NZ561998A - Marker assisted selection of bovine for milk fat colour - Google Patents

Abstract

Provided is a method of determining genetic merit of bovine with respect to milk or tissue colour or â-carotene content or with respect to capability of producing progeny that will have increased or decreased milk or tissue colour or â-carotene content, which comprises the step of determining the BCO2 allelic profile of the W80Stop G/A polymorphism in the BCO2 gene or one or more polymorphisms in linkage disequilibrium with the W80Stop G/A polymorphism in the BCO2 gene of said bovine, and determining the genetic merit of the bovine on the basis of the BCO2 allelic profile.

Description

NEW ZEALAND PATENTS ACT, 1953 No: 561998 Date: 26 September 2007 COMPLETE SPECIFICATION MARKER ASSISTED SELECTION OF BOVINE FOR DESIRED MILK OR TISSUE CONTENT We, VIALACTIA BIOSCIENCES (NZ) LIMITED, a company incorporated under the laws of New Zealand of 9 Princes Street, Auckland, New Zealand, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: 1 2 MARKER ASSISTED SELECTION OF BOVINE FOR DESIRED MILK OR TISSUE CONTENT FIELD OF THE INVENTION id="p-1" id="p-1"

[0001] This invention relates to an application of marker assisted selection of bovine for a quantitative trait loci (QTL) associated with milk and tissue colour and P-carotene content, particularly by assaying for the presence of polymorphisms in a gene which is associated with the QTL.

BACKGROUND id="p-2" id="p-2"

[0002] The genetic basis of bovine milk production is of immense significance to the dairy industry. An ability to modulate milk volumes and composition has the potential to alter farming practices and to produce products which are tailored to meet a range of requirements. In particular, a method of genetically evaluating bovine to select those which express desirable traits, such as desirable milk fat colour or composition, would be useful. Similarly, a method of genetically evaluating bovine with desirable tissue colour or composition would be useful. id="p-3" id="p-3"

[0003] Genetic bases for variations in the composition of milk, for example, the relative amounts of major milk proteins, and the effect of these variations on milk production characteristics and milk processing properties, has been the subject of considerable research, debate, and review. For example, PCT International application PCT/NZ01/00245 (published as W002/36824) reports that polymorphisms in the bovine Diacylglycerol-o-acyltransferase (DGAT1) gene are associated with increased milk yield and altered milk composition, and in particular that the presence of a K232A mutation in the DGAT1 gene results in a decrease in milk fat percentage, milk fat yield, solid fat content and milk protein percentage, while increasing milk volume and milk protein yield. In another example, PCT International application PCT/NZ02/00157 (published as 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. id="p-4" id="p-4"

[0004] The yellow colour of milk and milk fat, caused primarily by the presence of P-carotene, is considered a negative characteristic in some consumer markets. Conversely, 3 other markets prize the yellow colour, while foods enriched in P-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 etal., 1999). Similarly, different consumer markets variously perceive tissue colour. id="p-5" id="p-5"

[0005] Strategies to modulate milk colour or P-carotene content or tissue colour or P-carotene content could provide health benefits and are expected to be economically valuable. P-carotene and vitamin A deficiencies are still major problems (particularly in developing countries) leading to blindness and childhood mortality. Milk with increased P-carotene content would be of benefit, for example in markets where other dietary sources of P-carotene are scarce or not commonly consumed. id="p-6" id="p-6"

[0006] Marker assisted selection, which provides the ability to follow a specific favourable genetic allele, involves the identification of a DNA molecular marker or markers that segregate(s) with a gene or group of genes associated with or which in part defines a trait. DNA markers have several advantages. They are relatively easy to measure and are unambiguous, and as DNA markers are co-dominant, heterozygous and homozygous animals can be distinctively identified. Once a marker system is established, selection decisions are able to be made very easily as DNA markers can be assayed at any time after a DNA containing sample has been collected from an individual animal, whether embryonic, infant or adult. id="p-7" id="p-7"

[0007] It is an object of the present invention to provide a method for marker assisted selection of bovine with desired milk colour or milk P-carotene content or tissue colour or tissue P-carotene content; and/or to provide animals selected using the method of the invention as well as milk produced by or tissue(s) derived from the selected animals; and/or to provide the public with a useful choice.

SUMMARY OF THE INVENTION id="p-8" id="p-8"

[0008] This invention relates to the elucidation of the role of the gene encoding P-carotene oxygenase 2 (BC02, formerly known as P-carotene 9', lO'-dioxygenase 2 (BCD02)) [EC: 1.14.99.-] in milk or tissue colour or P-carotene content, particularly milk fat colour and P-carotene content. In particular, the invention relates to the identification of the W80Stop G/A polymorphism in the BC02 gene, and the association of the A allele with 4 production of milk and particularly milk fat with increased P-carotene content for the first time. id="p-9" id="p-9"

[0009] This gives rise to numerous, and separate, aspects of the invention. id="p-10" id="p-10"

[0010] In one aspect the invention provides a method of determining the genetic merit of a bovine with respect to milk or tissue colour or P-carotene content or with respect to capability of producing progeny that will have increased or decreased milk or tissue colour or P-carotene content, which comprises determining the BC02 allelic profile of the bovine, and determining the genetic merit of the bovine on the basis of the BC02 allelic profile. id="p-11" id="p-11"

[0011] In one embodiment, milk P-carotene content is milk fat P-carotene content. id="p-12" id="p-12"

[0012] In another embodiment, milk content is milk colour, more preferably milk fat colour. id="p-13" id="p-13"

[0013] In still further embodiments, the tissue is selected from liver or blood. id="p-14" id="p-14"

[0014] In one embodiment, the genetic merit with respect to milk or tissue colour or P-carotene content is production of milk or tissue with increased colour or P-carotene content. id="p-15" id="p-15"

[0015] Accordingly, in various embodiments the invention provides a method for identifying or selecting a bovine that produces milk, tissue, milk fat, or tissue fat with increased P-carotene content, or capable of producing progeny that produce milk, tissue, milk fat, or tissue fat with increased P-carotene content, comprising determining the expression or activity of the BC02 gene product, and identifying or selecting the bovine on the basis of the determination. id="p-16" id="p-16"

[0016] In another embodiment, the genetic merit with respect to milk or tissue colour or P-carotene content is production of milk or tissue with decreased colour or P-carotene content. Accordingly, in various embodiments the invention provides a method for identifying or selecting a bovine that produces milk, tissue, milk fat, or tissue fat with decreased P-carotene content, or capable of producing progeny that produce milk, tissue, milk fat, or tissue fat with decreased P-carotene content, comprising determining the expression or activity of the BC02 gene product, and identifying or selecting the bovine on the basis of the determination. id="p-17" id="p-17"

[0017] In one embodiment, the method comprises determining the expression or activity of a BC02 gene or gene product, and preferably identifying or selecting the bovine on the basis of the determination. id="p-18" id="p-18"

[0018] In one embodiment, expression or activity of the BC02 gene product is determined using BC02 mRNA, for example by determining the presence or amount of BC02 mRNA. In other embodiments, expression or activity of the BC02 gene product is determined using BC02 protein, preferably by determining the amount of BC02 protein, for example the amount of BC02 protein, or by determining the activity of BC02 protein, for example the enzymatic activity of BC02 protein present in a sample obtained from the bovine. It will be apparent that the activity of BC02 protein may be determined by determining the ratio of BC02 substrate: BC02 product, such as that present in a sample obtained from the bovine. In still other embodiments, the expression or activity of the BC02 gene product is determined using BC02 DNA, preferably by determining the presence or absence of one or more polymorphisms associated with decreased or increased BC02 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. id="p-19" id="p-19"

[0019] In another embodiment, the BC02 allelic profile of the bovine is determined together with the allelic profile of the bovine at one or more genetic loci associated with milk or tissue colour or P-carotene content. id="p-20" id="p-20"

[0020] In one embodiment, the one or more genetic loci is one or more polymorphisms in one or more genes associated with milk or tissue colour or P-carotene content. id="p-21" id="p-21"

[0021] The one or more polymorphisms can be detected directly or by detection of one or more polymorphisms which are in linkage disequilibrium with said one or more polymorphisms. id="p-22" id="p-22"

[0022] 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.) id="p-23" id="p-23"

[0023] It will be apparent that as used herein, the phrase "BC02 allelic profile" contemplates data indicative of the presence or absence of one or more alleles at one or more polymorphisms in the BC02 gene or which affect expression from the BC02 gene or the expression or activity of a BC02 gene product or which are associated with variation in the expression from the BC02 gene or in the expression or activity of a BC02 gene product. In preferred embodiments, the BC02 allelic profile comprises data indicative of the presence or absence of one or more alleles at one or more polymorphisms associated with increased or decreased milk or tissue colour or P-carotene content. For example, in preferred embodiments the BC02 allelic profile comprises data indicative of the presence or absence of 6 the A allele, or of the presence or absence of the G allele, at the W80Stop G/A polymorphism in the BC02 gene. In other embodiments, the BC02 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 BC02 gene, or in a regulatory region of the BC02 gene, or in an intron of the BC02 gene, and preferably comprises data indicative of the presence or absence of one or more alleles which affect expression from the BC02 gene or the expression or activity of a BC02 gene product or which are associated with variation in the expression from the BC02 gene or in the expression or activity of a BC02 gene product. id="p-24" id="p-24"

[0024] It will further be appreciated that the BC02 allelic profile may comprise information correlating the presence or absence of one or more polymorphisms as described above with milk or tissue colour or P-carotene content. id="p-25" id="p-25"

[0025] 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. id="p-26" id="p-26"

[0026] In yet a further embodiment, the allelic profile is determined with reference to the amino acid sequence of expressed BC02 protein obtained from said bovine. id="p-27" id="p-27"

[0027] In another embodiment, the allelic profile is determined with reference to the amount or activity of BC02 protein obtained from said bovine. id="p-28" id="p-28"

[0028] Conveniently, in said method the presence or absence of DNA encoding wild type BC02 in said bovine is determined, directly or indirectly, for example using an expressed gene product. id="p-29" id="p-29"

[0029] Alternatively, in said method the presence or absence of at least one nucleotide difference from the nucleotide sequence encoding wild type BC02 in said bovine is determined, directly or indirectly. id="p-30" id="p-30"

[0030] In one embodiment, the method comprises determining the BC02 W80Stop G/A allelic profile of the bovine. id="p-31" id="p-31"

[0031] More specifically, in said method the presence or absence of one or more of the A allele or G allele at the W80Stop G/A polymorphism in the BC02 gene is determined, directly or indirectly. For example, the presence of the A allele or G allele at the W80Stop G/A polymorphism in the BC02 gene may be determined using a polymorphism in linkage disequilibrium with the A allele or with the G allele at the W80Stop G/A polymorphism. id="p-32" id="p-32"

[0032] 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 7 protein "(A)" having biological activity of wild type BCD02 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 P-carotene levels, particularly with the production of milk with, inter 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 P-carotene levels, particularly with the production of milk with, inter alia, decreased fat colour, or decreased tissue colour. The presence of both the DNA encoding (A) and the DNA encoding (B) indicates an association with intermediate relative P-carotene levels, particularly with the production of milk with, inter alia, intermediate fat colour, or intermediate tissue colour. id="p-33" id="p-33"

[0033] The allelic protein (B) lacking the activity of (A) may be a truncated form of expressed bovine BC02 protein, preferably a truncated form of BC02 protein consisting of the first 79 amino acids encoded by the bovine BC02 gene. id="p-34" id="p-34"

[0034] As used herein, biological activity of wild type BC02 protein refers to both expression levels and activity characteristic of BC02 protein encoded by the wild type BC02 gene. id="p-35" id="p-35"

[0035] In another embodiment, the method includes ascertaining, from a sample of material containing DNA obtained from the bovine, whether the wild type BC02 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 BC02 gene product, preferably by determining the presence or absence of one or more polymorphisms associated with decreased or increased BC02 expression, for example one or more promoter polymorphisms associated with increased or decreased expression. id="p-36" id="p-36"

[0036] In another embodiment, the invention includes ascertaining whether mRNA encoding a protein "(A)" having biological activity of a wild type BC02 is present, or whether mRNA encoding a protein "(B)" at least partially lacking the activity of (A) is present, or whether mRNA encoding (A) and mRNA encoding (B) are both present. The absence of the mRNA encoding (A) and the presence of the mRNA encoding (B) again indicates an association with high relative P-carotene levels, particularly with the production of milk with, inter alia, increased fat colour, or increased tissue colour. The reverse association again holds true. Again, the presence of both the mRNA encoding (A) and the 8 mRNA encoding (B) indicates an association with intermediate relative P-carotene levels, particularly with the production of milk with, inter alia, intermediate fat colour, or intermediate tissue colour. id="p-37" id="p-37"

[0037] In another embodiment, the method includes ascertaining the amount of BC02 mRNA present in a sample of material containing mRNA obtained from the bovine. In another embodiment, the method includes ascertaining the BC02 expression profile of the bovine, preferably including ascertaining the BC02 mRNA expression profile of the bovine. id="p-38" id="p-38"

[0038] In another embodiment, the invention includes ascertaining whether a protein "(A)" having biological activity of a wild type BC02 is present, or whether a protein "(B)" at least partially lacking the activity of (A) is present, or whether (A) and (B) are both present. The absence of (A) and the presence of (B) again indicates an association with high relative P-carotene levels, particularly with the production of milk or tissue with, inter alia, increased fat colour and/or high relative P-carotene content. The reverse association again holds true. Further, the presence of both (A) and (B) indicates an association with intermediate relative P-carotene levels, particularly with the production of milk with, inter alia, intermediate fat colour. id="p-39" id="p-39"

[0039] In another embodiment, the method includes ascertaining the amount or activity of BC02 protein present in a sample of material containing protein obtained by the bovine. id="p-40" id="p-40"

[0040] In another aspect, the invention is a method for determining the BC02 genotype of a bovine, as may be desirable to know for breeding purposes. id="p-41" id="p-41"

[0041] In one embodiment, the method includes ascertaining, with reference to a sample of material containing nucleic acid obtained from the bovine and uncontaminated by heterologous nucleic acid, whether the sample contains (i) nucleic acid molecule encoding a protein having biological activity of wild type BC02 and optionally ascertaining whether the sample contains an (ii) allelic nucleic acid molecule encoding a protein lacking biological activity of wild type BC02. id="p-42" id="p-42"

[0042] In another embodiment, the method includes ascertaining, with reference to a sample of material containing protein obtained from the bovine and uncontaminated by heterologous protein, whether the sample contains (i) a protein having biological activity of wild type BC02 and optionally ascertaining whether the sample contains a protein lacking biological activity of wild type BC02. id="p-43" id="p-43"

[0043] In a further embodiment, the invention provides a method of determining genetic merit of a bovine with respect to milk or tissue P-carotene content which comprises 9 determining the BC02 allelic profile of the bovine, together with determining the allelic profile of the bovine at one or more genetic loci associated with milk or tissue P-carotene content. id="p-44" id="p-44"

[0044] In one embodiment, the one or more genetic loci is one or more polymorphisms in one or more genes associated with milk or tissue P-carotene content, preferably one or more polymorphisms in one or more genes involved in P-carotene uptake or metabolism. id="p-45" id="p-45"

[0045] In a further aspect, the invention includes a probe containing a nucleic acid molecule sufficiently complementary with a nucleic acid sequence present in SEQ ID NO: 1 or a nucleotide sequence encoding a wild type bovine BC02, or its complement, so as to bind thereto under stringent conditions, as well as a diagnostic kit containing such a probe. Particularly contemplated are probes that comprise one or other of the alleles at the W80Stop G/A polymorphism, for example a probe that includes an adenosine at the site corresponding to the W80Stop G/A polymorphism. id="p-46" id="p-46"

[0046] The invention also includes a primer composition useful for detection of the presence or absence of DNA encoding wild type BC02 and/or the presence of the DNA encoding a variant protein at least partially lacking wild type activity. In one form, the composition can include a nucleic acid primer substantially complementary to a nucleic acid sequence encoding wild type BC02, or its complement. 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. id="p-47" id="p-47"

[0047] Particularly contemplated are primers comprising or substantially complementary to a nucleic acid sequence present in SEQ ID NO: 1 and within approximately 1 to about 2000 bp of the W80Stop G/A polymorphism, more preferably within approximately 1 to about 1000 bp, or within approximately 1 to about 500 bp, approximately 1 to about 400 bp, approximately 1 to about 300 bp, approximately 1 to about 200 bp, approximately 1 to about 100 bp, approximately 1 to about 50 bp, or approximately 1 to about 20 bp of the W80Stop G/A polymorphism. id="p-48" id="p-48"

[0048] It will be appreciated by those skilled in the art that a pair of such primers can be used to determine the identity of the nucleotide at the W80Stop G/A 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. id="p-49" id="p-49"

[0049] The invention further includes an antibody composition useful for detection of the presence or absence of wild type BC02 and/or the presence or absence of a variant protein at least partially lacking wild type activity, particularly a truncated form of the BC02 protein, as well as a diagnostic kit containing such an antibody together with instructions for use, for example in a method of the invention. id="p-50" id="p-50"

[0050] The invention further provides a diagnostic kit useful in detecting DNA encoding a variant BC02 at least partially lacking wild type activity in a bovine which includes first and second primers for amplifying the DNA, the primers being complementary to nucleotide sequences of the DNA upstream and downstream, respectively, of a polymorphism in the portion of the DNA encoding BC02 which results in increased P-carotene levels (particularly increased P-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 BC02 gene. The kit can also include a third primer complementary to a naturally occurring mutation of a coding portion of the wild type BC02 gene. Preferably the kit includes instructions for use, for example in accordance with a method of the invention. id="p-51" id="p-51"

[0051] Thus, in another embodiment the invention provides a method of assessing the genetic merit of a bovine with respect to milk or tissue P-carotene content which comprises the step of determining the presence or absence of one or more polymorphisms selected from the group comprising: the W80Stop G/A polymorphism in the BCD02 gene, or one or more polymorphisms in linkage disequilibrium with the W80Stop G/A polymorphism in the BCD02 gene. id="p-52" id="p-52"

[0052] In another embodiment the invention provides a method of assessing the genetic merit of a bovine with respect to milk or tissue colour or P-carotene content which comprises the step of determining the presence or absence of one or more polymorphisms selected from the group comprising: the A allele at the WSOStop G/A polymorphism, or the G allele at the WSOStop G/A polymorphism. id="p-53" id="p-53"

[0053] 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. id="p-54" id="p-54"

[0054] Thus, in another embodiment the invention provides a method of assessing the genetic merit of a bovine with respect to milk or tissue P-carotene content which comprises the step of determining the presence or absence of the G allele at the W80Stop G/A polymorphism in the BC02 gene. The invention further provides a method of assessing the 11 genetic merit of a bovine with respect to milk or tissue P-carotene content which comprises the step of determining the presence or absence of the A allele at the W80Stop G/A polymorphism in the BC02 gene. id="p-55" id="p-55"

[0055] In another aspect, the present invention provides a method for selecting a bovine with a genotype indicative of desired milk colour or of desired milk P-carotene content or of desired tissue colour or of desired tissue P-carotene content. The method comprises determining the bovine BC02 allelic profile of said bovine, and selecting the bovine on the basis of the determination. id="p-56" id="p-56"

[0056] In one embodiment, the invention provides a method for selecting a bovine with a BC02 allelic profile indicative of increased milk colour or increased milk P-carotene content, preferably of increased milk fat colour or increased milk fat P-carotene content,. id="p-57" id="p-57"

[0057] In another embodiment, the invention provides a method for selecting a bovine with a BC02 allelic profile indicative of increased tissue colour or increased tissue P-carotene content. id="p-58" id="p-58"

[0058] Preferably the method comprises determining the presence of the A allele at the W80Stop G/A polymorphism in the BC02 gene, and selecting the bovine on the basis of the determination. Alternatively or additionally, the method comprises determining the absence of the G allele at the W80Stop G/A polymorphism in the BC02 gene, and selecting the bovine on the basis of the determination. id="p-59" id="p-59"

[0059] Preferably, the method comprises determining the presence of the AA genotype at the W80Stop G/A polymorphism in the BC02 gene, and selecting the bovine on the basis of the determination. id="p-60" id="p-60"

[0060] In a further embodiment the invention provides a method for selecting a bovine with a BC02 allelic profile indicative of decreased milk colour or decreased milk P-carotene content, preferably of decreased milk fat colour or decreased milk fat P-carotene content. id="p-61" id="p-61"

[0061] In another embodiment, the invention provides a method for selecting a bovine with a BC02 allelic profile indicative of decreased tissue colour or decreased tissue P-carotene content. id="p-62" id="p-62"

[0062] Preferably the method comprises determining the absence of the A allele at the W80Stop G/A polymorphism in the BC02 gene, and selecting the bovine on the basis of the determination. Alternatively or additionally, the method comprises determining the presence of the G allele at the W80Stop G/A polymorphism in the BC02 gene, and selecting the bovine on the basis of the determination. 12 id="p-63" id="p-63"

[0063] Preferably, the method comprises determining the presence of the GG genotype at the W80Stop G/A polymorphism in the BC02 gene, and selecting the bovine on the basis of the determination. id="p-64" id="p-64"

[0064] In a further embodiment the invention provides a method for selecting a bovine with a BC02 allelic profile indicative of intermediate milk colour or intermediate milk P-carotene content, preferably of intermediate milk fat colour or intermediate milk fat P-carotene content. id="p-65" id="p-65"

[0065] In another embodiment, the invention provides a method for selecting a bovine with a BC02 allelic profile indicative of intermediate tissue colour or intermediate tissue P-carotene content. id="p-66" id="p-66"

[0066] Preferably, the method comprises determining the presence of the AG genotype at the W80Stop G/A polymorphism in the BC02 gene, and selecting the bovine on the basis of the determination. id="p-67" id="p-67"

[0067] In one embodiment, the presence of the A allele or of the G allele is determined with respect to a BC02 polynucleotide (genomic DNA, mRNA or cDNA produced from mRNA) obtained from the bovine. id="p-68" id="p-68"

[0068] In a preferred embodiment, the presence of the G allele is determined by detecting the presence of a codon encoding tryptophan at a position corresponding to residue 80 in the BC02 sequence represented in SEQ ID NO:3. id="p-69" id="p-69"

[0069] In a preferred embodiment, the presence of the A allele is determined by detecting the presence of a codon encoding termination of protein synthesis at a position corresponding to residue 80 in the BC02 sequence represented in SEQ ID NO:3. id="p-70" id="p-70"

[0070] In one embodiment, the presence of the A allele or of the G allele is determined by sequencing a BC02 polynucleotide obtained from the bovine. id="p-71" id="p-71"

[0071] In a further embodiment the determination comprises the step of amplifying a BC02 polynucleotide sequence from genomic DNA, mRNA or cDNA produced from mRNA derived from said bovine, for example by PCR. id="p-72" id="p-72"

[0072] Preferably the determination is by use of primers which comprise a nucleotide sequence having at least about 12 contiguous bases of or complementary to the sequence of SEQ ID NO:l or SEQ ID NO:2 or a naturally occurring flanking sequence. id="p-73" id="p-73"

[0073] In one embodiment at least one of the primers comprises sequence corresponding to at least one of the allele-specific nucleotides described herein. 13 id="p-74" id="p-74"

[0074] In an alternative embodiment, the method comprises restriction enzyme digestion of a nucleotide derived from the bovine. Such digestion may also be performed on a product of the PCR amplification described above. id="p-75" id="p-75"

[0075] In a further embodiment, the presence of the A allele or of the G allele is determined by mass spectrometric analysis of a BC02 polynucleotide obtained from the bovine. id="p-76" id="p-76"

[0076] In an alternative embodiment, the presence of the A allele or of the G allele is determined by hybridisation of a probe or probes comprising a nucleotide sequence of or complementary to the sequence of SEQ ID NO: 1 or SEQ ID NO:2. id="p-77" id="p-77"

[0077] Preferably the probe or probes comprises 12 or more contiguous nucleotides of or complementary to the sequence of SEQ ID NO: 1 or SEQ ID NO:2. id="p-78" id="p-78"

[0078] Preferably the probe or probes comprise sequence corresponding to the A allele-specific or the G allele-specific nucleotides described herein or complements thereof. id="p-79" id="p-79"

[0079] In an alternative embodiment, the presence of the A allele or of the G allele is determined by analysis of a BC02 polypeptide obtained from the bovine. id="p-80" id="p-80"

[0080] In one embodiment, the presence of the A allele is determined by detecting the presence of a truncated form of a BC02 polypeptide, preferably a truncated form of a BC02 polypeptide 79 amino acids in length, more preferably a truncated form of a BC02 polypeptide consisting of the first 79 amino acids of SEQ ID NO. 3. Alternatively, the presence of the A allele is determined by detecting a reduction in the amount of or an absence of full length BC02 polypeptide. id="p-81" id="p-81"

[0081] Preferably the presence of the G allele is determined by detecting the presence of tryptophan at the position corresponding to residue 80 in SEQ ID NO:3, more preferably by detecting the presence of full length BC02 polypeptide. id="p-82" id="p-82"

[0082] In a further aspect the invention provides a bovine selected by a process of the invention; milk produced by the selected bovine or the progeny thereof as well as dairy products produced from such milk; and semen produced by or tissue from the selected bovine. id="p-83" id="p-83"

[0083] 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. 14 id="p-84" id="p-84"

[0084] In a still further aspect, the invention provides a method of determining genetic merit of a bovine with respect to one or more milk or tissue colour or P-carotene content traits, or with respect to capability of producing progeny predisposed to or with one or more milk or tissue colour or P-carotene content traits, the method comprising providing data about the BC02 allelic profile of said bovine, and determining the genetic merit of the bovine on the basis of the data. id="p-85" id="p-85"

[0085] Preferably, the data about the BC02 allelic profile comprises data representative of the presence or absence of the A allele or the G allele at the W80Stop G/A polymorphism in the BC02 gene. id="p-86" id="p-86"

[0086] Preferably, the method additionally comprises providing data comprising the result of at least one analysis of one or more genetic loci associated with one or more milk or tissue colour or P-carotene content traits, wherein the data is representative of the genetic merit of the bovine. id="p-87" id="p-87"

[0087] Preferably, the one or more genetic loci are one or more polymorphisms associated with an increase or decrease in expression or activity of a BC02 gene product. id="p-88" id="p-88"

[0088] Preferably the genetic loci is the BC02 gene (including all regulatory elements such as the promoter, introns and 3 'UTR). id="p-89" id="p-89"

[0089] In one embodiment, the one or more milk or tissue colour or P-carotene content traits is selected from the group comprising production of or capability of producing milk with increased milk colour, production of or capability of producing milk with increased milk P-carotene content, increased tissue colour, or increased tissue P-carotene content. id="p-90" id="p-90"

[0090] In another embodiment, the one or more milk or tissue colour or P-carotene content traits is selected from the group comprising production of or capability of producing milk with decreased milk colour, production of or capability of producing milk with decreased milk P-carotene content, decreased tissue colour, or decreased tissue P-carotene content. id="p-91" id="p-91"

[0091] Accordingly, in one embodiment the invention provides a method of determining genetic merit of a bovine with respect to milk or tissue colour or P-carotene content, or with respect to capability of producing progeny that will have increased or decreased milk or tissue colour or P-carotene content, the method comprising providing data about the BC02 allelic profile of the bovine, and determining the genetic merit of the bovine on the basis of the data. id="p-92" id="p-92"

[0092] Preferably, the data about the BC02 allelic profile comprises data representative of the presence or absence of the A allele or the G allele at the W80Stop G/A polymorphism in the BC02 gene. id="p-93" id="p-93"

[0093] Preferably, the method additionally comprises providing data comprising the result of at least one analysis of one or more genetic loci associated with one or more milk or tissue colour or P-carotene content traits, wherein the data is representative of the genetic merit of the bovine. id="p-94" id="p-94"

[0094] Preferably, the one or more genetic loci are one or more polymorphisms associated with an increase or decrease in expression or activity of a BC02 gene product. id="p-95" id="p-95"

[0095] Preferably the genetic loci is the BC02 gene (including all regulatory elements such as the promoter, introns and 3 'UTR). id="p-96" id="p-96"

[0096] In a further aspect the invention provides a method for identifying or selecting a bovine with respect to one or more milk or tissue colour or P-carotene content traits, the method comprising providing the result of one or more genetic tests of a sample from the bovine, and analysing the result for the presence or absence of one or more polymorphisms associated with increased or decreased expression or activity of BC02 gene product, or one or more polymorphisms in linkage disequilibrium with one or more polymorphisms associated with increased or decreased expression or activity of BC02 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 P-carotene content traits; and identifying or selecting the bovine on the basis of the result. id="p-97" id="p-97"

[0097] Preferably, the one or more polymorphisms associated with increased or decreased expression or activity of BC02 gene product is one or more polymorphisms in the BC02 gene. id="p-98" id="p-98"

[0098] In a further aspect the invention provides a method for selecting a bovine with one or more desired milk or tissue colour or P-carotene content traits, the method comprising a) providing the result of one or more genetic tests of a sample from the bovine, and b) analysing the result for the presence or absence of one or more polymorphisms selected from the group comprising: the W80Stop G/A polymorphism in the BC02 gene, or 16 one or more polymorphisms in linkage disequilibrium with the W80Stop G/A polymorphism in the BC02 gene, wherein a result indicative of the presence or absence of one or more of said polymorphisms is indicative of a bovine with one or more desired milk or tissue colour or P-carotene content traits. id="p-99" id="p-99"

[0099] In other aspects, the invention provides a system for performing one or more of the methods of the invention, said system comprising: computer processor means for receiving, processing and communicating data; storage means for storing data including a reference genetic database of the results of genetic analysis of a bovine with respect to one or more milk or tissue colour or P-carotene content traits and optionally a reference milk or tissue colour or P-carotene content trait database of non-genetic factors for one or more bovine milk or tissue colour or P-carotene content traits; and a computer program embedded within the computer processor which, once data consisting of or including the result of a genetic analysis for which data is included in the reference genetic database is received, processes said data in the context of said reference databases to determine, as an outcome, the genetic merit of the bovine, said outcome being communicable once known, preferably to a user having input said data. id="p-100" id="p-100"

[00100] Preferably, said system is accessible via the internet or by personal computer. id="p-101" id="p-101"

[00101] Preferably, said reference genetic database comprises or includes the results of one or more analyses of one or more genetic loci associated with one or more milk or tissue colour or P-carotene content traits, more preferably the one or more genetic loci are one or more polymorphisms in one or more genes associated with one or more milk or tissue colour or P-carotene content traits. id="p-102" id="p-102"

[00102] In yet a further aspect, the invention provides a computer program suitable for use in a system as defined above comprising a computer usable medium having program code embodied in the medium for causing the computer program to process received data consisting of or including the result of at least one genetic analysis of one or more genetic loci associated with one or more milk or tissue colour or P-carotene content traits in the context of both a reference genetic database of the results of said at least one genetic analysis and optionally a reference database of non-genetic factors associated with one or more bovine milk or tissue colour or P-carotene content traits. 17 id="p-103" id="p-103"

[00103] Preferably, the one or more genetic loci are one or more polymorphisms in one or more genes associated with one or more milk or tissue colour or P-carotene content traits. id="p-104" id="p-104"

[00104] Preferably the gene is the BC02 gene (including all regulatory elements such as the promoter, introns and 3'UTR). id="p-105" id="p-105"

[00105] Preferably, the one or more polymorphisms are one or more polymorphisms associated with an increase or decrease in expression or activity of a BC02 gene product. id="p-106" id="p-106"

[00106] In still another aspect, the invention provides a method of determining genetic merit of a bovine with respect to milk or tissue colour or P-carotene content, or with respect to capability of producing progeny that will have increased or decreased milk or tissue colour or P-carotene content, the method comprising determining milk or tissue colour or P-carotene content of the bovine, determining the BC02 allelic profile of the bovine, comparing the BC02 allelic profile of the bovine or the milk or tissue colour or P-carotene content of the bovine with that of a bovine having a known BC02 allelic profile; determining the genetic merit of the bovine on the basis of the comparison. id="p-107" id="p-107"

[00107] It will be appreciated that for the purposes of the comparison, the milk or tissue colour or P-carotene content associated with the known BC02 allelic profile is known. It will further be appreciated that the association of milk or tissue colour or P-carotene content with a particular BC02 allelic profile may be established by the methods described herein. id="p-108" id="p-108"

[00108] 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:l and comprising the W80Stop G/A polymorphism; or (b) at least 12 contiguous nucleotides of SEQ ID NO:2 and comprising the W80Stop G/A polymorphism; or (c) at least 12 contiguous nucleotides of a variant of SEQ ID NO:2; or (d) any one or more of SEQ ID NOs:4 - 25; or (e) a complement of any one of (a) to (d); or (f) a sequence of at least 12 contiguous nucleotides and capable of hybridising to the nucleotide sequence of any one of (a) to (e) under stringent conditions. id="p-109" id="p-109"

[00109] In one embodiment, the nucleic acid molecule encoding a BC02 of the invention comprises the W80Stop G/A polymorphism. 18 id="p-110" id="p-110"

[00110] The invention also provides a genetic construct comprising a BC02 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 BC02 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. id="p-111" id="p-111"

[00111] 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. id="p-112" id="p-112"

[00112] 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 P-carotene concentration determined as described in the Example. Data for peak (35 days post calving), mid (late November) and late (late February) lactation is shown. Figure 2 shows a graph depicting the detection of major QTL for milk fat colour on bovine chromosome 15. The maximum F-value (left-hand y-axis) occurred at 21 centimorgans (cM) and was 6.45. The 95% confidence interval the predicted gene location (0 - 25 cM) is shown by the grey horizontal bar at the top of the figure. The bootstrapping interval (corresponding to the right-hand y-axis) is shown by the histogram. The BC02 gene is located between the two markers indicated by arrows (BR3510 and MP076).

Figure 3 is a schematic showing a graphical representation of the W80Stop G/A polymorphism in bovine BC02. A: Graphical representation of the predicted exon structure of bovine BC02 gene. B. Identification of the G/A polymorphism at position 39304 (SEQ ID NO:l). The polymorphism (highlighted) was present in 3 of the six F1 sires (sires 2, 3, 6). Predicted 19 translation of the BC02 sequence comprising the A allele results the formation of a premature stop codon.

Figure 4 is a graph showing the effect of BC02 genotype on P-carotene concentration at peak, mid and late lactation. AA genotype, open bars; AG genotype, shaded bars, GG genotype, striped bars. Data shown are means ± s.e.m..

Figure 5 is three graphs showing the sire and seasonal effects of the BC02 polymorphism on P-carotene concentration. A: peak lactation, B: mid lactation and C: late lactation. White bars, AA genotype animals; Light grey bars, AG genotype animals; Dark grey bars, GG genotype animals.

Figure 6 is four graphs showing the effect of the BC02 genotypes on concentration of P-carotene (a, c) and retinol (b, d) in serum (a, b) and liver (c, d). Data shown are means ± s.e.m. Significant differences are indicated by asterix (*: p < 0.05, *: p<0.01, ANOYA, n=14 per genotype).

DETAILED DESCRIPTION OF THE INVENTION id="p-113" id="p-113"

[00113] The present invention recognises for the first time that a polymorphism in the BC02 gene in bovine is associated with a QTL for variations in milk colour and milk P-carotene content, and for variations in tissue colour and tissue P-carotene content including serum, blood, or liver P-carotene content. id="p-114" id="p-114"

[00114] For the sake of clarity, the phrase "milk or tissue colour or P-carotene content" is to be read as referring to milk colour or milk P-carotene content or tissue colour or tissue P-carotene content. Grammatical equivalents or components thereof are to be read likewise, such that the phrase "milk or tissue colour" is to be read as "milk colour or tissue colour". id="p-115" id="p-115"

[00115] It will be apparent to those skilled in the art that milk or tissue colour can readily be determined qualitatively or quantitatively. For example, a visual comparison may in many cases be sufficient to qualitatively determine a sample of milk or tissue having increased colour, or decreased colour, relative to another sample. Methods for quantitative determination of milk or tissue colour or P-carotene content are also known in the art, and examples are provided herein. id="p-116" id="p-116"

[00116] The invention provides methods of assessing the genetic merit of a bovine with respect to milk P-carotene content, more particularly milk fat P-carotene content. One such method comprises the step of determining the BC02 allelic profile of said bovine. Another such method comprises the step of determining the level of the BC02 gene product of said bovine. id="p-117" id="p-117"

[00117] The invention also provides a method for selecting a bovine with a genotype indicative of desired milk P-carotene content, particularly desired milk fat P-carotene content. One of the major applications of the present invention is in the selection of bovine having the A allele or the G allele of the WSOStop G/A polymorphism in the BC02 gene, which are associated with increased milk fat P-carotene content and milk fat colour, and decreased milk fat P-carotene content and milk fat colour, respectively. Accordingly, one method comprises determining the presence or absence of the A allele or of the G allele at the W80Stop G/A polymorphism of the BC02 gene, and selecting the bovine on the basis of the determination. id="p-118" id="p-118"

[00118] 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. id="p-119" id="p-119"

[00119] 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. id="p-120" id="p-120"

[00120] Similarly, the production of a wide range of tissue products, such as but not limited to meat, organs, pelts, fluids and the like, is well known in the art. Particularly contemplated herein are meat, organs, blood and serum having increased or decreased colour or P-carotene content. id="p-121" id="p-121"

[00121] The present invention recognises that mutations in the gene encoding BC02, as well as BC02 levels or activity, may be used as a selection tool to breed animals with higher or lower milk concentrations of P-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 P-carotene. 1 BC02 id="p-122" id="p-122"

[00122] BC02 is a key regulatory enzyme for the metabolism of P-carotene to vitamin A. BC02 catalyses the asymmetrical cleavage of P-carotene, resulting in the formation of two molecules: P-apo-10'-carotenal and P-ionone. Subsequent processing of P-apo-10'-carotenal 21 is believed to occur to produce retinol, and retinoic acid (RA), an important cell signaling molecule. This activity differs to that of another regulatory enzyme, BCMOl, which catalyses the symmetrical cleavage of P-carotene resulting in the formation of two retinal molecules. The conversion of P-carotene to retinol (vitamin A) in animals is usefully reviewed in von Lintig, J, and Vogt, K, Vitamin A Formation in Animals: Molecular Identification and Functional Characterization of Carotene Cleaving Enzymes, J. Nutr. 2004, 134:251S-256S. id="p-123" id="p-123"

[00123] BC02 was formerly known as P-carotene 9', lO'-dioxygenase 2 (BCD02), and was referred to as such in the provisional specification accompanying the Applicant's New Zealand patent application NZ 561998 filed 26 September 2007, the priority of which is claimed herein. It will be apparent that reference to BCD02 within that specification should be considered as a reference to the present nomenclature, BC02. id="p-124" id="p-124"

[00124] The present invention relates to the identification that a mutation in the BC02 gene leads to variation in milk or tissue colour and P-carotene content, particularly variation in milk fat colour and milk fat P-carotene content. id="p-125" id="p-125"

[00125] The genomic sequence comprising the bovine BC02 gene is presented herein as SEQ ID NO:l. The predicted coding sequence of bovine BC02 is presented as SEQ ID NO: 2, and is also available as NCBI accession number NM_001101987 (GI: 156120622). This coding sequence is derived from a cDNA clone, the sequence of which is available as NCBI accession number BC151703 (GI: 154757630). The amino acid sequence encoded by this coding sequence is presented herein as SEQ ID NO: 3, and is itself available as NCBI accession numbers NP_001095457 (GI: 156120623) and AAI51704 (GI: 154757631). id="p-126" id="p-126"

[00126] An earlier version of the predicted amino acid sequence for the bovine BC02 polypeptide was deposited as NCBI accession number XP 591937 (GI: 119907036). The 575 amino acid polypeptide of XP 591937 shares substantial similarity with human BC02 isoform A. In the residue numbering of this predicted amino acid sequence, the G/A polymorphism identified herein would result in a W102 Stop mutation. id="p-127" id="p-127"

[00127] As described herein, the BC02 mutation was closely associated with milk fat colour phenotype. Animals homozygous for the variant enzyme (AA; stop codon) produced milk with approximately 80% more P-carotene than animals homozygous (GG) for the wild type enzyme. This effect was observed at three stages of lactation, and within three sire families that carried the mutation. The animals that were heterozygous for the mutation produced milk with only approximately 17% more P-carotene. 22 id="p-128" id="p-128"

[00128] The AA genotype for BC02 was present in only 3.6% in the Holstein-Friesian x Jersey crossbred trial. Removal of these animals from the herd would reduce the herd average P-carotene concentration by approximately 3%. Removal of both the AA and AG animals would reduce the herd average P-carotene concentration by approximately 7%. Conversely, removal of AG and GG animals to produce yellowest milk would increase herd average P-carotene concentration by approximately 71%. id="p-129" id="p-129"

[00129] A reference bovine BC02 nucleotide sequence referred to as "wild type BC02" 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. Accordingly, as used herein the term "wild type" recognizes the characteristics of the BC02 nucleotide sequences presented as SEQ ID NOS:l and 2, and of the protein product encoded thereby. For example, when used with reference to enzymatic activity, the term "wild type" denotes activity associated with the wild type BC02 protein. Similarly, when used with reference to expression level, the term "wild type" denotes a level of expression associated with the wild type BC02 promoter. id="p-130" id="p-130"

[00130] It will be apparent that the term"activity" may refer both to the inherent enzymatic activity of a single molecule of BC02, 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 BC02 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). id="p-131" id="p-131"

[00131] As used herein, such as when used in reference to an allelic protein lacking the activity of wild type BC02, 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 BC02 may be a variant BC02 protein of greater or lesser enzymatic activity than that of wild type BC02. id="p-132" id="p-132"

[00132] Methods to assay the activity of BC02 are well known in the art. For example, one such method utilises an assay of the BC02 substrate P-carotene equivalent to that described in Winkelman et al., 1999, where the disappearance of P-carotene (and the associated reduction in absorbance at 450nm) correlates with BC02 enzymatic activity. Similarly, methods to indirectly measure the activity of BC02 are available, and include a 23 determination of the substrate:product ratio (for example the ratio of P-carotene to P-apo-10'-carotenal or to P-ionone), such as that present in a sample obtained from a subject. id="p-133" id="p-133"

[00133] The genetic polymorphism identified in the bovine BC02 gene is identified as a variant in SEQ ID NO:l and 2, and is reported in Figure 3. The nucleic acid and proteins sequences of the BC02 G and A alleles at the W80Stop G/A polymorphic site are shown in Figure 3B. 2 Identification and analysis of polymorphisms id="p-134" id="p-134"

[00134] It will be apparent to those skilled in the field that the convention of identifying polymorphisms effecting a coding change by their amino acid position in the gene in which they occur is followed herein. Accordingly, the W80Stop G/A polymorphism described herein occurs in codon 80 of the BCD02 gene. id="p-135" id="p-135"

[00135] The W80Stop G/A polymorphism can be detected directly or by detection of one or more polymorphisms which are in linkage disequilibrium with the W80Stop G/A polymorphism. Linkage disequilibrium is a phenomenon in genetics whereby two or more mutations or polymorphisms are in such close genetic proximity that they are co-inherited. This 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.) id="p-136" id="p-136"

[00136] Various degrees of linkage disequilibrium are possible. Preferably, the one or more polymorphisms in linkage disequilibrium with one or more of the polymorphisms specified herein are in greater than about 60% linkage disequilibrium, are in about 70% linkage disequilibrium, about 75%, about 80%, about 85%, about 90%, about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% linkage disequilibrium with the W80Stop G/A polymorphism of the BCD02 gene. (Devlin and Risch 1995; A comparison of linkage disequilibrium measures for fine-scale mapping, Genomics 29: 311-322). id="p-137" id="p-137"

[00137] 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. id="p-138" id="p-138"

[00138] In another aspect, the present invention provides a method for determining the genetic merit of a bovine with respect to milk or tissue P-carotene content, and particularly 24 with milk fat colour or P-carotene content. The method includes ascertaining, from a sample of material containing DNA obtained from the bovine, whether a sequence of the DNA encoding (a) a protein having biological activity of wild type BC02 is present, and whether a sequence of the DNA encoding (b) an allelic protein lacking the activity of (a) is present. id="p-139" id="p-139"

[00139] An example of another art standard method known for determining whether a particular DNA sequence is present in a sample is the Polymerase Chain Reaction (PCR). A preferred aspect of the invention thus includes a step in which ascertaining whether a sequence of the DNA encoding (a) is present, and whether a sequence of the DNA encoding (b) is present includes amplifying the DNA in the presence of primers based on a nucleotide sequence encoding a protein having biological activity of wild type BC02, 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 P-carotene levels, and particularly in milk having inter alia a higher P-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 P-carotene levels, and particularly in milk having inter alia a lower P-carotene content. id="p-140" id="p-140"

[00140] 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. id="p-141" id="p-141"

[00141] Accordingly, a preferred embodiment of the invention thus includes the step of amplifying a BC02 polynucleotide in the presence of at least one primer comprising a nucleotide sequence of or complementary to, the BC02 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 W80Stop A allele-specific or W80Stop G allele-specific nucleotides described herein. PCR methods are well known by those skilled in the art (Mullis et al., 1994.) The template for amplification may be selected from genomic DNA, mRNA or first strand cDNA derived from a sample obtained from the bovine under test (Sambrook et al., 1989). id="p-142" id="p-142"

[00142] Primers suitable for use in PCR based methods of the invention should be sufficiently complementary to the BC02 gene sequence, such as SEQ ID NO:l 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:l or SEQ ID NO:2, or naturally occurring flanking sequences thereof. Examples of such PCR primers are presented herein as SEQ ID NOS:4-25. id="p-143" id="p-143"

[00143] Suitable PCR primers may include sequence corresponding to the WSOStop A allele-specific or W80Stop G allele-specific BC02 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 BC02 gene of the test bovine. id="p-144" id="p-144"

[00144] Other methods for determining whether a particular nucleotide sequence is present in a sample may include the step of restriction enzyme digestion of nucleotide sample. Separation and visualisation of the digested restriction fragments by methods well known in the art, may form a diagnostic test for the presence of a particular nucleotide sequence. The nucleotide sequence digested may be a PCR product amplified as described above. id="p-145" id="p-145"

[00145] Still other methods for determining whether a particular nucleotide sequence is present in a sample include a step of hybridisation of a probe to a sample nucleotide sequence. Thus, methods for detecting the W80Stop A allele-specific or W80Stop G allele-specific nucleotides may comprise the additional steps of hybridisation of a probe derived from the BC02 sequence of SEQ ID NO: 1 or SEQ ID NO:2. id="p-146" id="p-146"

[00146] Such probes should comprise a nucleic acid molecule of sufficient length and sufficiently complementary to the BC02 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. id="p-147" id="p-147"

[00147] With respect to polynucleotide molecules greater than about 100 bases in length, typical stringent hybridization conditions are no more than 25 to 30° C (for example, 10° C) below the melting temperature (Tm) of the native duplex (see generally, Sambrook et al., 1989; Ausubel et al., 1987). Tm for polynucleotide molecules greater than about 100 bases can be calculated by the formula Tm = 81.5 + 0. 41% (G + C-log (Na+). 26 id="p-148" id="p-148"

[00148] With respect to polynucleotide molecules having a length less than 100 bases, exemplary stringent hybridization conditions are 5 to 10° C below Tm. On average, the Tm of a polynucleotide molecule of length less than 100 bp is reduced by approximately (500/oligonucleotide length)0 C. id="p-149" id="p-149"

[00149] 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. id="p-150" id="p-150"

[00150] Such probes would typically comprise at least 12 contiguous nucleotides of or complementary to the sequences presented SEQ ID NO:l or SEQ ID NO:2, and may comprise sequence corresponding to the allele-specific nucleotides described herein. id="p-151" id="p-151"

[00151] Such probes may additionally comprise means for detecting the presence of the probe when bound to sample nucleotide sequence. Methods for labelling probes such as radiolabelling are well known in the art (see for example, Sambrook et al., 1989). id="p-152" id="p-152"

[00152] In another aspect, the invention provides a method for determining the genetic merit of bovine with respect to milk or tissue P-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 BC02 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 P-carotene levels, particularly with the production of milk with, inter alia, increased fat colour. The reverse association again holds true. id="p-153" id="p-153"

[00153] Again, if an amplification method such as PCR is used in ascertaining whether a sequence of the mRNA encoding (A) is present, and whether a sequence of the mRNA encoding (B) is present, the method includes amplifying the mRNA, for example in the presence of a pair of primers complementary to a nucleotide sequence encoding a protein having biological activity of a wild type BC02, or in the presence of a pair of primers complementary to a nucleotide sequence encoding a variant BC02 protein. It will be appreciated that in embodiments of the invention reliant on assessing the amount of BC02 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. 27 id="p-154" id="p-154"

[00154] Other methods to quantitate or otherwise assess the amount of nucleic acid, particularly the amount of mRNA are well known in the art. These include Northern analysis using probes able to hybridise to the target BC02 mRNA. Such probes should comprise a nucleic acid molecule of sufficient length and sufficiently complementary to the BC02 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 BC02 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. id="p-155" id="p-155"

[00155] In a further aspect, the invention provides a method of determining genetic merit of a bovine with respect to milk or tissue P-carotene content which comprises determining the BC02 allelic profile of said bovine, together with determining the allelic profile of said bovine at one or more genetic loci associated with milk or tissue P-carotene content. id="p-156" id="p-156"

[00156] In one embodiment, said genetic loci is a polymorphism in a gene associated with milk or tissue P-carotene content, preferably a polymorphism in a gene involved in P-carotene uptake or metabolism. id="p-157" id="p-157"

[00157] 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. id="p-158" id="p-158"

[00158] 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. id="p-159" id="p-159"

[00159] 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 28 number of phenotypic traits (including latent traits), such as for example, disease propensity and severity, wellness propensity, drug responsiveness including, for example, susceptibility to adverse drug reactions, and as described herein association with desirable phenotypic traits. Knowledge of the association of a particular SNP with a phenotypic trait, coupled with the knowledge of whether a subject has said particular SNP, can enable the targeting of diagnostic, preventative and therapeutic applications to allow better disease management, to enhance understanding of disease states, to develop selective breeding regimes, and to identify subjects of desirable genetic merit. id="p-160" id="p-160"

[00160] 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. id="p-161" id="p-161"

[00161] 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 109 base pairs, while current estimates of the size of the haploid bovine genome are in the range of 2.6 - 2.7 x 109 base pairs), and the associated sensitivity and discriminatory requirements. id="p-162" id="p-162"

[00162] 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 29 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. id="p-163" id="p-163"

[00163] 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. id="p-164" id="p-164"

[00164] 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. id="p-165" id="p-165"

[00165] 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). id="p-166" id="p-166"

[00166] 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. id="p-167" id="p-167"

[00167] 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. id="p-168" id="p-168"

[00168] 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. id="p-169" id="p-169"

[00169] The technique of Lynx Therapeutics (Hayward, Calif.) using MEGATYPE™ technology can genotype very large numbers of SNPs simultaneously from small or large pools of genomic material. This technology uses fluorescently labeled probes and compares the collected genomes of two populations, enabling detection and recovery of DNA fragments spanning SNPs that distinguish the two populations, without requiring prior SNP mapping or knowledge. id="p-170" id="p-170"

[00170] 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 W80Stop G/A polymorphism in the BC02 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. 31 id="p-171" id="p-171"

[00171] 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. id="p-172" id="p-172"

[00172] 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. id="p-173" id="p-173"

[00173] 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 ProteomlQ™ system from Proteome Systems, provide high throughput platforms for proteome analysis combining sample preparation, protein separation, image acquisition and analysis, protein processing, mass spectrometry and bioinformatics technologies. id="p-174" id="p-174"

[00174] The majority of proteomic methods of protein identification utilise mass spectrometry, including ion trap mass spectrometry, liquid chromatography (LC) and LC/MSn mass spectrometry, gas chromatography (GC) mass spectroscopy, Fourier transform-ion cyclotron resonance-mass spectrometer (FT-MS), MALDI-TOF mass 32 spectrometry, and ESI mass spectrometry, and their derivatives. Mass spectrometric methods are also useful in the determination of post-translational modification of proteins, such as phosphorylation or glycosylation, and thus have utility in determining polymorphisms that result in or are associated with variation in post-translational modifications of proteins. id="p-175" id="p-175"

[00175] 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. id="p-176" id="p-176"

[00176] It will be apparent that the presence or absence of the A allele or of the G allele at the W80Stop G/A polymorphism in the BC02 gene may also be determined by analysis of a polypeptide sample, derived from a bovine. id="p-177" id="p-177"

[00177] 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. id="p-178" id="p-178"

[00178] A large number of methods reliant on the conformational variability of nucleic acids have been developed to detect SNPs. id="p-179" id="p-179"

[00179] For example, Single Strand Conformational Polymorphism (SSCP, Orita et al, PNAS 1989 86:2766-2770) is a method reliant on the ability of single-stranded nucleic acids to form secondary structure in solution under certain conditions. The secondary structure depends on the base composition and can be altered by a single nucleotide substitution, causing differences in electrophoretic mobility under nondenaturing conditions. The various polymorphs are typically detected by autoradiography when radioactively labelled, by silver staining of bands, by hybridisation with detectably labelled probe fragments or the use of fluorescent PCR primers which are subsequently detected, for example by an automated DNA sequencer. id="p-180" id="p-180"

[00180] 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 33 (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). id="p-181" id="p-181"

[00181] 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. id="p-182" id="p-182"

[00182] 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. id="p-183" id="p-183"

[00183] Yet further methods to detect SNPs rely on the differing susceptibility of single stranded and double stranded nucleic acids to cleavage by various agents, including chemical cleavage agents and nucleolytic enzymes. For example, cleavage of mismatches within RNA:DNA heteroduplexes by RNase A, of heteroduplexes by, for example bacteriophage T4 endonuclease YII or T7 endonuclease I, of the 5' end of the hairpin loops at the junction between single stranded and double stranded DNA by cleavase I, and the modification of mispaired nucleotides within heteroduplexes by chemical agents commonly used in Maxam-Gilbert sequencing chemistry, are all well known in the art. id="p-184" id="p-184"

[00184] Further examples include the Protein Translation Test (PTT), used to resolve stop codons generated by variations which lead to a premature termination of translation and to protein products of reduced size, and the use of mismatch binding proteins. 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. 34 id="p-185" id="p-185"

[00185] 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. id="p-186" id="p-186"

[00186] The above methods of detecting and identifying SNPs are amenable to use in the methods of the invention. 3 Polynucleotide and polypeptide variants id="p-187" id="p-187"

[00187] 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. id="p-188" id="p-188"

[00188] 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. id="p-189" id="p-189"

[00189] 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. id="p-190" id="p-190"

[00190] Preferably fragments of promoter 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 promoter polynucleotide of the invention. id="p-191" id="p-191"

[00191] 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. id="p-192" id="p-192"

[00192] 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. id="p-193" id="p-193"

[00193] 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. 36 3.1 Polynucleotide variants id="p-194" id="p-194"

[00194] 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. id="p-195" id="p-195"

[00195] Polynucleotide sequence identity can be determined in the following manner. The subject polynucleotide sequence is compared to a candidate polynucleotide sequence using BLASTN (from the BLAST suite of programs, version 2.2.10 [Oct 2004]) in bl2seq (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250), which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The default parameters of bl2seq are utilized except that filtering of low complexity parts should be turned off. id="p-196" id="p-196"

[00196] The identity of polynucleotide sequences may be examined using the following unix command line parameters: id="p-197" id="p-197"

[00197] bl2seq -i nucleotideseql -j nucleotideseq2 -F F -p blastn id="p-198" id="p-198"

[00198] 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 = ". id="p-199" id="p-199"

[00199] Polynucleotide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs (e.g. Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). A full implementation of the Needleman-Wunsch global alignment algorithm is found in the needle program in the EMBOSS package (Rice,P. Longden,! and Bleasby,A. EMBOSS: The European Molecular Biology Open Software Suite, Trends in Genetics June 37 2000, vol 16, No 6. pp.276-277) which can be obtained from http://www.hgmp.mrc.ac.uk/Software/EMBOSS/. The European Bioinformatics Institute server also provides the facility to perform EMBOSS-needle global alignments between two sequences on line at http:/www.ebi.ac.uk/emboss/align/. id="p-200" id="p-200"

[00200] Alternatively the GAP program may be used which computes an optimal global alignment of two sequences without penalizing terminal gaps. GAP is described in the following paper: Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235. id="p-201" id="p-201"

[00201] Polynucleotide variants of the present invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polypeptides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.10 [Oct 2004]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/). id="p-202" id="p-202"

[00202] The similarity of polynucleotide sequences may be examined using the following unix command line parameters: id="p-203" id="p-203"

[00203] bl2seq -i nucleotideseql -j nucleotideseq2 -F F -p tblastx id="p-204" id="p-204"

[00204] 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. id="p-205" id="p-205"

[00205] Variant polynucleotide sequences preferably exhibit an E value of less than 1 x 10"10, more preferably less than 1 x 10"20, less than 1 x 10"30, less than 1 x 10"40, less than 1 x 10"50, less than 1 x 10"60, less than 1 x 10"70, less than 1 x 10"80, less than 1 x 10"90, less than 1 x 10"100, less than 1 x 10"uo, less than 1 x 10"120 or less than 1 x 10"123 when compared with any one of the specifically identified sequences. id="p-206" id="p-206"

[00206] Alternatively, variant polynucleotides of the present invention hybridize to a specified polynucleotide sequence, or complements thereof under stringent conditions. 38 id="p-207" id="p-207"

[00207] 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. id="p-208" id="p-208"

[00208] With respect to polynucleotide molecules greater than about 100 bases in length, typical stringent hybridization conditions are no more than 25 to 30°C (for example, 10°C) below the melting temperature (Tm) of the native duplex (see generally, Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Ausubel et al., 1987, Current Protocols in Molecular Biology, Greene Publishing,). Tm for polynucleotide molecules greater than about 100 bases can be calculated by the formula Tm = 81. 5 + 0. 41% (G + C-log (Na+). (Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Bolton and McCarthy, 1962, PNAS 84:1390). Typical stringent conditions for polynucleotide of greater than 100 bases in length would be hybridization conditions such as prewashing in a solution of 6X SSC, 0.2% SDS; hybridizing at 65°C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in IX SSC, 0.1% SDS at 65°C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65°C. id="p-209" id="p-209"

[00209] 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. id="p-210" id="p-210"

[00210] With respect to the DNA mimics known as peptide nucleic acids (PNAs) (Nielsen et al., Science. 1991 Dec 6;254(5037): 1497-500) Tm values are higher than those for DNA-DNA or DNA-RNA hybrids, and can be calculated using the formula described in Giesen et al., Nucleic Acids Res. 1998 Nov 1;26(21):5004-6. Exemplary stringent hybridization conditions for a DNA-PNA hybrid having a length less than 100 bases are 5 to 10°C below the Tm. id="p-211" id="p-211"

[00211] 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 39 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. id="p-212" id="p-212"

[00212] Polynucleotide sequence alterations resulting in conservative substitutions of one or several amino acids in the encoded polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306). id="p-213" id="p-213"

[00213] Variant polynucleotides due to silent variations and conservative substitutions in the encoded polypeptide sequence may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.10 [Oct 2004]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/) via the tblastx algorithm as previously described. id="p-214" id="p-214"

[00214] Polypeptide Variants id="p-215" id="p-215"

[00215] 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. id="p-216" id="p-216"

[00216] Polypeptide sequence identity can be determined in the following manner. The subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (from the BLAST suite of programs, version 2.2.10 [Oct 2004]) in bl2seq, which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The default parameters of bl2seq are utilized except that filtering of low complexity regions should be turned off. 40 id="p-217" id="p-217"

[00217] Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs. EMBOSS-needle (available at http:/www. ebi.ac.uk/emboss/align/) and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity. id="p-218" id="p-218"

[00218] Polypeptide variants of the present invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polypeptides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.10 [Oct 2004]) from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The similarity of polypeptide sequences may be examined using the following unix command line parameters: bl2seq -i peptideseql -j peptideseq2 -F F -p blastp id="p-219" id="p-219"

[00219] Variant polypeptide sequences preferably exhibit an E value of less than 1 x 10"10, more preferably less than 1 x 10"20, less than 1 x 10"30, less than 1 x 10"40, less than 1 x 10"50, less than 1 x 10"60, less than 1 x 10"70, less than 1 x 10~80, less than 1 x 10~90, less than 1 xlO" 10°, less than 1 x 10"uo, less than 1 x 10"120 or less than 1 x 10"123 when compared with any one of the specifically identified sequences. id="p-220" id="p-220"

[00220] 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. id="p-221" id="p-221"

[00221] 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). id="p-222" id="p-222"

[00222] 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. 41 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 id="p-223" id="p-223"

[00223] The invention further provides diagnostic kits useful in determining the bovine BC02 allelic profile of bovine, for example for use in the methods of the present invention. id="p-224" id="p-224"

[00224] Accordingly, in one embodiment the invention provides a diagnostic kit which can be used to determine the BC02 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 one of the naturally occurring mutations described herein. Such a kit could also include a primer for amplifying the corresponding region of the normal gene that produces a functionally wild type BC02. Usually, such a kit would also include another primer upstream or downstream of the region of the gene. These primers are used to amplify the segment containing the mutation of interest. The actual genotyping is carried out using primers that target specific mutations described herein and that could function as allele-specific oligonucleotides in conventional hybridisation, Taqman assays, OLE assays, etc. Alternatively, primers can be designed to permit genotyping by microsequencing. id="p-225" id="p-225"

[00225] 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 BC02 mutation such as described above. Primer (b) encodes a region upstream or downstream of the region to be amplified by a primer (a) so that genetic material containing the mutation is amplified, by PCR, for example, in the presence of the two primers. Primer (c) is based on the region corresponding to that on which primer (a) is based, but lacking the mutation. Thus, genetic material containing the non-mutated region will be amplified in the presence of primers (b) and (c). Genetic material homozygous for the wild type gene will thus provide amplified products in the presence of primers (b) and (c). Genetic material homozygous for the mutated gene will thus provide amplified products in the presence of primers (a) and (b). Heterozygous genetic material will provide amplified products in both cases. id="p-226" id="p-226"

[00226] In one embodiment, the diagnostic kit is useful in detecting DNA comprising a variant BC02 gene or encoding a variant BC02 polypeptide at least partially lacking wild type activity in a bovine which includes first and second primers for amplifying the DNA, the primers being complementary to nucleotide sequences of the DNA upstream and downstream, respectively, of a polymorphism in the portion of the DNA encoding BC02 which results in 42 increased P-carotene levels (particularly increased P-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 BC02 gene. The kit can also include a third primer complementary to a naturally occurring mutation of a coding portion of the wild type BC02 gene. Preferably the kit includes instructions for use, for example in accordance with a method of the invention. id="p-227" id="p-227"

[00227] In one embodiment, the diagnostic kit comprises a nucleotide probe complementary to the sequence, or an oligonucleotide fragment thereof, shown in SEQ ID NO:l or SEQ ID NO: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:lor 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 (<100bp), lower temperatures must be used even with 100% homology. In general, one starts washing at low temperatures (37°C to 40°C), and raises the temperature by 3-5°C intervals until background is low enough to be a major factor in autoradiography. The diagnostic kit can also contain an instruction manual for use of the kit. id="p-228" id="p-228"

[00228] In another embodiment, the diagnostic kit comprises an antibody or an antibody composition useful for detection of the presence or absence of wild type BC02 and/or the presence or absence of a variant protein at least partially lacking wild type activity, particularly a truncated form of the BC02 protein, together with instructions for use, for example in a method of the invention.

Sample preparation id="p-229" id="p-229"

[00229] 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. 43 id="p-230" id="p-230"

[00230] 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 id="p-231" id="p-231"

[00231] 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. id="p-232" id="p-232"

[00232] Both the SNPs and the results of an analysis of the SNPs utilised in the present invention may be "provided" in a variety of mediums to facilitate use thereof. As used in this section, "provided" refers to a manufacture, other than an isolated nucleic acid molecule, that contains SNP information of the present invention. Such a manufacture provides the SNP information in a form that allows a skilled artisan to examine the manufacture using means not directly applicable to examining the SNPs or a subset thereof as they exist in nature or in purified form. The SNP information that may be provided in such a form includes any of the SNP information provided by the present invention such as, for example, polymorphic nucleic acid and/or amino acid sequence information, information about observed SNP alleles, alternative codons, populations, allele frequencies, SNP types, and/or affected proteins, phenotypic effect or association, or any other information provided by the present invention in Tables 1 and 2 and/or the Sequence ID Listing. id="p-233" id="p-233"

[00233] 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 44 media such as CD-ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media. A skilled artisan can readily appreciate how any of the presently known computer readable media can be used to create a manufacture comprising computer readable medium having recorded thereon SNP information of the present invention. One such medium is provided with the present application, namely, the present application contains computer readable medium (floppy disc) that has nucleic acid sequences used in analysing the SNPs utilised in the present invention, together with derived amino acid sequence, provided/recorded thereon in ASCII text format in a Sequence ID Listing. id="p-234" id="p-234"

[00234] 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. id="p-235" id="p-235"

[00235] A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon SNP information of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the SNP information of the present invention on computer readable medium. For example, sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, represented in the form of an ASCII file, or stored in a database application, such as OB2, Sybase, Oracle, or the like. A skilled artisan can readily adapt any number of data processor structuring formats (e.g., text file or database) in order to obtain computer readable medium having recorded thereon the SNP information of the present invention. id="p-236" id="p-236"

[00236] By providing the SNPs and/or the results of an analysis of the SNPs utilised in the present invention in computer readable form, a skilled artisan can routinely access the SNP information for a variety of purposes. Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium. Examples of publicly available computer software include BLAST (Altschul et at, J. Mol. Biol. 215:403-410 (1990)) and BLAZE (Brutlag et at, Comp. Chem. 17:203-207 (1993)) search algorithms. id="p-237" id="p-237"

[00237] The present invention further provides systems, particularly computer-based systems, which contain the SNP information described herein. Such systems may be designed 45 to store and/or analyze information on, for example, a number of SNP positions, or information on SNP genotypes from a number of subjects. The SNP information of the present invention represents a valuable information source. The SNP information of the present invention stored/analyzed in a computer-based system may be used for such applications as identifying or selecting subjects, in addition to computer-intensive applications as determining or analyzing SNP allele frequencies in a population, mapping disease genes, genotype-phenotype association studies, grouping SNPs into haplotypes, correlating SNP haplotypes with response to particular drugs, or for various other bioinformatic, pharmacogenomic, drug development, or selection or identification applications. id="p-238" id="p-238"

[00238] 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. id="p-239" id="p-239"

[00239] As stated above, the computer-based systems of the present invention comprise data storage having stored therein SNP information, such as SNPs and/or the results of an analysis of the SNPs utilised in the present invention, and the necessary hardware and software for supporting and implementing one or more programs or algorithms. As used herein, "data storage" refers to memory which can store SNP information of the present invention, or a memory access facility which can access manufactures having recorded thereon the SNP information of the present invention. id="p-240" id="p-240"

[00240] 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. id="p-241" id="p-241"

[00241] 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 46 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. id="p-242" id="p-242"

[00242] 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. id="p-243" id="p-243"

[00243] Accordingly, the present invention provides a system for performing one or more of the methods of the invention, said system comprising: computer processor means for receiving, processing and communicating data; storage means for storing data including a reference genetic database of the results of genetic analysis of a bovine with respect to one or more milk or tissue colour or P-carotene content traits and optionally a reference milk or tissue colour or P-carotene content traits database of non-genetic factors for bovine milk or tissue colour or P-carotene content traits; and 47 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. id="p-244" id="p-244"

[00244] Preferably, said system is accessible via the internet or by personal computer. id="p-245" id="p-245"

[00245] Preferably, said reference genetic database comprises or includes the results of one or more analyses of one or more genetic loci associated with one or more milk or tissue colour or P-carotene content traits, more preferably the one or more genetic loci are one or more polymorphisms in one or more genes associated with one or more milk or tissue colour or P-carotene content traits. id="p-246" id="p-246"

[00246] In yet a further aspect, the invention provides a computer program suitable for use in a system as defined above comprising a computer usable medium having program code embodied in the medium for causing the computer program to process received data consisting of or including the result of at least one genetic analysis of one or more genetic loci associated with one or more milk or tissue colour or P-carotene content traits in the context of both a reference genetic database of the results of said at least one genetic analysis and optionally a reference database of non-genetic factors associated with bovine milk or tissue colour or P-carotene content traits. id="p-247" id="p-247"

[00247] Preferably, the one or more genetic loci are one or more polymorphisms in one or more genes associated with one or more milk or tissue colour or P-carotene content traits. id="p-248" id="p-248"

[00248] 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. id="p-249" id="p-249"

[00249] 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. id="p-250" id="p-250"

[00250] The invention consists in the foregoing and also envisages constructions of which the following gives examples only. 48 EXAMPLE -Analysis of the genetic basis for milk fat colour id="p-251" id="p-251"

[00251] 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 id="p-252" id="p-252"

[00252] 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 F1 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 F1 cows with these F1 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 id="p-253" id="p-253"

[00253] All milk measurements were taken during the animals' second lactation. Cows were milked twice daily; milk volume was recorded at each milking. Milk fat colour was measured at three time points during lactation: peak lactation (35 days post-calving), mid lactation (mid November) and late lactation (late February). On each collection day, samples were collected from the a.m. and p.m. milkings and combined to make a single composite sample for each animal. Milk fat colour was measured as previously described (Winkelman et al, 1999). Briefly, nonsaponifiable material (including carotenoids) was extracted from fresh milk samples and the absorbance at 450nm was measured. Fat colour (|_ig P-carotene/mg milk fat) was calculated (Winkelman etal., 1999). 3. Genotyping id="p-254" id="p-254"

[00254] Genomic DNA was prepared from whole blood from a total of 1665 animals within the trial pedigree (850 F2 daughters, six F1 sires, 796 F1 dams, and 13 selected F0 sires). An initial whole genome scan was conducted by genotyping each animal for 285 microsatellite markers, obtained primarily from published marker maps. Subsequently, the 49 pedigree was genotyped using the Affymetrix Bovine 10K SNP GeneChip. A total of 6634 informative SNP markers were placed on the map. 4. Candidate gene sequencing id="p-255" id="p-255"

[00255] BC02 was identified as a candidate gene for the milk fat colour QTL on chromosome 15. Intron/exon boundaries were determined by homology with the human gene sequence. Exons were amplified using the primers presented as SEQ ID NOS: 4-25 and sequenced in both directions. The determined wild-type coding sequence for the BC02 gene is shown in SEQ ID NO:2.

. Statistical analysis id="p-256" id="p-256"

[00256] The dataset consisted of milk fat colour phenotypes collected during the animals' second lactation for two cohorts of F2 animals. Data manipulation was performed using SAS (version 9.1). Phenotype data for milk fat colour was recorded at three time points during lactation (peak, mid and late lactation). These data were matched with the following covariates: cohort (cohort 1 or cohort 2), sire (sires 1 - 6), milk fat%, milk protein%, lactose%, milk solids%, milk yield, condition score, live weight (average taken for ± seven days around each of the milk fat colour time points at peak, mid and late lactation), somatic cell count (threshold of 200,000 cells during ± seven days around sampling times), free fatty acids (as an indicator of milk fat quality, measured in the same sample as milk fat colour at peak, mid and late lactation), calving week, and estrus week. Animals with missing data points for any of the measurements were excluded and the final datasets included 597, 648 and 632 observations at peak, mid and late lactation, respectively. id="p-257" id="p-257"

[00257] Analyses were conducted using both raw and log-transformed data. ANOVA was conducted for the milk fat colour phenotype at peak, mid and late lactation. The final ANOVA models for each of the lactations were produced using a backward elimination process; all the covariates were included in the model at the first stage of the modeling process and the least significant covariates removed at each subsequent stage until all the remaining covariates were found to be significant (sig. level set at 0.1). Thus, the final models were as follows: peak lactation (sire, cohort, protein%, calving week), mid lactation (sire, cohort) and late lactation (sire, cohort, protein%, milk solid%, and somatic cell count). 6. QTL detection id="p-258" id="p-258"

[00258] The data used for QTL detection were the residuals from each model for both non-transformed and log-transformed data. The raw phenotype data (no covariates or modeling) was also used to detect QTLs. Since the same results were detected with each kind 50 of data, results presented below used non-transformed, modeled data. QTL detection was conducted using a line of descent model and a half-sib model. Subsequently, the BC02 mutation was included as a covariate into the models for peak, mid and late lactation, and ANOVA was performed to test for an association between the BC02 mutation and P-carotene concentration of milk. 7. Tissue p-carotene and retinol measurements id="p-259" id="p-259"

[00259] Subcutaneous adipose tissue biopsies (~500mg tissue), liver biopsies (-100 mg tissue), and serum samples (10 mL) were taken from a subset of 42 cows (14 animals each AA, AG and GG genotypes). P-carotene and retinol measurements were determined using HPLC with commercial standards, based on a published method (Hulshof et al., 2006). The inter-assay variation was on average 5%.

Results 1. P-Carotene concentration in milk is influenced by stage of lactation. id="p-260" id="p-260"

[00260] The effects of stage of lactation on P-carotene concentration in milk are shown in Figure 1. P-carotene was significantly affected (p<0.0001) by stage of lactation with the highest concentrations occurring at peak lactation (average 10.94 ± 0.15 |_ig/g fat; range 4.1 -23.9 jLig/g fat), and the lowest at late lactation (average 7.18 |_ig/g fat ± 0.08; range 3.4 - 18.6 |u.g/g fat). The concentration of P-carotene in milk at mid lactation was 9.07 ± 0.11 |_ig/g fat (range 3.6 - 22.6 ju,g/g fat). 2. Detection of a major QTL for milk fat colour on bovine chromosome 15 id="p-261" id="p-261"

[00261] Analysis of the P-carotene data within the half-sib model of QTL analysis showed the presence of a significant QTL on bovine chromosome 15 (Figure 2). The maximum F value for the QTL was 6.47, and the most likely position was estimated at 21 cM. Bootstrap analysis (n = 1000) showed that the 95% confidence interval for the QTL was 0-23 cM (Figure 2). The marker information content for chromosome 15 in this analysis ranged between 0.70 and 0.82 and averaged 0.75. There were a total of 233 markers (8 microsatellite markers and 225 single nucleotide polymorphisms) used for this analysis on chromosome 15. 3. Identification of BC02 as a candidate gene and detection of a polymorphism causing a premature stop codon id="p-262" id="p-262"

[00262] P-carotene oxygenase 2 (BC02 ) catalyses the asymmetrical cleavage of P-carotene to vitamin A (Kiefer et al, 2001) and was mapped between markers BR3510 and MB076 on bovine chromosome 15 (Figure 2). This was identified as a candidate gene for the chromosome 15 milk fat colour QTL effect. To determine whether this gene explained the observed variation in milk P-carotene content, the BC02 coding region in the six F1 sires was sequenced to identify any genetic polymorphisms that could potentially alter the function of this enzyme. Intron/exon boundaries were determined using the UniGene bovine gene prediction Bt. 64006 and by sequence comparison with the human BC02 gene sequence (UniGene Hs.647227). 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. id="p-263" id="p-263"

[00263] An G to A substitution at genomic nucleotide position 39304 (see SEQ ID NO:l) was heterozygous in three F1 sires (sires 2, 3 and 6). The mutation results in the substitution of a tryptophan (G allele) with a premature stop codon (A allele) at amino acid position 80 in the BC02 protein (see Figure 3). The remaining three sires were homozygous for the G allele. To determine whether this mutation was associated with the QTL effect, the polymorphism in the remainder of the FJXB trial pedigree was genotyped. The frequency of each BC02 genotype is shown in Table 1 below. The AA genotype was present in 3.6% of the F2 population, while the AG and GG genotypes were present in 33% and 63% respectively.

Table 1: Genotype frequencies of F2 population, within each F1 sire family Sire Genotype Frequency AA 0.036 All Sires AG 0.333 GG 0.631 AA N/A1 1 AG 0.254 GG 0.745 AA 0.059 2 AG 0.402 GG 0.539 AA 0.071 3 AG 0.505 GG 0.424 AA N/A 4 AG 0.08 GG 0.92 AA N/A AG 0.207 GG 0.793 AA 0.084 6 AG 0.527 GG 0.389 1. N/A = not applicable (for offspring of sires homozygous for the G allele). 52 4. BC02 Polymorphism Has Significant Effect on BC02 mRNA id="p-264" id="p-264"

[00264] It is generally recognised that premature stop codons in eukaryotic genes often result in rapid degradation of mRNA transcripts. The effect of the W80Stop G to A substitution on liver mRNA levels was analysed, both by microarray (see Table 2), and realtime PCR (see Table 3).

Table 2: BC02 mRNA expression analysis by microarray Fold change Std error P value BC02 expression 3.86536 0.30524465 0.000003 Data generated from 5 individual array experiments (GG genotype vs. AA genotype). BC02 expression 3.86 fold higher in GG animals.

Table 3: BC02 mRNA expression analysis by RT-PCR Genotype Mean CT N AA 31.70485 11 GA 29.30833 6 GG 29.29467 Fold change = 2AACT = 2A2.4 = 5.27 fold higher in GG animals. Mean CT is mean cycle time to threshold. id="p-265" id="p-265"

[00265] As shown in Tables 2 and 3 above, four- to five-fold less BC02 mRNA was observed in AA genotype animals compared to GG genotype animals.

. BC02 polymorphism has a significant effect on P-carotene concentration in milk id="p-266" id="p-266"

[00266] The effect of the BC02 polymorphism on P-carotene concentration is shown in Figure 4. Animals homozygous for the A allele produce milks with significantly greater (p < 0.0001) concentrations of P-carotene than both heterozygous (AG) and homozygous (GG) animals. The effect of the BC02 polymorphism on P-carotene concentration in each of the sire families carrying the mutation was similar (Figure 5), with AA animals producing milk with approximately 80% more P-carotene than GG animals. The effect of the AA genotype was similar at each stage of lactation. id="p-267" id="p-267"

[00267] Animals homozygous for the premature stop codon (AA genotype) produced milk with approximately 78%, 78% and 68% more P-carotene than animals homozygous for the wild type protein (GG genotype; p < 0.0001) at peak, mid and late lactation, respectively. Animals heterozygous for the mutation (AG genotype) produced milk with approximately 17%, 15% and 16% more P-carotene (p < 0.001) at peak, mid and late lactation. 53 6. BC02 polymorphism has a significant effect on retinol and P-carotene concentrations in serum and liver id="p-268" id="p-268"

[00268] The effect of the BC02 polymorphism on retinol concentration and P-carotene concentration in both serum and liver is shown in Figure 6. P-Carotene and vitamin A concentrations were measured in serum and liver tissue samples from a subset of 42 F2 cows (n=14 each genotype). Serum P-carotene concentration was significantly higher in BC02" " (AA) cows than in heterozygous or homozygous wild-type cows (16.86 vs. 13.35 or 11.38 (xg/ml P-carotene, respectively; p < 0.05, Figure 6A). Thus, the effect of the BC02 stop mutation on P-carotene was similar for both milk and serum, showing that the effect of the mutation was not confined to the mammary gland. Vitamin A concentration was higher in serum from BC02"" cows (P < 0.05, Figure 6B). Liver P-carotene concentration differed between genotype groups (see Figure 6C), and liver vitamin A was significantly lower in BC02"" cows compared to BC02+/+ cows (686.81 vs. 1023.75 (xg/g, p < 0.05; Figure 6D). id="p-269" id="p-269"

[00269] Discussion id="p-270" id="p-270"

[00270] The present invention recognises that the BC02 polymorphism described above, together with polymorphisms in linkage disequilibrium with it, are useful as a selection tool to breed animals with higher or lower milk concentrations of P-carotene, with increased or decreased milk fat colour, or with increased or decreased tissue colour or P-carotene content. Such a strategy may allow the production of, for example, milk products more suitable to particular markets, depending on the preference for white or yellow milk and milk products, or the dietary or health requirements prevalent in a market.

Publications von Lintig, J, and Vogt, K; Vitamin A Formation in Animals: Molecular Identification and Functional Characterization of Carotene Cleaving Enzymes, J. Nutr. 2004, 134:251S-256S. Reich DE et al; Linkage disequilibrium in the human genome, Nature 2001, 411:199-204. Winkelman et al., Estimation of Heritabilities and Correlations Associated with Milk Color Traits, J Dairy Sci. 1999, 82:215-224.

Mullis et al., Eds. 1994 The Polymerase Chain Reaction, Birkhauser.

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

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

Rice P. Longden,! and Bleasby,A., EMBOSS: The European Molecular Biology Open Software Suite, Trends in Genetics, June 2000, vol 16, No 6. pp.276-277. 54 Huang, X., On Global Sequence Alignment. Computer Applications in the Biosciences 1994, 10, 227-235.

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.

Bolton and McCarthy, PNAS 1962, 84:1390.

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

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

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

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

Orita et al., PNAS 1989, 86:2766-2770.

Kiefer C. et al., Identification and characterization of a mammalian enzyme catalyzing the asymmetric oxidative cleavage of provitamin A. J Biol Chem. 2001, 27;276(17): 14110-6. Devlin and Risch 1995; A comparison of linkage disequilibrium measures for fine-scale mapping, Genomics 29: 311-322.

Hulshof, Paul J.M. et al., "Variation in retinol and carotenoid content of milk and milk products in the Netherlands", Journal of Food Composition and Analysis 19(2006) 65-75.

INDUSTRIAL APPLICATION id="p-271" id="p-271"

[00271] The present invention is directed to methods of genotyping bovine to facilitate the selection of animals with altered milk or tissue colour or P-carotene content. In particular, such traits include desired milk fat colour or P-carotene content, or desired tissue colour or P-carotene content. It is anticipated that herds of bovine selected for such traits will produce milk fat and tissue of more desirable colour (whether more or less yellow), or more desirable P-carotene content, and therefore be of significant economical benefit to farmers. 55 SEQUENCE LISTING <110> Vialactia Biosciences (NZ) Limited <12 0> MARKER ASSISTED SELECTION OF BOVINE FOR DESIRED MILK OR TISSUE CONTENT <130> 565328 JBM <150> NZ 561998 <151> 2007-09-26 <160> 25 <170> Patentln version 3.2 <210> 1 <211> 67896 <212 > DNA <213> Bos taurus <22 0> <221> <222> <223> <22 0> <221> <222> <223> misc_feature (31708) . . (31757) n is a, c, g, or t variation (39304)..(39304) G to A substitution <400> 1 agtcagcgtg ttttctcaga tctatgatat ttttcctgaa tcccagtctg tgagtcagga catacagggg cactatttag tgttcttcta ttacaccgtg aacttcctta gaaataaaga aagcagggac ataggatgag ttacgcggga ggcatccttc aagcccaggc agtgagagag ttttccatgg tcctgacttt tacatcatct ggtcagtcaa caaataaagt tacttagcct tggcctagac aagggcaggg accaacacaa tgtttttaga aggatttggg tctttctacg tacgccggat cttaggactc tgtggaaaag tttgttaacc caggattcag gactttctca tttgcattct cagcctctta gggatagact aatagttgga ggcgtcggcg gatagctcag gaccaataaa cgttctcccg ataattagta agagcgacat tctactttta tatcaaagtc tctggccagg tcaggacgct tacattccta aaggtctaac gtaacgtgat gatatggaga tttctaatta gggtttaaag aatcccggga ttctgtcagc cccaggtagg tgtgcctctt attcaggcag tgtaagacaa ggtttagaag actaaaaata ggtggacttc aagagagtag agtgggaagg tttgtgccgg acctatttat taggaaaatt acttcaagac aaggagagga agcatggtgg ggggagacca tacactgaga aggtgcttca ggggcctgct tatttctcca cagggtgagg gtgattaata taatatcaaa cttagcaaca gcccactata catctcgtgc tctgctgctg gacttttcta cgatttgccc tcttgaagaa caacactgta acagcctttc ttctcttgga gtactgctac tttaaaaact catttgagat acatttaggg ggtcctgccc ttaaatattt cagtggagaa aagaagtttg gacactgagg gttccgcgct gtattccgag tgttatgcaa tacaaatgta gacaatttac gggctgatta gtgtagtggg tcagagagat ggttaattct aacattggct cttatagttt ctctcacggt cagaactcaa ctattgcagt cttccttctc taccaggggc gaggtggtgt tgtttataga tctgcaatag atgttctaat ctatttaaca agactttgaa tgaaagtgaa cggctgatcc tatcaaagat aagaggctga caccacttac cctccccggt ccagatggag aaactgatcc aagtcacgcg tacagaggtc gaccctctcc ttctcaaaac ttttagttaa cgggctcgtt atatattcat caacaaatga tctaacttct tgggaggctg cacaaaaatg tgatttcctt ctctctctcc ttctgaagtt taaaggagag ttttatgcaa gaaaaatatt gtaaacattt cttctaagta caacagataa ttttctttgg agggtattca ataaagagta gtagcttggt gtaggccgca cggatcttag actcaaccag caattcttta gggtcagcag ttaggggtgt ttgtgacagc agacgccacc ggaaagaatt cagggtggta taatgtgtgt aaaacccttc ctaaagaacc tgagcaatca 60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 56 gacgagcctg atcacgccca ggtgggggac ggcaggctct atcccgcctt tcactctggc ctaatggcaa cctggagaag ggaacctggc gtgctttgag ttttaaagaa ccctatattt taaaatgaaa taggcaatac tctattttaa agtgcattat taccattaaa aatctttaga atcgaatggt atcttagttc ttaaccgcta attagcacat ctaagagaaa caggaacaca aagagttgat gtttacattt agactgataa aaagactcat acttctaacc tcctgagtgg ggataagtaa gtaatgtgca cacagctaag aaaagtcctg ctaaaagaga cctggaggaa atggtggctc tggctggctt cacatccttt ttttaattct tttctcagta cagtatctag agaaacccag atgtgaagaa tagaattttt accaagtgag gatctttccc ttagtgatgt atatattgtg actgctaagt ggctcccccg tccaatgcat accgcatgga tggagtgggt tttcttatga acttgtgtct ggattttcca acccagggat ctagagccac tttattaact tcaggcaggc ggaattatta agccccccgt ggttatgggg tgcgtatgtc ccccaacagg agaaccagat gaaatggcaa aggctatagt cctacatctc gtgggataat tagtttgatt gattatctta tgcctgcgaa aataattttt ttaatttata acagaagaaa atcacattct ggttatttat cccaccaggg gactgctagg ctgtggttaa tggtagcaat aaaaggattg ggtctgagaa tccttaccag aatttttgtg cagaaaacag acggtggaag ctcagtggct gccttgattt taatatctgc gaaactgtag acttctgctt agtgtcagaa ggggcgatgt tttcagttcc atctctcttc gagctttccc ctgcattatc gatggcagtt gatagtgctc caactcattg atcattgtgg tgttttcatt ggaaaagtgt ccatctctat ataattgaca aaattatcat cgcttcagtc tccctgggat gaaagtgaaa ctgcagccta tgccattgcc tgagaagttt gactttttgc ggcaagatac caaacctgct ctgggaagcc ataatctgac tagagaacct ttaactggag aaagtcagag gtagacgctc gagcctcctt tttgacagaa gtaagagatg gccactcaag ccatagggtt tgagaaggtc agatacaatg ttctagttta tagtatctaa cactgaatac ttgtggcttt acagttcttt ttaaattgca agtctgtcaa atttctttat atcaaaccca gaagtcctgg catgtagatt ggtaattaaa tggttcacaa taaggaagga tagatgagac cttttgattt tgatctttgg agactccgca atctacttcg tggagaacag ttcctctcag cctacaggaa tgcagactag actatcttta cttcacttca tcaaaccagg ccaagtctga tgatctccca aatagctagt tcatgatggc tgtagttaaa atgtatcatt tcaggaagaa gtgaaagttt gtaccacatg agatttgttt tgttagtttc cacaagtcta gtgtccgact tctccaggca agtgaaagtg ccaggctcct ttctccgatt taagatttac ggccctatgg tggagtgggt tctcttatgt aaagatttac tctgtatttt tcagaggagt ctctagatta atgtaggtaa gaggatttcc cctcatgacc ttggttattt tggggttcta tgttcttgcc gcaaagagtc tcaagtgctc ttgtaaaata gacttctata ttagttatgt atatgaaata tgaatttgag cctaggaaat gatcctatta aagtactgag ttaatttttt tacccctgga gcatttttta cagtcttgtg gtcctgagtt atggagaact gacgaatttg tgtctataga cctcttcaca tcaggagaag ggtcatctca tgtgggacct tttagttttc aagctaacat aaaaaaaaat tataaatttc tgactactga tacctctctc aatgtttatt ttcttgtcat atctaaaggt atatttttgc agggaccttt tagcactcaa tctctgtgtg atctactttg ttacctgtat ctctgttagc taaataatta aggtgtacaa gttaacattc ctgtgtgacc agaacactgg aagtcgctca ccatccatgg cctaaccata tgttgtacat agtgcagcct tgccatgccc ctcctctatt tgttagcaac acatccctta ttgtaggtta actccttctc gagcacaaag agggggactc tttgccatgg acggggcttc tccctgggtt tggagaatcc agacacgact taaactgaat ttttgctaaa tcactattag agttgaccac atagggacaa gtaatttcag gccgtgtaca aattcacata attaatgtaa aagctgtgtg gtgaaagctc ttaaacaatg aaaaggcctg aattaagcag gttggagttc gagactgtgt gaaacaggca gttttgaaga actcttccat gctggagtcc gggaaattcg ttagcatggg tgctacacaa ggagatttta aagtcgtctg gctctgtttc ttacccagta tttgctcagg tcagatatca gctgtttttt ttgtttactt tcttgattac tatatatatg actactaggg ctttaaacat taaacttttt actttgaaat caatcatatg cataatgatt ctaaccatgc ccttagatgg agtgggttgc gtcgtgtccg gattttccag cattttaaca gctgtgtgct accaggctcc tcctccaggg ggcaggcagg tttcaaatat tttattttat 5340 5400 5460 5520 5580 5640 5700 5760 5820 5880 5940 6000 6060 6120 6180 6240 6300 6360 6420 6480 6540 6600 6660 6720 6780 6840 6900 6960 7020 7080 7140 7200 7260 7320 7380 7440 7500 7560 7620 7680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 8460 8520 8580 8640 8700 8760 8820 8880 57 gaattttcat ctgtgtctgt attctacata aggataatgc gtggattaat tccattcatc ctgcagtaaa ttaatatcca cttggccccc gcttttgtgt gcatcagaag ccaaagaatg aagataacac taacttttta tggtatataa agaattccag atgactctct gtcgttctcg ggcaacatat gctggaggaa catgcggcta caaaacaacc tttccaaagt gtggttagta atgaataatg ttgtaaaacc agaatcctgt tgtagttccc ccttgcccac tataaagcca ccatgcccat ctgagatatc ggaaaacacg atcattgtaa agaggcagta ttgataaacg atagtcattg agagtacaaa aggcatcgca aagtggggat ccagcagtta gtcttgagga ggttatcagg aagtcactta caattcctgg agagaggctc gctcaaatag ttaacgtctg ggtctataac gtccctcttt atttgtcctg tttcaacttt gagctttaga ggctccgtgg tagcctgttc cttgtaagca ctccaggata tatttttttt ttaccttcaa tgatgtaaac cccatttacc gagcttgttt taagtgaaat cctcaaactc attctatgat catccatggg catgagggtg gtggatcata ggaggagaag agtaaagttt ggggcagaaa tctggaggtt tgtcctcagg aaataacaga tcctaaggaa acccctctct cattcccccc ttccataact tctcctaatc gctacagcag gaaacaaatc agaattattg gaagcaattg acatgagtaa gcacaagttc acctttctaa aatgcctgtt aaagagggaa cgagttctaa tgagtaaagg aggtaagagc cagtcccaat ggaatgatta ttaagttgtt gatattaaac tcaggtaaaa aaccgagaca gtgttgcacc tgtgattgtt gtccaccacg gaccgattgt tccaacggca cccatctaaa attttggtca cactttgaca gagggatggc aggcctgctt ttgaaaagct aatgtctgtg ttttggagta cgtctcttga cccggaggat taccccctga cagcccaaac actacgacag agaatatcca cttcccactt gacaaatctc acaaacgaga ccttaagaat tcttgcaata ttctttcttt catatggtac tactcacgtt gtgtgtgtgt cacttaggtt catgcatctt ttgaaaggtt aattcaatcc tattaaagta gagtacccgc gtaaagacct caagatacat aggttgaatc tgtggagaaa ctttggggac ttttctttta gcttccaagc ctcatattga tagcaggagt cagttataca taattaagat aggcttcagg cattcgtgct ctccttgtgc tttgtgcttg gagtaaaatt caaacgctgc ggtggggtaa ctagacccag cacatatcca tcgtgcctgg cattgcatgt ggctaacttt gtcctttttc agccattaga atgtataatc agtccatttt gtcgaaggta ggaagccttc ggaatgcagc gactcaggat aggtacaaag gggtgccacc gctgtgggag cccccagatc gactcggagg gagagctgag cgtaagaaag gttcgagccc gttaatttgc tggggtctcc gttacagcag ctggggataa ggaaaagcct ttagcttttg cgttgtaatc acaccttttg aaccatttgg tttccactga accaatctct ctttttactc ttgtctttct aaaaatggca gtaaaattta gtttccatat ttataaggta gttgttgaat ggggccagag taaaggagat ttgctagtgt cctccgacct ccttgtaaag caaagactgt gaaaaaaagt ccctagactc ggagaattat tgataagggg ggatatctga ttgagcaacc attacagatg agttgtccac agtgtccatg catatcaggt agctgtcaga ttcagcatta agtcaaagca agccaaataa attagcaggc ggtgcatctc ataagtcccg ccagacaaag ttcctgagac tggggacggc aggagttagc tctagaccta aaaattaaaa agggttggta ttcacagact cactgacgaa ataggagtga atttggagtc tcagagcata tcttaactcg aggaaagaat ccactgtttt ctgggtcagg ttacataatt gccttccata tcattaaagc gcagatgcct aggaacagtg ctttaaaggc tttcatgaat caatccatcc gcatatgagt cagattttgc ataatattct taagtactct gcattttcag ttcaaccacc tttatgattt ctgtctgact acatttcctt tatatatcac ctcagctatt gtgtttttta cacgcgaata acgaggcttg agagaaagct taataatgag actcccataa accaggtata ccttaggcag ttgtcctttt cctatcaacc gttgcctagg cgctgtccct tccaggggcc atttgtaact cagggagcaa catggggaat gccctaatca atatatgtac atatctagag agagctgaaa tccactcgta tcataccaat ttttctggaa cctatccaac tttggagcaa ggagaaccgg aaatatccca tctgtttgtt catgtaacct tttaccttat gtcacattat gatgtcatca tggagaaagt gaggggagcg gcccaggaca agcagaagta gaaagtaaag agtatgatgt aacctggtaa tatgaggacc agtcttctcc agttaattcc aatacattta tatgggtagg tttgataatg taagtgatat ggagccattg taaaatcttt agtaaaggta aaaatcaatt tagcttttca ttaaagtttt cagcacccaa gaattattaa 8940 9000 9060 9120 9180 9240 9300 9360 9420 9480 9540 9600 9660 9720 9780 9840 9900 9960 0020 0080 0140 0200 0260 0320 0380 0440 0500 0560 0620 0680 0740 0800 0860 0920 0980 1040 1100 1160 1220 1280 1340 1400 1460 1520 1580 1640 1700 1760 1820 1880 1940 2000 2060 2120 2180 2240 2300 2360 2420 2480 58 tcggactgta tcttcctcag tgtagtgaag ccttcggcta ttaggacaat cctgttgctg taagcatact tgggtcagag ttcaaaacct aaagaactgc tcctcccaag ttttccttct actggtcctt ttaaccttag ccattaatta cagtggggcc tggtgaggcc aacaaattaa tgaagaacct tgctgagttt caaatgtgac agggggtgat atgattaggc ttgtaaccac tctgtctcag ttaaagtttt ccctggggca aataggaatt actgagaaat acggggtata ttaccatttg aatagtccct ttcagtggat ggaatagcat ttttgttcaa accttgctca tcgtgtgaaa ttggcttgtc tcagtaagca acaactatta cccgggcacc gggcagtctc gatgcctgag cccttttcac agccatggcg atattcccta tggtccatac tctatccttt aaggccttct atctgccaat acatctaaca ttctatagtt ccctactgat agtctttgac gtcataaagc gtctcccaga cacagactga ttcctcagtc tttttcctct ttctactgaa accatccttt tatattgtgg gggtttcgta ttttactccc atatagcagt ttttaaagtg tagaatctgt ccacaagctc gttcagcagt catctgtaaa ctgcatagtt caggaaggag ctaacaacaa aatttaagat tttttaaagc acccacatgc ctcggggttg attctgaccc taaaagcctt cagctataag agtaacctgt ttagtatagg ccagatatct agcctgccag cacagagcaa gtagatcccg aaactgtcca gactttcttc atttggcccg aaggaattac atttctttat gttcctttaa actgcttctt tttgtgctcg ctaaagggag gtatatccct acagcacaga cagacagtcc cagaataagt atacctgggg ttcagtcctg tcttccagtg ggcaatcccg ctgggcccct aaggcttcca ccataataga gcctgcttta aagatcactt ctcagtctat ttgccatagt aaatgactct gggaccgcct tcattaccaa tcagcagtca agagtaacac tcctccttga ttatttctcc tctctggctg tctctttgtc accagagtag atcagtaatc tctttcctgt aagtgccgct atccctgctg taaaagtctc tctctctttc atagatattt cgctaacaga caccacggcg tatttccatg taaagttagg agtggcagga tgactgatac tccactcaca ttcaggtgct aactacatct tgtcaaaact tgtgggcaag ttgagtttct tttatataaa gattcccaaa tttaattctc aaccgattgt aaagtttaag aatatcatct tgacaaactt ggttaactga cgccaaaggc ttcaggaatt agcctcattt acccaaaata attttcgatg atgtggaaat acccacagat agaaagggag tcccaaaagg atcccagttg cattacggaa tgccccagta ctcctcaggt attgtcttga tggtcctttg cttgaggtgc ctgggcattt ccttttcctt ctgtctgagc atagcttacg ttccctcttc ctaggaattt ttaaaggctt gatcccatct gatgcccagt gccactcatc gcgtttgtcc ctttaaaagc ttctttgtat cggtgggtgt ctctgcgcat tcctgtcctc tctgaggtcg tttgctcctc tccacaggac tttctggctt tgccctttgc tcgatctctc catattgcag acccactgcc gcactggttc taacctgctt tcaggattgt aattgagaac tttaaatttc ttaagaagcc tcatgagaag aataaagccg aattctctgc cccaatgcca ctcagagctg ggaggccaaa ggccgggcaa aatcctctca tcagggccta tgacaaagct aaatcttctg acatattgta tgtccgaata gaagctggct actgaataga tcagacaata attattcgta ggagtgttgc atgggtttta acgtgtgggt tttccatcag ggctccatat ggtgagggag caagataaag gcggatcggg tctaaaagga gtaattagga gagtccgacc cagaccggac ccctcctttc ttctcaggct tgtctttttt cagttgtaac gcggctatct actttcggga accaggagct agaacgcgcc tcctttagct ggggagggcc tccataagca caagatatac tctgatatat ttcttgataa ttcataaaga atgatcccag catctcatcg tactgagaca gtttctcata ctgtccagat gacagtcagc agtgcataac tgaaatgctt catgagcatg ctttgcttaa cctaggggag tacgctttcc ctaatggggt aatcgtgatt cacaaacttt tactgtctgt tcagtacagt ctgccccaat ttagataagc caccttttct gagcttgcag ccagtttgtc gttccccata attgtcttaa tggccctagt gagccttttc aggctcgcga acaccactgc agtgaggact gcgtagggtc aggcatcctt gagtgtaagg agtcttgaac aaggactgtt acccttcttt ctttgagctt cccatactct tcatgaaaac attctggcac aataactgaa aagaaagtgg aatcgacgga tgggagcttg ccggagacct atggagccgg cacagccgta gtttaagaac tgcctttctt agagtgtcta ggagccgact tcaatctcag tctttctctt tgcctaagtc gtgttgtagt gctatctcat accgcttttc atcacatcct ttttctgggt gaaaaaggct gacaacagct ggatagattg cttacttcga ggagttgttt 12540 12600 12660 12720 12780 12840 12900 12960 13020 13080 13140 13200 13260 13320 13380 13440 13500 13560 13620 13680 13740 13800 13860 13920 13980 14040 14100 14160 14220 14280 14340 14400 14460 14520 14580 14640 14700 14760 14820 14880 14940 15000 15060 15120 15180 15240 15300 15360 15420 15480 15540 15600 15660 15720 15780 15840 15900 15960 16020 16080 59 gtgggcagtt gggagctgaa cagagagaaa cctgtctaat gtcctccaat taagccctgg taatttatgt acttgttatt ctgtagtttt tgttgaccat aaataggttt agatattaac atctgtgttg tatttaaaaa gaacatatga tctactatat catatcaagc cctgaaaagc gcaggataca cccagggaca agtctgttat ataacccaca aagccttaat tttaaatgag atttatttgg ggctggatga ccatctaaaa actgatgtgc tatccattaa gcctggtggg ttagggcgtg gagaagaact tagaccttag ggaatgtaac tgtcctgatt cggccagaag ctttaataaa aaaaacagac tgtcgcgctc actaattatg gggagaacgg ttattggtct tgagctatcc ttcctgatct cccggcaatt cctacccatc aagaaccaca cagagggcag tcacaacctt ttgtcccctg aagcacaagc acaccaccct aggagagtga gacccatcat ttggactcca ttgctccttg tactttgcca atgtgagagt cctgtggtat ccatcttaac tgaatcccag ggtttcacac aagggcaaca tgtagaacag ggataccgtg gcaattttat tttcaccaac tcttgtcttt gattttcatt ctgtatgtct atggtttttt ggaatttttt ctacaaatct ctttcttgta taacacattt attacaatta tctcaaaaat actatgaaca aattctattc taaaacaaga cttatcactg cattttcaag aaagctaaaa aaattaatat aaattacatg tattccagaa tttgccaact tgttatcacc acagtaactc aatctggtca tttaagatat cagcccatac gctaagtaaa tttatttggg gactgaccgc atgatgtaac agtccaggac tctggaaaat tctctcactc cctgggcttc aaggacgcct cccgcgtaaa tcattctatt tcgcctacgc accctcacca agcagaaaat tctagtccct acagaatgaa gtctaattca cttatttaac tgaaatcaag tatggcagaa aaaagttggt ttcatggcaa aaatcactgc gaagaaaagc acaaaggtcc aggactataa tgggagaaga ggaaatattc tttggagtcc ccaaatctat catatgctac tattatactt gccatgcagt gaggattctc aatacgtgag ttgataataa tccctgctga tttttgtaaa ttgctattga gtccttattt gaatgattta tttaatttat ataggagact tttttttaaa taatagaatg agttcagcat aagtttccat cacaaacatt tggaattatg tgcaatgtga gactgaaaaa aaaaatgaga tccactttta aacttttttt atttttgatt accatccatc ctcattctcc tggccatact atttcttaat caccctgaac ttctttcctt tggcgtctgt tgtcacaagg acccctaaga tgctgtcccc aaagaattgg tggttgagtc taagatccga gcggcctacg ccaagctact actctttata agtctctcct cagtttggcc tggatcaaaa cccatcagga agtcaaaatt tgaaactatg ttatatgcag actgctgtga agcgaagaag ttaaaactca atagatgggg agatggtgac tatgaccaaa atctagtcaa agaaagcctt ctcttgagag aatcctgaat caggtatggg acccttagga ttctacccat aagagaccct atcacatagg tattttttgt tgttctcttt ccattttaac ttagtgatgt atgtctattc gttgtatgag taaaaatttc cttttttagt ttatgtttaa tggaaaatac tagataaatt aatagacaga aattaagatt ggactatgaa tcatgatcta ttggaaatca catttcatga acacagaggg aattaaaatc aatgatggat aaaaatgtga gtacagttca tacttttcat cttcccccaa gtgcttctcc atatctttga gagcccgatc aactaaaacc tttgagaaaa agagggtcgt cctctgtata acgtgacttt gatcagttcc tccacctgga ccggggaggc taagtggtgc cagcctcttt ttcttaatta tcgaataccc tcaggccaaa atttactgag agattccaca acagaaaact agccatgtca agtacatcat gaaatatcaa aactaaagag acattcagag aaacaatgga tgaatgaaac ctagacaaca agctgtggtt ctttaagtgc tcccttggac attcattgga ggcaaagtaa cataagtctg ttcccttgtt ccaactggcc aagaccaggt ggctttgata ttttccacgt aggtgtgatg tgagcatctg agatctttgg tttttaaaaa atactgtctt gactatctaa ttttttattt agaacaaagt aagattttta aaagtggaag tatacaattt ccaggagaca aaggtcctga atatcaaaag atctttgact tgactgaagc aaagacactt gtggatgact taaaatatac gtagcattaa cttgtgaaac gtttagtcat acctgttgtt aggccagtgg tcgtctgata cactacaccc atcagccctg aaattgtcag catttgtctg tgtataacat tcgaaatact gcgtggaccc ctcagtgtct aaacttcttg tctccactga acatttaatt tggatcagct ctacagggag catggcctgc agcctcttat aaccaagctg tatatgtcaa gtgaaacacc taaccccaga cctcttgata aactaagatc aactgacaga tgaatgaaat tattaaaaaa tttcctgtag caaagaattg agcaaggaga aggactgatg 60 ctgaagctga accctgatgc ttggatggca gacagggaag tactgaactg aaaatatgta ccatatacag aggtgcccaa tgagcgaaag atgctgtaaa attggaagtg actaaaatgg gcaagaatcc gtacttgggt ttcatatcac aatggttcta gtgtcctttc tgacaggcaa gccaagagaa acacatggac aaagatggag tcatgaactc tccctaaggt acgggattag aagaggcaag ctgagaaggc aaaaatatat tcctcagtga tcttcaggaa agaatctgcc agaagggaaa ccatggggtc caagggaata agaaacagaa tcttaatgac agtgaagtca attccagctg ctggaaaact gaaaggcaat aaagtaatgc gatgttcaag tgaatcatag tctaaatcct ataccagacc agaactggac atattgtcac gataaagcac gatgatacca agaggagagt cagtcccatc tttcttgggc gatccttgga ttgccaacaa tgagagttgg ctggagaaga ggaaatgaac tggccacctg gaggagaagg gagtttgagg tgaggtcaca aactccagta tgcgaaagat tcaccgactc cctggcgtgc aactgaatgt cacagtagaa tatgcaaggc tatgttactg cagaaacaat gaacaatatt gtcaaacagg accagaatgg cttagaagag gcaatctcaa agttatccaa tgaggatcta catcacagga agttggccgt cacactgatc atcaccagat aagctcttac cttattgcaa ataacctaaa atctgataga gatcaaaacc tttacaaata ccatctgaat tcaatgcaaa aattagagat tgcaatgtgg ggatgcccac ccaaagagtt tttcatataa tacattaaga ccagataacc actgggcctt agctatttca cagcagtggc gccaaagaat tcaaaatttt ctggatttag gaaaaggaag ttgactgtgt accttacttg atagaacaac cctgcttatt aagctggaat ccttatggca gaaaaacctg acttcatggc tccaaaatca agaaaagcta aggttcttct accataagga ctcttgagaa cctgaatatt atgtgaagag ggacgacaga aagccctggg aagagtcaga gtttggccac tgagggcagg gatggacaca tgcagtccat agggccaccc gggaatggca aaaaagatat gagaagagca gcccagctgt gcatagaaac agatgccaag gcaaatttaa atggaatagc aaacaacaga gtctatgccc caagaccttc gactggaatg ggagtacaac atagcaaaca ggtcaatacc agtcagaaaa aattcagact tcaaatccct gtgcctgaag atcctcaaaa gctgagaaaa gcagagttcc gaaatagagg accaaaggaa gagatctgag tccagtattc ggatacgact agatgggcac agaagtggca acaatggtgt aggaggcatc aatcctaaaa tacaggacgg gttcaaatat ccaagctaag aagaggcaga agaattccaa ggttcacaac cctcctgaga ggactggttc taacttatat aaagattgct gaaagtgaag gcttaaaact aaatagatgg cagcagatgg tgaccaacct agtcatagct aaactgagca tcccttggac cattagaagg ctgactcact ggatgagatg agttggtgat cacgactgag ctgatgcaaa aggagaaggg ggtttgggtg ggagtcacaa aagacagact aaccacttca gacacagaaa gagacatagc ggatgtgtct ctggaatgtt agtgaacatc ttcagatgac cctcaggggc atgatcttgg caaccactaa cagaactaac caaaagtaaa atgaagcatg ccctcctcca gaaatcagat aacaagacct taaattgaag tattatacag aaatatggat aaaagaaatg gaagagaagc aaagaatagg aaaacaacaa tatgagcttc tttgatccct tggcctggag gagtgacttt aataaaagac aagatatata gatcactcac actatgaaca gaaagtgctg gaaaaggtca tgcaaaattg cttcaatagt ggaaacagag gaaaacacct aaactgtgga aacctgtata aaaattggca acagagtata gcgagaaata aggaactaaa caatattcaa ggaaacaatg tgactatagc agacagcata ataacttttt ctgaagaatt tgcaaggaaa actgatgctg gactctgatg gttgggtggt gaacagggaa tgactaaact gagctgattc tacaacagag gacttcggga agagtcgaac gtcttgatgg gcattcttac gatgaactcc tccaaaagga ggtggtgaaa aggttcatga aacattttag cattgtatct aacaaagagt ttcatttcca tgctgaagaa acacacacac aagtcaagag gcaaaaacta acaacaaaag tgattacatt ggagctgaat aaaggaggga tggtagtaac ggaggtttgt caaaaaggca aaaaggtaaa aaggagagag agtgttaaag ccttgtggct gggttgggaa aatttcatgg catttcactt agaagtggta gaagaactgt ctagaaccaa aagctagtgg cactcgatat gttttcattc tactcatttc acatgaatca atcaaattgc acttctgctt aaattcttga caggtcaaga aaggagtaca tcatgtgaaa tcaataacct agcctcttga aaaacaaata gaaacaggga catgaaatta ttgaaaagca ccagtagccg gatgcttttg ccaaaccagt aagctgaagc ctgggaaaga atcaccgatt gcctggcaag gaactggact actggaaaag gataagatgg gttggtgatg atgactgagc agagttctgc cttgagaacc ccaggttggt atgaagaagc gtaaagtctg atcaaggtaa gaatacgtga gctactatgg ccgaaatgca aagcaaacca gtggaaacca acacaaaaag aaacctggag acagactttt agacaactct ctttgcagtc gtggctcaga aaaccactag aaatggattc gacgttgtac aaatggtcat ggagaaaagg aagaaagcct actagagatc cagatggtaa gatcccctgg actctatagt cagttcactt tggacctaac acaaaaaaga acatttggag aggtgatgga gccagcaaat caatcccaaa acatgctagc agaacttcca caacatccgt cattgactat agcaatggga agcaacagtt tcaaggctgt ttctggcctg cgtatatgca tgaagatgaa tcatggcatc cagacttcat aaagatgctt gagaattgct tgcatgcatg aactgtggtg ccattctaaa tccaatactt ttgaaggcag tgattgacat ctgcagttca gaaatactgt 16140 16200 16260 16320 16380 16440 16500 16560 16620 16680 16740 16800 16860 16920 16980 17040 17100 17160 17220 17280 17340 17400 17460 17520 17580 17640 17700 17760 17820 17880 17940 18000 18060 18120 18180 18240 18300 18360 18420 18480 18540 18600 18660 18720 18780 18840 18900 18960 19020 19080 19140 19200 19260 19320 19380 19440 19500 19560 19620 19680 19740 19800 19860 19920 19980 20040 20100 20160 20220 20280 20340 20400 20460 20520 20580 20640 20700 20760 20820 20880 20940 21000 21060 21120 21180 21240 21300 21360 21420 21480 21540 21600 21660 21720 21780 21840 21900 21960 22020 22080 22140 22200 22260 22320 22380 22440 22500 22560 22620 22680 22740 22800 22860 22920 22980 23040 23100 23160 23220 23280 61 acaatctgca tgaccgtaag ctgttcaagg ggaactacta ggtcacagga ggaatctaca gggcattgaa aagtgaagcc attattaatc tgaacatgga caaataaagc caacacttga accagaggga ttaggacatt tcatttaaca gctcagcagt atgggtcagg agaatcccat cacgactgaa tggaagtgaa gggtattagt tgcaggctga tgcggcgagg agtaactctc attgcccctc cagagtgaag cctggcagca ctatatattc ctcaacaaat tttctaactt gttgggaggc cttcagagga agctctaggt aggtgtaggt tcgaggattt ttcctcatga gtaacgggga tattcagtgc cagacactca gtcctggtgc agtccttttg agaacttctt ggggctaatg ctgccactgg cactgaaaca ctttatcctg tatttgattt gttcaaggat ttttatgtcc ggtggtgtta tcttccacac acatctctac cttttctccc ttccggtgtg ctgacttatt ttctctcact agagtgacac tgctttcctg actaatttgt tttttttttc gttggttgtt ttgtagacag gtcaaatata tcaccatagc caggcaagtg aaagtaaggt gggaaagaag ctttatacgc ccatttttca atctgaaccc acacaatgaa agggacaagt caggagaaga gacaagaagg cttaataggc aaaagaatcc aagatccctt ggacagagga gcaacttagt ctgaaaagac tggagaagaa catgagggat tttcagagga tggggaatta tatccataat gaagttttgg gtaaggactc attaatgtgt gaaaaaccct ctctaaagaa tgtgagcaat gtttgtaggt taactccttc aagagcacaa ccagggggac cctttgccat cctccttcaa ccctgacccc ccagcaattc acacaaggtt ttggatcccg tggtataatt gtgacttcct agccgctgtc ctcaaaggca gtgcacacaa cgcaagtata caactgtata tggtgacatg ttccagagcc tacatccatc tctgatgatt aacatttgct attttttgga tctgcagttt tgaattatta ctttctaaga ttgaagctca taactggctg tttcttttaa aaaaacccac tttcccacta taaatattta atacatgtgg gcagaacaag gaactagaag agaaagaaaa attatctctt catgggaaaa actccatctt agaaaaggaa gaagtaaatt atcagcaaag tcaaatgtag tttggtgacc atctgcaagg ggaggagggc gcctggcggg acacatgcac ctcaggaagc ggtgcgaagg tagtgaagga ggggaagagg gggaggtaag gaagtgggca gaaagggacc agatggggct gtaagggcag tcaccaacac cctgttttta cacatgtggc taaaacactc tccgaaagag agtagtaaag tcctgaggct gggcggagta aaggacgtat gcagccagtc cggctcagtc ttgctgtgcc ggaaatctgt gttctccagg ccatgaggac cccgccgcag ggtccggctc ggttttattt tatttcataa tacacattat cccagcacag atccctattc tcttctatct tgcgaatggc cttcctgtat ctccctctct gtccttttct cctggttgtt tacaggtttc cccctttctc cttcttcaag tcacctaatc agatgtgaag tgcagaggtt ttcccctgac aaaccaaggt gatctcagtc ttacttatcc tgcactaagt taatcctcat cttgctcaag attctaagga ggaatactca gagcagcaga tgttatgtgg aaaagtagac tttttgagaa taggaacctc atgaaaaaac ctacagtcca acatgtagca aggaaaacag aacgagggga gaaacagata cctctttctg gaagaaggca ggtctgtctg ccgtgggaag gagtaacgtg gggatatgga aatttctaat gagggtttaa cggacgagcc ttgtcacacc gtggtggggg taggcaggct cgatctcgcc cctcactctg gttggcatgc gctgttaacc ccttgttggg ctccaagagt tttgggtcct ttgtgtgtca ttacgccact gtcactgctg agtctcttgt gtaccctctg aaatatatat gtacatacgt cacccaacac gtgtcacaca taaacctgta tcagactctt tctccatctc ctgttactgc cattgtactg catctttctg actgtttcac catcatcatt ataccatata ctaagatacc gaaccatagg tggcacgcct ccaacattga tcacagcaac cagcgctgac ggaaggggaa gttcattcct actcagtcat attatagtgc atatactatt ggtcagaggc aaattatttt gagtatagga tgaggactgg ctgataccac aggacacgca actccagtaa tagggtcaca tggtggggtg aaacaatgag tttcaggatg aaaagggagg ctctgatgat gttcaaggag ttgacagtca ctggaaagag attaatatca gacttagcaa tagcccacta agcatctcgt tgtcaggcag caagagtttt acagccccct ctggttatgg tttgcgtatg gcccccaaca agttgctgcc cacacctccc tcactgctcc ctattttcct agaacttttg tctgctcagt tgctgtgcct atccatgcct gggggtcact attgtctctg attttatata atcttttcat acagtaagtt caggtactct ttttgccagc gttcaaaact agttaaagag ctacctcatg tttcatttta tcccatacgc ttttctgttc gcctgccact ccttcaggtt tttttactct 3340 3400 3460 3520 3580 3640 3700 3760 3820 3880 3940 4000 4060 4120 4180 4240 4300 4360 4420 4480 4540 4600 4660 4720 4780 4840 4900 4960 5020 5080 5140 5200 5260 5320 5380 5440 5500 5560 5620 5680 5740 5800 5860 5920 5980 6040 6100 6160 6220 62 8 0 6340 6400 6460 6520 6580 6640 6700 6760 6820 6880 62 gcaaacaccc tccattctgt ttccattgtt ctgccagtcc taggaaatgg ggagggctac atgatagtta gactcaagaa gatcttctga cagttattac ggggtttgct gaaggaaaga ataggtttgg ttgaaactgt gctagctaat aatgaaggaa cagtgtgatc aaaaaaaaag tttgtgggcc aaatacagat aaacaagaaa ttcacagaga tttttgttca aggcttcctt tgatacaatc ccagcagggt tcagcttcag gtttgatctc agcatcaatt tactggaaaa ttttaatatg tcatggctgc gtttccattt gttttttgaa aggcttagtt ttgatatttt atgatgtact cttttcccag gatgcacagg ttttcagttt agatgttttt ctgtttcctc tgttggagaa tgtacaatgt tgaatctatt atggcctagt catgatctga gtcttcaact tgtgtagagt tggcaaaagt tcactccagg catctttttt cagcttcttc tgccttggaa tttggactct agtagcaaat tgatttccta ccttgattca actttacttt tcttcattcc ttaaagaatc tgttcctcct gcagtaatca aggagacaca caacctactc agcccatgga tttatacgcc acgttgtttg gatggaatct aaaatgagca tgacagctca aaaaaatatg ctctgtgtat tttgttttta gttatcaagg ttctctggca cctggtcggg aaatgacaat tctatgcaaa taccaaaact cttattgagt aattctacca gttgctcagt gtccttcacc caaccatctc cttttctaat cattagtcct cttgcagtcc cttcgctgct atcatagctt ttgtctaggt agtcaccatt tttccctgtc tgctgagttt cctcttcgct tcctggcaat ctgcatagaa ttttgaacca tttcccagga gttgtggtcc ctggaattct tgccttttct ttttgagcat tacaaacctg tgtcacttcc agttttctct gccatggtca gaaagaatat catctcttgt ctgttagttt tatctcttga tggtatttgt agcattagtg aagaacagag attgttgact ataatggtca aaatgtcaat tggacctaac caccaccaga ttttggagct atgttcttcc ttgcatagac ttatatactt agacgtgagt cagtattctt gttgcaaaga tataatttct atttgactaa aacaagttaa tggaagagat gtgtgagtca aataaatgag catgatttta atgttttttg aaaaaaagga gtccagtggt gaactaggat gaaaaagaag tatgcttgaa gacagaggta aactaactgc aacttttgaa catgtccagc atcacccaga atcctctgtc gagtcagctt tccagttaat atgggactcc cagctttctt tgattataca ttgtcatagc tgcagtgatt tatttgacat taagccagat ttctgtcata cttgattcca gttaaataag atctgttgtt gacagagagg acacagtcca cttgcctttt aaatccagct tgctttgcta tgtccatggt actgtataat actttcttca gctcccagtc tgtcagtctg gttgttggag ggccctgctt cttcttactt tctagaaggt cttggggcat accatgctgt atgagggctg tctgaattaa gttcactctt attccaggtt cacttccaca atctctctgc ctcttgactt agttttttta cccaggtggt ccacccctgg gcctgggaaa atcagatgtg gggcttggac ataaatacct ttatctaaaa tggcttaata acaatatgag gaagctgagt ttgggattta aaaaaaatta gaaagcccaa tagactccat cccacaagtt tcattgaata aacctcaagg gaaagtatag ctctttaaaa tatcagatag tctttgcaac gcctactcaa atcatcttct tttgcataag attcaggatt caagagtttt tttgatccaa gacctttgtc ttttcttcca ttggagcccc gaagggatgg ttttcattct aaggtggtgt gcttgtgctt cagggtgaca ccatgtctgt atggtctagt aaggtttaga ctatgatcca tgaacatctg gcatgtgaaa tcttcaggca cataagggat atttaagtct ttgtttttgc atttcggtat aaagagtttg cattttgtac ttgcattcca cttgtaggtc agacttggat tgtttttgag ctccattcct atttgcccat gccatctcct cctatgcaat gctgggcgtt tcttgtccag ttgtctgatg aaaaaaaaaa gttagtggtg ggtgggaaga tcccaagaac attgaataca aatgcctctc agaatgacag gtaatttggg gcagaacgtg gtggtaacaa gtctgaattt gactggaata ccaaaacaca atgtacaaat gcatccatgg gtacgttgca gataaaaaat aaatgaataa acaccagttt aagtactagg tttcagtgct cccatggact actcatgtcc cctcctgcct atggctgaat gatttcttta ctccaacacc ctctcatatc agtaaagtaa aggagcaagt ccaaaataaa gaccagatgc cctctttcac catctgcatg tatccagcct atatatagcc ttctaacttt attcccatct atattcaatg acagatgttg gaagttctca tggttttgat ctccatcaaa ttgatttagg gaattttgca tgactgtata tgacaatctg ctatgaccag tccaaggcca gtcccctgtg ttcatagaac tactgtgata attgtaccca tctaagagat tctggtccat gtttgaccac attgttcttt gtgtctgctt tagcatgttg 6940 7000 7060 7120 7180 7240 7300 7360 7420 7480 7540 7600 7660 7720 7780 7840 7900 7960 8020 8080 8140 8200 8260 8320 8380 8440 8500 8560 8620 8680 8740 8800 8860 8920 8980 9040 9100 9160 9220 9280 9340 9400 9460 9520 9580 9640 9700 9760 9820 9880 9940 0000 0060 0120 0180 0240 0300 0360 0420 0480 63 gacctggggg aggtaagaat ctctccatca gagttagaca tcattctatc gtgactggct ctttaatcca ggtcaaactg agcatgcagt gttgccatgc gtggggtcac aagagtctgt tcgaattcgc gggccctagg tgtgtcatct ataccacacg cactgctgac tctcttgtgg cttctctgat tgtattactg cccatcgatg tgtacatccc gaaaatgcag catgacaaag aaagtacaaa ccaacactac atggtaggtt aaaataatca agcccaccac atttcctact tgcattggca atatacacat agccagtact tttttattga aatcagccat catcccaccc attcccattg acatttcacc tccattgctg gttagaatat ggttcatcta ttcactgtgt tgcttccatg ttttcagttt ggttttattc ttacattccc ttgtttgtag gttttgattt gccatctgta gagttgtttg cctttgtcag tgcttatagt cttttgtttt tgtcatcgaa ttaggtcttt tcattctttt tgccccattg ttatttctgg atattctctt tagctccatt gttcatcttt gctgaagtta atacccgtcc aagttgtgct tgccctctga gtggttaaaa gttttctgct tgataagggt tgctcccagg ctcggttgga ttctcctttc ttctccagtc tgggggttct acttttgcta gctcagtggc ccacgcctcc ctgtgccttc ggatcactgc tttctctggc gggcttctcc gttgttcagc tctactctgc ggaagtttcc ttgatacata atatatagta attaaaagca cataattcag tgtgtgtgca gctcttctgt ccagaggatc ggcagattct atgtatatac actaattggg aggataattg aggtacacat ctctagattg gctatctatt ctctcctcca ccctgcaaag gatatttatg cctcatcaga atatgtacca ttctagctat tggtttcctc ctagtttttt accaacagtg actttttgat acatttctct tgtcttcttt tttttctggc ttgtttcact ttcctttgct tatttccatt tgttctgcct aattcatttt acatgtagct tatattcttg attttctatc gatgactgta cttctttctc ccatttcata tttgccattc ctcttgagtg ccatgtgaac tggatgagga cttgttcttg gatggctggg agtggcagta actcttgtat gactcccaga tcctggatcc ctgtggaagt cagtcctttt cagtgcgagt tgtatggtgg cagggctgct acaagagaca tcctttccct ggatctgaga tttgcccttg agccagttgt catcttactt cacttctttt aaagaaaaca ctaatagaag caattcacta aaatccattt tgctcagtca ccatgggatt ttcttgaccc ttaccaccgt atacacacat aaacactaca ctttacagaa atatcccctc atacagaggg ttacatatgg ctctctccat aaattcttca tttctctttt actgactcat caacttcttt cgtaaatagt aaggtatatg aaggactctc caagagcatt gatggccatt aataatgagt ggagaaatgt attgagttgt tgctattatt atgcaaaagc actctgggag atgttttcct gagtttatct gtcttgtttt cctcctttgt ttgttccatt gcttttcagt aagagtgctt tctttttgca cctactccag gccctacatg agtttggtta taagaggctt ctctggtggg gctgtgttcc atggggacct tcagtgcccc cgctcaccag ttgtgtgccc tctgtaatca gctggatccc acttctttgg ggctaacggc gcagccggag ctcagaccct tcatcctggt tttttttaaa gattcggggt gattttggtg acagcagatg tatgaagcaa attattccta gctaataaat tgatcaggta tgtcattcac tatctgactc ctacaggcaa agggattgaa gcaacctggc ataaattgat agtgtttttt ttttgttgtt ccttttgaaa catgtttgag taatgtaagt gtccataagt gtaccatttt tgactcattt atgtgttcct atccaatcat gctgcagtga cctgggagtg cataccaact cccttttctc ctgaccagta gatgttgagc ctgtttaggt atgagctgct tttgccattc ttttaagttt gtgggtcata ctaagagttt ttgtgtatgg cccagcaccg caaaaataag ggcctatttt ataatctgaa tggttatttg tactgttcaa tggaccatgt gcatggctca gttttctgta gtggaggctt cagggccatg ctccctgtag ccttcaaatg tgaccatgtg aaattctggc aaggttttgt aatcccactg caggttggga tataactctt gacctcctcc ccgctgtccc caaatacagg gtgcacaaag tgtgactgtg atcttttttt ttctcacagg tgagattaat gtctaatatt ttatttatga attttcttaa ggtaatccta catattaata tttgtgaccc gactattgga cccacatctc aagccacgca atatttcatc tttttttttt ttctgtcaaa ctccctccca tttcctgagc ttccatgtta ctattctcta tctagattct cactctgtat tttttatggc ctgtcgatgg acaatgggat ggattgctgg tccatagtgg cacaccctct tgaggtgata atcttttcat ctttttccca tgtatatttt tgaggattgt aatcaggtca gaggatcttt tatagtttct tgttaggaag tttattgaag gtacccatag tctgtttttc gtcaggaaag ggatcttctg 30540 30600 30660 30720 30780 30840 30900 30960 31020 31080 31140 31200 31260 31320 31380 31440 31500 31560 31620 31680 31740 31800 31860 31920 31980 32040 32100 32160 32220 32280 32340 32400 32460 32520 32580 32640 32700 32760 32820 32880 32940 33000 33060 33120 33180 33240 33300 33360 33420 33480 33540 33600 33660 33720 33780 33840 33900 33960 34020 34080 64 tgaattgtga ttgcactgaa cttttgtctg atgggattga gtgatttctg gtaatttttt atgagagctt tgctgtagct ttttgttcct ctgtaggctt tttgaagtgt ttgagatgat ttttgcatat ctttttgatg cttcatcagt atcaggatga gaaagagttt tgaagcctgg taattgggtt ctaagaatct gtctcctata nnnnnnntct gatgagtctg tttattaatt tgtgatttct tttggtgtaa tcaatgtttt tccttttgct aaagtttttg gtttaatctc tagtgctgtg ttgatatgtg aggtgtattc atctaatgta cattggtgcg ttttatgtct atttacagtt cttatctctt tccagctttc tgtatgtgtc tttgtatcca aggtaactat attttttttt gctggtttgg catcaatttt tcagtacttt cagctgttaa ctttctttgt gggtttatcc atgttgggga tcttcttctt cagaggtctc gaagttattt ctgctattga tgtttattct ttttcaaggt tctatttcct gtatttttct caggattcaa tggtttgtgt aattttttgt tctgtagatt cttaggtaag ttccttaatt tgtattgatt gatattatct tacttcttct aggacttcca gatcttaggg atcatatatg tttaatcata catatggttt attgaagaat tgttgctgaa gatattggcc tggtggcctt tagaaggata gcttttgttt gcttataatt gtccatttct atcctttnnn cctttttcat gctaaaggtt tttactattg ttccttctac agttaggttg tcttatttct gcattccata atttcccttt catgtgtttg gtcgaagaag acccaagatg ttctgcattt tcatttggga agtggggttt gttaatgttt gttatgtctt gtaatcttct ttttgcttcc ttgaggtctg ttcagccagt tgatatatat cttctctggt tggtactgaa gaatgagatc aaatatatcc gcatatgggg gtttagtctt tatatgggac aattttcaac cttcttctgg tgagactatt ctaccatttt ttctttccag ttaattcttc ttttgatcat cttcatttat gccttttcat gattgaattc aagcttcctg tctagttcta gcatttggta tttattccta tctctttctg ttgtatcctg ttagggtttt tttctgatct gaactacgtt ggaatgcttt gcctttacta aatgggtgct ttatctttaa ccttgcattc ttctgtttgc tgtagtttct gtagaatgag ggcattagct ttggggagat tctgtttctt tccaagttag nnnnnnnnnn ttctaatttt tgtcaatttt tttctttcat taattttgag ttttttctaa tgaggtagga gattttgagt tgatttcttc tgtttcttag atactagata tggtgtatcc ggatggagac cttgtatttc taaagtctcc gtcttatgca ccttgtggat ttattttaag catttgcatg aagtgggttt ctgtgtcttt gttcctattg atttttggac ttctcttaac cttgccgggt tgccactccc tttccctgta tggtagtttg tctttgtgtc tataatctct gacccctata ctcagttctt atcttccagc agtattttta taggtctttg ctttactatc ttggacttct tattatattt tttcttcctt tggggtgagt tgaaaaatgc atatagtcat gatatttaat atttttcatt caactttgct ctatgtacag ggattccttt gaataatagt cggtttttcc tgttgaggta gaattttgtc atttgttaac ctggagtaaa taaaattttg ttttttgtgt ttggaagtgt cttttctaaa tttagatcac cctggttcag ccattttatg nnnnnnnnnn gttgatttga gtttatcttc ttctttttca gtctttttgt acaaagttta ttgtattgct tgttgtgttt agtaacctgt tttttttctt tgatttcagt tggagagtgt tgtcctgaag cttattaatt tactattact ttgaggtgct tgatcccttg gtctatttag gaatattttt cttgtagaca tggttagagc ccattttctt tgtgtaagtc ttttgcttgt acagtaatct ttctggcctg tgttacttct attagtgtgt tcttggactt tcaaaaattt attcaactgt ttcattcttt tcactgattc atttcagtaa ttaattgatt attattctga gtgtttctag taacttattg ttagtttctg ttttgtttgt cattggtaat tttcacaata tctttttgtt cttagtatat aaattcactg tatcatgtga atttcttttt ggtgaaagtg accattgaga ggttccttcc aaaagctttt atggtgtatc cccaacttga ttgaggattt tgtctttgtc tccttcctct tgtttgatgg agcttcaatt tcttgtaaga ccatatagtt nnnnnnnnnn ttcttctcta tcaaagaacc cttatttctg tctttttttt gatgtttttc attagcttcc tcattgtcat tggttgttta gtaattgata tttcttaaat tccatatgca atatcaatga ttctgttttg gtgttactgt cctatgttgg atcattacat tctgatatga ccatcctctc gcatatatat atttaatctg aattggggtt cctttaacat ctgagaagct tggttgtaga cagagtttct cccttgcttc gtcttggtgt gactgactat tctcctatcc tggtgcattt ctactttttt attcttctgc ttgtgttgtt cttacatttt attctttttc tttgttcctt tgtttgaggt ccctcctaag tttttgtgct 65 gagtttttgt ctgctgatgt gggtgctact tttctcacta tgctcccact gtttgttatg accccagaca acatatacat gattgacctg ctaaagcaca aaaacaaaac gttaaataga cagtaggaaa aaaaattaaa aagaaaaatg caacagtaaa gcagtcctac acatctgcca tggggaataa agaatagata aactgcatgg ataataggtt ctgcaaaagc ggaaaataaa caacagaggg tcaaaacata ctgggagtca gacctgctgt cagtgtccac tatatattcc tacagctggt tgtggtttta ggtttgcccc tggcagcacc gaagggtatg gtccctgaca ctgaagtgac gtgagttgag tatgcaggga gcctcttgta ttcaggcact ggggcctcct cttgggtggg ggagtcctgc tcatgctggt cagcagtatc aacctccttc ggattgctcc gtccctgtcc ctgccacaga cactttgatg gtcctactaa ctatatattc ctgtatgcac tatccaccta atgattcaat cttaataaaa aaaaataaat gagactagcc tggactggag ttgtttgttt tgggtttgta gggagttgtg tttgatactc ccaaaggctc gcattaagtg aatggcagtt atatacctat gcaaacaaag aactgaaaaa taagaaacat gatagatgaa ggcagacaaa attataaaaa gaaaactcca aagtgtgact aaaagtaaag aaacaaaact agcaatgaaa ggcaaagtta ggaaaagaac taaaatagta ccaacataga aagctcaaaa gggaaaaagg agaataataa cagttcacct gggggcagct agctatcaga atagacacag gggaaggttt tttccacctc tgtgagggcc agattctcag gcaacaagta gagaagcctg cctgtgccca tgtgaaggtg cagactgcct gctggcaatc ttgacggctc ggctgggatc gtcctactct tctgtagttc gtgtggggag gccttgcttc ctacattccc acagtcccca agggtatgta tcagctgttt tggggattgg cactcctgtt tttacctctg ttctgtgtct ctcctccacc atcacaaaga attctagttt atacaagagt ctgttgttgt actagccttg ttcctctgat tttttgtttt tgatgcctgg ctagggttag agggcttaat agagtaaaac acaaaatcag gagcaaagtc gaaattaaaa cagaactaaa gttgagagga gatttataca ggaataaatg agagaaaaga cagaactgca gaatataccc tacaatagat aaagcacaaa aaaaactaac aatagaggta agtaggaaaa aaattaaaga ggcagaggtt gcttaattag aaaagaaaaa atgtttttct cacctcccaa cagattctaa actagtgcat agtctggcta tggattttct tgcatgtggg agaagtggag gggagttggt ttggccaata gcaggccaca tagatgcaag gcgcaactgc ctgctgcagg gaaggcctct ccttgcctgg ctactctgta gtagatctgc agtgcctcag agagaggcta catggctgcc tcaatccatc aacaccagct tggctgcagc cgctctcagc accccagctt tcccccacta gtcaggtatt gaaggtatat attttgttct tgtcggttct ttaaaggaaa agtcaagaaa tgtccagttg ctgctaagct gggcaaggct gtttgttgtt tcatgtattc ttctctggta ctctggtcat aaatatccaa gcaaataaat aaaacagtcc attttattta gcaaggtacc aagggaagaa tgttaaaatt tagaaaaaat aaaaaaaatg aaagcccaac atattaatat tgactcagtg ctggaaagca aagtatgttg gataaagaag acaaacaaag aagagaaaaa cataacaaca atttcatagt agaaaaaaat gagtcactgc ggatgctctc tctggtcctt tttcttttgt gttgattgtg tccttagcaa ttgtccactg atggtgcagt ggctagggca tgctccagta gcctacaggg acatgttttg ttggcttgcg agttatggtc ttggctagtc agtccttctc gatcagcatg acatcagtca gcgttttatg tggtgatggc tggctttcct tttctacaat cccaaccact aaggactgtc cttaaatgtt cagttccccc gttccttgtc cctgtctgct tcctgatgtt cttggatcct ccgtaagtca ttagatgaat acactaaaaa ctcagtcgtg gcttctaagt gagtgaggtg tagatgaaga agttggattc gtctagggtc gaatgaagat aaatgagaag aattaaataa aagaaaaata ccagttaaga aaatggggaa agaaagaata taactgcaag aaaatatatt ggagaagaaa atagagccag acacccataa aacaaaggaa aaactaaaga agaggaaagg atttatatac gaataaatgt aaaagggaaa ataaaaaatg gccaataaaa ccaaagaatc tgtcagagtc caacactgtg cttctgtgtg gggaactctc tggatttaat cactgcccct gggtttgccc tgcttgggtc gcaggaaaca tgcttgcctg ttgcaaagag cttgtggcag gggaccctgg ttctcagagt tttcttcata tgttgttcag tcaggcactt cttaaaggaa gggcagcctc tccaccccct ccacaggcat caacagcagc gtgccttcca tggttctcat tctcctctga acccactgag ctgctgagtt ctcagctggt ccatggagag atcttctttg atttttaaat gaataccaag agaaaattgt tctgacactt tgcttcagtc ataatcctgt atcctgcaca ctttatgaga ttggagttag tccacaagtg ccaaagatga tggagtatac aagtatagta acaaaactaa taaagcaatg gatatgcaaa gggaaaagaa ttaaaaaatt gaaaaaagga aggtttataa gcaaaaccaa accaaatatt aggtgccaac aaagaaagaa attaaggatt aaaaaaaata ctccacagaa tgactgaggg ttgacagcta tacagaaaag ctttccctcg ctgatatcca ttcttgcctc aatgaccttt ctgcagcttg aggtttcagc tgaaggactg acagggtttc tagtgctcta gagaccctcc tcagacacta ctctgcccca cagtgacaag cttttttcca gctctgcctg cgcatcggga aaaggggcac catcctgggt tctactgttc atgcgtgagt ttcccaccat ccttgccctg ggggacctgt tctatttagg ctcagacaat ggcagatcca ttgcgtgatt gttctgcatt atgtactcca agtaggatgg ttaatacaac gttcatgtgg ctgaggggtg tgcaagccca gtgtctgact 34140 34200 34260 34320 34380 34440 34500 34560 34620 34680 34740 34800 34860 34920 34980 35040 35100 35160 35220 35280 35340 35400 35460 35520 35580 35640 35700 35760 35820 35880 35940 36000 36060 36120 36180 36240 36300 36360 36420 36480 36540 36600 36660 36720 36780 36840 36900 36960 37020 37080 37140 37200 37260 37320 37380 37440 37500 37560 37620 37680 7740 7800 7860 7920 7980 8040 8100 8160 8220 8280 8340 8400 8460 8520 8580 8640 8700 8760 8820 8880 8940 9000 9060 9120 9180 9240 9300 9360 9420 9480 9540 9600 9660 9720 9780 9840 9900 9960 0020 0080 0140 0200 0260 0320 0380 0440 0500 0560 0620 0680 0740 0800 0860 0920 0980 1040 1100 1160 1220 1280 66 ccatagatgg agtgggttgc gtcgtgtccg gattttccag cagatattaa tatgatatca tggacccaag aaagagggcc aataaaattt tcagaacttt ttccccctct ggctttagat tttcaccatg attatatgtt ccttttggtt cctgctttca cttccttttc caaatgttaa catcatttag gacattcatc taaacagaaa ccattttaat gatacaatta gtttgtgata ggatatactc aggaacccat tccttcctaa ggaatggctt tttctacaga tttggtacat tttgaaaagc tctttaaatt ttattttgat cctatagtaa caccaatgtc tatgaataaa atataataga agaaaaaatt cctctgtctt cttccttagt tttctgtcat ccattctgcc atgaacggag agcgactaac gccagaattt catttcttgg tttctttttc aaactatagt ctaaatagag ctggacaaaa aacaacgtac tgaaaaaata ttatatgaaa caaatatgta tttctatttg acatgcattg tactatcaga catacagagg agtttggttg cctagctgtc cagcccacca catttccttc actcttagcg gcaagagtac tatataattt atatattaat aacttacaga ttttataaaa accatctcag ttcatcttga gctgagcccc acctcatgta ttatagcatg aaattttgtt attgtaaata agacagtaac catgctgaag acccttcaaa atacatcatt ttatttgctt atataataca ttgagaaata tcttagaagt aggggttagg aaaatctgtc cccacttcct tacttatttt tacttcacca gtgatacata tggctctccc ctggtaagca attatgacat atgagaggat gttgtggtgt aactatgtgc gtggacattg ccctaaaatt gtattcttaa cattagaatg ccaactgtct ttcattttct ctggagaagg gagcctggtg acatactgcc taatgcttat tggtggtggt tttagtagga ttttatatct acctacccat tttatcgctg aacatgggga actgggagtt ctcatttgaa atttattgtt atcttttcat tagagatttt aattttaaat aatttatttt catttgcagc tgttaatctg ggctcccccg tccaatgcat accccatgga tggagtgggg tatattattt ggacaaatgg aattgattta actttcttat ccatatttaa aaaactgaaa tgagaatcac agtgctattg tgacagaatt tatcccattc atgctgctat cttttaagac acattgaaca aagtaaagtg tctagtgtga actttagcat taaaagatat tcaagcattc caacagctta atcaagagaa atccctcaat tatctctata tctatttttg gttcaaggtg taaggccaac tgacccatgc ccctttatta atgtcaatct gtttcaactg ttaaataact tgtacaaggg aaaccctgga tctctccatt acaaaatcaa gcctgtggta gaaggctagg gcttagctct aaatggcaac ggctacagtc ttcatgttat tcttttgaac aggagggatt aaaataattt tattttaaaa gtaatttaag tgaacggagc actcctatgg aactgtcttc aatttcagaa atgttctgtg catgactcac cagtatatat ggcacagcaa taccatggga tacttgtatc ttccagaagg tccctgggat gaaagtgaaa ctgcagccca tgccattgcc taattaaata gcaagttata tgataaagat ttcttgcagt atgtacaggt ctctatactc cattctactt tatagtattt tctctccttt ttcagctgat aagatgggtg aaggctttga ttcaatctat aaaaattttc taatatggac agttttgata gaagataact catatgctat gggttttcct tgtaagttgg ttcaagttct tttgcaatct ctcctaccaa gagaagggca agtgatcgtg aagaatgttt tgcccagaat ttttttataa ctttctccct ttggggtttg tgattactac aaaaacagaa caaatacttt aattacccac ttggttatcc gcaaaggaga aggtagaatt ccaatccagt catggggtcg acattaattt tttcttgtga ttgtttggtt tgatttcaca gtatgtgata tctctgaatg aactgcacat gaaacatggt ccagagttcc agaaatctct gtctttttgt atttttatgt ttctcaaact agagataaag gtaatacact acatcccctt ttctaatggc tctccaggca agtgaaagtg ccaggctcct ttctctgaaa tgaacatgta tagaatagaa ggcatcttaa ttaacacaat tagtgttgtg attaaacaat catgtttcca gtctttttgt ttaaggctga ggatgcttag tacatatatc gataagacca caagcatacg cttacagtgt aaaaatgatt aatttgacaa atgtctaaaa tatcttggct ttagccctct tctttcattg ttgactaatc ctccttttga ggtacaatca cagtgacata atcgaattgt ttgaacgttt tttcaggtaa atatggatct tgattgtgtg atttcagcaa cttagtacgg aaggtaaaat ttagagtagt tgattcaggt ttaaagataa cacagcatag ggactggaga attcttgcct taaaaagtca aaagcaaaat tctggataat ttcttgttgt gaggtgagac catccctact gtcttaaatt cctcattacg aacattgcgg attacggcta gaaattgcat gtatatactt tccattatgc tgaattttta agaaggcaca acaaagtaag cctgttcatc taataatgat 41340 41400 41460 41520 41580 41640 41700 41760 41820 41880 41940 42000 42060 42120 42180 42240 42300 42360 42420 42480 42540 42600 42660 42720 42780 42840 42900 42960 43020 43080 43140 43200 43260 43320 43380 43440 43500 43560 43620 43680 43740 43800 43860 43920 43980 44040 44100 44160 44220 44280 44340 44400 44460 44520 44580 44640 44700 44760 44820 44880 67 tatagagtaa tctctgagaa tgtgttagtt cctgtgctat ctaatcccat tttcaatgtc tcccatataa taatatctag gggcagtgaa ccaggcttgg accatcttaa ttatcatata aagtttctga tagattaact ctctccatat tactggctca ttgttacctg tctttttagg ttggtccatt aaccaagcga cccaggttca atcaggtaca ttcgaatttt tcactcctaa aaaactttgg agattgaggg actcaatgga gtgctgtagt ccattccagt tctatggagt tgtttaacca cctttagaac catcttcaga aacccttcac tggcctatga cctcaatttg gagcaaatcg ccttgtactt tagtaaattg actctaatct tgaggaattt atatgtgttt ttctaccttt agccctttta atttaaaata tattggaact atataattgt tcaagtgttt ttttaatctt ctctgtcatt atccacgtct cagctatgtc ttgaattatt tttattctca ctctctgctt acttattctt agaatccacc agaagggata gaatctgcct gaagggaaag ctagtatgtg aggtacagtt tcccatattc acagcaggac actcctaatt tgagtctatt atgatatcat gtccatccat ccttgagaag caccaagcct taattattta cccaaatata ctgtctatct tttgaatagg ttacacatat agaagagata acctcaggta taaaatcttt tttttttttt ggcttccctg gtccctgggt gatgtgaaag ggtactggag aggaaataaa ccacctgatg caggaggaga catgagtttg ccatgtagaa gttcttacct tgcaaagagt tttttattta accccaaagc tattctggtt tccttatctt atctctgaga atgtttatgc tttctctggg cctgcaagac agtaatcgtc caagtaaaac ttgaactaga gggggacatt acaaaataaa aaattaacag atgtaatctc gtagagttta ttctcaaaac ttactaggat tctccctaaa ctacttgctt aatgggtcaa atatcacctc gtaatagctt gtatatcagc gctatgggca ggctgagaat tgcaatgaag ggctacccac gcaatgcagg gctacccact ctcgtgttaa atttttattt ttttccatta cttgttgttt tattccccac tctgttttgt atgatatttg ggcattaaga atttaagatg gaataacaca tcactgagta catctccctc tagctgtcaa ttaatgtaag aagtatatat gggtaccctg gggatgtatt aagcaaatgg tgatcattaa gtggctcaga tgggaagatc ttggaccata aagattcttg tcctttccat caaagagctg agggggaaac agtaaactcc agagctggac gaagaatttc tatagataac tttttctaaa catctggaat cagtatactt gatgcatttt aaccttgcac aaagtcctga cgttcagtcc ttacagttag atttaatcca tggctttact acacaaatat tagtgatttt aagcttttaa aaatcctcta ttagatagac tctttcataa atatagtatc atctaataga tttctctgct aagatgatga aaagtaattt cactgccccc tttaaccagt cagttatccc gggcagcact ctttgcattt gagaccccag tccagtattc agacctgggt ccagtattct ccactttttc tttaattgag tggtttactg atctattttg ccatttcccc aaataagttc cctttctctg aaggtattat cttgcttaag gaaaaagctt cagaggattc tggaaaggaa tctatcattt gaattggctt gaacgtgaag ggaacttcat cttcaaatct gtacactgac aaactcattt cagtaaagaa ccatggagaa aagaaggctg agagtcttgg ttgaatgact actcattgga agagggtgaa aggagatagt atgacttagt atgtacagag tttgtatata ataaatcctc gctttccagc gaaaaggggc aacaagatct cagtcattat ggcgtctccc tctgctgtgt gacagttcat attgctgaga tcttttttct atgtgtagct tttttggtaa ttgttttcac aacatttaat acttacagta ctcatcactg ttaacctaga ggttacaaaa taaaatcttc ccttggaatc cagcaatgcc actctggttt ctccttgctt tttaacctac gtcctcactg gctcttctca tttgattcct ttgggcttcc ttgatccctg ggcctgagaa ccatatagtt gtatagttga caagatattg tatacagtag tttggtaacc atttgtatca tcttactttg tactcccatt gaagcctcaa gctattagtc tctcagggca tgtgtaagta ataactatga tttgggttgt acttcaggtt cacccagacg gttataggaa tcagcttttt cttaataaat tctgcctgga aatggctacc agtgccaaag gcagcaggag gatgctgaag aaagaccctg atgattggat gaaggacaag gactgaacaa aggcgcctgg gtccataata attagatcag aatgtaagat taagaatctg ccaggtggtt gagttgtact agagttaggc gtttcctggg caactagtag taggaaattc tacttgatta gcagagagct ataaagtgct ttttccggat ctagctagtt gatggagatg tataaagcat cctccccact ttaatttttt cccatctcag atgttttata ttcaaaatat gggccaccat ctccctttac cccacctcat cagttattta cctcatagct gggtcaagaa cttgtggctc ggttggaaag ttccatggac 44940 45000 45060 45120 45180 45240 45300 45360 45420 45480 45540 45600 45660 45720 45780 45840 45900 45960 46020 46080 46140 46200 46260 46320 46380 46440 46500 46560 46620 46680 46740 46800 46860 46920 46980 47040 47100 47160 47220 47280 47340 47400 47460 47520 47580 47640 47700 47760 47820 47880 47940 48000 48060 48120 48180 48240 48300 48360 48420 48480 68 atgggattgc tttctcccag caccttctta acatccttat atctgatgca gaagagcctt gcttaggact tggtgctagc tccctggatc tagaaaatcc agatgtgact tggttatcct caggacctac ttcattcatt tggtttacct ctgctccaga tgaaataaat tctttggatg taatagtcag aaagtgaccc gactgtagtg tggctgctat gggaaccatt tttcaaaacc aaataacccc gcctagtccg acaggacttt ctgaccctgg ggatgagacc ggacctgcct ggtttatcag ctgtttagtc aagcagttac tgtcagttta agatttagtc caggatgctc ttgttttgta gtgtttccac cagctataat atggggatgg aaattgctcc gatagaattt tttgggaggg tctgcatcat caaaaggatt aaaccaaaac attcaaccca ggacctatga aattacttta acacgtgttc catcccagtg atttcatata cctctccctc ctcgcataca gtattggtgt cctcattaga tggaagttcc aagaattatt agtccccata acttgaacca aaagagttgg atatcctcaa gtaaagcctt acactcactt ttatattttt ttgtctattt cctagaagag agtaaagagc aggaagatcg catggacaga gaagtgactt gttccaaaag gttggcactt gagttattta acaggtcagt agagcctagt actctgacag ggaaaatctg atgaataatt caaggatata tttggaactg cctatctttt tgtgaatatt ttagctaata ttatgaaata caaatacaag ccattttcca ggagacaatc ttcttactac taatgtcact agtatgtgat atgtgttgct gaaaaaagtg ggaaattatc acccagccat caggatgaaa ttgtgttgtc aatagaagct aaatggacca cggtatatgg aaaaaataaa agtattttag aaattcatat aaacttttat aagaaatgaa aatttgaaaa agctattcta ctatttattt caatattgta cccatcctga caccagcccc tgatattata tcccacagag gggttattgt ttttctttct actgattcaa tgtttttatg tctgattggc actgttgcat ttttaggaat acacaactga ggctctcttc ctcagaccac ctaattccca acttattgta tgtacactat tgcctgggat cctcctgcca cctggaggag gtaacctggt agctctcaag aggtgaagag ctaacaccag acaaacatat gtttggtact ctgctaggaa tgtagaatat cataaggact gcatgagtga gaagactgaa gggagtgggg ccttgaagct aaaatacaat ctgtttataa tatgtgtcat gactatcttt ggttcctgct catggagccc cacagctttg gttgaagggg aagagaccaa taccctagcc ttaacaagtt agttccttcc aattccactt caatgaataa tgtcattgaa atgggctttg ctctggtgga caaatctctg gtcttaatta gtgattttaa tccacataga agccaatctg gctccagatc ggaagggaaa agaagttcct tttctttttt ttggttttgc acccccctcc aagcatcctg catgtttcaa tccaaaagac taccatcttt ggcttacttc atgtattctt taggaacaga gtttgtttag ttcagacatc gacaaggaac gcgactttca ctcactccct cataatttaa ttcccttgtt tgatttttta tttgctgtac ataactgaca atgttggaga gaaatggcaa gggctatggt gagctgctga attgtgagat aaagataatt gttcttagtc gaggatgcaa acagataaat aatcagagca gggtacctga atgttgatag tggaagaagt agaaggacag gggattgagt agctgttggt agttaatata gcatattgca tattcttcaa ataatgttat aggtgatatg gtgagcccag aactagtttt ggttgtgttt aactgactga tttcattaat cagcacattg aagttctcta ctcctagtca taaatacaaa ggtgattatg ggaaattgat tactttcccc aaaaaaaaaa agaccacaaa ttgcttcagt ctcctggatc ataaatgaca aaaaagattt gttttttgtt taaaaaattt catacatcaa cacctccctc tatcctgcat tgccattctc tgttctatac ctaaattcca actctgtata tttaatggct aaatatatac gtcacagtaa tctattttgc tacatcatct ctttcatcac tcaggtcttg gttgcaaata tattcctcat aaaatttgag acaagtgtct ctcagtaagg cttaagagat cccaccctag tcacagggtt taaatgagta tctacaagtt caatatgatc aatccaacaa agatgtgtca acacagaaaa taaaggaggg gttgattatt gataggagga tggacataaa atagaatttg tcatcttcag tagttacttt tttttgtatc taagtctttt atgtatgtaa tcgggttcct ttctattgct aaataagaca ggaaaataat tgatgtgaat tggaagagat tgactgctct agtcagggag tgaagaacag tagaaatcag ggaaaaatat aagtgtcaat aaagtgggag ttggttttgt gaatttgacc taataatcat ttttttcaga cttacctcca cttttaatat aaaaaatgca ttttgttttt atttatttta catgaatccg cccataccgt cgaacctgga ccaaatcaac atctgtgtct tatatatgcg ataggctcca gagtactact agcaaagctg tatgtccaac caaataacat tcattgaaca 8540 8600 8660 8720 8780 8840 8900 8960 9020 9080 9140 9200 9260 9320 9380 9440 9500 9560 9620 9680 9740 9800 9860 9920 9980 0040 0100 0160 0220 0280 0340 0400 0460 0520 0580 0640 0700 0760 0820 0880 0940 1000 1060 1120 1180 1240 1300 1360 1420 1480 1540 1600 1660 1720 1780 1840 1900 1960 2020 2080 69 atcaatttgt agctgggaac gagaattttg cttgttgggt ggagggctag atggttttat agaaatcctc actgttcatg gacattttga ctttctatag gaaatagtat ttttagtaat tagaagtatt tattgggcca aaatagcctc cttctacatt atcttaattt gtgttcctgc atcaatgcct ggaattttag gtagaattct tttgggcttg ttaaatcatt atatgtactt cacttgtgat gatagtaaaa cttagttcct ttccttggat aaacattgct tggtaggtct gattgcctca tttttgtaaa gatctgtcta cttccaactc taaaaagtta cttctgggct tctcattgca gctgtggctc agctgtggtg ggaatcttcc ccatggtacc tttcagtgtg ttgaaactgt atgaagttga tttgatctgt agagttttat ctgtactagc ctcatttgga tagatacctt atcaatagtt caaggtcata atctatatat tttattccat gctattacca ttcaaatctt agtaggtcct tcccagttta gtcacgtctg actggagtag gtctcttatg ggagaatcat cccagtataa actatgaatg atttcttaca ctatttatta cctgtaaatt ttggcatgtt cagaggtctt tatacaagta atgcctgatg cccaaattta gacagcattt ttctatttaa tatttcatga agtttattga ccatattctg gcactttgtg agcttcttcc ttgaggacca aagtctacca ctgatttgtc gaatttgcag gaagacataa aatgcttagt tagttcctta tggtaagcaa cagcttgaag tatgtaatga tttaaggttt tgatatcttg gtttttgttg agcaaacaag gattaatttg atgaacaggg catttacatt ttctctagtt gcggcttctc atgggctcag cacaggctca tggatcaggg accaggaaag aggatattac tgtaaatttt ttttgtatat aaattacttt ttctttcttt taagacttca aaatgtttaa tatcagatta tcctttatga aaaatatttt atttatattc tttatttctc gcctgggctc ttcccattta tgttggttat tcccctctac actctgcaaa gttgccatgc tctcctgcat ctcttctaaa tacacggttt attggacata taagccactc attaatacag atctgattca agcatggatt ctgtaacttt tgataatatc aatagcatta ctgtagtccc tccagaggat ccatgtacta gaactttgaa ttcatatgag gttttcatac gatctgtacc agggatgtac gggctgtgtt actacagaat aagagtggtc tttctcttgc gtttgtaatt aaaaattata ttacacttga agcaaaacta ggattcattt aaattgtttt acttacattt atatttacag gccttttgca caaacaaaca gacataactg aatctctctc aacaaaattt gcagtgagca ttgttgcagg taattacagc gaagttgtgg attgaaactg tcctgaatct atatctttag atctttaaaa tgatctcata ggattttcta acaattctta acttgaaaag taaatcacca tcttaatggt gtaatacatt atgttatctt tgtttctgga tctctccttt agtgggcaaa ggttattaaa ctattttaaa catctttccc cccatggact cctcctccag tgactggcag attcgaggaa catgtggtgg aaggcaaatt ttaattaaag tgaattttgc attacaaaat tttctgataa tgcaattata accaaagtcc ttatatttct gcatataatt tagtcactta aagagtttag gttagttaaa atgagatgat ctaccaggcc ctaaaggaaa ttcagtaaac gtcattgatt cttaggaaaa aaaataattt ctctctggca tatttattta ttaaagaaaa gcactaaaca taatattaat tttcctgata ctgctgtagt acttagctta tataagtact tttccataag aaaaaaacca acatctttac catttatttt attcttttta ggggctactc gcatgggttc ccataggccc tgcactgact tatcccctgc tttaaaattt ttcaagtcat ctttcattct tttagagagc tgtatacact tgcatcttat aagtgctgat ctaagtatga accttttgat gtgaatcctg atagaagaat cttcattttt ccaccttctt atcagtttct caatattgag tattatagtc ctttgtaaac atagactgcc aggatcttct gttctttacc aggccttttc ataagcatac tccatatcac gatattgcct atatcagaaa gacaataaaa taatatttgc tttttattag aaagcgtatt ctacttttta tcattaccaa atttattata gatttattta tcagttgatt tttaaatcta tattacttaa taaaatatct cttttgttac tgtgctgtca ctgggaaaga tgatctagcc aataaagagg atacttcaga ccaagaaata tatttacatt ccttcgataa atcaatctaa tttattttta cttatgttat ttagctcctt ttttagaatc tggttgggat aataatgaga aatcttttaa tttatttggc ttcattgcag tgggcgcata tagagagtgc ttagttgtgc agtggcaggc cttgaaggaa cattagggag gtttttgacg ttgctaaatt catattatct tttattcttc agcagtaatt aattgctgca aaacagacgt ttttggacat ttttaattct tagcatatca ttggttgaaa tttacatata cagagttccc tgtatatgtc catgtatgct aggctcctct ggacccaggg actagtgcca 52140 52200 52260 52320 52380 52440 52500 52560 52620 52680 52740 52800 52860 52920 52980 53040 53100 53160 53220 53280 53340 53400 53460 53520 53580 53640 53700 53760 53820 53880 53940 54000 54060 54120 54180 54240 54300 54360 54420 54480 54540 54600 54660 54720 54780 54840 54900 54960 55020 55080 55140 55200 55260 55320 55380 55440 55500 55560 55620 55680 70 cccttgtaaa gttcatttgt tatctgattt gtctttttaa tcttctgttg cagatggtaa tatcccctgg cagaggagcc ctaatgcttt acattggaat attcccaggg taccattctt cttttctcca gagtggtatg tgttgagcat tttcagttat tacaatatac agttggagtt atgatatagt aacagtaggc caggggacac agttactgaa aaagatattt gctttgactt gattgcagag tacttttaaa taagaagtca atttatcaga gtcttgagta acaaaatttg ctctttgtga gagtttgctc ctcccctctc ttttctcatc gggttgattt agtcttctcc caactctcac cagcaaagtg caaggagcaa aagaaaataa caatgatctt ccctcatcaa cataactgag gtctgatgtt gccctggcat tgttacttct catctcttaa ttgatgaagc cattggcaat tagtatttct taaatcacaa atagaactat ttagtcaaac tttctaatat tttataaata gcttttatac agtgggaaaa acaaactaga cattagaatc aaaaacttcc ctataaattt atcatatttt acttcactat tggctgagta atggactttt agaatctgcc agaagggaat tggcaggcta cacacttcat gcatgtatcc attgcaagat catagtggct caccttgtcc aggtgatatc cgtttcatgt aaaagtatga tcccactata tcaatatcaa agacctgaaa gacaaattct cagagcatat atcacataga gaagataact agttattgaa aagtatttaa atgccatgta taacaagaaa atttgttgca aggtataaaa tactttagtt tcccatggac aaatccatgt cttttgcttt aggtggtcaa cctttaggat agtatcacaa atttacatga atatctttgc gctttttaaa cagctgtcac agttatttga gaagctcttt gttgttgata ttgcatgatg actcctttcc tgacccacat gaattttcca agaagtagat ttgctgtcta ttattttctc agataataag atcaaagtat aggtggaatc atatatatta tatgtgtata tacactcaat cataaactaa taaacataaa atagctggag aagattttgc gttttctatg ttagattcca gatcatctct atattccatt aggttgcttc tgcagtgcag ggctacccac cagtccatag gtcttggctt ttttggactg catatggtag ataccaattt agaatttctt tcattgtagt gtctcttggc caacacattt tattacaatt actctcaaaa agcaatatga attcaattct ccagggacat atctgttctc cgcatatttt agcctcaata atgaggaatt tttggaaatt aattagaaaa tgcagctgaa acaaacgata attaactgtt tgtggcatgc tgattgagtc caatatttcc attattggag tgactggttt ttcagaaaca ctactggaaa ttttttaata atttcagact tgtttgcact aagttgagtc agttcctctt tttctcctgg tgctctgaat caattttgaa acatatttct cactttgttg gttttctgga tcatatgtta agaattgcat ttttctagac taaacaaata aagatgtcat tcttatacat tatatgtata aaaggtttga atgaaatggg agatcatcat cttcagagag atctggattt tctgtgagtc tatataagta atccatgttg gcatatatgt aatgtcttgg gagacccagg tccagtattc gattgcaaag taatttgtat tgtccttttt ctctattttt acattcccac gtttatagtc tttaatttgt tatctgtata acaagagact atctttttaa actaatataa accagttcaa attctattca aaaacaaggt ttatcactgt caagtgcaat aaattaaaag aatatcaaaa aaatgtatac tatttgtaac gctgctcagt gacactagca gttgttgttc caggcttccc ggtgttgcta tagtatcgag ctcagcatca gatctccttg tcagttctgt aatcatagct cactgtctag acaatcacat ttttcccctt ctaagccagc tactttctgc caatcttgat aaagttaaat ccagtccatt caggagacag tgatccatac attcccttgc atctcctgtt ttgaagcttc tttcagcagt taaatgataa aaattttttt atatatatgt caaaagcttt aaaagaacaa aacactgaat atagtccagt ataaaaaaca taaaaagtga tgtttttgtt gtgtcttgtg ctgcaaatgg tacacatctt ctgaggcttc tttgatctct ttgcctggag agtcagacac tgtaaacagt ttttttcttc ggttttttaa cagtggtgta tttttgatga atttctctaa tcttctttct tggaaaatac gtagataaat tgactagaga cataattaag agtacccata gcaaaaattt tatactggaa gtgacatttc actgaaaaaa tgagaaatca ttttaaatga agcataaaaa gcaattaaat taaaattttg agttgctaag ttgtccttca tctaaccatc gtcttttcca gtccttccaa cagtccaaag gctcagcctt ttaactatat atttgtccta cagcaatgat caatgtgcct tttttcactc tgttagagtg tccagctttt gaacagggtg ctcccatgtc gtaaggtgat agtcaaaggc tttctttatg gttgtttttt aaaccttatt aataccagat atctagcatt agctagacaa gtatatatat tttttaatat caaaaggaga atgaaataga tggttttaaa cctgggtatt ttacaagttt 55740 55800 55860 55920 55980 56040 56100 56160 56220 56280 56340 56400 56460 56520 56580 56640 56700 56760 56820 56880 56940 57000 57060 57120 57180 57240 57300 57360 57420 57480 57540 57600 57660 57720 57780 57840 57900 57960 58020 58080 58140 58200 58260 58320 58380 58440 58500 58560 58620 58680 58740 58800 58860 58920 58980 59040 59100 59160 59220 59280 71 tggtagtttc attaaatgaa agaccaaaaa acaatataaa ggaatggaaa tacagaatga caaacaccct aacactgaaa cagcaaaaac tcagacttga aaatccctta atagagtgcc agaccatccc aaataactgt tgaatgcaga gcaaagaaat gagataccaa acctaacaga aaaagacctt ggacatcctg tggaggtgat gctgcactca gacagttttc aattgtactc gcaatatgtg cagagatcaa catctattta gtggaaactt gtatgcaggt aggaaaagga gtacatcatg aatatcaatc aagagcctct cagaaaacta gtggaaacag agccatgaaa atattaaaat ttccagtagt tgatgctttt atccaaccag gaagctgaaa cctgatgctg ggatggcatc cagggaggcc ctgaactgaa cgtggtaagt taatccattt tgttctgaat cctatagcat ctggaaactt tggttgtagg tagagtttct ttgtgttatt tgtcagttca tctctgtact ttttatttct taactcaaat tttttttttc ttccaggtca attgttcaaa agtattacag taatagttac aaaaaaaaga cctgctagaa agtaggaagt agcagggcaa cttccaacaa tcagattaat aagacaggga actgaagaaa tgattataca tgatgaacta tgtggaaaag gaaaagaaga gttccaaaga agaggaaaac aggaacattt agcagaagat cacgacccaa gaatgtgaag ggaattccag atatgtcagc attccaatcc atctcacacg aaccatgaac attgccaaca tgctttattg ctgaaagaga caggaagcaa gtatgtcaag agaaaggctg acctcagata tgatgaaagt agatcatggc tgtcagaatt ttaaaggacg cggagacatt gatgtatgga gaactgtggt tccatcctaa ctccaatact ggaaagattg accgacgtaa tggcatgctg ctgaacaata ggaagcgcat acatttaagg ttattttttg ttcttgtgaa ttcatttcta ttcttcgctt ttctacaagc tgtcgttctt attactgtgt ttctggactt tcaaatattt gttggtgcat attcttttct cttatctgtt tctgtttgtt aatcctatta ataatggaaa taattataat ctaacatccc caagaaacac aagctaatag cacaagagaa tatattcttt gctgactgtg gtgggaaaaa gtggaagtga tggacggagg aaatgcaaaa gaagtgaaaa atagcaagga aacagaatgg catgcaaaga attaagaaga ataatcatga tcaggtgggc ttgagctatt aaatttggaa caaagaaagg ctagtaaagt ttccagttgt tctgctggat actatgccaa tgggaatatc cagttagaac gctgtatatt ggctggagaa tgcagatgac gaaagaggag atctggtccc tattttcctg cttgctcctt actttgccaa tgtgagagtt gttggagaag aggagattag ttggccacct agggcaggag tggacatggg cggttcatgg taaaataatt acatgcacac taattattga taggtctttt gttggtttgg catcaaatct tcatcactct cagatttgag cccttgttgc gtctcagtgt ggctgactat tcctgagtcc ttaatgttgt ctatattctg cttctgtttc tgatctttag ttttctgttg agatgttatc tgcaagccat aaaaagatgt ctggagtaac agttttgcca gactctacac gcagccaaag gctcagatca ccactagacc gaaatagatt tttgtgacat aagcaaaatg gcaaaggaga gagataagaa gaaagactag ggggctcaat ggtggtaaaa aggtgtgatc cttaggaagc tgaaatccta aactcagcag caatgccaaa aatgctcaaa tcaagctggt catggaaaaa agcctttgac agaccacctg tggacatgga gtcaccctgc gcacaagctg accaccctta agtgaaaaag atcacttcat ggctccaaaa ggaaggaaag caaagatcca ggactataaa actcttgaga tcctgggtat gatgtgaaga gagaagggga tttgggtgga ggtcgcaaag acatagaatt attttcatct tatatatgct ccttctctta tggtgctaaa gaacgagagt agatatacca aagtcagttg ttttaacatt attcctcctg ttcctttccc tttctctctc cctagaggtc ttttgtggca agttattctg ttcttctagg accaatcatg agatttcaca atggaaatat ccttttcatt aggcaaattt agagaatgca atggacatca atggagaagc tgaactcctt attcaggtat taagggccca tgtacaggag gctgtctgag aaaggaaaga agcctttctt agacctcttc aaaggacaga tacacagaag attcacactc atcactacaa aaagatgata tgaccacagg gaatgctcaa attctccaag tttagaaaag gcaagagagt tgtgtggatc acctgcctct acaacaaact ttatttaact gaatcaagat tggcagaaag ttggcttaaa gggaaataga tcactgtgga ttatgaccaa tctagtcaag gaaagctgag gtcccttgga tcattggaag gctgactcat cgacagagga ctctgggaac agtcggacat tgaggggtta gcccagccgg cctattacca tgtttcctgc ttctcttaac cttgttgggt tgccgtctcc ataacctgat ttatctttgt ggggttatcc atgtaaggga ttctctttct tcttaggctg gtgatgtcca ctattgattt tctttggtaa 59340 59400 59460 59520 59580 59640 59700 59760 59820 59880 59940 60000 60060 60120 60180 60240 60300 60360 60420 60480 60540 60600 60 660 60720 60780 60840 60900 60960 61020 61080 61140 61200 61260 61320 61380 61440 61500 61560 61620 61680 61740 61800 61860 61920 61980 62040 62100 62160 62220 62280 62340 62400 62460 62520 62580 62640 62700 62760 62820 62880 72 catcttctct atcgccattc ccctctgcct ggattttggt agttttgatt caccgtctgt gttatttgtt tttgttagtt gtgtatagtt attctttcca tttatatgct attattatca agtaccttct tctttctgac atgaaatctc atgttttcat ttttattgtc gaagttgatc tttttcagta tgtctttcat atcttttctc gctctctgtt ttggctcttt ctcttaaatc ataccttctc ccgatgtatt gtttctatca cttaagtgct aatatttgga gctaaggtac gaaacatgac acatcttaaa gactttgaac taggatcaag ccttataaaa gtggtggtga taaatgaatg agtttcctgg aaggctcatg aaatttacca ttgtgcaacc tgtacccttt tcctactttc caatatttct catgctgtag gtatatactc gctattatga ttcaattctt ctaactgttt accagcaatg tctggttttt ctcattgtca atttattcac agatcaattg aatgaaaaag agtagctgat cttttctttc tcaaacagct atccatgctg aaagaccaaa gttctttttt atttagttgt tttcattttg tcttcttgct tgcatttctc ttgcttttga tttttggtat gtttcatttg ttcattactg gacaatggag actgttcatt atattgtttt cttgctctta tgaagaacat tatttctgtt tggataaaga ttctgatttc cattaccccc tgatttcctt tagctttgaa tcttttcttt gcttatgttc tcttcttatc taggcactat ttttataatt cagaatagtt tctgttgctt gactaagttt gactctgttg attatcaatt acagttgctg gggtagtgac tacaactttt tgatgaggga gtgtgacatg tcatataagt cgtgttaaat ttgtgatatt agagctctct ttagacaatt agcactgcgg aaataacagc tgtttctata tcttttgtgt catgtatcag attttgttta ataatgctgt tttggcatat gaggaactgc tacaaaggtt tttttttttt ttttgatttg tttgtatatc tttttctgta tttaataaaa gtccttagtg caaatctatg gttttaaaca cctaactcac catgtttaaa cactagcatt ttctctggga attaactttc tcttctctct taataataag gaaatgcctg tgagttgcat gtattatttt acttagcaca ggagtctgga atatatttga atgtaaacat attccttctt cttttactgt tgtcagaaaa atctaagttt tgttgagaaa caaagatttt ttgatttatt aaatttcacc ctggggctag ttttttagta ttaccattca ggtcttaaac tgtgattctc aaagtttgat atcttttttt tattctacac tcttccttca gcagatattc caagagctgg caaaagagct gtccctctag gtcgatgtgg aaggatcttg ttacatatga ggataaaatc gtagtgtagt tcattgtttc ttatgtgtat ttcattttca ttcctatagc aatttgtcta ctggcttact aatctcattc ttcatctgtt tatgaatatt tcctagaagg caaattgttt ccactttctt tgccttgata catttcccaa ttggtgaaat ttgtttctta tatttctatc gaaatttgcc ctctatcgtt ttttgtccag cactattgga taaatttaaa attctgaatt ttttatcttg tgtaatgtgg gccctctggt cgatgttgag tttaggtctt gaactgcttg ttcccattct atatattata acactttaac attctctctt aattacccat gcctctctgg cttctttagt atgtctttat ttattttctt tcagttgtca ctctgatttt tttttaagga cattattttt agtacacata tcttccatcc aactttcttt tcattgcttt tgctataacc acctctatta tttttccttt agaatattgt gagaagattt ttaatctagt actgtttttg agaggtttat cttttcaaaa caatatcctt gtagtgatat taaaatttac tgaataatgt tatgtaagag ttttttgtgt aattcagtga gaacttgttc ctccatctcc ttttatgtac taatttatca ctttaaaagg cagggacatg ggtgtacaag gaattgctgg ttaacagcaa tacattctca ataaccatcc tgactaataa gtctaggtga cagctgcata ttcttagcct cccaatatgt ctttgttggc cttttctatc actctctttt agtaaatttt ctttttctga tccattcatc ttttcattct gtgaggtgat catcttttca ttgcctattt tatgttttgg gagggttgtc ttgataaatt tccatttatt tatattaacc actacccatt gtgtccatgt ttagatatgc tataccttct tcaacactgt gttaaaagct ctgaagttag tttttgaatc tcaaatattg tgataccctt tttgctctaa cttcagctgt tttcagttct ccaatttaaa tatggatccc gaattatact gtttgaaaaa tcatttgttt tataaagatt tttattctta caagacaaac ctatcaaaat ataaatatcc aactgttctg acacacacac tggtggatta atcgttgttg ggtaaaatat cattaaatat ttgatcctac caaggttgtg ctcatataaa aaatcttttc tttaataata tgggttgttt tatctgtgtg atcatatgat atgcaccatt aaccacaaac taataagtgt tgttcagcat aatgcccttt tgggtctgca ttcaactgcc agtttctatc tttttagctc tgttatcaat tgttcacctt taaaggctaa 62940 63000 63060 63120 63180 63240 63300 63360 63420 63480 63540 63600 63660 63720 63780 63840 63900 63960 64020 64080 64140 64200 64260 64320 64380 64440 64500 64560 64620 64680 64740 64800 64860 64920 64980 65040 65100 65160 65220 65280 65340 65400 65460 65520 65580 65640 65700 65760 65820 65880 65940 66000 66060 66120 66180 66240 66300 66360 66420 66480 73 aattatttat gttgtagcat tagttccctg actggaccac atctttactt ggtatataag atgtgcttaa aaacatcatc tgattcacta atgaaattta ttgctccagc gctcgttgcc aatttcaatc acccatttta tctgacttga ttcttatcta ccagtgtcta tgaatgaatg tctcaattat tttctcctaa ccttacttgg cagccagtgc actttgaccc gctgttctcc gatcatggag ttatctatcc tctaagtatt tcactgtata tgccattaga aaaagagaaa acttttatat tcttttgtgc aagaggaagg atcagttctt acgtggtgaa gacctgttct aatagataag agcccattaa tacttgtatc agagtttctt cacatgtcaa gtttgttctc tctgtttaga tcgctcagta ctattaaatc cacaagccca tctatttccg taccactgca ggactgttgt agcttataac tggcttttat tggggtcatt cagaaagaaa tattttgatt cttttatcat tttacatttg atcaaaacag aaagagactt cttctagtcc aatctagtgt tttttggcca gcgtgatctt accagggatc caggggagtt ggtaggagta acaaaatgat accattctta tctgcagagc gcagggctca gaggaaacca aactgattgt cagagtgttt acggaagttc ctctctagtc gctgtataat ttaccattct aaacaatgct aaaatgattg tgggatgtgc acaggtctat caatttgaat tgtgaaacag tcagcagtgc acaatgtttg catggcttgc cttttgtgtt taaggttggc gctatcagcg caacttagtc caatattttt ggtattagcc cctcttttag aagtattgaa ctatggctgt caagacacgg gccctttgaa cagctactta caatgttgtg cattctctct gtagtaacat tcccaaacac taagtctgtg ttccacatac tgacaatctc tgagaaccat gaaaaataat gttgatggga aaggctccct actggcagcc cagtcttcta ccctcagaac ctttctgtgg actagcactg ccagtttgat aattagtact gataggaatt ctttgtgggt gcatcagatc aatattttat gatagcctcc cactaggtct tcgttgtggc aaacctggaa cccaaagcta ttgaaaacat acagtttatt ctgtcagaaa ggctgtgttg taggtctcag ggggtgtgct gacatgtgtg gttggggatc cagagcccca cctcaccttt cattatttat tcctccacta tggtctagag tttatgcagt tgttattcta aatttaatag gcccctgagg gctgatggaa ttctttattc gtgcctagta aaaagagttg ttgaaagagg gctgaccatc tctcagctct acaaaaccca aaaaatgatg cccacactga atttggtgct tttccgcaga ggcttccggc agggtaatga ttaattactg cttttttaaa atagtttcag caagttcccc taggaccttg cctaactatc agtctgtttc aaggaatgtc taggtccatc ggaattaggg tctatgcttg aattataact ctcctatatt aaattcatgc tcaaatactt ctgtttttgt tgatcacccc ggcattttga accttctccc ttattacagt tatttacaaa atcattttac tcgtagttgt accaccctct gacctagtgt tagttgtggc atgtgaacgc ctcttgcttt attctttatg ttctgcatgt ccccatctta agacacttat aggggccccc aaaaatcaac tccaagagcc aaatgctgcc agcacataag tagagaaaga aagtgtgttt ttggtactac gaatataagg aaggtactca tacttagaaa tgcatgacag ccagaaattc gagagaacct aggtatgcta actagaaaag agttttataa cagaattcta tttatacagc cccacatttt agtaaaataa ataatctttt tagaacctca cctgaagaaa cttatgaaaa tcaactatgg atttggtggg aattccgttt ttttgtttac aaaaagactt gaggacagca tcccatccag ttggttgtcc ccttccccat tgcttcataa aaatgatagt cctgttgctc gtatgttccc atggtatttt ctacaacttg cagttcagta tggggctgac ttttggggac tccggtacca taaccaggta gggttatatt ttttgtttct tttaaaattc ctactttcat acatgttgaa aaagccagta gtgctgccag ctcaataatc acgtgggatc ttagtgtggc gggagcgtgg aagcttaaaa tatggagata gagtatttcc tttattaggg aaaactgttc agaaaggaaa ctctcctagg tgccagggaa catatagcct ccaaacctga cttaatggca ttaagtagca ccagaaactg actgatgttt tgccaataat ttgtctcatc ccctcgaagg cagcccgttg tgaatggata tagtgtggct cattcagctg ggtgggagtg tttcaaactg aggtggtttg aagcccatgt atgtgttctt gttctcaagc gtaagaatta tctatatcct ccaattcaat tgattctctg ccttttctga ggtagatctt tttattttat aggggactca cctgccacct attttaatga attcctttcc ataagttcat tctcctctgt aaatgggcag tctagggaat gagaggaatc tgtttgatga agcctgattc aaaagctata ttacaggttt ggagccagta aatgtttttc tagtgatgct ctctgtatta tacttgtaga acatattatc tttgtaactt ttgaaatccc ttttaattga ttgatatcta 74 atttcttgtc tttataagta tgtaatcagt ccctgtaatt ccagaataac gagatggaaa agatataaat tttgtttagt tgagaatgga gaggaaacag aacaaaaaaa gaagtgcctg agccacaagc agtgtacctt ttcatgaaat ggattcctaa gctcccagga tacgacaaaa gtgaccatca tttatcattg gcacctttgc gctgactcct tctcttgaga gagagacgca tagagaagcc gctgcctagg aatgtctgcc agaccagggc acaaaatgga aggagcaggt gtaatttctt tcatcattct tatgaagatg atttctgata attttcttct tgcaaatgcc tctgggaact accttccaaa aatcatggtg gtgctgtttc attggggtca ctgcttagtc tcatcccttg acaaaagaat tccatcagag tggagcgcag aggaaccctg agtcctgtgt ttgtggctga tataggactg atgggcttag aaatctcaac ggcattttat aggaagatcc tgctctaggt gcacttagtg agaaagatgg gaaacaaaga tgtcttggat atatgggttc aggtttgaat cagtagacat ttatattttc tcacaagtcc ttgtgtgaga tcgatcccct ttctcttggt atatatacag attgggtccc aaacccatct tgccttcacc caaggacttt tagatgcact tgacactttt gacttagtcc ctgccttgca tttgggagaa ttaaacccga atggacttct aatgaatcag tctctccctt gaattttcag gccaagaact catggcacct gaatgtgctc ctgcttttaa ttagggattg tttgtttctt caggcttaca ttcttgagat aacggttact actatatata tgtgtctttc tgctcactct cctcctcgag attggt ttgagttcca taaagttttg tcatgcagta atgctacctt agaattttta agagattcta tcagctataa caagccactg aggaatttta tattgtcttt ttgaagaact tcgtgaccat gctacataag aatttctgtt tcagggaatg tttaagcgga tctttagtaa atggattgtc gtacgtttga ctcacagaaa tttctgtctc cctgcccggg agggtgcccg actttgatca gtgagaaaag tatttgctac ttgaacagct tatctcgtag tgaaggtgat tttccttttt tgcccagtga cagcccacta ctttttctag gggccgagcc ctgatggaac agagagcaaa aaaaattgaa tttagcagga acagcacact agattctctt tcctgacctt ttttctgata aatcattaaa tctctctccg cttctaagac gtgccttcgt 66540 66600 66660 66720 66780 66840 66900 66960 67020 67080 67140 67200 67260 67320 67380 67440 67500 67560 67620 67680 67740 67800 67860 67896 <210> 2 <211> 1593 <212 > DNA <213> Bos taurus <22 0> <221> CDS <222> (1) . . (1593) <22 0> <221> variation <222> (240)..(240) <223> G to A substitution <400> 2 atg ata tta cgc egg ate cca gtt ttg aag aag tac atg gaa aag act 48 Met lie Leu Arg Arg lie Pro Val Leu Lys Lys Tyr Met Glu Lys Thr 15 10 15 cat cag aaa aca gtg ate ttt ggt cag gag aag act ctt cca tgc att 96 His Gin Lys Thr Val lie Phe Gly Gin Glu Lys Thr Leu Pro Cys lie 20 25 30 gca cca ctt eta ace acg gtg gaa gag act ccg cag gtc ate tea get 144 Ala Pro Leu Leu Thr Thr Val Glu Glu Thr Pro Gin Val lie Ser Ala 35 40 45 gga gtc cag gga cat ttt cct gag tgg etc agt ggc tat eta ctt cgt 192 Gly Val Gin Gly His Phe Pro Glu Trp Leu Ser Gly Tyr Leu Leu Arg 50 55 60 gtg gga cct ggg aaa ttc gag ttc ggg aag gat aag tac aat cac tgg 240 Val Gly Pro Gly Lys Phe Glu Phe Gly Lys Asp Lys Tyr Asn His Trp 75 65 70 75 80 ttt gat gga atg get tta ctt cac cag ttc aag gtg gag aag ggc aca 288 Phe Asp Gly Met Ala Leu Leu His Gin Phe Lys Val Glu Lys Gly Thr 85 90 95 gtg aca tat agg age aag ttt eta cag agt gat aca tat aag gcc aac 336 Val Thr Tyr Arg Ser Lys Phe Leu Gin Ser Asp Thr Tyr Lys Ala Asn 100 105 110 agt gat cgt gat cga att gtg ate tea gaa ttt ggt aca ttg get etc 384 Ser Asp Arg Asp Arg lie Val lie Ser Glu Phe Gly Thr Leu Ala Leu 115 120 125 cct gac cca tgc aag aat gtt ttt gaa cgt ttc atg tea aaa ttt gaa 432 Pro Asp Pro Cys Lys Asn Val Phe Glu Arg Phe Met Ser Lys Phe Glu 130 135 140 aag cct gca att act gat aac ace aat gtc aac tat gtg ctg tac aag 480 Lys Pro Ala lie Thr Asp Asn Thr Asn Val Asn Tyr Val Leu Tyr Lys 145 150 155 160 ggt gat tac tac ctt agt acg gaa acc aac ttt atg aat aaa gtg gac 528 Gly Asp Tyr Tyr Leu Ser Thr Glu Thr Asn Phe Met Asn Lys Val Asp 165 170 175 att gaa acc ctg gaa aaa aca gaa aag gtt aac tgg aca aaa ttt ate 576 lie Glu Thr Leu Glu Lys Thr Glu Lys Val Asn Trp Thr Lys Phe lie 180 185 190 get gtg aac gga gca act gca cat cct cat tac gat cca gat gga aca 624 Ala Val Asn Gly Ala Thr Ala His Pro His Tyr Asp Pro Asp Gly Thr 195 200 205 acg tac aac atg ggg aac tcc tat ggg aaa cat ggt tcc tgc tat aat 672 Thr Tyr Asn Met Gly Asn Ser Tyr Gly Lys His Gly Ser Cys Tyr Asn 210 215 220 gtt att egg gtt cct cca gaa aaa tct gac cct ggg gag aca ate cat 720 Val lie Arg Val Pro Pro Glu Lys Ser Asp Pro Gly Glu Thr lie His 225 230 235 240 gga gcc cag gtg ata tgt tct att get tct gag gag ggg atg aga cct 768 Gly Ala Gin Val lie Cys Ser lie Ala Ser Glu Glu Gly Met Arg Pro 245 250 255 tct tac tac cac age ttt gga atg aca agg aac tac ate ate ttc att 816 Ser Tyr Tyr His Ser Phe Gly Met Thr Arg Asn Tyr lie lie Phe lie 260 265 270 gaa cag cct eta aag ate aat ttg tgg aga ate ate tct tct aaa att 864 Glu Gin Pro Leu Lys lie Asn Leu Trp Arg lie lie Ser Ser Lys lie 275 280 285 cga gga aag gcc ttt tea gat gga ata age tgg gaa ccc cag tat aat 912 Arg Gly Lys Ala Phe Ser Asp Gly lie Ser Trp Glu Pro Gin Tyr Asn 290 295 300 aca egg ttt cat gtg gtg gat aag cat act ggt cag ctt ctt cca ggg 960 Thr Arg Phe His Val Val Asp Lys His Thr Gly Gin Leu Leu Pro Gly 76 305 310 315 320 atg tac ttc agt aaa cct ttt gtt act ttt cac caa ate aat gcc ttt 1008 Met Tyr Phe Ser Lys Pro Phe Val Thr Phe His Gin lie Asn Ala Phe 325 330 335 gag gac cag ggc tgt gtt gtc att gat ttg tgc tgt cag gat gat gga 1056 Glu Asp Gin Gly Cys Val Val lie Asp Leu Cys Cys Gin Asp Asp Gly 340 345 350 gga att tta gaa gtc tac caa eta cag aat ctt agg aaa act ggg aaa 1104 Gly lie Leu Glu Val Tyr Gin Leu Gin Asn Leu Arg Lys Thr Gly Lys 355 360 365 gag ctt gat cag gtc tat aat tta ata gcc aga aat tcc cct cga agg 1152 Glu Leu Asp Gin Val Tyr Asn Leu lie Ala Arg Asn Ser Pro Arg Arg 370 375 380 ttt gtt ttg ccc tta ctt ggc aat ttg aat gcc cct gag gga gag aac 1200 Phe Val Leu Pro Leu Leu Gly Asn Leu Asn Ala Pro Glu Gly Glu Asn 385 390 395 400 ctc age ccg ttg acc tat tct tea gcc agt get gtg aaa cag get gat 1248 Leu Ser Pro Leu Thr Tyr Ser Ser Ala Ser Ala Val Lys Gin Ala Asp 405 410 415 gga aag att tgg tgc tct tat gaa aat eta tat cct gag gac eta aaa 1296 Gly Lys lie Trp Cys Ser Tyr Glu Asn Leu Tyr Pro Glu Asp Leu Lys 420 425 430 gag gaa gga agt att gaa ttt ccg cag ate aac tat ggc caa ttc aat 1344 Glu Glu Gly Ser lie Glu Phe Pro Gin lie Asn Tyr Gly Gin Phe Asn 435 440 445 gga aaa aag tat cag ttc ttc tat ggc tgt ggc ttc egg cat ttg gtg 1392 Gly Lys Lys Tyr Gin Phe Phe Tyr Gly Cys Gly Phe Arg His Leu Val 450 455 460 ggt gat tct ctg ate aag gtt gac gtg gtg aac aag aca egg agg gtt 1440 Gly Asp Ser Leu lie Lys Val Asp Val Val Asn Lys Thr Arg Arg Val 465 470 475 480 tgg aga gaa gat ggc ttt tat ccc tea gaa cct gtt ttt gtt ccg gta 1488 Trp Arg Glu Asp Gly Phe Tyr Pro Ser Glu Pro Val Phe Val Pro Val 485 490 495 cca gga gcc agt aaa gaa gat gat ggg gtc att ctt tct gtg gtg ate 1536 Pro Gly Ala Ser Lys Glu Asp Asp Gly Val lie Leu Ser Val Val lie 500 505 510 acc cct aac cag aac aaa aaa aaa ttt tct tct tgt ctt gga tgc caa 1584 Thr Pro Asn Gin Asn Lys Lys Lys Phe Ser Ser Cys Leu Gly Cys Gin 515 520 525 gaa ctt tga 1593 Glu Leu 530 <210> 3 77 <211> 530 <212> PRT <213> Bos taurus <400> 3 Met lie Leu Arg Arg lie Pro Val Leu Lys Lys Tyr Met Glu Lys Thr 15 10 15 His Gin Lys Thr Val lie Phe Gly Gin Glu Lys Thr Leu Pro Cys lie 20 25 30 Ala Pro Leu Leu Thr Thr Val Glu Glu Thr Pro Gin Val lie Ser Ala 35 40 45 Gly Val Gin Gly His Phe Pro Glu Trp Leu Ser Gly Tyr Leu Leu Arg 50 55 60 Val Gly Pro Gly Lys Phe Glu Phe Gly Lys Asp Lys Tyr Asn His Trp 65 70 75 80 Phe Asp Gly Met Ala Leu Leu His Gin Phe Lys Val Glu Lys Gly Thr 85 90 95 Val Thr Tyr Arg Ser Lys Phe Leu Gin Ser Asp Thr Tyr Lys Ala Asn 100 105 110 Ser Asp Arg Asp Arg lie Val lie Ser Glu Phe Gly Thr Leu Ala Leu 115 120 125 Pro Asp Pro Cys Lys Asn Val Phe Glu Arg Phe Met Ser Lys Phe Glu 130 135 140 Lys Pro Ala lie Thr Asp Asn Thr Asn Val Asn Tyr Val Leu Tyr Lys 145 150 155 160 Gly Asp Tyr Tyr Leu Ser Thr Glu Thr Asn Phe Met Asn Lys Val Asp 165 170 175 lie Glu Thr Leu Glu Lys Thr Glu Lys Val Asn Trp Thr Lys Phe lie 180 185 190 Ala Val Asn Gly Ala Thr Ala His Pro His Tyr Asp Pro Asp Gly Thr 195 200 205 Thr Tyr Asn Met Gly Asn Ser Tyr Gly Lys His Gly Ser Cys Tyr Asn 210 215 220 Val lie Arg Val Pro Pro Glu Lys Ser Asp Pro Gly Glu Thr lie His 225 230 235 240 Gly Ala Gin Val lie Cys Ser lie Ala Ser Glu Glu Gly Met Arg Pro 245 250 255 Ser Tyr Tyr His Ser Phe Gly Met Thr Arg Asn Tyr lie lie Phe lie 260 265 270 Glu Gin Pro Leu Lys lie Asn Leu Trp Arg lie lie Ser Ser Lys lie 275 280 285 78 Arg Gly Lys Ala Phe Ser Asp Gly lie Ser Trp Glu Pro Gin Tyr Asn 290 295 300 Thr Arg Phe His Val Val Asp Lys His Thr Gly Gin Leu Leu Pro Gly 305 310 315 320 Met Tyr Phe Ser Lys Pro Phe Val Thr Phe His Gin lie Asn Ala Phe 325 330 335 Glu Asp Gin Gly Cys Val Val lie Asp Leu Cys Cys Gin Asp Asp Gly 340 345 350 Gly lie Leu Glu Val Tyr Gin Leu Gin Asn Leu Arg Lys Thr Gly Lys 355 360 365 Glu Leu Asp Gin Val Tyr Asn Leu lie Ala Arg Asn Ser Pro Arg Arg 370 375 380 Phe Val Leu Pro Leu Leu Gly Asn Leu Asn Ala Pro Glu Gly Glu Asn 385 390 395 400 Leu Ser Pro Leu Thr Tyr Ser Ser Ala Ser Ala Val Lys Gin Ala Asp 405 410 415 Gly Lys lie Trp Cys Ser Tyr Glu Asn Leu Tyr Pro Glu Asp Leu Lys 420 425 430 Glu Glu Gly Ser lie Glu Phe Pro Gin lie Asn Tyr Gly Gin Phe Asn 435 440 445 Gly Lys Lys Tyr Gin Phe Phe Tyr Gly Cys Gly Phe Arg His Leu Val 450 455 460 Gly Asp Ser Leu lie Lys Val Asp Val Val Asn Lys Thr Arg Arg Val 465 470 475 480 Trp Arg Glu Asp Gly Phe Tyr Pro Ser Glu Pro Val Phe Val Pro Val 485 490 495 Pro Gly Ala Ser Lys Glu Asp Asp Gly Val lie Leu Ser Val Val lie 500 505 510 Thr Pro Asn Gin Asn Lys Lys Lys Phe Ser Ser Cys Leu Gly Cys Gin 515 520 525 Glu Leu 530 <210> 4 <211> 20 <212 > DNA <213> Artificial <22 0> <223> Synthetic <400> 4 gctaggaacc catcccactt <210> 5 79 <211> 19 <212> DNA <213> Artificial <22 0> <223> Synthetic <400> 5 ctgaaatcaa accccaaag 19 <210> 6 <211> 21 <212 > DNA <213> Artificial <22 0> <223> Synthetic <4 0 0 > 6 ttcaactgct ttctcccttg a 21 <210> 7 <211> 22 <212 > DNA <213> Artificial <22 0> <223> Synthetic <400> 7 gggcagaatg gatctaggta aa 22 <210> 8 <211> 24 <212> DNA <213> Artificial <22 0> <223> Synthetic <400> 8 atgcatctaa atagagacct accc 24 <210> 9 <211> 20 <212 > DNA <213> Artificial <22 0> <223> Synthetic <400> 9 acaatgcatg tgagccaaaa 20 <210> 10 <211> 20 <212 > DNA <213> Artificial <22 0> <223> Synthetic <400> 10 tgcctagtcc gcaaatacaa 20 <210> 11 <211> 21 <212> DNA <213> Artificial <22 0> <223> Synthetic <400> 11 tccatcagtc agttggctag g <210> 12 <211> 22 <212 > DNA <213> Artificial <22 0> <223> Synthetic <400> 12 tgacaagtcc ccataactgt tg <210> 13 <211> 24 <212 > DNA <213> Artificial <22 0> <223> Synthetic <400> 13 ttttctgata tgcaaaattc actg <210> 14 <211> 22 <212> DNA <213> Artificial <22 0> <223> Synthetic <400> 14 cagattggct tctacattcc at <210> 15 <211> 20 <212 > DNA <213> Artificial <220> <223> Synthetic <400> 15 atttgccaga gaggcaagag <210> 16 <211> 20 <212 > DNA <213> Artificial <22 0> <223> Synthetic <400> 16 tgccaataat gttgactgga <210> 17 <211> 20 <212 > DNA <213> Artificial <22 0> <223> Synthetic <400> 17 tctgcaactc ttttgcaagc <210> 18 <211> 20 <212> DNA <213> Artificial <22 0> <223> Synthetic <400> 18 ggtattagcc cccacactga <210> 19 <211> 20 <212 > DNA <213> Artificial <22 0> <223> Synthetic <400> 19 ttcaaagggc agaacaggtc <210> <211> <212 > DNA <213> Artificial <22 0> <223> Synthetic <400> ctgttgtact ggcagccaaa <210> 21 <211> 16 <212> DNA <213> Artificial <22 0> <223> Synthetic <400> 21 aggtatcaaa ctggaa <210> 22 <211> 20 <212 > DNA <213> Artificial <22 0> <223> Synthetic <400> 22 aatgaatcag caagccactg 82 <210> 23 <211> 23 <212 > DNA <213> Artificial <22 0> <223> Synthetic <400> 23 ttcattcaaa cctagttccc aga 23 <210> 24 <211> 21 <212> DNA <213> Artificial <22 0> <223> Synthetic <400> 24 cagcccacta gaggaaacag a 21 <210> 25 <211> 20 <212 > DNA <213> Artificial <22 0> <223> Synthetic <400> 25 tgatggtcac aaggtcagga 20

Claims (1)

1. A method of determining genetic merit of bovine with respect to milk or tissue colour or P-carotene content or with respect to capability of producing progeny that will have increased or decreased milk or tissue colour or P-carotene content, which comprises the step of determining the BC02 allelic profile of the W80Stop G/A polymorphism in the BC02 gene or one or more polymorphisms in linkage disequilibrium with the W80Stop G/A polymorphism in the BC02 gene of said bovine, and determining the genetic merit of the bovine on the basis of the BC02 allelic profile. 2.The method as claimed in claim 1, wherein the milk P-carotene content is milk fat P-carotene content. 3.The method as claimed in claim 1, wherein the allelic profile is determined with respect to DNA, mRNA and/or protein obtained from said bovine. 4.The method as claimed in any one of claims 1 to 3, wherein the allelic profile is determined by determining the presence or absence of the A allele at the W80Stop G/A polymorphism of the bovine BC02 gene. 5.The method as claimed in any one of claims 1 to 4, wherein the allelic profile is determined by determining the presence or absence of the G allele at the W80Stop G/A polymorphism of the bovine BC02 gene. 6.The method as claimed in any one of claims 1 to 5 wherein the allelic profile of the W80Stop G/A polymorphism in the BC02 gene is determined by the use of one or more polymorphisms in linkage disequilibrium with this polymorphism. 7.The method as claimed in claim 1 wherein the allelic profile is determined by determining the expression or activity of a BC02 gene or gene product. 8.The method of identifying or selecting bovine having a desired BC02 allelic profile comprising determining the allelic profile according to the method of any one of claims 1 to 7 and identifying or selecting said bovine on the basis of the determination. 9.A method of determining genetic merit of bovine with respect to milk or tissue colour or P-carotene content, or with respect to capability of producing progeny that will have increased or decreased milk or tissue colour or P-carotene content, the method comprising providing data about the BC02 allelic profile of the W80Stop G/A polymorphism in the BC02 gene or one or more polymorphisms in linkage disequilibrium with the W80Stop G/A polymorphism in the BC02 gene of said bovine, and determining the genetic merit of the bovine on the basis of the data. 10. A method for identifying or selecting bovine with respect to milk or tissue colour or P-carotene content, or with respect to capability of producing progeny that will have one or more desired milk or tissue colour or P-carotene content traits, the method comprising providing data about the BC02 allelic profile of said bovine, and identifying or selecting the bovine on the basis of the data, wherein the the BC02 allelic profile comprises data indicative of the presence or absence of one or more alleles at the W80Stop G/A polymorphism in the BC02 gene, or one or more polymorphisms which are in linkage disequilibrium with the W80Stop G/A polymorphism in the BC02 gene. 11. The method as claimed in claim 10, comprising the step of amplifying at least a fragment of the bovine BC02 gene sequence to determine the presence or absence of the one or more alleles. 12. The method as claimed in claim 11, wherein the primers used in the amplification are selected from the group consisting of SEQ ID NOs: 4 to 25. 13. The method as claimed in claim 11 or claim 12 wherein the presence or absence of the one or more alleles is determined by determining the expression or activity of a BC02 gene or gene product. 14. A probe or primer having about at least 12 contiguous bases of SEQ ID NO: 1 or SEQ ID NO: 2, and comprising an adenosine at the position corresponding to the W80Stop G/A polymorphism in the BC02 gene, or comprising a nucleotide capable of hybridising to a nucleotide capable of hybridising under stringent conditions to an adenosine at the position corresponding to the W80Stop G/A polymorphism in the BC02 gene. 15. A probe or primer having about at least 12 contiguous bases of one of SEQ ID NO: 4 -25. 27.85 A probe or primer comprising a nucleotide sequence having 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 1 to about 2000 nucleotides of the W80Stop G/A polymorphism in the BC02 gene. A pair of primers comprising two primers as claimed in claim 16. Collected semen produced by bovine identified by the method of any one of claims 8 or 10 to 13. A method of selecting a herd of bovine, comprising selecting individuals by the method of any one of claims 8 or 10 to 13, and segregating and collecting the selected individuals to form the herd. A herd of bovine selected by the method of claim 19. A herd of bovine comprising two or more bovine, wherein the bovine are the progeny of bovine selected by the method of any one of claims 8 or 10 to 13. Collected or pooled milk produced by bovine as claimed in claim 20 or 21. Collected or pooled milk produced by bovine as identified by the method of any one of claims 8 or 10 to 13. Collected or pooled milk as claimed in claim 22 or 23 having increased or decreased milk colour or increased or decreased P-carotene content when compared to milk produced by a bovine having a BC02 gene comprising the nucleotide sequence of SEQ ID NO: 2 or a functional variant thereof or capable of expressing a functional equivalent of the BC02 gene product of SEQ ID NO:2. A dairy product made from the milk as claimed in claim 22 or 23. A kit for genotyping a bovine with respect to one or more milk or tissue colour or P-carotene content, comprising a probe or primer as defined in any one of claims 15 to 16 or a pair of primers as defined in claim 17. An isolated, purified or recombinant nucleic acid molecule comprising nucleotide sequence selected from (a) at least 12 contiguous nucleotides of SEQ ID NO:l and comprising the W80Stop G/A polymorphism; or (b) at least 12 contiguous nucleotides of SEQ ID NO:2 and comprising the W80Stop G/A polymorphism; or (c) at least 12 contiguous nucleotides of a variant of SEQ ID NO:2; or (d) any one or more of SEQ ID NOs:4 - 25; or 86 (e) a complement of any one of (a) to (d); or (f) a sequence of at least 12 contiguous nucleotides and capable of hybridising to the nucleotide sequence of any one of (a) to (e) under stringent conditions. 28. A vector comprising the nucleic acid of claim 27. 29. A method of determining genetic merit of a bovine with respect to milk or tissue colour or P-carotene content, or with respect to capability of producing progeny that will have increased or decreased milk or tissue colour or P-carotene content, the method comprising determining milk or tissue colour or P-carotene content of the bovine, determining the BC02 allelic profile of the W80Stop G/A polymorphism in the BC02 gene or one or more polymorphisms in linkage disequilibrium with the W80Stop G/A polymorphism in the BC02 gene of the bovine, comparing the BC02 allelic profile of the bovine or the milk or tissue colour or P-carotene content of the bovine with that of a bovine having a known BC02 allelic profile; determining the genetic merit of the bovine on the basis of the comparison.