 341 N and 278G. Indeed, this activity accords well with the milk J3-carotene content data presented above and in Figure 4. Animals homozygous for the 34lD amino acid produce milk that is higher in 13-carotene (figure 4B).

Further, the N34 1 D polymorphism explains more variation than the G278R polymorphism when fitted as a fixed effect in the QTI. analysis (Figures 5 and 6). The BCMOI enzyme cleaves 13-carotene resulting in colourless retinol, and consequently reduced enzyme activity would be expected to result in higher 13-carotene milk content (increased yellow colour).

Discussion 1003031 The present invention recognises that the BCMO1 polymorphisms described above, together with polymorphisms in linkage disequilibrium with these polymorphisms, are useful as a selection tool to identify and breed animals with higher or lower milk concentrations of 13-carotene, and thus milk fat colour. Such a strategy may allow the production of 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.

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INDUSTRIAL APPLICATION

1003041 The present invention is directed to methods of genotyping bovine to facilitate the selection of animals with altered milk production phenotypes. in particular, such phenotypes include desired milk colour and desired milk composition. It is anticipated that herds of bovine selected for such traits will produce milk fat of more desirable colour (whether more or less yellow), or more desirable composition, and therefore be of significant economical benefit to farmers.

SEQUENCE ID LISTING

<110> Vialactia Biosciences (NZ) Limited <120> Marker Assisted Selection of Bovine for Desired Milk Content <130> 576277 JBM <150> NZ 564717 <151> 2007-12-24 <150> NZ 566978 <151> 2008-03-26 <160> 17 <170> Patentln version 3.5 <210> 1 <211> 32605 <212> DNA <213> Bos taurus <220> <221> variant <222> (135)..(135) <223> - 1054 CIT polymorphism <220> <221> misc_feature <222> (3158)..(3207) <223> n is a, c, g, or t <220> <221> variant <222> (17117).. (17117) <223> G15929A (G278R) polymorphism <220> <221> variant <222> (19256).. (19256) <223> A18068C (N341D) polymorphism <400> 1 agtgcctctc acttttctca ctccacccag agaggcaaca gctggttctt tcttgaagca 60 acgtaagtaa agtgaggtca gtaactcatc atccaacgta gcaacgtgag tgtggatttc 120 tggtqataac atcacctcga gattcagaaa tgcaggggaa ccttcagtac tattccttac 180 agttcgtgcg tagtttttgg ttcccagtat ttattattta aaagggtaca tgaaacgttt 240 tatgtttact tcttcaaaaa gtgaaatata attaagttgc tctttacata ggattctttt 300 tcttgctggg aggtaggqgg tggtgaggag actcagagac ctttagtcaa aataaaacca 360 aacaattttc cctaaatgat cttgccagtt tttttttttt tttttttttc atttttattg 420 tagtatagtt gatttggggc ttccctagtg gctcagtgat aaagaaccca cctgctaatg 480 caggagatgt gggtttgatc cctgqgtcag gaggatgccc tggagaaaga aatggcaaca 540 cactccagta tttttgcctg gacagaggag tctggcggcc tacagtccat ggggtcgcaa 600 aagaactgga cacaacacag cgactaaagc aatagcgtag ctgatttact aaagtaagag 660 cataacatag cgttatgtta atttcaggta.tacagcaaaa cgaatcagtt atacctatac 720 agtatagaca ctcttttttt aggttctttc ccatataggc cattatagag tattgagtag 780 agtttcctgt gctatacaga agctccttgt tatctatttt atatattagt gtgtatgtat 840 caatcccaat cttccaattt atccttcccc ccatcccacc agtttaaacc catcaagaaa 900 gttgtttgtt agttttggtt cagccaaatg tggaagtgga aagggagagc tggggatgta 960 atcggaggat taaagattag ctgccacaca tggccgggga actgaccttt gaccaaatat 1020 aagtcacgcc agcacagctt ctccctgtgc aggaagagat cccaggccct cgcagtgcca 1080 tctgaaggga gggagatgta aaggaagctg cagggaggga agacaaggag tggccaagag 1140 cagtccctga acacggacga gcatcgctct cgctcagagc cctgcacaat ggaaataata 1200 tttggcagaa ataagaagga gcaactggag cctgtgaggq ccagagtaac aggtgagcat 1260 tttgtataaa ccacgggtac ttacatgttt agaLgtacac tttttttttt cctgcttata 1320 aaagtaatgc ttactgtaaa tcatttttgg aaaatctgga aaaccgtaaa tagaggtgac 1380 atgatccata ttctaggcat attccctgtg aaaatctcag tgtgctttta tgcattataa 1440 ctcagctttc aaggggagtc agtggttctc actacttcca ggagaaaagt ccgaggacta 1500 ctgctattgg ctcatagcca gtgggattat cctgcaaact gtgctctgtc cccagggtgc 1560 ggtatttgta aaagtgggtg catcctaaat taatttcaaa atgatacgag aagcgtgtgc 1620 gagggcccgt tcttgtgcat gcgcggtgcg ggagggcacc gcagatcccg atgaaatttc 1680 ctcaaaggtg cggtttcagg gtgcgagcac tgcagagcag atgagacagc tccagggtgg 1740 gagctgccac atgaaccctc cttgcatagc tttctcctcc tcttctgcat ggccactttc 1800 ctgggaccat cttgtttaga ctgtacaggg cagagagtgg tctgcacttc gggtcccagg 1860 agccgtgctg agaacaagga agggggtcag ctcagccaca ccactgtttt ggacagatgg 1920 gcagttttca caaggacgag acagcactgc cctctgagag gcaagtcaca ttggagcttt 1980 acagcagacg aattgccttg gaaggctttt ggaagaaaga aatagcatgt atgtgtattt 2040 tttctgatta cagaggtaat gtgcaatcta tatatgtatg tatattcagt tcagttcagt 2100 cactcagtcg tgtccgattc tttgtacttt atatccagag ggatataaaa atcagctgta 2160 tttcttctgt ctcttaagga ctgttaatat ttgagtctat tttctttaag tttttaaaag 2220 aaacatcaat atagatagta tatgcatgcg tcctccgtcg cttcagtcat gtctgaccct 2280 ttgcaaccat atggactgta gcccaccagg ctcctctgtc aatgtgattc cccaggcaag 2340 aatactggag tgggttgccc tgccctcctc caggggatct tcccaaccca gggattgaac 2400 ccgcgtctcc tgcattgcaq gtggattctt taccactqag ccacccggga aacccataag 2460 attaatctga tgataggtag actttatata caaatacttt ttcaaaaaac aaatgtacac 2520 ttactattaa ctttttattt ttaaaaaaag tggatctgtg atacccatca ttgtatgctc 2580 tggggtacgg tgggcatttt gccattatac tcccttgcag ctcatgttta ttggctgcat 2640 gtcaatactt ggaaatttta gagaacaaga atcccgggac ctgagatggt gaatgaacct 2700 ctagatctaa gtccgttgtt ttcacaggat ggtccccagc cctgcagcat gagcactccc 2760 caggctcttg ttagaatctc aaattcctgg acccacttca gaccctctga atcagacacc 2820 caggggtgga gcccagaaat gtgtgttttc agagccctct ggctgcgtgc aggtggggga 2880 ccactgctgt aaactgggcc cccttccttg acaggtgtga agctgcgggc ctgggagaag 2940 catcgacttg cccgaggcca ggcqgccagc agaggacagg ctccagccca gtgccagcct 3000 ccctgcaggg tcacgtctaa tccaccagtc agqgtctgct ggcaggcagc tcactacaca 3060 cqgtgctgtg tggccgcatc tgtccagqga agccaqgcca aggctctggc ctcacatccg 3120 gcagggcttt cgtgactctg attcttcctt tacaaganflfl flflflflflflflflflfl flflflflflflflflflfl 3180 nnnnnnnnnn nnnnnnnnnn nnnnnnngca attgccacat tattagaaac caaatgtgaa 3240 tgcatcttgc tgaatggaat gaagccaata tctcctatgt cctctaatct ctctctcctt 3300 cctaatqaat ttqaattcct taggqtgtga aaagggggga gaagagtaga gagagatgtg 3360 gtcatgtttt cactgtccct gagaggyacc aaaagtacta ggaagtcacc catttgagta 3420 tccaqqaaac ttcagaaaaa ggaaggactc aagcttttta ttttaaatga ccaaccaaat 3480 tcagtcttgt ggaggtgggg ggaagtcaga tttaacagag gaagttagtt aaataagttt 3540 cacaatgtgg aagggaagct aacatgacct tgtqggtggt gccctgtgag ccaactcaaa 3600 ttatagtttt agccacttct gccaggtcgc gctagtgata aagaacctgc ctgccaatgc 3660 aggagacaaa agagatgcag gtttgatccc tgagtgggga agatctcLtg gtggagagqg 3720 tggtaaccca ctccagtatt cttgcctgca gaatcccatg ggcagaggag cctggtggcc 3780 tatagtccac agggtcgcaa qa9ttgaaca cgactgaagc ggcttagcac gcatgctgat 3840 tgcataatgc ttttgtttta cttgcttcct ctctgatgta ctaaattata gagaggattc 3900 tttttgatcc ggaagaattc atgtataaaa gtgctaatgg gataaaagga tagctccaat 3960 tttttttccc aattcagtct tgggcagtcc aatatacctt gtggctcagc tggtaaagaa 4020 tccgcccgca atgcgggaga cctgggtttg atccctgggt ggggaagatc ccctggggaa 4080 gtgcatggca acccactcca gtattcttgc ctggagaatc cccatggaca gaggagcctg 4140 gtgggccata gttcatgggg ttgcaaagag atgggcatga ctgagcgact aagcacagca 4200 caaatgttct ttttacatct agggctttac ttttaagctc agattcatct ttccctgctc 4260 tgcctcaacc tgctccttct cctgtgtccc aggtcactgt gaatagtcac acccagggca 4320 ggagactcgg ctggcacttt ttcagtqttc cccttccatt tcccctacaa ttagtcaaac 4380 tctaagtccc tttgttttat cagtttctct ctgtcccact gctactatct( tcttcctctc 4440 tcaggtcttc ccacctctct ctagcttctc ctccaaccca gagtccacac catagccaga 4500 aataactttt ggtaccccaa atctgatcac attccattag tatgagaaga ggtaggtcct 4560 tagagacaga acgtggatta ggggttgcta ggagctgggt ggcatatgca gactaactgc 4620 taatgagcac agggcttctt ttgggggtga tggaaatgct cctgggttgg tggtatggtt 4680 gcacaacatt gtaaatatac taaacacaca cacacacaca ccctgaattg tatactttga 4740 aatgatggat ttatgttatg tgaattatat ctcaattttt aaaattgcaa aagaaaaaaa 4800 ttaatcctat tattccctgt cacacccatc aaaagtgtct ctttcttcta agcaccaaat 4860 caaagctaga tttggtctta ctgatcttgc cctctactta gccttgcagc ctcaatactt 4920 gctataccct cacttgaaag ttacaggcca gctcttacta aacagcttgc atacttcttg 4980 gtctttgaac cagtctctgg tcLctgggtt caggtttttc cagagataag aattcaggag 5040 taagtcattt atttgggatg tgcctcccag ataatcacga tgatgtgatc actgacctag 5100 agccagacat cctggaatgt gaagtcaagt gggccttaga aagcatcact acaaacaaag 5160 ctagtggagg tgatggaatt ccagttgagc tgttcccaat cctgaaagat gatgctgtga 5220 aagtgctgca ttcaatatgc cagcaaattt ggaaaactca gcagtggcca caggactgga 5280 aaaggtcagt tttcattcca atcccaaaga aaggcaatgc caaagaatgc tcaaactacc 5340 gcacaattqc actctctca cacactagta aagtaatgct caaaattctc caagccaggc 5400 ttcagcaata tgtgaactgt gaacttcctg atgttcaagc tggttttaqa aaaggcagag 5460 gaaccagaga tcaaactgcc aacatccact ggatcatgga aaaagcaaga gagttccaga 5520 aaaacatcta tttctgcttt attgactatg ccaaagcctt tgactgtgtg gatcacaata 5580 aactgtggaa aattctgaaa gagatgggaa taccagatca cctgatttgc ctcLcgagaa 5640 atttgtatgc aggtcaggaa gcaacagtta gaactggaca tggaacaaca gactggttcc 5700 aaataggaaa aggagttcgt caaggctgta tattgtcacc ctgcttattt aacttatatg 5760 cagagtacat catgagaaac gctggactgg aagaagcaca agctggaatc aagatgcLg 5820 ggagaaatat caataacctc agatacgcag atgacaccac ccttagggca gaaagtgaag 5880 aggaactaaa aagcctcttg atgaaagtga aagaggagag tgaaaaagtt ggcttaaagc 5940 tcaacattca gaaaaccaag atcatggcat ccggtcccat cacttcatqg gaaatagatg 6000 ggaaacagtg gaaaccgtgt cagactttat ttttctgggc tccaaaatca ctacagatgg 6060 tgactgcagc catgaaatta aaagacgctt actccttgga aggaaagtta tgaccaacct 6120 agatagcata Ltcaaaagca gagacaLtac LLLgccaaca aaggtttgtc tagtcaaggc 6180 tatggtcttt cctgtggtca tgtatggatg tgagagttgg actgtgaaga aggctgaggg 6240 ccgaagaatt gatgcttttg aactatggtg ttggagaaga ctcttgcaag tcccttggac 6300 tgcaaggaga ttcaaccagt ccattctgaa ggagatcagc cctgggattt ctttggaagg 6360 aatgatgcta aaqctgaaac tccaqtactt tggccacctc acgcaaagag ttgactcata 6420 ggaaaagact ctgatgctgg gagggattgg gggcaagagg agaaggggac gacagaggat 6480 gagatggctg catggcatca ctgactcgat ggacgtgagt ctgagtgaac tccaggagtt 6540 ggtgatggac agggaggcct ggcgtgctgc gattcatggg gtcgcaaaag agtcagacac 6600 gactggcaa ctgatctgat ctgatcccaa ggaacactgg gcgaaggagg cgttgtcaag 6660 ccattgacca ccaggagtta ttg.gagctgc atcctccggg caaagcataa accacgcctc 6720 acattgtgtc caccgagggc aaggaagtgg tggccttgga cccacagcac cccatctgtc 6780 gtcgggtgag agcagcttcc aggggcatta actctccaac gtggttctgg cttgtccatg 6840 tgtgtgcaga gctgctccac agtgagaaca aagccctcag gtgaagagct ttaggtattt 6900 gcagaaagca gccttcaggg agagatgaaa gccaagggga cgtgggcagg gcgccgacag 6960 cacctgcaac actgtcccaa cagccgtgga atgcctgcct cacgtataat ttaaactttt 7020 ctaatagccc cgtaccaaaa agaacaaaga aacagatggc attaatttta atagtgtttt 7080 tcctttaatg ccacatatcc aaaatgttat cagttcaaca tatagctaat gttcttaaat 7140 tgctaatgag atacatttct ttttcatcct aagtctctgg aatctggtgt gcatttcaca 7200 attactgtac atctcagttg ggactagcca agtttccagt acccagttcc cctacatggg 7260 acagcacagc tctagaatgt tcLitcctca tgcctttgca aggcaaacac caactctccc 7320 tcaaaactca accaggtcac ctcctccagg aagtgttcct caccctcctg ctctcaaccc 7380 accttggagc ggagttctgc tccacccagc acccttgctg agtctgtgga actgccttta 7440 tcacagtgta ttttgggtat tttccttagt ctttgtctct taatggatga ggagttcctt 7500 ggaagcagtg tgcatgggtg ctcagttggg tccaactctt tgtgtctcca tggactgtaa 7560 cctgccaggc tcctctgtcc atgggattcc aggcaagaat accggagtgg gttgccatgc 7620 cctcccccag gggtcttccc aacccatgga tggaacccgt gtcttacgtg tcctgcactg 7680 acaggcaggt tctttcccac tagtgccacc tgggaagccc cacttgaaca taggcatgct 7740 gcctgttcat ctcggtaccc tcagcgccca cacaatgcca ggcacattct ttctggaagt 7800 qaacagattc agtgagtggt agaggggctg tgtggacact tctttgagct ttttgcjttta 7860 ggtgcagagc tgqaaactaa acattctttc tctcatcagg caaqattcca gcctggctgc 7920 aggggatcct gctccgcaat gggcctggga tgcacacggt gggcgagacc agatacaacc 7980 actggttcga tggcctggcc ttqctccaca gcttcaccat caqagatggt aagagcaccg 8040 catggtttyc cytgygLgct gtgacaaaLg accacagaLg gagtatctta aagcaacaCa 8100 caattctatt tctcttctgg tttagtagct aaatcatgtc tgactctttt gtgatcccat 8160 gcacggtaac ccaccaqgct cctctgtcca ggggattttc caggcaagaa tactqgagtg 8220 ggctgccatt tcctcttccg ggggtcttcc tgacccaggg atccaacctcj catctcctgt 8280 ggctcctgca ttgcaggtgc agcctttacc actgagccac ctggaaagca cctaaaagtt 8340 tctagtqagt ctcagactaa aactttactc tcaqaggtaa gcagctgttt cctgttcttt 8400 tctgagatgq gcagtcaggt tgccagtgtt ggtgcagcac agcctggcct ggtctctcct 8460 ctcctctaga gctctcaatg cctcccctgc gtctcacagt ggaaatqtaa acaaaaacat 8520 taagtacttc tatacacaat ttaagagggc acacataagg caatcagggc cctccgattt 8580 gcaagtaaca taaagccagt acttctcaaa agatggtctg tgggtttctg catcagaatc 8640 ccccaggatg ctcatttaag ctgtgaatcc tgagaccctg cccagggctc aaaqggaagg 8700 atagagcatc tgtgttccca gatgaccccg agcacactgg tggtgtgggc ctccggcctc 8760 cgtggtcttc aaaaccaggc ggttctaqtg cacagttggc atgaggaacg gaaatcagtg 8820 ccaaaccggg aagacaagaa ctggagtaag. aacagcaaag acagaaaaat qactcataac 8880 ccttagcaga gyccaccaga agataatgct ttcaaacact aacaaagatt tcaatcataa 8940 aggccaagtg gagatgggga agagtataca ggagcgcttt tttcaagtct aaaattcttt 9000 caaaattaaa attttttata aagcagattt gaagatgagt caacccaggt cccccttagc 9060 gatagatttg attgttttcc aataagaaac caagccgtca tcccatgagg atcgaacgtg 9120 tgaattccaa gagtcaaagt cacagagaga ttttttttaa attatagttg atttacaatg 9180 ccgtgttagt ttcaggtgtg cagcaaagtg aàtcagttat acacatacat gtatctactc 9240 ctcttttttt tttggagaag qaaatggtaa cccgctccag tactcttgcc aggagaatcc 9300 catggacaga ggagcctgct ggcatgctgt agtccatggg gtcgaaaaag agtcggacac 9360 aactgagtga ctgaacaact ctttttttag attcttttcc catataggct ataacagagt 9420 gttgagtaga gttcccgttc tatacaggaa gtccttatta gttatctact ttatacatat 9480 agtagcacat gtaggtcaat ccctgggcga gaattccctg agatgcagcc tctccttgaa 9540 gccatccttg tgacagtgga tatgtgatgg gcatttctct gatgggaacg agttgtcagt 9600 gtgtcttggg ccccactttt tggcatatta taattgcacc aaccatggaa gcataggtta 9660 tactacagca aggaatgtga aaagtcaaca taatgtcatg tgctaattaa tggcaaaaaa 9720 accccaccaa actctagagg tttcaaatta cccaccaaaa tgaaggcccc aggcctaagt 9780 agtcacagcc ctgaagacct ggtggggtcc ccacgggtcc cccagcctcc ccccattcct 9840 gctgctctca agcacctaac tqgtgcttca gcctctgtgg actcagccct cacaggaagt 9900 gggctgtgct ccccaacccc acgtcttgac ttacagactc tggggagact gactgggaac 9960 acctcgccct ctttcctagc cccacctccc cgattcgtgg ttcagttcca cttatccttc 10020 aagggctttg aaaagttgtg gcaagqgaat cagagatcag agtgacacag ccacaaacca 10080 aggaaaacag actctccctg cgagctccaa gaggaacccg tcctgctgac actttgattt 10140 ttqccctgaa aaacccactt caqacctqtc tttcaaaaca gataacaaat ttgtgtttcg 10200 tgaagccact acatttatga taaLLtgtLa cagcagccac aaqaaatLaa Lacggtcta 10260 gatcatcta gtatattttc acgttggtct tggcagactg gtcaagggtc catgcttttc 10320 tggaqtgagt gagattctct ccagtgtcat caacacatgt actgaccctc tcctgtgtgc 10380 agaatgggta taagagtgct ccctggttgc agagtacggc agaagccaac acaatattgt 10440 aaaacaaata tcttccaatt aaaaattttt taaagatttg aaaaataatt ttagtgagag 10500 caaaaagtct tctgtaccat taataattaa aaatgaaacc atgtttatt caattaaaaa 10560 aatgttactc caggggctaa attgttaatg taagqctccc agaactgaac tgggtccttg 10620 ggaacccag cctgagtaLg gggccttctg qtcagtcct ctaaacctgg accttacctg 10680 gagggactcc accgtggtgg aaggcatggt. cagtggcctq ggccagcagg tctctgggtg 10740 atgctqqaca aggcgatgga acafttctga gcctcagttc cgccatacgt aqatggagct 10800 gggactagga atacttacct tgccgtattg ttgtgagtca accttgccaa aatgtatgtg 10860 agagttcaca ccagcqctta ttgtatccag cacacacatt taagagatgg tctcagtaag 10920 ccacccccac ccccaccccg accccttcac tccctggaa tggctcaaat aagttcagat 10980 aagtttcaaa taagcctgtg atgattctgc tgcttctctt tcaggtgaag tctactacag 11040 gagcaaatac ctgagaagtg atacctacac tgccaacatc gaagcaaaca ggattgtggt 11100 gtcagaattc ggaacaatgg cctatccaga cccctgcaaa aacatatttt ccaagtaact 11160 gtccgttttg tatctagtcc cgctttccaa tatccttgac ggtcgagaca accagcgttt 11220 gctcctctga cggtgtgttt agggtggatg qaaaacttcc catgatggcc tttgaccatg 11280 caggctacgt acttctggat tacagatagc aggcaaggca aagagcatct ggcagattgt 11340 cagatgttct cccacatggc tggtgggaga gcactttgaa gtcacctatc aaaattacaa 11400 acacacattc ccttcagtcc agagaggtac ttgcctgttt ctgaaatgac aaatgtaca 11460 ggttattcgt ggctgaatga tttgttatga tgaggaactg aaaataactc aggtgatcat 11520 taacagaaga gtgttaaata aatcacagta acactataca ggaaaacacc aggcagatat 11580 aaaaagcact gaggatgctc tctatgtact gaatggaaaa ttccccactg ctaagtgggg 11640 aaaaaggaaa aaaaagctaa gtgtagaaag agcatataat atgctgtaat ttgtgctata 11700 aggcagggga tatctaggtt tgtttttgaa tgtqcagaaa gcctgggaga aggcatgggc 11760 gaggtaggga accaacggga ggaaggacgg gggagatttt cagttgtaca catttaaaac 11820 catctgggtt gtgatgcata tgaatgcctt acgcgttctg aattgttggc caggqaatca 11880 ggccagtggt catcatctcc tttctgatag gctttagaca tagaaggggt acattagggg 11940 tagactggcc tggagaggga agctggtttc tgcttctcca gaactgacat catctctcca 12000 atcccagctt ccagcgctgg gggtcccgca aatagcaggc tgggaacagc cattgcagac 12060 gggggaggag ccagggggca gcgggaggga aggaattgaa accgcagctt gtgcggcatc 12120 cttgctqtta cagtgattgc cagtaggggg agcactcacc agtactcggg gccaagactt 12180 cctgctttga gtgtgccttc taataggatg atcagttctt aactcagttt actttccttt 12240 ttcaaagaaa cgaatttctt ggacaaagtt gaggtaacga gggtggattg tcattcctgc 12300 tcaaagcaga gatcaactgc ttttctaqtt tcqggggagc cattatgggt atgtgtatga 12360 tttctataga gcttcttaaa atacttgaga actgtccacc agctcttqat ggctctggtg 12420 ggqqagacct ttggatctgg ggagctgggg agggctcagt gqgcagcctt gctgtcccag 12480 ctgactccct tcttagctga ttggttgtgg tggtgctttg ggttaactcg atctctggac 12540 tgtcagattc ctcaaatgac cagttgtgac ccagcacttt ccctccctta gttqaagtag 12600 aqtgttttaa gtggtaggcg aactaaaqcc atctqgagta gaaccttcca gctgagtgga 12660 cacaqtggtt gacctgctga cttgctttcc tccccagagc tttctcctac ctgtcccaca 12720 ctatccccga tttcacagac aactgtctga tcaacatcat gaggtgtgga gaagacttct 12780 acgcgaccac agagaccagt tacatcagga ggatcaaccc ccagaccctg gaaaccctgg 12840 agaaggtatc agtgagtcta caagcagcct cagtgcctga cttaaggaat ggaccctctg 12900 cagggaggcc aggaggctgc aggcactcag agaggagccc cctctgagcc cctcgtctcg 12960 ggcatctlag cactctgccc tgccatctct gtgtctgaat tcgttcctag cactcccgtg 13020 acaaaggaac acaaactagg tggcttaggt caacagaaat gtgttccccc acagttctaa 13080 aggccggaag tctgaaatca ggtgctggca ggccctgttc tctctgaagc ttctagggaa 13140 ggaaacttct ttgcctcttc ctgcttctgg tggtcgcccc ttggtttaca gatgcctcgt 13200 tccaatctct tgtaaagata caaatcaccc accctattcc agaatgacct cattttaacc 13260 ctatttccaa aatatagaca cgagcggtta ggacctattt caacttgcct ttttggaagg 13320 tccagttcga cccctgacaq tgtcgacagt cctcggcact gagtggcccc tcagtggaaa 13380 tttgaatgaa tgacagacag tgctgggctg tccaggaccc ccagcatccc tggagaggaa 13440 ggcattaggc agagagcatg ggtgcaaaaa tcagcgccca cgtggatacc tttcctgggc 13500 aqaagcgagt ctacgttttc acaaagatca gtagacccac tttcagtttt ctccagctgq 13560 ctgttcaaag ccttgatgtc tttgcaatac atcttaacga tctgttcaag ggagtgcttt 13620 tcttagccat gaaatcatgt ccagctcctc tgaagctagg cagcataggt ccaactgagg 13680 agcactttcc acaaagctgc agagaggaat cactaggtga gaaggaggta gcgcccgact 13740 tttggctgaa ggttgtgccg ttctcaggga ggaagggcgg acagctggtt ccctggccac 13800 cccattgctc tatgattgtg gcctgtttgg agtcgggatc gttactgagg tcgttagtca 13860 gtgtttccct ttgaccaaat gctttttgta acctctccgt tcccagaata accctaggag 13920 agaggcctgg ggatttatgc ctctgtggga tacaaaaggg gaggatttaa atgacttcac 13980 cagggccggg attagcaccc aggctgcctc tctgcccacg atgttacttg gagctctctg 14040 cggtgaggca tcgggtttcc acaccgaggc cacccggtgg ggccagagag ggacaaccgt 14100 cagagaacac acgtagctca ccaagccccc tgcagcctgt gtgctgtggg agcccccacg 14160 aggttacatc aggtcgcaag aggaagcgtc tcctttaaag gcaatgtctg ccttattttc 14220 aggttgggag acatggaaaa atagccatca cctctctgag cctggagtca ggcagctctt 14280 ggcgggactt tgccctctcc cctttttaaa agcagctgac tgtcgtgttc tgcaggttga 14340 ttttcgtaaa tatgtggctg taaatctggc aacttcacat cctcactacg acgctgctgg 14400 aaatgttctc aatgttggca cgtccatcgt ggacaagggg aagacaaaat acgtgatctt 14460 taagatccct gccccagtcc caggtgggtc atcctggggc tgggatgagg cgtgaacacc 14520 aaggtggctc tttgggaaga gttctggttc tggttgatgg cacaacactc aacctgatac 14580 ttgcccacat ccatgactca gaggaccatg gactcgcttt tctcctttag gagaaaaggg 14640 aggatgctac gtttcctttt ggaagaagtg aatccctttg ctctttcaac ctccttttct 14700 ttcgattcct gttcattatt tcattaattt atctgttaat catgtattta tcaatctatc 14760 cactgatgtt gtcatccatc catcatccat ccttcctttc attcattcat tttttagttt 14820 tctgacaaac ctccatctgt cagggctgtg ctgctttttc tcatccaagc cccaaaagcc 14880 aggctccccg ggttgttcag ggagctcagg cctgctggca tccactgcag attccgccct 14940 gttccctgag ccttctagac attcttcact caggcccygg agcctggcat ggcgcccagg 15000 ctgattgtga ttgcgtgttc ctgcggggga gaagggtgqg actcctagcc cttatttggg 15060 caggaccctc tcgcaaccac acccaggaga ccaggcctcc gagggtggct qtgcgggctc 15120 cggggatggt gcaggacttc gagggtgctc agctagacct tctggtttag qaagacaggt 15180 ttccatagga gattqcccct ggcccctccc ttaccattct gtagagcacc cccgtccagc 15240 aaattccagg ctccactggc agacgggaat ccctgggtcc catggtgcag tgagtgctct 15300 tgttaaatgg aacccccqtt aattaagcta ataaagggat gcagggtccg gctcaggatc 15360 ctagaatgtc ccacctgagg gtaqgctagc catttgaqtt tgccacctca tgttcctgag 15420 gaccgggagc cccaggqgat cgggtgacqt ccccacgqct cacagcaggt accctccgca 15480 cctgctccag gctggcgctt caccaggatt cacacctgcc tcccgcctcc caggccaggg 15540 tgcctttcct tcccgtttgc tttgtgttcc atttgtttag tgaagaaaag acctacctac 15600 cccacaaccc gacagcctgc ctcccaccag ccagctgttt acaqggtgca caggtggcca 15660 caggaatggc agggacgctt gcatgtaact cattatiacc cagccgcagg cctgggtggg 15720 gaggcctttt ggccagcagc tgactcatcc ttgcagagga ataaggaaac caagcagtcc 15780 caagcacctc tgtgctgggt gccgggtgag gcgcctggcc tcctgcagta tttccttcaq 15840 ttcccccctt ccctccaagg gttgtccctc caagaacag aggaggtggc caggctgagg 15900 gyactgtgtc ctttggctta accctgtgcc tctggctgcc ccgccaagcc tcggtttccc 15960 atctgtaaaa tgggaatcat acagccgacc ctgactgcca cagtctgtca catcaacaca 16020 ggcacttggg ctcactccta ggtccttcag aacaggaaat gccaagagaa attcacagaa 16080 accctgtgaa atgcatgtca gatgatggac ctcagcagcg tgtcaaagcL Lcatcttggg 16140 aagctagccc acggcccgqq cgtggctqtq ctcagtcgct cagtcgtgtc cgactgtttg 16200 cgaccccatg gactgtagcc caccaggctc ctctgtccat qqggattctc cagqcaggaa 16260 cactggagtg ggttgccatg cctcactcca gggcatggcc tgggagactg acggaaatac 16320 agagccgtgg gccctgcccc acaggctgag tcagcctcca cactttgtca tgacccccag 16380 acaatctgcq tgcacgttca atgcttcagg agcattgctc cagagccggg gttgcaaatg 16440 ttttctgtaa agggccccag agtaactatt tgagacattg ccggctgtgt gqtctqcgcc 16500 acgtctgctq gactctgaat cctgaaggca aagcagctgt ggactctgca ggaggcggca 16560 gcgtggccct cgctccagat aaacctttat tttggaagca ggcggaggcc agcttagccc 16620 caggccagag ttactgacic ctggccctaa ggcccagaga cccaggggcc ttgagcctgg 16680 ggaggcaqcg gtttcaqatg ctaaaccagg cccctgggtc gggtgggggg aggtccagtc 16740 cctqcctcag ccaggcagca gcccttcacc cgqtttgctg tcgactccca aagattcttc 16800 tcagcggggc gggctgaaag gctqggctgt aggatgcggt gqccctqtgq tgtccttggg 16860 aggcagtcag gtcccagctg ctcctgaggc tggcttctct ccagggggca ggaaggaggg 16920 ccggagcccc ctgaaggaca cggaggtctt ctgctccatc gccgcccact ccctcctctc 16980 cccgagctat taccacagct tcggagtcag cgagaactac atcattttcc tcgagcagcc 17040 tttcaagttg gacatcctca agatggccac ggcttacatt cgggqtgtga gctgggcttc 17100 ctgcctggcc ttccacgggg aggacaaggt aaggcctagc caggcaaccc cccaccccgc 17160 agccccagga cagccagagc cagccggIgg tcagcaggca ggggcgggcg gctcagcggg 17220 ggaggaagcg agctcccagg cagtgtcccc agaggcacca gctagccttc cagagacttc 17280 tgaggctggg tgggccttca caggggtccc gagttagggc aaggggctca gcatcatctg 17340 ttcaaacagc acacgagtgt agaaagcagc tgccaagccc cctacttctc cccatccagc 17400 cctgccagag ggccccctcc gtcgLccatt cagcaccgct ctgctcccct tcctgtgcac 17460 cacgagcatc cttgggcatc cgtacagagg tgtctttgtg gccaccccct ctttaatgcc 17520 tgcattctca gggaggagag gaagcaaagc agctccccqa ggtccctccc ctgctcagaa 17580 ccttctgctg gttccccgtc ttccccagaa tcggacccac aggcctctct cactggcctq 17640 ggaaggccct catcatgccc tcagcccagc ctctgqctcc ctcctacacc tcagacctgc 17700 tgcacacacg ttcctgcctc agggcctctg cactttctgc tccctccact ggaacattct 17760 tccccagctg qctccctccc cttgtgaatc caggtctgqc tctaaggccc tcacctccat 17820 caccaaggtc cccctagctg tcacagaccc ctccccacta ccaggctttc tccacctccc 17880 acctcatttt cccagtcgtg ctcagccatc atctttcaca tgttgtacat ttggtcactg 17940 acccttggcc tcacccccac tggactataa actgcatgaa agtgtggggg gtttatctct 18000 ctagttcccc tgaaaatatg tcagtgtttg tggcacgaat. gagctgggca agggggccat 18060 cagggggccg gcggaagtgg gtttyccctg ggygaccacc tggagcatca gggccccatt 18120 ggcctccagg ctgcagggat cccccaggga tctctggccc cagagagtcc cacagtgaga 18180 gcccccgagc cctggcaggg ctttgaatgc atttgtagag ttttctgagc agtccctqca 18240 gctgacccct ggattgggtt tatgggaaag agtgcctgt cacttcaccc tgtgttcatc 18300 tcccactgtc ccttctcctc tgcaacactc agccctcatc acccccactt aacttcagag 18360 cttgattttg gggaaagaga gaaagagacg atgagccaga gaaggtggct ggagcctcct 18420 ctccctcctt tcagaggagg atggttaggc cagagctgcc aaaagaaaga aaacacccct 18480 tcaccctcgg attaagcaac tagggctgta gatacggcta attgccaccc agagcgatac 18540 cgcctatgac acccagaggt gtgtacggag gcctactggg ctgatatcag cttgactaga 18600 ctggtcactc agcactgcag ccctccaggg cagcctggtt gctgggacaa tctgcagtgg 18660 gccgqggctt tggtgggagg tccaggacgg ggtttcagqg gctctggcca tgggtgggac 18720 ctgaggaagc tgagagggct ggtcctcccc aacttggcca ttgccaggga gctggccact 18780 tgccgcccac ctggatctca gttgaatgaa ggtcggggag acgctgcact ttcagggccc 18840 tccctgaaag gtctqqtcct ccacgtgagg ccgagacccc atgcggcaaa gcttccctcc 18900 tccccatggc Ltctggtgag qtttgccctc tggagtggga cagagaggct cagaaagggg 18960 aggggttgct tcagggtccc gagcaggtga accgttggct ggggcttgtc ccccacccag 19020 tgctgcagcg tggggctggc agagggagcg ctgagccccg ggctgcatct cctttcagac 19080 tcacatccac atcatcgacc gaaggacgcg gaagcccgtg ccgaccaagt atcacacqga 19140 ccccatggtg gtatttcacc acgtcaatgc ctacgaggag gacggctgcc tcctgttcga 19200 tgtcatcacc tacgaggacg gcaqcctcta ccagctcttc tacttqgcca acctgaacga 19260 ggactttaaq gagaactcca ggctcacctc catgcccacc ctcaagaggt tcgtgcttcc 19320 cctccacgtg gacaaggtaa cggcttgagt tcttggggag gtgagcccac tcgggaggtg 19380 gctttggttc tgatttcatt gacattgaaa ttcaaaatga ggtgttctta gagaaaggca 19440 gaaaatgctt tcattatttt tccaaatatt gaacatattc agggtagaaa ttttagaaaa 19500 tacagattaa gtgaaaagga aaaaaatcac cataagccga ttgcaaatat atttgtattg 19560 cctcccaagt ctctctctct ccgtagatag acaggcaaat agacaggaag atatacatac 19620 atattagtat ataatqatat atatqcatat aacatatgca tattgtatat atacatcaaa 19680 gctgttaaaa agtatgtaca gtatgatttt cttttctgaa actaatatat agcatataaa 19740 tatgtatttc ttaattttag agaaaaattg cgctgtgaat aatttttaac agccttaatg 19800 aaatataact cacgtaccat actattcacc cattttcact catttaaaqt gtacagctca 19860 atggctttta gtatattcac aqagttgcac tacaatcaat tttacaatat tttcatttcc 19920 cccagaagag cctcccccat tccttccacc cccaggtaac cactgcccta ctttctatct 19980 ctgtggattt gcctattttg tttcatataa atggaatcat accgtaagca gtctttgtgg 20040 ctggctcctt tcacttagca tgttttcaaa gttcgtcagc accgtggcac gtatccgtgc 20100 ct.cttttttt atgactgaat atgtacacac tgttttttgt actgcttttc cgtcctcctc 20160 atatatctaa acatctttcc aagacattaa acattcttct qtgacatcat tttaaatatt 20220 tgcctgacaa tctgcggtgg gattcatct ccagttgctt acccatttcc ctgatgttqa 20280 acagtcctgt tcttgcattc aaaatgacca ccaggccaga cattaccgtc aattagqgtg 20340 ctgctgccta ggctctgggc ttggttttaa aggctacaga ggcttctggc tccagaactt 20400 ccttacttca tgacttaatt cgtttaaatg aaacccagtt gagttcaatt caattttgat 20460 ggctcatcty tactgaacac actcLaLqta ccagqtacgt tgctaagcat tttattttct 20520 ttacctcact tagtgatcaa aacctatgca agagatccac acgttagcct cattttacag 20580 atgaagaaac tgaggctcag agaggttaag gaatgtgccc aagytcacac agcctggtag 20640 caaagccaag actggctctg agcccttcct tggagccggt aaagggtata actctgcaga 20700 tcgccccagg gggtcacctt gacccctcac aggqcccccc tgcaggccta gggaacagga 20760 gacactccta catgcccagg gaqqcttccg ccacccccag acccctccca gccagaggcc 20820 ctggggcctg catttgagcc cagctgcgtc agaccacaga gctgggtcct ttctcctgtc 20880 cagcatttcc cccgtctgca gtcacttgtg tatctctgtc atgatgttca ctgtatttcc 20940 taccacttct gttaaattta caaaatcaca ttttcattta aatcaactca ctctttccct 21000 agctcatttg gaagggaacc tatgacaacc cagggcaaag ccagcgttgc gcgctgccaa 21060 aggagaaagt gatgagcgcc gagtgctgtg atgaagcaca ggtcccgacjc actggcctga 21120 tgctgttgct cttgccaaga ccccatctct cccttaaaga gaacactggg aaagtattca 21180 gtgttaaggg tgtagtggca cctaactgag acgctctcct tgacccggct gggaagactg 21240 gaagagaaga aaggattaac tttctcctgg tatgattcag ggctaacgag tgaacagcag 21300 ggcccacaga atctccacct gagcccctgg cctccgcgcc tcccccccca cictgggaag 21360 ttccacagtg cagcgcttgg agcatttcct ggctccaggg tgcagtgggg gcggggtggg 21420 gggctgtcac ccagagggcg agtccccagg ccgacccttc cccctgcctc acagacctca 21480 gcagagagga agcactctcc cccgacgccc gcagcacggg attctgtcac ctcttaacaa 21540 gctcgagaaa caatctgctt ccttggaacc aagctaagaa gtttagcctg tacagtttaa 21600 acaggcgctg cgtgaaccat ctgcaaagaa aagtgagcqc tgggaagggt agctctggcc 21660 acgcttttta attgctattt attgtccctt aaaaaaatgt cttcttgctt cactgcatgt 21720 gaacagggga aggttgagca ggcagacatg gcctgtgtgt tcagatccag cagctccgat 21780 tccatcccat ccacagtcta cattacggag gggccttggg ggcgggcgat gccaccgttt 21840 ctcaaatgcc cttcattctg gggcctttgc acatcgctgt ccctctgttg tcctctttcc 21900 atgagtcctc cattctcgtc ccggccgcag gtcggactgg cctgggggct ccgggaccac 21960 cagtccactg aaggcagaac ctctagggga ggggttggga tgggcatggc tcccaggcac 22020 cccccacccc caccccagga tcctagttcc tgctgatcct tcaggtctgg cgtcctgact 22080 ccctcagtgg gtttgtacat tccagtggca cctacagctc tttgctcaca ttgatgtcag 22140 gagtaatgag cttgttgggt cacctcgtcc tgtgaaaggt cccagggggc ccggactctt 22200 qgtcLtgcca 9ccatttatc ccctgcaccc atcacgggt ctggttctcc gcaggttctc 22260 ttgaagccct tagatgtttt aaatgaatga atgagaaagg tqatgcaaqg tctctcaaag 22320 gacacaaggc cttcctttag cagttggaac acttccaaag tggaaagcgc cttccacgta 22380 gaagyycta aagtgaatga ggtgtgttca gaagtctgag gLcLcccagc tgtcccagat 22440 tctctggcat ttgggccaqc tggggtcaga ggaaggaggg gcccgttggt acccttggcc 22500 tgtLtgttgg ggtggcgtgg gggttgtcta aggcggagct aqqcctccag gggcctgggt 22560 ggtgtygccc atcigLgcca gctgccctgc cctcaccctg cagggctccg Lgcqcgcca 22620 tgggggtggc atctttgacc ttgaaggggt ggctccaact cctggctccg tgaatttgat 22680 ttgtcttgtg ttcaaatagc aagagaagcc ccgtttctcg agggccccct ctgcgctggg 22740 ccctgtgtta gacgcctttt cttctcttag acccaggcca gaggggcccc tgctctggct 22800 tttctccagt ggcccttctc cacagggtga qgaacccacg qggccacagg gtgcccaccc 22860 agaagggcac ccttccgacc ccagccatac ctgggatccc tgagttcctt gcccagatgt 22920 cccacacctg cctccagcgc ctgcctctgt cctcctqaqg gcagacagtg ctctggtgtg 22980 tcggcctcag gcccacgagg aaqgcaqgga gaaaaggggq cggggagcct gggcccagag 23040 gccggccttc attctcccct gcagcccatt ccagaaggcc aaggagtcct ggaattctaa 23100 attcacacqc gtccttccag acccagcact gtgcactaga aacctgccgc gatgaagcag 23160 tgttctctgt ccaacgcagc ggccgctgga cgggatgcct gggctattga aatgcagcca 23220 gtgaagccga ggaatggaat tttaaatttt actgaattta aatttaaata gccacctgtg 23280 gctgggggtg gctqcctcac cccgggcagc acagttccac aacatgttga agctctgtgt 23340 ataaagtaag ggaagataaa atggtatttq acttaaccat tcaatatatg tggacaaagg 23400 gttggtgggc ctctgcgctc atcctgggtc ctgcaaatgt tgtgtgagtg ctgagtcact 23460 tcggtcgtgt ccgactctgt gcaaccccat ggactgtggc ccaccaggct cctctgtcca 23520 tgcqggttct ccaggcaaga atactgqagt ggggtgccat gcccccctca aacgtgtggg 23580 ggcgtgtaat ggtctgcatt ctcctcactg aatctgcatg tgagtgcttg aagttatttg 23640 tcatccccat gacaacagat gagaaagaaa gtaacgtgcc aaacatcgca gaatcatcgt 23700 aggatttgag ctgggatttg ggtgcttgtc tgtctcattc caagttacta actacagtgg 23760 gtttggccct gqaccaagtq ttttcataac cattatttaa tttaatccta accacqgtgc 23820 tcagacgctg gtcgaaagaa ccttcgcttg cactcgccat ctgtccagaa gttacagatt 23880 gcccccagtt ttacaaacaa ggaagcagqc tccagttcac ctagtctcag ggaggcctgc 23940 cggaactcac gaagaaacct atagagtcag atttcacacc caggactgcc aaacttcaaa 24000 gccagtgttt tqgggggatg actgagatta acttcttctc ctgggacgtc cctcqcatcg 24060 aagaatttca aagccaqtct gcaagatttt attgaattct ggctggagag cgatttcttc 24120 ccaaagtcat gcacactggc cacagaaagc agccgagtca tgctcgttac tgctatgtgt 24180 ggaaaaattc atggtccatg cctcaaccag tctgaccagt ttcatgctgg agctagaaaa 24240 catgtccgca gaccgcctcc ctgagaatca agcccaggac atycactaga ggcccgtggc 24300 tgctgcctcg tgactacaag gcaacgggca gggcctctca ccgtctgtga ggctgcgcgc 24360 acaggcggag gcaaacaaga gctqctccca acaagacgcc cgcjtgtgccg ggcgctggac 24420 gactgattct tacaaccacc tgcaaagcac gtgttctgcc tctcgagcgt tcacacattc 24480 ctttagcagt tgcttcttag gagctgcctg tgtgccatgc acagggtgaa atgtggtcac 24540 tgtcttcaca gggaagacaq catcaaaqaa ggagccaggt ccataaagga gataattaca 24600 gtgtgggaca gacaccttgg agacactgc gactgagctg tcaqggaaaq ggacgctgga 24660 gctgagacct qtgggaqggg aatqqqccag cctcagqqtc tttgcacctq ctttccctgc 24720 tgcctgcagt gcttctgttt ctgagcagaa gaaccccagt gtggaggtct qggctcagct 24780 tccgctcccc tagtgcqacc ttgggcaagc cactttccta ccctgggcct cagtttcctc 24840 ctcLgtcaac tgggattgca ctaaacacat ctcaaaattc aqcLLctaag atgcagLaaa 24900 tgtcctgcct tcctagcaaa aagctgctgt tgttcaaatt tgttgttggg tagttgctaa 24960 gtcatgtccg actcttttgt gaccccatgg actttagctc accaggccct tctgtccatq 25020 ggatttttcc aggcaagaat actggagtgg gttçjccattt atcgatttaa ttataaaaac 25080 ttgtttggag acatttggaa agtcgagaaa agtttaaatg agaagtcagg catggcaqta 25140 attctccttc tqgaatttca cagttgccgg tggtcttgct cctcatcccc tcccaggtgc 25200 ccaactgaac tcacccgcca qgggtaatgg caataacaat aataataata atacaattat 25260 gtgatcaaca gcagttaaca tttgtttagc acttactgtt aacctggcct tcttgtaagc 25320 caacagcatg ggttatttta tttaatttgc atgataactc agagatgcat atgctattta 25380 tacaatgttt gtgctatacc tcattataca ggtggggaaa ctgaacttca gagatgtcaa 25440 gtaacttgtt caagtaccat ggcaagtgtq tggggacaca gggagtaact gctgctcgat 25500 agatatttaa ttagttgtgt tacgtctctc tcccgggtta aaqtactgaa ccaagtcttq 25560 actgccactt aagaccaaga gacgtccttc ataqatattt tttcctccca agttattttt 25620 atctcatgtt tgtttttctt tcctgctggt ttgtttttct cctatttttt cataggagaa 25680 aatactgaaa atctagaaaa acatagacaa agaaattaaa gtctgcatca cccgctgcat 25740 gctctattgt tactatttgg cacatttctc ccaggctctt tccactgtga tttcttttct 25800 tttttaacac ggctgagctc agaccgtgtc tgcggttctc tctgctgctc tttcactcqg 25860 tgttctctgt gttattatga actctttata aacacctttt taataactga tgcagccttt 25920 gacaagtgac tcatacagta tttaaaaagc gagaacttgt aagtacttcc atttcactta 25980 taagtacatc catttcacct gtgagggatc aggcagccca gagcaggaga aaagttccag 26040 ggtttcggag cccccttcca caacccacca gctttgcctt cttcgqggag tcacctccag 26100 tccccgattc tcatttttaa ctgagaactc caatcatgta atagctgctt ctggtggcta 26160 gcatgaggcc aaaacaagac actgaagcat tcttttaaaa atgtgcacat aaagccagtg 26220 tttgctctgg acaacgtttt gatttatttg gaaaaactga tctctttcag aatgcagaag 26280 tgggctccaa tttaatcaaa ctqtcgtcta caacagcacg agccctaaag gaaaaagatg 26340 accaggtcta ctgtcagccg gagttgctct qtgaaggtaa aacgcatctt ctctcctgtg 26400 ttcagaggaa qgggtggatg tcctctttac gtaacgcctc cttccagtgc agaggagctg 26460 tgtgtggacc agggaggcaa gggaaggctg tgggactcta qgcattgtac tggagacgct 26520 tctctctcat gtaccttcat acccctccca gcactggggt qgtctctaga cccaggaact 26580 ttcacggctg gccttttgaa tyctgccatg taattttttt gctttctttt aaadcattta 26640 tttatttgtt gcatttaaat ttttatacac tttttaaagg ttactttcca cttagttatt 26700 ccaaaatatt ggctgtgttc cccatgttgt acaatcgtcc ttgagcctgt cttacatcca 26760 atagtctgta cttccctgtc cccaccccta tatcatcccc tccttccccc tcactggtaa 26820 ccactaactt gttctctgtc tcaaatgctg ccatgcattt gcaaaaaatg ctcgacatcg 26880 cttattatta gagaaatgga aatcaaaact gcagggaggt atcagctcac accagtcaga 26940 atggccatca tcaqccaaaa aacaaacaaa aaaaaatcta taaaaaaaat gctggagagg 27000 gtatggagaa aaqggaaccc cctcttgcac agttggtggq aatgtaaact gatatagcca 27060 ctatggaaaa cagtatggag atttctttga aaactaqqaa taaaactacc acgtqacaca 27120 gcaatcccac tgcagggcct acaccctgag gaaaccacag ttccgatgct gccatgtgct 27180 ggacagcaca gcatacgcgg gtctccacct gggcacactg agcaccctgc agaacagqcg 27240 ccatctcctc tgctggccag aggagaaaac tgggcctcag aaaggctgtg cgccacgcag 27300 aacgtaaaqc ccctcggaga gaagccacag cccccgqagc aaqgcttccc tgcccagcac 27360 tgcggctgcg gggcgqaggg ggcagcgctg ggcattgcag ctgttgagca gcgtccgqgg 27420 cctgcaaccc cjactgggaac cactgtccta gagcagaagc cLgtqqagca gggcctgtgc 27480 cccgtgctca gtgctgtcac cagcacgaga caggaaccag gctcagagca gggtgtgctc 27540 aqtqcgtagg tgaggagcga actggcctgc ccaqgacacg gctggggagt ggaggggcag 27600 ggctccagqc tcqccacatc gctqccccat gccatggtgg gggggacttg gaaatttaat 27660 ttgacacagc ggacatatgt aaatatgtat acatggacaL ggalgaaatt tgaaggctLt 27720 tttggattac aaaagtgata gacataatga aattttcaca tgagaaactc agaatggaca 27780 atataaacta cggccacaa taagaaagtt atcagcccaq tagcacttct aaatttttcc 27840 ttlacacttg taacatagtg gtgataattc tctaggcagc cttttacqtc'. taacgttatc 27900 atggggacat tttcccatgt tactaaaatt ctttgtaaat attacttttt attatacgat 27960 ttcattagat tcatctaact attccccact gatgcttcct ccatgcttca gaagtttctg 28020 ttcacgccca catacatttg cacgcacata cttttttttt tttaagagct tttaaaaagt 28080 tgcagtggcc tctgatttta atcatgtaat tctctttgat ttctatcttg gataattaaa 28140 gatqtttata cacttaaaaa ccaaggtcta tcttaccttg ctcccgaagc ttctaataga 28200 gctggggtaa ttatctgaag ttccctcctc ctgccctttg accacccttg atccaactca 28260 gggcctgagt gatgttcaqg gatcctggct tqgaagccat ttttgtagaa aqgatgcctg 28320 gcctcgatac cacgaagggc gggtggtccc accctcctga aggggqcttt gtcagccatg 28380 tcqtctcttt ccaggcttag aactqcctca catcaattat gcccacaatg ggcagccata 28440 ccgctatatc tttgctgctg gagtccagtg gagccccatc ccaaccaagg tactggtccc 28500 tgqcgggtgg cagcctggga tggtcatgtc tgtctgcaaa tctgagttct cagggatgLc 28560 aggactgaga tgcccttggg qatgggggta ggtcagagag tgagctcaqt ttgggacaaa 28620 atatgctgga igcaatgacg gagcatccag gagagggaac tgctcctaat gggcttcact 28680 caggtaagac gtggtggctg gagtttatcc agtgatttct ttattggatg catatttact 28740 gagcaccagc ttaggcacac aggaaaagca agtgaacagg aggatcctgq ttcctgccct 28800 caaggaactc atagtctcaa gcagagaaaa cagagctgaa acttggagga tttattttgc 28860 tttttaaaac ttttttattc aagtatagtg gctcagtggt aaagaattca cctgccaatq 28920 taggagacga gggttcaatc cctgggtccg gaagatcccc tggagaaaga aatgggaacc 28980 cactccagtg ttcttacctg ggaaatctca Lagacagayc agcctggcag gctacagtcc 29040 atggggtcac agagtcggac acaacttggc aactaaacag ctgtaacaac ggggctgatt 29100 tacaatatca tgttagtttc aggtgttcag cacagaggtt catattttgg ttcctaaact 29160 gctatttgtt gaacacttgc tgtttcccag ggaccatgct gagtgctgta cagacatcac 29220 ttttccttag tagcttcagg ttttagtaag aaaatggaaa atcttttttt aaaaaacgca 29280 atcagtgcag gaatgtacaa tgagaaaaat taaggttccc acatgctcat ccacacgccc 29340 cgagtcactt cccattaatc ccacacccta aggggaaaqa qgcttattag agacagagag 29400 aaggccaggg actcacgctt ggaaagcaaL gaagaaagca cattatgctt taaaggctgg 29460 gcaatgaata tgttagatta tctgatacgt ataaaaattg caatatagtg cccaatatca 29520 gataaatcca gggttcttat ttcttaaaga attcagagtg tatatcagct ctqctgtggt 29580 ggggggacct tagcttaaag ccggagaagg aagggagctg ctttcctgtt tttgtgctga 29640 ccagaggcag gtgtcatctg gggcctatgc tcgctggtgL gaggtcccca tctqaggcgc 29700 tcaccccttc tctggggcct tgacgccttc ctattcqccg ggaatgagca tgctctgcaa 29760 ccattcattL cgcgtttctc ccctttgcat gcgcagataa taaaatacga cattctcacc 29820 aagtcctcct tgacgtggag aqaggagcac tgctggccgg cggagcccct gttcgtgccc 29880 acaccaggtg ccaaggacga ggatgatggt aaggcccggg aagggcaacc caccttctgc 29940 actgtgcttg tggtcacccc tcaaactcag acaccgttca cacagctggt gcagccatgc 30000 t.tgaagtcc aggcggttct gagtgcaaag cagtagggcg agggagtccc caagcccacc 30060 cccacttctg acatcagctq caagttcagg ggtccccaag accaccctca gcttcactag 30120 gagcacccac agaagactct gaaagctgtt acactcgtgg ggacagttat ctcagcggaa 30180 gggtacagac tgaaagagaa aaggcgagtg ggatggaatc cgqgagaatc ccatgggtgg 30240 tctccggtgg tcctccctgc gggagtcacg gaccactccc acttccccaa cçjagccccac 30300 ggagcgcggc caaccagggg tgctccctca gcctcggagc ccatgtgggg cgtggtcgtg 30360 cggaccgcgc tgacttctag tcttcacccc tctggaggtc gagctgacgg ctgtggccca 30420 agtactaaat tgcttccgtc gtgtcccact ctttgcgtcc ccatggactg ttgccgacca 30480 ggctcctctg tccatcggat tctccaggca ggaacactgg agtgggctgc caLgccctcc 30540 tcctggggat cttcctgacc caaggatcca acctgcatct cttacgtctc ctgcattggc 30600 aggtgagacc tttaccatta gtgcctcctg ggaagccctg tggcccaagq ccgggttgga 30660 tggcacagcc tcaggtaaac aaagacctct cgtcagaccg

acagtccaag gactcgggcg 30720 tcacctccca ggggcagagg agagacctcc ttttggggga gactaacaca aaagcccact 30780 tccttgccac agggcaacac tgccctggcc cagagtgtgg aatcctggtt ttaaaattca 30840 ttgcagtggc tgggtcagaa cttgtaactc ctctgagcac ataaactttc cagggagccc 30900 cttaaaaagc ttgtgggact cctctctcat ccaggtttcc acctgaccta aaggtccttt 30960 ccctccttga ttttgcccca gaaatgccct gcccgtcccc ctggggatgc caaggagggt 31020 aaatagaagc ctgggtgggg ctgccaacac ctggggggag ggcaggctgc ccgctgagcc 31080 tgccagctgc ttccctcggg cgagaaggga gtggttttct acctgggagt gtcttcctat 31140 tgacctgcac ggcacagcgt gccaggcagg ggacccgtgt cactgtcgcc tctaaggtag 31200 ggqacggttg gtgccacctc ctgccgagqc tggcctcccc cgggttggqc gtqtacgccc 31260 tgcatattct cctgtggaca ggaagcagcc ttgtcttcat tccaccagaa gaaaggctca 31320 gagcttgagg tcacttaccc acggttgcac gcccagtaca tacacagggg agggtttgag 31380 tccaggctga tttgacctca gttgtgctca gccacLctga aacccgggag gcctcgggaa 31440 gaatcaggaa tatttgctgg aagtttattg ctctattttg tttagccctc tgtccctttg 31500 agagtgaata tttagactat tctcatttca caaaggggaa agctgcacac gcatgaggtg 31560 gttgcactta ttcaaacgcc atttgaatgc agcaggtctg agtttaggct aagtgcagtg 31620 ttgaagctga ttcgctgtaa ctggaatttc taaatgtag gaaaatagcc atttttctgt 31680 atgtgcaata attcacaaag tgctttcata actgaccgtg caagatcctc tcagcaaccc 31740 cctgaqagtg gggagattat ccccactcta cagatgaaga gactgaggct cagaaaggtg 31800 aggggatggg cccagccaag ccttccgatt tccctggcct ttcccttcag ctcacacaga 31860 atatcaaatt cacttccctt ctgtttcgat tcaattcagt tcagttcagt tcagtcgctc 31920 agtcatgtcg actctttgcg accccatgaa tcacagcaca ccaggcctcc ctgtccatca 31980 ccaactcccg gagttcaccc aaactcacgt ccatcgagtc agtgatgcca tccagccatt 32040 tcatcctctq tcatccttct cttcctgccc ccaatccctc cccagcatca gggtcttttc 32100 caatgagtca actcttcgca ttaggtacaa agaacaggga gacagagctg agaaacttag 32160 cacccccctc cccaccccag ctggaagagg ttcgagggtg cacttttaca aaatgtctct 32220 ctttgtttcc ccaaagggat. tatcttatcg gccatagtct ctaccgatcc ccagaagtcg 32280 ccttttctgc tggttctcga tgccagaact tttacggaac tggcccgcgc gtccgtcqat 32340 gtggagatgc acctggattt ccacgggctg ttcatcccag atgcaggcag gqacccgggg 32400 aagcaggccc cttcccagga ggcgccggcc agggctgccg ccggacgtgc ggccccaaga 32460 acctgacagc ctggaggctt tggtcctggg gaccagctcc gcccagctca cggctgtccc 32520 cgccccgggg gaggtgcggg agaggtggtc cttcgttcca tttcgcactt attctttccg 32580 cagctgcttt gagtcaacat tctga 32605 <210> 2 <211> 1791 <212> DNA <213> Bos Laurus <220> <221> CDS <222> (1).. (1791) <400> 2 atg gaa ata ata ttt ggc aga aat aag aag gag caa ctg gag cct gtg 48 Met Glu lie lie Phe Gly Arg Asn Lys Lys Glu Gin Leu Giu Pro Val 1 5 10 15 agg gcc aga gta aca ggc aag att cca gcc tgg ctg cag ggg atc ctg 96 Arg Ma Arg Vai Thr Gly Lys lie Pro Ala Trp Leu Gin Gly lie Leu 25 30 ctc cgc aat ggg cct ggg atg cac acg gtg ggc gag acc aga tac aac 144 Leu Arg Asn Gly Pro Gly Met His Thr Val Giy Giu Thr Arg Tyr Asn 40 45 cac tgg ttc gat ggc ctg gcc ttg ctc cac agc ttc acc atc aga gaL 192 His Trp Phe Asp Gly Leu Ala Leu Leu His Ser Phe Thr lie Arg Asp 55 60 ggt gaa gtc tac tac agy agc aaa tac ctg aga agt gat acc tac act 240 Gly Giu Val Tyr Tyr Arg Ser Lys Tyr Leu Arg Ser Asp Thr Tyr Thr 70 75 80 gcc aac atc gaa gca aac agg att gtg gtg tca gaa ttc gga aca atg 288 Ala Asn lie Glu Ala Asn Arg fle Val Val Ser Glu Phe Gly Thr Met 90 95 gcc tat cca gac ccc tgc aaa aac ata ttt tcc aaa gct ttc tcc tac 336 Ala Tyr Pro Asp Pro Cys Lys Asri lie Phe Ser Lys Ala Phe Ser Tyr 105 110 ctg Lcc cac act atc ccc at ttc aca gac aac tgt clg atc aac atc 384 Leu Ser His Thr Tie Pro Asp Phe Thr Asp Asn Cys Leu Tie Asn lie 120 125 agg agg tgc gga gaa gac ttc tac gcg acc aca gag acc agt tac atc 432 Arg Arg Cys Gly Glu Asp Phe Tyr Ala Thr Thr Glu Thr Ser Tyr lie 135 140 agg agg atc aac ccc cag acc ctg gaa acc ctg gag aag gtt gat ttt 480 Arg Arg lie Asn Pro Gin Thr Leu Glu Thr Leu Glu Lys Val Asp Phe 150 155 160 cgt aaa tat gtg gct gta aat ctg gca act tca cat cct cac tac gac 528 Arg Lys Tyr Val Ala Val Asn Leu Ala Thr Ser His Pro His Tyr Asp 170 175 gct gct gga aat gtt ctc aat gtt ggc acg tcc atc gtg gac aag ggg 576 Ala Ala Gly Asn Val Leu Asn Val Gly Thr Ser lie Val Asp Lys Gly 185 190 aag aca aaa tac gtg atc ttt aag atc cct gcc cca gtc cca ggg ggc 624 Lys Thr Lys Tyr Val Tie Phe Lys Tie Pro Ala Pro Val Pro Gly Gly 200 205 agg aag gag ggc cgg agc ccc ctg aag gac acg gag gtc ttc tgc tcc 672 Arg Lys Glu Gly Arg Ser Pro Leu Lys Asp Thr Glu Val Phe Cys Ser 210 215 220 atc gcc gcc cac tcc ctc ctc tcc ccg agc tat tac cac agc ttc gga 720 Tie Ala Ala His Ser Teti Leu Ser Pro Ser Tyr Tyr His Ser Phe Gly 225 230 235 240 gtc agc gag aac tac atc att ttc ctc gag cag cct tic aag ttg gac 768 Val Ser Glu Asn Tyr lie Tie Phe [eu Glu Gin Pro Phe Lys Leu Asp 245 250 255 atc ctc aag atg gcc acg gct tac att cgg ggt gtg agc tgg gct tcc 816 lie Leu Lys Met Ala Thr Ala Tyr lie Arg Giy Val Ser Trp Ala Ser 260 265 270 tgc ctg gcc ttc cac ggg gag gac aag act cac atc cac atc atc gac 864 Cys Leu Ala Phe His Gly Glu Asp Lys Thr His lie His lie lie Asp 275 280 285 cga agg acg cgg aag ccc gtg ccg acc aag tat cac acg gac ccc atg 912 Arg Arg Thr Arg Lys Pro Val Pro Thr Lys Tyr His Thr Asp Pro Met 290 295 300 gtg gta Lit cac cac gtc aat gcc tac gag gag gac ggc tgc ctc ctg 960 Val Val Phe His His Vai Asn Ala Tyr Glu Glu Asp Giy Cys Leu [eu 305 310 315 320 ttc gat gtc atc acc tac gag gac ggc agc ctc tac cag ctc ttc tac 1008 Phe Asp Vai lie Thr Tyr Glu Asp Giy Ser [eu Tyr Gin Leu Phe Tyr 325 330 335 ttg gcc aac ctg aac gag gac ttt aag gag aac tcc agg ctc acc tcc i056 Leu Ala Asni Leu Asn Glu Asp Phe Lys Glu Asn Ser Arg Leu Thr Ser 340 345 350 atg ccc acc ctc aag agg ttc gtg ctt ccc ctc cac gtg gac aag aat 1104 Met Pro Thr Leu Lys Arg Phe Vai Leu Pro Leu His Val Asp Lys Asn 355 360 365 gca gaa gtg ggc tcc aat tta atc aaa ctg tcg tct aca aca gca cga 1152 Ala Glu Vai Gly Ser Asn [eu lie Lys Leu Ser Ser Thr Thr Ala Arg 370 375 380 gcc cta aag qaa aaa gat gac cag gtc tac tgt cag ccg gag ttg ctc 1200 Ala Leu Lys Giu Lys Asp Asp Gin Vai Tyr Cys Gin Pro Glu Leu Leu 385 390 395 400 tgt gaa ggc tta gaa ctg cct cac atc aat tat gcc cac aat ggg cag 1248 Cys Glu Gly Leu Glu Leu Pro His lie Asn Tyr Ala His Asn Giy Gin 405 410 415 cca tac cgc tat atc ttt gct gct gga gtc cag tgg agc cct agg cca 1296 Pro Tyr Arg Tyr lie Phe Ala Ala Gly Val Gin Trp Ser Pro Arg Pro 420 425 430 ttg att tac gcc gcg att cgc ctt gcc aag tcc tcc ttg acg tgg aaa 1344 Leu lie Tyr Ala Ala lie Arg Leu Ala Lys Ser Ser Leu Thr Trp Lys 435 440 445 gag gag cac tgc tgg ccg gcg gag ccc ctg ttc gtg ccc aca cca ggt 1392 Glu Glu His Cys Trp Pro Ala Glu Pro Leu Phe Val Pro Thr Pro Gly 450 455 460 gcc aag gac gag gat gat ggg att atc tta tcg gcc ata gtc tct acc 1440 Ala Lys Asp Glu Asp Asp Gly lie lie Leu Ser Ala lie Val Ser Thr 465 470 475 480 gat ccc cag aag tcg cct ttt ctg ctg gtt ctc gat gcc aga act ttt 1488 Asp Pro Gin Lys Ser Pro Phe Leu Leu Val Leu Asp Ala Arg Thr Phe 485 490 495 acg gaa ctg gcc cgc gcg tcc gtc gat gtg gag atg cac ctg gat ttc 1536 Thr Glu Leu Ala Arg Ala Ser Val Asp Val Glu Met His Leu Asp Phe 500 505 510 cac ggg ctg ttc atc cca gat gca ggc agg gac ccg ggg aag cag gcc 1584 His Gly Leu Phe Tie Pro Asp Ala Giy Arg Asp Pro Gly Lys Gin Ala 515 520 525 cct tcc cag gag gcg ccg gcc agg gct gcc gcc gga cgt gcg gcc cca 1632 Pro Ser Gin G}u Ala Pro Ala Arg Ala Ala Ala Giy Arg Ala Ala Pro 530 535 540 aga act gac agc ctg gag gct ttg gtc ctq ggg acc agc tcc gcc cag 1680 Arg Thr Asp Ser Leu Glu Ala Leu Val Leu Gly Thr Ser Ser Ala Gin 545 550 555 560 ctc acg gct gtc ccc gcc ccg ggg gaa ggq cgg gag agi ggt cct tcg 1728 Leu Thr Ala Val Pro Ala Pro Gly Glu Gly Arg Glu Ser Gly Pro Ser 565 570 575 ttc cat ttc gca cat att ctt tcc gca gct gct ttg agt caa aat tct 1776 Phe Ills Phe Ala His Ile Leu Ser Ala Ala Ma Leu Ser Gin Asn Ser 580 585 590 gaa acg gaa aca taa 1791 Glu Thr Glu Thr <210> 3 <211> 596 <212> PRT <213> Sos taurus <400> 3 Met Glu lie lie Phe Gly Arg Asn Lys Lys Glu Gin Leu Glu Pro Val 1 5 10 15 Arg Ala Arg Val Thr Giy Lys lie Pro Ala Trp Leu Gin Gly lie Leu 25 30 Leu Arq Asn Gly Pro Gly Met His Thr Val Gly Glu Thr Arg Tyr Asn 40 45 His Trp Phe Asp Gly Leu Ala Leu Leu His Ser Phe Thr lie Arg Asp 55 60 Gly Glu Val Tyr Tyr Arg Ser Lys Tyr Leu Arg Ser Asp Thr Tyr Thr 70 75 80 Ala Asn lie Glu Ala Asn Arg lie Vai Val Ser Glu Phe Gly Thr Met 90 95 Ala Tyr Pro Asp Pro Cys Lys Asn lie Phe Ser Lys Ala Phe Ser Tyr 105 110 Lcu Ser His Thr lie Pro Asp Phe Thr Asp Asn Cys Leu lie Asn Tie 120 125 Arg Arg Cys Gly Glu Asp Phe Tyr Ala Thr Thr Glu Thr Ser Tyr lie 135 140 Arg Arg lie Asn Pro Gin Thr Leu Giu Thr Leu Glu Lys Vail Asp Phe 150 il55 160 Arg Lys Tyr Vai Ala Vai Asn Leu Ala Thr Ser His Pro His Tyr Asp 170 175 Ala Ala Gly Asn Val Leu Asri Val Gly Thr Ser lie Val Asp Lys Gly 185 190 Lys Thr Lys Tyr Val lie Phe Lys lie Pro Ala Pro Val Pro Gly Gly 200 205 Arg Lys Glu Gly Arg Ser Pro Leu Lys Asp Thr Glu Val Phe Cys Ser 210 215 220 lie Ala Ala His Ser Leu Leu Ser Pro Ser Tyr Tyr His Ser Phe Gly 225 230 235 240 Val Ser Glu Asn Tyr lie lie Phe Leu Glu Gin Pro Phe Lys Leu Asp 245 250 255 lie Leu Lys Met Ala Thr Ala Tyr lie Arg Gly Val. Ser Trp Ala Ser 260 265 270 Cys Leu Ala Phe His Giy Glu Asp Lys Thr His Tie His lie lie Asp 275 280 285 Arg Arg Thr Arg Lys Pro Vai Pro Thr Lys Tyr His Thr Asp Pro Met 290 295 300 Val Val Phe His His Val Asn Ala Tyr Glu Glu Asp Gly Cys Leu Leu 305 310 315 320 Phe Asp Val lie Thr Tyr Glu Asp Gly Ser Leu Tyr Gin Leu Phe Tyr 325 330 335 Leu Ala Asn Leu Asn Glu Asp Phe Lys Glu Asn Ser Arg Leu Thr Ser 340 345 350 Met Pro Thr Leu Lys Ary Phe Val Leu Pro Leu His Val Asp Lys Asn 355 360 365 Ala Glu Val Gly Ser Asn Leu Tie Lys Leu Ser Ser Thr Thr Ala Arg 370 375 380 Ala Leu Lys Glu Lys Asp Asp Gin Val Tyr Cys Gin Pro Giu Leu Leu 385 390 395 400 Cys Glu Gly Leu Glu Leu Pro His lie Asn Tyr Ala His Asn Gly Gin 405 410 415 Pro Tyr Ar Tyr Ile Phe Ala Ala Gly Val Gin Trp Ser Pro Arq Pro 420 425 430 Leu Tie Tyr Ala Ma Tie Arg Leu Ala Lys Ser Ser Leu Thr Trp Lys 435 440 445 Glu Glu His Cys Trp Pro Ala Glu Pro Leu Phe Val Pro Thr Pro Gly 450 455 460 Ala Lys Asp GUi Asp Asp Gly lie lie Leu Ser Ala Tle Val Ser Thr 465 470 475 480 Asp Pro Gin Lys Ser Pro Phe Leu Leu Val Leu Asp Ala Arg Thr Phe 485 490 495 Thr Glu Leu Ala Arg Ala Ser Val Asp Val Glu Met His Leu Asp Phe 500 505 510 His Gly Leu Phe lie Pro Asp Ala Gly Arg Asp Pro Gly Lys Gin Ala 515 520 525 Pro Ser Gin Glu Ala Pro Ala Arg Ala Ala Ala Gly Arg Ala Ala Pro 530 535 540 Arg Thr Asp Set Leu Glu Ala Leu Val Leu Gly Thr Ser Set Ma Gin 545 550 555 560 Leu Thr Ala Val Pro Ala Pro Gly Glu Gly Arg Glu Ser Gly Pro Set 565 570 575 Phe His Phe Ala His Tie Leu Ser Ala Ala Ala Leu Set Gin Asn Set 580 585 590 Glu Thr Glu Thr <210> 4 <211> 20 <212> DNA <213> Artificial <220> <223> Synthetic <400> 4 tctcactcca cccagagagg 20 <210> 5 <211> 20 <212> DNA <213> Artificial <220> <223> Synthetic <400> 5 agtcgctgtg ttgtgtccag 20 <210> 6 <211> 20 <212> DNA <213> Artificial <220> <223> Synthetic <400> 6 Ltgcctggac agaggagtct 20 <210> 7 <211> 20 <212> DNA <213> Artificial <220> <223> Synthetic <400> 7 aggctccagt tyctccttct 20 <210> 8 <211> 20 <212> DNA <213> Artificial <220> <223> Synthetic <400> 8 ggccctcgct ccagataaac 20 <210> 9 <211> 20 <212> DNA <213> Artificial <220> <223> Synthetic <400> 9 gacactgcct gggagctcgc 20 <210> 10 <211> 18 <212> DNA <213> Artificial <220> <223> Synthetic <400> 10 ggcagaggga gcgctgag 18 <210> 11 <211> 20 <212> DNA <213> Artificial <220> <223> Synthetic <400> 11 tttgcaatcg gcttatggtg 20 <210> 12 <211> 29 <212> DNA <213> Artificial <220> <223> Synthetic <400> 12 tgtgcggccg ccatggaaat aatatttgg 29 <210> 13 <211> 20 <212> DNA <213> Artificial <220> <223> Synthetic <400> 13 gatagtcctc acggccaaaa 20 <210> 14 <211> 27 <212> DNA <213> Artificial <220> <223> Synthetic <400> 14 cctggccttc cacagggagg acaagac 27 <210> 15 <211> 27 <212> DNA <213> Artificial <220> <223> Synthetic <400> 15 gtcttgtcct ccctgtggaa ggccagg 27 <210> 16 <211> 35 <212> DNA <213> Artificial <220> <223> Synthetic <400> 16 tctacttggc caacctggac gaggacttta aggag 35 <210> 17 <211> 35 <212> DNA <213> Artificial <220> <223> Synthetic <400> 17 ctccttaaag tcctcgtcca ggttggccaa gtaga 35

Claims

1. A method of determining genetic merit of a bovine, the method comprising determining the BCMOI allelic profile of the bovine and determining the genetic merit of the bovine on the basis of the BCMOI allelic profile.

2. The method as claimed in claim 1, wherein the genetic merit is with respect to a milk content phenotype or a milk colour phenotype.

3. The method as claimed in claim 2, wherein the milk content phenotype is milk l-carotene content.

4. The method as claimed in claim 3, wherein the milk n-carotene content is milk fat J-carotene content.

5. The method as claimed in any one of claims I to 4, wherein the allelic profile is determined with respect to DNA, mRNA andlor protein obtained from said bovine.

6. The method as claimed in any one of claims I to 5, wherein the allelic profile is determined by determining the presence or absence of one or more of the following: the C allele at the C-1054T promoter polymorphism in the BCMO1 gene, the T allele at the C-1054T promoter polymorphism in the BCMO1 gene, the G allele at the G15929A (G278R) polymorphism in the BCMO1 gene, the A allele at the G15929A (G278R) polymorphism in the BCMOI gene, the A allele at the A18068G (N341D) polymorphism in the BCMOI gene, and the 0 allele at the A18068G (N341D) polymorphism in the BCMOI gene.

7. The method as claimed in claim 6 wherein the presence or absence of one or more of the alleles is determined by the use of one or more polymorphisms in linkage disequilibrium with the allele.

8. The method as claimed in any one of claims I to 7, wherein the allelic profile is determined by determining the expression of a BCMOI gene or the expression or activity of a BCMO 1 gene product.

9. The method as claimed in any one of claims 1 to 8, the method comprising determining milk colour or milk J3-carotene content of the bovine, determining the BCMOI allelic profile of the bovine, comparing the BCMO1 allelic profile of the bovine or the milk colour or milk f-carotene content of the bovine with that of a bovine having a known BCMO I allelic profile; and determining the genetic merit of the bovine on the basis of the comparison.

10. A method of identifying or selecting a bovine having a desired BCMOI allelic profile comprising determining a BCMOI allelic profile of the bovine according to the method of any one of claims I to 9 and identifying or selecting said bovine on the basis of the determination.

II. A method of determining genetic merit of a bovine with respect to milk colour or milk J3-carotene content, the method comprising providing data about the BCMOI allelic profile of said bovine, and determining the genetic merit of the bovine on the basis of the data.

12. The method of claim 11 wherein the genetic merit is a capability of producing progeny that will have increased milk colour or milk LI-carotene content.

13. The method of claim 11 wherein the genetic merit is a capability of producing progeny that will have decreased milk colour or milk 3-carotene content.

14. A method for identifying or selecting a bovine with respect to milk colour or milk I-carotene content, or with respect to capability of producing progeny that will have increased or decreased milk colour or milk n-carotene content, the method comprising providing data about the BCMO1 allelic profile of said bovine, and identifying or selecting the bovine on the basis of the data.

15. The method as claimed in any one of claims 11 to 14, wherein the data about the BCMO1 allelic profile is indicative of the presence or absence of one or more polymorphisms selected from the group comprising: the C-I 054T promoter polymorphism in the bovine BCMO 1 gene, the G15929A (G278R) polymorphism in the bovine BCMOI gene, the Al 8068G (N34 ID) polymorphism in the bovine BCMO I gene, or one or more polymorphisms in linkage disequilibrium with one or more of the group comprising: the C-i 054T promoter polymorphism of the bovine BCMO 1 gene, the GI 5929A (G278R) polymorphism of the bovine BCMO1 gene, or the A18068G (N341D) polymorphism of the bovine BCMO1 gene.

16. The method as claimed in any one of claims 3 to 15, wherein the milk colour or milk content is increased milk colour or increased milk 3-carotene content.

17. The method as claimed in claim 16, the method comprising.determining (a) the. presence of the I allele at the C-1054T promoter polymorphism in the bovine BCMOI gene, or (b) the absence of the C allele at the C-1054T promoter polymorphism in the bovine BCMOI gene, or (c) the presence of the G allele at the G15929A (0278R) polymorphism in the bovine BCMOI gene, or (d) the absence of the the A allele at the G15929A (G278R) polymorphism in the bovine BCMOI gene, or (e) the presence of the 0 allele at the Al8068G (N341D) polymorphism in the bovine BCMO1 gene, or (1) the absence of the A allele at the Al 8068G (N341D) polymorphism in the bovine BCMOI gene, or (g) any combination of two or more of (a) to (f), and identif'ing or selecting the bovine on the basis of the determination.

18. The method as claimed in any one of claims 3 to 15, wherein the milk colour or milk content is decreased milk colour or decreased milk n-carotene content.

19. The method as claimed in claim 18, the method comprising determining (a) the presence of the C allele at the C-1054T promoter polymorphism in the bovine BCMOI gene, or (b) the absence of the T allele at the C-I 054T promoter polymorphism in the bovine BCMOI gene, or (c) the presence of the A allele at the G15929A (G278R) polymorphism in the bovine BCMO 1 gene, or (d) the absence of the the G allele at the G15929A (G278R) polymorphism in the bovine BCMO I gene, or (e) the presence of the A allele at the A18O68G (N341D) polymorphism in the bovine BCMO 1 gene, or (f) the absence of the 0 allele at the A18068G (N341D) polymorphism in the bovine BCMO1 gene, or (g) any combination of two or more of(a) to (f), and identifying or selecting the bovine on the basis of the determination.

20. The method as claimed in any one of claims I to 19, further comprising the step of amplifying at least a fragment of the bovine BCMO1 gene to determine the presence or absence of one or more polymorphisms associated with increased or decreased expression or activity of a BCMOI gene product.

21. The method as claimed in claim 20, wherein the primers used in the amplification are selected from the group consisting of SEQ ID NOs: 4 to 17.

22. The method of any one of claims 11 to 21, wherein the method additionally comprises providing data indicative of the presence or absence of one or more of the group comprising the C allele at the C-321 0 promoter polymorphism in the SCARBI gene, the 0 allele at the C-321G promoter polymorphism in the SCARBI gene, the 0 allele at the W8OStop 0/A polymorphism in the BCO2 gene, the A allele at the W8OStop G/A polymorphism in the BCO2 gene, or one or more polymorphisms in linkage disequilibrium with one or more of the group comprising the C allele at the C-321G promoter polymorphism in the SCARBI gene, the G allele at the C-32 1 G promoter polymorphism in the SCARB I gene, the G allele at the W8OStop G/A polymorphism in the BCO2 gene, or the A allele at the W8OStop G/A polymorphism in the BCO2 gene.

23. A probe or primer comprising a nucleotide sequence selected from the group comprising: (a) from 12 to 10000 contiguous nucleotides of SEQ ID NO: 1 and comprising a cytosine at the C-i 054T promoter polymorphism or a nucleotide capable of hybridising to a cytosine at the C-i 054T promoter polymorphism; or (b) from 12 to 1791 contiguous nucleotides of SEQ ID NO:2 and comprising a cytosine at the C-1054T polymorphism or a nucleotide capable of hybridising to a cytosine at the C-1054T promoter polymorphism; or (c) from 12 to 10000 contiguous nucleotides of SEQ ID NO:l and comprising a thymine at the C-1054T polymorphism or,a nuclcotide capable of hybridising to a thymine at the C-I 054T promoter polymorphism; or (d) from 12 to 1791 contiguous nucleotides of SEQ ID NO:2 and comprising a thymine at the C-I 054T polymorphism or a nucleotide capable of hybridising to a thymine at the C-1054T promoter polymorphism; or (e) from 12 to 10000 contiguous nucleotides of SEQ ID NO:1 and comprising a guanine at the G15929A (G278R) polymorphism or a nucleotide capable of hybridising to a guanine at the G I 5929A (G278R) polymorphism; or (f) from 12 to 1791 contiguous nucleotides of SEQ ID NO:2 and comprising a guanine at the G15929A (G278R) polymorphism or a nucleotide capable of hybridising to a guanine at the G15929A (G278R) polymorphism; or (g) from 12 to 10000 contiguous nucleotides of SEQ ID NO: I and comprising an adeninc at the 01 5929A (G278R) polymorphism or a nucleotide capable of hybridising to an adenine at the 01 5929A (G278R) polymorphism; or (h) from 12 to 1791 contiguous nucleotides of SEQ ID NO:2 and comprising an adenine at the GI 5929A (G278R) polymorphism or a nucleotide capable of hybridising to an adenine at the 01 5929A (G278R) polymorphism; or (i) from 12 to 10000 contiguous nucleotides of SEQ ID NO:1 and comprising a guanine at the the A 180680 (N34 I D) polymorphism or a nuçleotide capable of hybridising to a guanine at the A18068G (N341D) polymorphism; or (j) from 12 to 1791 contiguous nucleotides of SEQ ID NO:2 and comprising a guanine at the A18068G (N341D) polymorphism or a nucleotide capable of hybridising to a guanine at the Al 8068G (N341 D) polymorphism; or (k) from 12 to 10000 contiguous nucleotides of SEQ ID NO: I and comprising an adenine at the A18068G (N341D) polymorphism or a nucleotide capable of hybridising to an adenine at the Al 8068G (N34 1 D) polymorphism; or (1) from 12 to 1791 contiguous nucleotides of SEQ ID NO:2 and comprising an adenine at the A18068G (N341D) polymorphism or a nucleotide capable of hybridising to an adenine at the A18068G (N341D) polymorphism.

24. A probe or primer having about at least 12 contiguous bases of any one of SEQ ID NOs: 4-17.

25. A probe or primer as claimed in claim 23 comprising a nucleotide sequence comprising at least about 12 contiguous bases of SEQ ID NO: 1 or SEQ ID NO: 2 wherein the about 12 contiguous bases comprise or are within about I to about 2000 nucleotides of one or more of the group consisting of the C-I 054T promoter polymorphism of the bovine BCMO I gene, the 01 5929A (G278R) polymorphism of the bovine BCMO1 gene, or the A18068G (N341D) polymorphism of the bovine BCMO1 gene.

26. A pair of primers comprising any two primers as claimed in any one of claims 23 to 25.

27. A bovine identified by the method of any one of claims 10 or 14 to 21.

28. A bovine as claimed in claim 27, wherein the bovine is a bull.

29. Collected semen produced by a bovine as claimed in claim 28.

30. A bovine as claimed in claim 27, wherein the bovine is a cow.

31. A method of selecting a herd of bovine, comprising selecting individuals by the method of any one of claims 10 or 14 to 21, and segregating and collecting the selected individuals to form the herd.

32. A herd of bovine selected by the method of claim 31.

33. 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 10 or 14 to 21.

34. Collected or pooled milk produced by bovine as claimed in claim 30 or by a herd of bovine as claimed in claim 32 or 33.

35. Collected or pooled milk as claimed in claim 34 having increased or decreased colour or increased or decreased n-carotene content when compared to milk produced by a bovine having a BCMO1 gene comprising the nucleotide sequence of SEQ ID NO: 1.

36. A dairy product made from the m1k as claimed in any one of claims 34 to 35.

37. A kit for genotyping a bovine with respect to one or more milk colour or 13-carotene content phenotypes, comprising a probe or primer as defined in any one of claims 23 to 25 or a pair of primers as defined in claim 26.

38. An isolated, purified or recombinant nucleic acid molecule comprising nucleotide sequence selected from the group comprising: (a) from 12 to 20000 contiguous nucleotides of SEQ ID NO:1 and comprising one or more of the C-1054T promoter polymorphism, the G15929A (G278R) polymorphism, or the Al 8068G (N34 I D) polymorphism; or (b) from 12 to 1791 contiguous nucleotides of SEQ ID NO:2 and comprising one or more of the C-1054T promoter polymorphism, the G15929A (G278R) polymorphism, or the Al 8068G (N34 1 D) polymorphism; or (c) from 12 to 20000 contiguous nucleotides of a functional variant of SEQ ID NO:1; or (d) from 12 to 1791 contiguous nucleotides of a functional variant of SEQ ID NO:2; or (e) at least 12 contiguous nucleotides of any one of SEQ ID NOs:4 -17; or (0 a complement of any one of(a) to (e); or (g) a sequence of at least 12 contiguous nucleotides and capable of hybridising to the nucleotide sequence of any one of (a) to (f) under stringent conditions.

39. A vector comprising the nucleic acid of claim 38.

40. A host cell comprising a vector as claimed in claim 39.

41. An isolated, purified or recombinant polypeptide comprising an amino acid sequence having at least 95% sequence identity with a sequence present in SEQ ID NO:3, wherein the polypeptide one or more of the following: (a) arginine at the position corresponding to amino acid 278 of SEQ ID NO:3; or (b) an amino acid other than glycine at the position corresponding to amino acid 278 of SEQ ID NO:3; or (c) aspartate at the position corresponding to amino acid 341 of SEQ ID NO:3; or (d) an amino acid other than asparagine at the position corresponding to amino acid 341 of SEQ ID NO:3; or (e) any combination of(a) or (b) and (c) or (d).

42. The polypeptide as claimed in claim 41, wherein the polypeptide comprises at least amino acids.

43. The polypeptide as claimed in claim 41 wherein the polypeptide has at least about 20% of the enzymatic activity of a BCMO1 polypeptide consisting of the amino acid sequence of SEQ ID NO:3.

44. An antibody capable of binding a polypeptide as claimed in any one of claims 41 to 43.