CN102869788A

CN102869788A - Marker assisted selection of a mammalian subject for desired phenotype

Info

Publication number: CN102869788A
Application number: CN2011800158255A
Authority: CN
Inventors: S·R·戴维斯; K·莱纳特; S·D·贝里; R·G·斯内尔; E·M·贝蒂
Original assignee: ViaLactia Biosciences NZ Ltd
Current assignee: ViaLactia Biosciences NZ Ltd
Priority date: 2010-01-27
Filing date: 2011-01-27
Publication date: 2013-01-09
Also published as: NZ582981A; WO2011093728A1

Abstract

The present invention provides methods of genotyping mammalian subjects for desired lactoferrin phenotypes by determining the lactoferrin genotype of the subject. The invention particularly provides methods wherein the presence or absence of the T allele or the C allele at the 30126 T/C polymorphism, of the A allele or the G allele at the 7447 A/G polymorphism, or of the C allele or the G allele at the -7 G/C polymorphism in the bovine Lf gene, is associated with increased or decreased lactoferrin production or secretion, including increased or decreased milk or colostrum Lf content.

Description

For the marker assisted selection for the mammalian object for expecting phenotype

Invention field

The present invention relates to mammalian object and lactoferrin (Lf) intake, produce, adjust or secrete, the application of marker assisted selection including such as breast or the related quantitative trait locus (QTL) of colostrum Lf contents, especially by the presence for determining polymorphism in the gene related to QTL.

Background

The information included below for being beneficial to understand the present invention.Do not recognize provided herein is any information and prior art or part common knowledge --- being related to presently described or claimed invention --- it is relevant；Or any specific or implicit disclosure quoted or file are prior arts, or formed such as application priority date common knowledge a part.All documents cited herein is all merged in its by quoting herein.

The hereditary basis that cow's milk and colostrum are produced is significant to dairy industry.Correspondingly, breast composition --- for example, relative quantity of main lactoprotein --- hereditary basis of change, and these influences changed to newborn industry characteristics and newborn working properties have turned into important research, the theme for discussing and commenting on.For example; PCT International Application Serial No. PCTs/NZ01/00245 (being disclosed as WO02/36824) reports; ox diacylglycerol-o- acyltransferases (DGAT1) gene pleiomorphism is related to increased milk production and the newborn composition changed; the presence that K232A is mutated particularly in DGAT1 genes causes milk fat percentage, fat yield, solid fats content and the reduction of lactoprotein percentage, while increasing newborn volume and lactoprotein yield.In another example, PCT International Application Serial No. PCTs/NZ02/00157 (being disclosed as WO03/104492) reports, bovine growth hormone acceptor (GHR) gene pleiomorphism is related to increased newborn volume and the newborn composition changed, particularly, the presence of F279Y amino acid variants causes to increase the butterfat and lactoprotein percentage of milk production and reduction, and live-weight (live weight) reduction.For breast composition other characteristics, change it is basic not clear.

The ability of regulation breast or colostrum composition has the potentiality for changing agricultural practice, promoting animals and humans health, and produces the special potentiality to meet the product of widespread demand.Particularly, ox is evaluated to select expression to expect phenotype in hereditary Shangdi, and the method for the ox of breast or colostrum lactoferrin content will be useful as desired.

Played a crucial role during for example, colostrum is by mother's protein, such as lactoferrin passes to offspring --- this growth and health important --- to neonatal mammal.Cow's serum and mammal gland secretion (it represents the secretion of these in mammal) contain Lf.Lf is considered as providing to protect in neonatal mammal resisting certain micro-organisms pathogen and their toxin.These anti-microbial effects are it is reported that provide passive immunity until the self immune system of new born bovine is ripe.

So, Lf is ideally suited for being used as nutraceutical or food supplement.Lf can suppress or reduce the ill-effect due to pathogen with probiotic combinations.--- such as nose, eye, ear, lung, the breast and vagina --- pathogen of infection that to target causes mucomembranous surface using nutraceutical composition is used as including Lf.And the product containing Lf is suitable to intestines and oral health application.

Health benefits can be provided and be expected to economically valuable by increasing or decreasing the strategy of breast or colostrum Lf contents.Lf is generally present in the emulsion of cow with low-level, and Lf commerical grade purifying usually requires cation-exchange chromatography, it is ultrafiltration and diafiltration afterwards, or ion-exchange chromatography, it is eluting salt scheme afterwards to provide the pure or relatively pure protein portion containing Lf.It will be appreciated, however, that these methods and the product for therefore utilizing these methods to prepare are relatively expensive.

Marker assisted selection --- it provides the ability for following special favourable genetic alleles --- is including recognizing that one or more related with to character or part limits the gene of character or the DNA molecular marker of genome isolation.DNA marker has several advantages.They are relatively easily measured and clear, and because DNA marker is codominant, the animal of heterozygosis and homozygosis can discriminatively be recognized.Once setting up Mk system, can easily formulate selection and determine because DNA marker can be in the sample containing DNA from single object --- whether embryo, childhood or adult --- any time after being collected determines.

It is an object of the invention to provide produce, adjust or secretion phenotype with desired Lf, the method of the marker assisted selection of mammalian object including desired breast or colostrum Lf contents, or the object for being recognized or being selected using the method for the present invention and the breast or colostrum that are produced by the object selected are provided, the purposes of such breast or colostrum, or it is supplied to the useful selection of the public.

Summary of the invention

The present invention relates to coding lactoferrin (Lf) gene pleiomorphism is illustrated to mammalian mucosal secretion, Lf concentration in such as breast or colostrum is particularly the effect of breast or colostrum Lf contents.Especially, the present invention relates to recognize ox Lf genes 7447A/G (I145V) polymorphism first.The invention additionally relates to by the C allele in 30126T/C polymorphisms and the G allele in 7447A/G polymorphisms and associated with the generation of the breast with reduced Lf contents and colostrum in the C allele of -7G/C polymorphisms, and by the T allele in 30126T/C polymorphisms, the A allele in 7447A/G polymorphisms and associated with having the generation of the newborn and colostrum of increased Lf contents in the G allele of -7G/C polymorphisms.

This produces many and single aspects of the present invention.

On the one hand, the present invention provides the method for determining mammalian object genetic state, the ability with the breast increased or decreased or the offspring of colostrum Lf contents is determined to the genetic state of mammal including determining the Lf allele distributions (allelic profile) of mammal and on the basis of Lf allele distributions on breast or colostrum Lf contents, or on producing.

In one embodiment, mammalian object is ox.

In one embodiment, the genetic state on breast or colostrum Lf contents is to produce breast or colostrum with increased breast or colostrum Lf contents.

In one embodiment, the genetic state on breast or the ox of colostrum Lf contents is the adaptability milked once a day.

In one embodiment, the genetic state on breast or the ox of colostrum Lf contents is that breast or colostrum with increased Lf contents are produced when milking once a day.

In one embodiment, the genetic state on breast or the ox of colostrum Lf contents is the ability that offspring's --- it will produce breast or colostrum with increased Lf contents --- is produced when milking once a day.

Correspondingly, in one embodiment, the method that the present invention provides identification or selection ox, this method includes expression or the activity for determining Lf gene outcomes, with the identification on the basis of the measure or the such ox of selection, it produces breast, colostrum, blood, serum, Mucosal secretions, or with the increased mucomembranous surface of Lf contents, or offspring can be produced --- it produces breast, colostrum, blood, serum, Mucosal secretions, or with the increased mucomembranous surface of Lf contents.

In another embodiment, the genetic state on breast or colostrum Lf contents is to produce breast or colostrum with reduced breast or colostrum Lf contents.

Correspondingly, in one embodiment, the method that the present invention provides identification or selection ox, this method includes expression or the activity for determining Lf gene outcomes, with the identification on the basis of the measure or the such ox of selection, it produces breast, colostrum, blood, serum, Mucosal secretions, or the mucomembranous surface with Lf content reductions, or offspring can be produced --- it produces breast, colostrum, blood, serum, Mucosal secretions, or the mucomembranous surface with Lf content reductions.

In various embodiments, increased Lf contents are increased total Lf concentration, such as example, increased [total Lf] mol.L^-1Breast, or increased [total Lf] mol.L^-1Colostrum.Similarly, in various embodiments, the Lf contents of reduction are the total Lf concentration reduced, such as example, [total Lf] mol.L reduced^-1Breast, or [total Lf] mol.L reduced^-1Colostrum.

In various embodiments, the newborn concentration of increased Lf is at least 150mg.L^-1, at least 160mg.L^-1, at least 170mg.L^-1, at least 180mg.L^-1, at least 190mg.L^-1, at least 200mg.L^-1, at least 210mg.L^-1, at least 220mg.L^-1, at least 230mg.L^-1, at least 240mg.L^-1, at least 250mg.L^-1, at least 260mg.L^-1, at least 270mg.L^-1, at least 280mg.L^-1, at least 290mg.L^-1, at least 300mg.L^-1, at least 325mg.L^-1, at least 350mg.L^-1, at least 375mg.L^-1, at least 400mg.L^-1, at least 450mg.L^-1Or at least 500mg.L^-1。

In the various embodiments related to milking once a day, the newborn concentration of increased Lf is at least 300mg.L^-1, at least 325mg.L^-1, at least 350mg.L^-1, at least 375mg.L^-1, at least 400mg.L^-1, at least 425mg.L^-1, at least 450mg.L^-1, at least 475mg.L^-1, at least 500mg.L^-1, at least 525mg.L^-1, at least 550mg.L^-1, at least 575mg.L^-1, at least 600mg.L^-1, at least 650mg.L^-1Or at least 700mg.L^-1。

In one embodiment, the genetic state on breast or the ox of colostrum Lf contents is to produce to have at least about 600mg.L when milking once a day^-1Lf breast.

In various embodiments, increased Lf contents are increased Lf concentration, or relative to the increased Lf concentration of another component, such as Lf concentration for example increased relative to lactoglobulin, such as in breast or colostrum.Similarly, in various embodiments, the Lf contents of reduction are reduced Lf concentration, or relative to the Lf concentration of another component reduction, such as example, relative to the Lf concentration of lactoglobulin reduction, such as in breast or colostrum.

In various embodiments, increased Lf contents are increased total Lf yield, such as example, increased Lf yield in breast, or increased Lf yield in colostrum.Similarly, in various embodiments, the Lf contents of reduction be reduce total Lf yield, such as example, in breast reduction Lf yield, or in colostrum reduction Lf yield.

In various embodiments, the adaptability milked once a day is the ability for producing such breast or colostrum, and it has relative to breast or the increased Lf concentration of colostrum lactoglobulin concentration, it is highly preferred that relative to breast or the increased Lf concentration of colostrum beta lactoglobulin concentration.

In one embodiment, when milking once a day, increased Lf contents are relative to breast or the increased Lf concentration of colostrum lactoglobulin concentration, it is highly preferred that relative to breast or the increased Lf concentration of colostrum beta lactoglobulin concentration.

It should be appreciated that including determining the expression of expression or Lf genes from Lf genes including determining the expression of Lf gene outcomes or the method for activity.

In one embodiment, the expression of Lf gene outcomes or activity are determined using LfmRNA, for example, pass through the presence for determining Lf mRNA or amount.In other embodiments, the expression of Lf gene outcomes or activity are determined using Lf albumen, it is preferred that the amount by determining Lf albumen, such as amount of Lf albumen, or by determining the activity of Lf albumen, for example it is present in the activity from the Lf albumen in the sample that object is obtained, or by determining Lf protein variants, derivative or the amount of fragment.It should be appreciated that the activity of Lf albumen, is such as present in the activity from the Lf albumen in the sample that object is obtained, it can determine by methods known in the art, for example the antimicrobial acivity by determining Lf mediations.In other embodiments, the expression of Lf gene outcomes or activity are determined using LfDNA, it is preferred that presence or shortage by determining one or more polymorphisms related to Lf that is decreasing or increasing expression or activity, such as one or more polymorphisms related to the expression increased or decreased as described herein or activity.

In another embodiment, the Lf allele distributions of object are determined one or more with the allele distributions of object with newborn or first milk content --- including breast or serum colostrum protein content --- together with related genetic loci.

In one embodiment, one or more genetic locis are one or more polymorphisms one or more of to gene newborn or that serum colostrum protein content is related.

In one embodiment, one or more polymorphisms are AA/BB polymorphisms in ox beta lactoglobulin gene.

One or more polymorphisms can be detected directly or be detected by detecting one or more polymorphisms in the linkage disequilibrium with one or more of polymorphisms.

Linkage disequilibrium (LD) is the phenomenon in science of heredity, and mutation two or more whereby or polymorphism are in so closely heredity is close, so that they are co-inheritances.This means the presence for the other polymorphisms of polymorphic sexual cue in Genotyping, detecting a presence.(Reich DE et al；Linkage disequilibrium in the human genome, Nature 2001,411：199-204.)

It should be understood that, as used herein, phrase " Lf allele distributions " considers such data, it indicates the presence of one or more allele of one or more polymorphisms or shortage in Lf genes, or expression or the activity of its expression for influenceing Lf genes or Lf gene outcomes, its to the expression of Lf genes or the expression of Lf gene outcomes or activity change it is related.In a preferred embodiment, Lf allele distributions include presence or the data of shortage for indicating one or more allele of newborn or related colostrum Lf contents one or more polymorphisms to increasing or decreasing.

For example, in various preferred embodiments, the Lf allele distributions of ox include indicating following data：

A) in Lf genes 30126T/C (rs43706484) polymorphism C allele presence or shortage；Or

B) in Lf genes 30126T/C polymorphisms T allele presence or shortage；Or

C) in Lf genes 7447A/G polymorphisms G allele presence or shortage；Or

D) in Lf genes 7447A/G polymorphisms A allele presence or shortage；Or

E) in Lf genes -7G/C (rs43706485) polymorphism C allele presence or shortage；Or

F) in Lf genes -7G/C polymorphisms G allele presence or shortage；Or

G) there is a) presence of polymorphism or shortage into the linkage disequilibrium of any one or more in f) above, particularly there is a) presence of polymorphism or shortage into 100% linkage disequilibrium (D '=1.0) of any one or more in f) above, or

H) a) to any two or multiple any combinations in g).

In other embodiments, Lf allele distributions include indicating the presence of one or more allele of one or more polymorphisms or the data of shortage in Lf gene promoters or Lf Gene regulations area or Lf gene introns, preferably include to indicate presence or the data of shortage of one or more allele, the expression of effecting allele Lf genes or expression or the activity of Lf gene outcomes, or its to the expression of Lf genes or the expression of Lf gene outcomes or activity change it is related.

It will be further understood that Lf allele distributions may include the presence of one or more polymorphisms as described above or lack the information associated with breast or colostrum Lf contents.

In one embodiment, allele distributions utilize the nucleic acid that is obtained from the object, and the DNA that preferably obtains from the object is determined, or alternatively, the allele distributions are determined using the RNA obtained from the object.

In yet another embodiment, on the amino acid sequence of the Lf albumen obtained from the object of expression, allele distributions are determined.

In another embodiment, on the amount or activity of the Lf albumen obtained from the object, allele distributions are determined.

Easily, in the process, encoding wild type Lf DNA presence or shortage in the object are directly or indirectly determined, for example, utilizes the gene outcome of expression.

Alternatively, in the process, the presence of at least one nucleotide difference or shortage in encoding wild type Lf nucleotide sequence are directly or indirectly determined in the object.

In various embodiments, this method includes the 30126T/C Lf allele distributions for determining ox, or determines the 7447A/G Lf allele distributions of ox, or determines the -7G/C Lf allele distributions of ox.It is highly preferred that this method include determine it is two or more in these polymorphisms, or all three in these polymorphisms Lf allele distributions.

More specifically, in the process, directly or indirectly determining the T allele or presence or the shortage of C allele in Lf genes in 30126T/C polymorphisms.For example, the presence of the T allele or C allele in Lf genes in 30126 T/C polymorphisms can be determined using with the polymorphism in the T allele of 30126 T/C polymorphisms or the linkage disequilibrium of C allele.

Similarly, can directly or indirectly determine in ox Lf genes 7447A/G (I145V) polymorphisms or -7G/C polymorphisms specific allele presence or shortage, for example pass through the analysis of one or more polymorphisms of the LD with one or more of these polymorphisms.

In one embodiment, this method includes determining that the DNA sequence dna of protein " (A) " of the coding with wild type Lf bioactivity whether there is from the sample of the material containing the DNA obtained from object, or the DNA sequence dna of coding at least partly allelic protein " (B) " of shortage (A) activity whether there is, or whether the DNA sequence dna of coding (A) and the DNA sequence dna of coding (B) are all present.The presence instruction for encoding the DNA of (A) shortage and the DNA of coding (B) is related to low breast relatively, colostrum, blood, serum, Mucosal secretions or mucomembranous surface Lf contents, related especially to the generation of the breast or colostrum of the Lf contents with reduction etc..Reverse correlation is applicable, the wherein DNA of the DNA of coding (A) presence and coding (B) shortage instruction is related to high breast relatively, colostrum, blood, serum, Mucosal secretions or mucomembranous surface Lf contents, related especially to the generation of breast or colostrum with increased Lf contents etc..The DNA and coding (B) that encode (A) DNA, which exist, indicates that Lf contents relative to medium are related, related especially to the generation of breast or colostrum with medium Lf contents etc..

As used herein, the bioactivity of wild type Lf albumen refers to the expression and activity characteristic of the Lf albumen by wild type Lf gene codes.

In another embodiment, this method includes determining that wild type Lf gene orders whether there is from the material sample containing the DNA obtained from object.In another embodiment, this method includes determining the expression of Lf gene outcomes from the material sample containing the DNA obtained from object, preferably passes through the presence for determining one or more polymorphisms --- such as one or more with expression that increase or decrease related promotor polymorphism --- related to Lf expression that is decreasing or increasing or shortage.

In another embodiment, the present invention includes determining that the mRNA of protein " (A) " of the coding with wild type Lf bioactivity whether there is, or the mRNA sequence of coding at least partly protein " (B) " of shortage (A) activity whether there is, or whether the mRNA sequence of coding (A) and the mRNA sequence of coding (B) are all present.Encode the mRNA of (A) shortage and the mRNA of coding (B) presence again indicate that it is related to low breast relatively, colostrum, blood, serum, Mucosal secretions or mucomembranous surface Lf contents, it is related especially to the generation of the newborn or colostrum of the Lf contents with reduction etc..Reverse correlation is applicable again, the wherein mRNA of the mRNA of coding (A) presence and coding (B) shortages again indicate that it is related to high breast relatively, colostrum, blood, serum, Mucosal secretions or mucomembranous surface Lf contents, it is related especially to the generation of the newborn or colostrum with increased Lf contents etc..Also, the mRNA of coding (A) and the mRNA of coding (B) are related to medium breast relatively, colostrum, blood, serum, Mucosal secretions or mucomembranous surface Lf contents in the presence of indicating, especially related to the newborn generation with medium Lf contents etc..

In another embodiment, this method includes the amount for determining the Lf mRNA present in the sample of the material containing the mRNA obtained from object.In another embodiment, this method includes the Lf expression overviews (expression profile) for determining object, preferably includes to determine the Lf mRNA expression overviews of object.

In another embodiment, the present invention includes determining that the protein " (A) " with wild type Lf bioactivity whether there is, or protein " (B) " of at least partly shortage (A) activity whether there is, or whether (A) and (B) is all present.(A) shortage and the presence of (B) again indicate that it is related to low breast relatively, colostrum, blood, serum, Mucosal secretions or mucomembranous surface Lf contents, especially to the Lf contents with reduction etc. breast or colostrum generation it is related.Reverse correlation is applicable again, the shortage of the wherein presence of (A) and (B) again indicate that it is related to high breast relatively, colostrum, blood, serum, Mucosal secretions or mucomembranous surface Lf contents, especially to increased Lf contents etc. breast or colostrum generation it is related.Again, (A) and (B) is all related to medium breast relatively, colostrum, blood, serum, Mucosal secretions or mucomembranous surface Lf contents in the presence of indicating, especially related to the newborn generation with medium Lf contents etc..

In another embodiment, this method includes the amount or activity for determining the Lf albumen present in the sample of the material containing the protein obtained from object.

It is being related to the another aspect of animal doctor or agricultural application, the present invention is the method for the Lf genotype for determining mammalian object, can such as be expected to know breeding objective.

In one embodiment, this method includes, on the sample containing the material that the nucleic acid not polluted by heterologous nucleic acids is obtained from object, determine whether the sample contains the nucleic acid molecules or gene outcome of (i) encoding wild type Lf genes as having the protein of wild type Lf bioactivity and optionally determining whether the sample such as lacks the protein of wild type Lf bioactivity containing (ii) allele nucleic acid molecules or gene outcome for encoding variant Lf genes.

In another embodiment, this method includes, on the sample containing the material that the protein not polluted by heterologous protein is obtained from object, determine whether the sample has the protein of wild type Lf bioactivity containing (i) and optionally determine whether the sample lacks the protein of wild type Lf bioactivity containing (iii).

In other embodiment, on breast or colostrum Lf contents, the method that the present invention provides the genetic state of measure object, it includes providing the Lf allele distributions of object and in one or more allele distributions to the object of genetic loci newborn or that colostrum Lf contents are related, and determines on the basis of allele distributions genetic state.

In one embodiment, one or more genetic locis are in one or more one or more polymorphisms to gene newborn or that colostrum Lf contents are related.

In a further aspect, the present invention includes the probe containing the abundant complementary nucleic acid molecules of nucleotide sequence or encoding wild type ox Lf nucleotide sequence with being present in NC_007320.3, or its complementary strand, with connection under strict conditions；And the diagnostic kit containing such probe.What is especially considered is such probe, it is included in one one or more in 30126T/C polymorphisms, 7447A/G polymorphisms and -7G/C polymorphisms in ox Lf genes or other allele, for example, be included in the probe of the guanine in site corresponding to 7447A/G polymorphisms.

Present invention additionally comprises for detecting the Primer composition that wild type Lf genes exist or lacked, include the presence of encoding wild type Lf nucleotide sequence and/or the coding at least partly nucleotide sequence of the variant proteins of shortage wild-type activity.In one form, said composition may include the basic complementary nucleic acid primer of nucleotide sequence with encoding wild type Lf, or its complementary strand.Nucleotide sequence can be identified completely or partially in Genbank refers to NC_007320.3 or NM_180998.Also include diagnostic kit, it includes such composition.

What is especially considered is such primer, it includes being present in NC_007320.3 and in 30126T/C polymorphisms, about the 1 of one of 7447A/G polymorphisms or -7G/C polymorphisms is in about 2000bp, in more preferably at about 1 to about 1000bp, or in about 1 to about 500bp, in about 1 to about 400bp, about 1 to about 300bp, in about 1 to about 200bp, in about 1 to about 100bp, in about 1 to about 50bp, or in 30126T/C polymorphisms, nucleotide sequence in one of 7447A/G polymorphisms or -7G/C polymorphisms about 1 to about 20bp, or with being present in NC_007320.3 and in 30126T/C polymorphisms, about the 1 of one of 7447A/G polymorphisms or -7G/C polymorphisms is in about 2000bp, in more preferably at about 1 to about 1000bp, or in about 1 to about 500bp, in about 1 to about 400bp, in about 1 to about 300bp, in about 1 to about 200bp, in about 1 to about 100bp, in about 1 to about 50bp, or in 30126T/C polymorphisms, nucleotide sequence in one of 7447A/G polymorphisms or -7G/C polymorphisms about 1 to about 20bp is substantially complementary.

The example of such primer may be from herein such as SEQ ID NOs：1 to 3 sequence provided.

It will be understood by those skilled in the art that primer as a pair can be used for producing amplicon by, for example, the Primer selection with one or more sequence specifics determining the homogeneity in the nucleotides of given polymorphism.Therefore consider to include the Primer composition of primer as a pair.

The present invention also provides diagnostic kit, and its presence for including the nucleic acid for being used for presence or shortage and/or the coded reference Lf for determining reference Lf genes or the Primer composition of shortage, the diagnostic kit include one or more primers as described herein or Primer composition.

Present invention additionally encompasses antibody compositions, its presence for being used to detect wild type Lf or the presence of shortage or amount and/or the variant proteins of at least part shortage wild-type activity or shortage or amount；And diagnostic kit, it contains such antibody and used --- for example in the method for the invention --- specification.

The present invention additionally provides diagnostic kit, it is used for the DNA or mRNA that detect the variant Lf gene outcomes that the DNA or coding that include variant Lf genes in object at least partly lack wild-type activity, the diagnostic kit includes the first and second primers for DNA amplification or mRNA, nucleotide sequence of the primer respectively with DNA the or mRNA upstream and downstreams of polymorphism in Lf genes is complementary, and the polymorphism causes the Lf levels (the Lf contents particularly increased or decreased in breast or colostrum) increased or decreased.The kit may also include the 3rd or the 4th primer, and its mutation with naturally occurring wild type Lf genes is complementary.It is preferred that the kit is including the use of --- such as the method according to the invention --- specification.

In one embodiment, at least one in nucleotide sequence is selected from the noncoding region with reference to Lf genes.

Kit may also include and the coding or naturally occurring mutation --- such as the mutation in Lf gene promoters --- complementary primer of noncoding region with reference to Lf genes.Preferably, the kit is including the use of --- such as the method according to the invention --- specification.

Therefore, in another embodiment, the present invention provides the method for evaluating the genetic state of ox on breast or colostrum Lf contents, the step of it includes measure one or more presence or shortage selected from following polymorphism：

30126T/C polymorphisms in Lf genes, or

7447A/G (I145V) polymorphism in Lf genes, or

- 7G/C polymorphisms in Lf genes, or

With one or more of linkage disequilibrium one or more in these polymorphisms polymorphism.

In another embodiment, the present invention provides the method on breast or colostrum Lf Content evaluation ox genetic states, the step of it includes measure one or more presence or shortage selected from following polymorphism：

In the C allele or T allele of 30126T/C polymorphisms in Lf genes, or

In the G allele or A allele of 7447A/G polymorphisms in Lf genes, or

In the C allele or G allele of -7G/C polymorphisms in Lf genes.

Further, one or more polymorphisms can directly be detected or detected by the detection in one or more of the linkage disequilibrium with one or more polymorphisms polymorphism.

On the other hand, the method that the present invention provides identification or selection mammalian object --- with genotype one or more in the desired Lf of instruction, generation, regulation or secretion phenotype ---.This method includes the Lf allele distributions for determining the object, and identification or selecting object on the basis of the measure.

In one embodiment, Lf allele distributions are determined by providing the analysis result of the sample from the object for the presence to one or more polymorphisms in following one or more related Lf genes or shortage：

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

(b) the Lf secretions increased or decreased, or

(c) generation of breast, colostrum, blood, serum, Mucosal secretions, or with one or more mucomembranous surfaces with the Lf contents increased or decreased, or

(d) there is one or more of linkage disequilibrium of one or more polymorphisms polymorphism in the Lf genes related to one or more of (a) to (c) above.

In one embodiment, object is ox.

In one embodiment, phenotype is desired breast or colostrum Lf contents.

In one embodiment, the method that the present invention provides the ox of Lf allele distributions of the selection with increased breast or colostrum Lf contents is indicated.

Preferably, this method includes determining the T allele in 30126T/C polymorphisms, the A allele in 7447A/G polymorphisms or the presence in one or more of the G allele of -7G/C polymorphisms in Lf genes, and selects on the basis of measure ox.Alternately or additionally, this method includes determining the C allele in 30126T/C polymorphisms, the G allele in 7447A/G polymorphisms or the shortage in one or more of the C allele of -7G/C polymorphisms in Lf genes, and selects on the basis of measure ox.

Preferably, this method includes determining the TT genotype in 30126T/C polymorphisms, the AA genotype in 7447A/G polymorphisms or the presence in one or more of the GG genotype of -7G/C polymorphisms in Lf genes, and selects on the basis of measure ox.

In other embodiment, the method that the present invention provides the ox of Lf allele distributions of the selection with the reduced breast of instruction or colostrum Lf contents.

Preferably, this method includes determining the C allele in 30126T/C polymorphisms, the G allele in 7447A/G polymorphisms or the presence in one or more of the C allele of -7G/C polymorphisms in Lf genes, and selects on the basis of measure ox.Alternately or additionally, this method includes determining the C allele in 30126T/C polymorphisms, the G allele in 7447A/G polymorphisms or the shortage in one or more of the C allele of -7G/C polymorphisms in Lf genes, and selects on the basis of measure ox.

Preferably, this method includes determining the CC genotype in 30126T/C polymorphisms, the GG genotype in 7447A/G polymorphisms or the presence in one or more of the CC genotype of -7G/C polymorphisms in Lf genes, and selects on the basis of measure ox.

In other embodiment, the present invention, which provides selection, has the medium breast of instruction or colostrum Lf contents, preferably medium breast or the method for the ox of the Lf allele distributions of colostrum Lf contents.

Preferably, this method includes determining the CT genotype in 30126T/C polymorphisms, the AG genotype in 7447A/G polymorphisms or the presence in one or more of the CG genotype of -7G/C polymorphisms in Lf genes, and selects on the basis of measure ox.

In one embodiment, the presence of allele is determined on the Lf polynucleotides (genomic DNA, mRNA or the cDNA from mRNA generations) obtained from ox.

In one embodiment, the presence of allele is determined by the way that the Lf polynucleotides obtained from ox are sequenced.

In other embodiment, the step of including from the genomic DNA from the ox, mRNA or from cDNA amplification --- such as passing through PCR --- Lf polynucleotide sequences that mRNA is produced is determined.

Preferably, measure is that, by using such primer, it includes the nucleotide sequence of at least about 12 continuous bases with NC_007320.3 or NM_180998 sequences or naturally occurring flanking sequence or the nucleotide sequence with NC_007320.3 or NM_180998 sequences or at least about 12 complementary continuous bases of naturally occurring flanking sequence.

In one embodiment, at least one in primer includes the sequence of at least one in the nucleotides corresponding to allele specific described herein.

In alternative embodiments, this method includes the restriction endonuclease digestion of the nucleotides from ox.This digestion can also be carried out to desired pcr amplification product above.

In other embodiment, the presence of allele is determined by the mass spectral analysis of the Lf polynucleotides obtained from ox.

In alternative embodiments, the presence of allele is determined by the nucleotide sequence including NC_007320.3 or NM_180998 sequences or with the hybridization of the probe (one or more) of the nucleotide sequence of NC_007320.3 or NM_180998 sequences complementation.

Preferably, probe includes NC_007320.3 or NM_180998 sequences or 12 or more complementary with NC_007320.3 or NM_180998 sequences continuous nucleotides.

Preferably, probe is included corresponding to the sequence of at least one in allele-specific nucleotide described herein or its complementary strand.

In alternative embodiments, the presence of allele is determined by the analysis of the Lf polypeptides obtained from ox.

Further, the present invention provides through the ox of method of the present invention selection；The breast or colostrum that are produced by the ox of selection or its offspring and the composition and dairy products that are produced from such breast；The composition produced from such colostrum, and the ovum or seminal fluid or the tissue of the ox from selection that the ox selected produces.

In also further aspect, the method for the invention for providing selection a herd of cattle, including individual is selected by the method for the present invention, and separate and assemble the individual of selection to form group.Include the ox of the ox generation by method described above selection the present invention further provides a herd of cattle so selected, and a group.

In also further aspect, the present invention provides through method of the present invention selection a herd of cattle and the milk rather than the more conventional method twice daily milked with therefore other concentrated milk Lf levels for then squeezing the group once a day.

In also further aspect, the present invention is provided on one or more breasts or colostrum Lf content phenotypes, or on the method for the genetic state for producing the ability of the offspring tended in advance or with one or more breasts or colostrum Lf content phenotypes and determining ox, this method includes：

The data of Lf allele distributions on the ox are provided, and

The genetic state of ox is determined on the basis of the data.

Preferably, the data on Lf allele distributions include the data for representing the C allele or T allele in 30126T/C polymorphisms, the A allele or G allele in 7447A/G polymorphisms or one or more presence in the C allele or G allele of -7G/C polymorphisms or shortage in Lf genes.

Preferably, this method comprises additionally in the data for the result for providing at least one analysis for including the one or more genetic locis related to one or more breasts or colostrum Lf content phenotypes, wherein the data represent the genetic state of ox.

Preferably, one or more genetic locis be one or more expression to Lf gene outcomes or activity increase or decrease related polymorphism.

Preferably, genetic loci is Lf genes (including all regulating elements such as promoter, introne and 3 ' UTR).

In one embodiment, one or more breasts or colostrum Lf contents phenotype are selected from the breast produced with increased Lf contents or the newborn ability produced with increased Lf contents or produce the colostrum with increased Lf contents or produce the ability of the colostrum with increased Lf contents.In another embodiment, one or more breasts or colostrum Lf contents phenotype are selected from the breast produced with reduced Lf contents or the newborn ability produced with reduced Lf contents or produce the colostrum with reduced Lf contents or produce the ability of the colostrum with reduced Lf contents.

Correspondingly, in one embodiment, the present invention, which is provided, determines on breast or colostrum Lf contents, or on producing the method for the ox genetic state of the ability with the breast increased or decreased or the offspring of colostrum Lf contents, this method includes：

The data of Lf allele distributions on ox are provided, and

The genetic state of ox is determined on the basis of data.

In a further aspect, the present invention provides the method for producing, adjust or secreting phenotype and recognizing or select mammalian object on one or more desired Lf, and this method includes：

The result of one or more genetic tests of sample from object is provided, and

The result is analyzed for one or more presence selected from following polymorphism or shortage：

(a) expression increased or decreased to Lf gene outcomes or active related one or more polymorphisms, or

(b) one or more of Lf genes related to the Lf secretions increased or decreased polymorphism, or

(c) it is related to the generation of breast, colostrum, blood, serum, Mucosal secretions, or with one or more with one or more of the Lf genes of mucomembranous surface of Lf contents increased or decreased polymorphism, or

(d) have to one or more of linkage disequilibrium of one or more polymorphisms polymorphism in one or more related Lf genes in (a) to (c) above,

The result for wherein indicating presence one or more in the polymorphism or shortage be indicate to produce with one or more desired Lf, the object of regulation or secretion phenotype；With

Identification or selecting object on the basis of the result.

Preferably, to Lf gene outcomes the expression increased or decreased or active related one or more polymorphisms is one or more of Lf genes polymorphism.

In one embodiment, one or more Lf are produced, regulation or secretion phenotype are one or more breasts or colostrum Lf content phenotypes, including increased breast or colostrum Lf contents.Further, the method that the present invention provides ox of the selection with one or more desired breasts or colostrum Lf content phenotypes, this method includes：

A) result of the genetic test of one or more samples from ox is provided, and

B) result is analyzed for one or more presence selected from following polymorphism or shortage：

30126T/C polymorphisms in Lf genes, or

7447A/G (I145V) polymorphism in Lf genes, or

- 7G/C polymorphisms in Lf genes, or

With one or more of linkage disequilibrium one or more in -7G/C polymorphisms in 7447A/G (I145V) polymorphisms in 30126T/C polymorphisms, Lf genes in Lf genes or Lf genes polymorphism,

The result for wherein indicating presence one or more in the polymorphism or shortage is to indicate the ox with one or more desired breasts or colostrum Lf content phenotypes.

In other side, the present invention provides the system for carrying out one or more method of the present invention, and the system includes：

Computer processor device for receiving, handling and transmitting data；

Storage device for storing data, data include reference breast or colostrum Lf content trait data storehouse of the object on the reference genetic database and optionally one or more object breasts or the non-genetic factor of colostrum Lf content phenotypes of one or more breasts or the genetic analysis result of colostrum Lf content phenotypes；With

It is implanted into the computer program in computer processor, once receiving the data of genetic analysis result are constituted or include by genetic analysis result --- the data for genetic analysis result are included in reference in genetic database, the computer program handles the data to determine in the background of the reference database --- as a result --- genetic state of object, once learn, the result can be conveyed to, and preferably input the user of the data.

Preferably, the system is accessed by Internet or by personal computer.

Preferably, the reference genetic database contains or comprises the result of one or more analyses of the one or more genetic locis related to one or more breasts or colostrum Lf content phenotypes, and more preferably one or more genetic locis are one or more of one or more genes related to one or more breasts or colostrum Lf content phenotypes polymorphisms.

In a further aspect, the present invention provides the computer program for being suitable to be used in system as defined above, it includes the available medium of computer, the medium has the program code in implantation medium, for causing computer program in the reference genetic database of at least one genetic analysis result and optionally the data received are handled to the background of one or more objects breast or the reference database of the related non-genetic factor of colostrum Lf content phenotypes, the data are made up of the result of at least one genetic analysis of one or more genetic locis related to one or more breasts or colostrum Lf content phenotypes includes the result of at least one genetic analysis with one or more breasts or the related one or more genetic locis of colostrum Lf contents phenotype.

Preferably, one or more genetic locis are one or more of one or more genes related to one or more breasts or colostrum Lf content phenotypes polymorphisms.

Preferably, gene is Lf genes (including all regulating elements such as promoter, introne and 3 ' UTR).

Preferably, one or more polymorphisms are that expression to Lf gene outcomes or activity increase or decrease related one or more polymorphisms.

In also further aspect, the present invention is provided on breast or colostrum Lf contents, or on producing the method that the ability with the breast increased or decreased or the offspring of colostrum Lf contents is determined into the genetic state of ox, this method includes：

The breast or colostrum Lf contents of ox are determined,

The Lf allele distributions of ox are determined,

Compare the Lf allele distributions of ox or the breast of ox or colostrum Lf contents and the Lf allele distributions of the ox with known Lf allele distributions or the breast or colostrum Lf contents of ox；

The genetic state of ox is determined on the basis of the comparison.

It should be appreciated that for comparative purposes, the breast related to known Lf allele distributions or colostrum Lf contents are known.Also, it is understood that the relevance of breast or colostrum Lf contents and specific Lf allele distributions can be set up by method described herein.

On the other hand, the present invention relates to the nucleic acid molecules of separation, purifying or restructuring, it includes being selected from following nucleotide sequence：

(a) at least 12 of NC_007320.3 continuous nucleotides and it is included in the guanines of 7447A/G polymorphisms；Or

(b) at least 12 of NM_180998 continuous nucleotides and it is included in the guanines of 7447A/G polymorphisms；Or

(c) at least 12 of the variant of (a) or (b) continuous nucleotides；Or

(d)SEQ ID NOs：At least 12 continuous nucleotides of any one or more in 1 to 3 and the guanine for being included in 7447A/G polymorphisms；Or

(e) any one complementary strand in (a) to (d)；Or

(f) sequence of at least 12 continuous nucleotides and can under strict conditions it hybridize with any one nucleotide sequence in (a) to (e).

In one embodiment, Lf nucleic acid molecules are Lf fragments as defined herein, and wherein Lf fragments include one or more of 30126T/C polymorphisms, 7447A/G polymorphisms or -7G/C polymorphisms, or its any two or multiple combinations.

The present invention, which is also provided, includes the genetic constructs of the Lf nucleic acid molecules of the present invention, carrier including genetic constructs as described above or nucleotide sequence, host cell including genetic constructs or carrier, by the polypeptide of the Lf nucleic acid molecule encodings of the present invention, optionally combine the antibody of the polypeptide of the present invention, and the method for being recombinantly produced the polypeptide of the present invention.

Further, the dangerous method of the one or more symptom related to the Lf expression decreased or increased or activity of measure object development is provided, including one or more polymorphisms in the expression for the Lf gene outcomes to increasing or decreasing or active related Lf genes, or the presence of one or more of linkage disequilibrium of one or more polymorphisms polymorphism or shortage in expression with the Lf gene outcomes to increasing or decreasing or the related Lf genes of activity, the analysis result of sample from the object is provided, the presence of wherein one or more polymorphisms or the danger for lacking the one or more symptom related to the Lf expression decreased or increased or activity of denoted object development.

In one embodiment, the symptom related to the Lf expression decreased or increased or activity is mastitis.

In one embodiment, the presence of one or more polymorphisms or shortage indicate whether that the symptom that object is subjected to is caused by the Lf gene product expressions or activity increased or decreased.

In another aspect, the present invention provides measure object and develops one or more and Lf generations, regulation, or the dangerous method of the related symptom of secretion, this method includes one or more polymorphisms in the expression for the Lf gene outcomes to increasing or decreasing or active related Lf genes, or the presence of one or more of linkage disequilibrium of one or more polymorphisms polymorphism or shortage in expression with the Lf gene outcomes to increasing or decreasing or the related Lf genes of activity, the analysis result of sample from the object is provided, the presence or shortage denoted object development of wherein one or more polymorphisms are one or more to be produced with Lf, regulation, or the danger of the related symptom of secretion.

In one embodiment, one or more symptom are related to the Lf secretions of reduction.

In one embodiment, one or more symptom are to the increased neurological susceptibility of one or more infectious diseases.

In various examples, infectious disease is the infectious disease of mucomembranous surface, or the infectious disease as caused by infectious agent by the entrance of mucomembranous surface.

Infectious agent can be microorganism such as yeast (including, some kinds of such as Mycotoruloides (Candida spp.)), virus --- including retroviruse, such as such as HIV, rotavirus, adenovirus, norovirus (norovirus), bacterium or fungi.

In one embodiment, infectious disease is mastitis.

In one embodiment, infectious disease is infectious diarrhea, including by virus, as norovirus belongs to some kinds (Norovirus spp.), some kinds of rotavirus (Rotavirus spp.), Adenovirus (Adenovirus spp.) or some kinds of Astrovirus (Astrovirus spp.), bacterium, such as some kinds of campylobacter (Campylobacter spp.), some kinds of Salmonella (Salmonella spp.), some kinds of Cryptosporidium (Cryptosporidium spp.) and giardia lamblia belong to some kinds (Giardia lamblia spp), some kinds of Shigella (Shigella spp.), infectious diarrhea caused by Escherichia coli (Escherichia coli) and clostridium difficile (Clostridium difficile).For example, in the embodiment that object is ox, deer, sheep or pig, infectious disease is poultry diarrhoeal diseases.

One or more polymorphisms can directly be detected or detected by the detection of one or more of the linkage disequilibrium with one or more of polymorphisms polymorphism.

In one embodiment, one or more polymorphisms are selected from：

30126T/C polymorphisms in Lf genes, or

7447A/G polymorphisms in Lf genes, or

- 7G/C polymorphisms in Lf genes, or

One or more of linkage disequilibrium with one or more of these polymorphisms polymorphism,

One or more presence or the danger of the one or more symptom related to the Lf expression decreased or increased or activity of shortage denoted object development in wherein described polymorphism.

This method can comprise additionally in one or more other polymorphisms that sample of the analysis from the object is absorbed to Lf, produced, adjusting or secretion is related, include the presence for one or more polymorphisms that the Lf to increasing or decreasing is produced or secretion is related.

In addition, the detection of one or more further polymorphisms can directly be carried out or carried out by the detection of the polymorphism in the linkage disequilibrium with one or more further polymorphisms.

On the other hand have there is provided treatment or be on the verge of to develop the method for the dangerous object of one or more symptom related to Lf generations, regulation or secretion, this method includes giving object newborn or colostrum product or composition as described herein.

In one embodiment, breast or colostrum composition are included or from breast or colostrum with increased Lf contents.In another embodiment, breast or colostrum composition include or breast or colostrum from the Lf contents with reduction.

In various embodiments, object is fetus, neonate, baby or children's object.

In some embodiments, composition is parent formula, infant formula, larger infant formula (follow-on formula), growth formula or diet product.In other embodiments, composition is nutraceutical.In other embodiments, composition is medicine.

In one embodiment, method comprises additionally in the Lf allele distributions of measure object.

It is being related in fetus object in the embodiment of holding or rise Lf levels, composition is suitable to orally give mother in period of gestation.

In some embodiments, composition is parent formula, infant formula, larger infant formula, growth formula or diet product.In other embodiments, composition is nutraceutical.

In another aspect, the present invention relates to separation, purifying or recombinant protein, its at least about 10 continuous amino acid for including NP_851341 and the valine for being included in the position of amino acid/11 45 (145 positions for being equal to NP_851341) corresponding to ox Lf.

In one embodiment, polypeptide is variant as defined herein.

In another embodiment, polypeptide is Lf functional variety, or its function fragment.

As used by this specification, term " including (comprising) " refers to " by least partly constituting ".When explaining that this specification includes each narrative tense of term " comprising ", also it may be present except by the feature in addition to that or those feature the term.Related term such as " including (comprise) " and " including (comprises) " is explained in an identical manner.

As described above, in this specification referred to other sources of patent specification, other external files or information, the purpose of background is provided generally for the feature that the present invention is discussed.Unless specifically stated otherwise, the reference to such external file is not construed as recognizing that such file or such information source are prior arts with any authority, or forms general knowledge known in this field part.

Brief description

Fig. 1 shows influence of the lactation stage to Lf concentration in cow's milk.The data of display are the average Lf concentration in each lactation stage, ± average stdev.On peak, minimum average Lf concentration is observed in lactation (after calving 35 days), and the average Lf concentration of highest is observed in late lactation.

Fig. 2 shows effect of the breeding stock to Lf concentration in cow's milk.The data of display are the average Lf concentration of the filial generation of each breeding stock, ± average stdev.By breeding stock numbering 1-6, these breeding stocks formation experiment pedigree, the basis of Friesian-Jersey cross experiments.

The QTL of breast Lf concentration and each mark and the figure of the association analysis of Lf concentration in cow's milk on Fig. 3 display description ox chromosome 22s (solid line).QTL maximum F values (y-axis) are that 10.19, QTL most likely location is estimated in 70cM.Show that QTL 95% confidential interval is 70-77cM from analysis (n=1000 repeats) is drawn.The related mark of topnotch is included in QTL.

Fig. 4 is the schematic diagram of ox Lf genes, and the sequence vestige of the polymorphism of interior identification and subsequent Genotyping is tested together with FJXB.

Fig. 5 is description in lactation stage peak, mid-term and late period lactation, figures of the polymorphism Lf_30126 to the effect of newborn Lf concentration.In each lactation stage, TT genotype is all related to higher newborn Lf concentration.The data of display are mean+SDs.

Fig. 6 is the figure for describing Lf gene pleiomorphisms to the statistics effect of ox chromosome 22 breast Lf QTL adjustment：Black diamonds；Initial QTL, cross；Polymorphism Lf_7447 effect, white square；Polymorphism Lf_30126 effect, white triangles shape；Polymorphism Lf_-7 effect.

Fig. 7 is the QTL (white diamond) for QTL (gray circular) and Lf the mRNA expression for showing breast Lf concentration on ox chromosome 22 figure.The position of two QTL maximum F values is all harmonious in about 70cM.

Fig. 8 shows the effect (black diamonds) that the effect of QTL (white square) and the LfmRNA expression of breast Lf concentration on ox chromosome 22 is adapted to breast LfQTL.The QTL that the QTL and LfmRNA of this prompting breast Lf concentration are expressed is closely related.

Detailed description of the invention

The present invention recognizes that the polymorphism in ox Lf genes is related to the QTL of for Lf changes of contents --- the particularly change of breast or colostrum Lf contents --- first.

For the sake of clarity, phrase " breast or colostrum Lf contents " will be understood as referring to newborn Lf contents or colostrum Lf contents.Those skilled in the art will be apparent to be that breast or colostrum Lf contents can be determined easily qualitatively or quantitatively.In some applications, breast or colostrum sample relative to another sample (individual for being preferred from known Lf allele distributions) it is qualitative compare can be enough to make it is associated with specific gene type.The method of the quantitative determination of breast or colostrum Lf contents is also known in the art, and provided herein is example.

The present invention provides the method for evaluating ox on Lf contents, more specifically breast or the genetic state of colostrum Lf contents.The step of one such method includes determining the Lf allele distributions of the ox.The step of another such method includes determining the Lf gene product levels of the ox.

The present invention also provides the method for recognizing or selecting to have the mammalian object for indicating the genotype that one or more desired Lf produced, and adjusted or secreted phenotype --- including desired breast or colostrum Lf contents, particularly expecting Lf content ---.The ox that one of main application of the present invention is identification or selection with the T allele in 30126T/C polymorphisms, the A allele in 7447A/G polymorphisms or the G allele in -7G/C polymorphisms in Lf genes one or more of --- these allele are each independently related to increased breast or colostrum Lf contents ---；Or with the C allele in Lf genes in 30126T/C polymorphisms, the G allele in 7447A/G polymorphisms or the C allele in -7G/C polymorphisms one or more of --- these allele are each independently related to the breast or colostrum Lf contents of reduction --- ox.Correspondingly, one method includes determining C allele or T allele of the Lf genes in 30126T/C polymorphisms, the A allele or G allele in 7447A/G polymorphisms, or in the G allele or C allele of -7G/C polymorphisms one or more presence or shortage, and select ox on the basis of the measure.

Moreover, it relates to the non-human subject of selection, the ovum or seminal fluid of such as ox and the non-human subject from the selection available for other breeding plan.The ox so selected will be used for breast or colostrum is produced.The invention further relates to the breast or colostrum that the ox of selection or its offspring produce, and the dairy products produced from such breast and the composition (including pharmaceutical composition, alimentation composition, veterinary compositions etc.) produced from such colostrum.

The generation of extensive dairy products is well known in the art, and the dairy products considered herein include daily iron supplement tablet including ice cream, yogurt and cheese, dairy beverage (such as the milk beverage and sour cheese drink including milk shake), milk powder, newborn base sports supplement, food additives such as protein spreading (sprinkle) and meal supplement product.

Similarly, the production of extensive colostrum product --- such as but be not limited to nutritional food replenisher and analog --- is well known in the art.What is especially considered herein is colostrum composition, and it has increased Lf contents or produced from the colostrum with increased Lf contents.The method for preparing the composition rich in Lf is also known in the art, exemplified by as discussed in the PCT International Patent Application PCT/NZ2006/000263 for being disclosed as WO 2007/043900, and this application is all incorporated herein with its by quoting.

Present invention recognizes that the mutation of coding Lf gene, and Lf level or activity can be used as select tools to breed the animal with the newborn concentration of higher or lower Lf.This can allow the generation for being more suitable for the milk product of particular market in turn, the generation of high such as Lf food.

The method that development is absorbed to Lf, produces, adjusts or secretion phenotype the dangerous object of one or more related symptom in --- Lf intakes or the Lf levels increased or decreased such as reduction --- is on the verge of the present invention additionally provides identification, identification can benefit the method for the object of (including for example increased Lf intakes or supplement) from specific therapeutic scheme, the method of prevention or the treatment symptom related to such phenotype in its object is needed, and composition and product for such method.

As used herein, phrase " milking once a day " and phraseological equivalent refer to milks once within twenty four hours period.Phrase " twice daily milking " and phraseological equivalent refer to milks twice within twenty four hours period.

Lactoferrin

Lactoferrin is the iron combination glycoprotein for being present in about 80kD in most of exocrine secretions --- including tears, bile, bronchorrhea, gastro-intestinal Fluid, cervical guide mucus, seminal fluid and breast ---.Lactoferrin most abundant source is the breast and colostrum of mammal.Bovine lactoferrin (UniProtKB/Swiss-ProtP24627) is made up of the single polypeptide chain with 17 disulfide bond.The three-dimensional structure of bovine lactoferrin includes the leaf (N- leaves and C- leaves) of two formed objects.Each leaf includes metal ion binding pocket；Each pocket have with CO₃Ion complex reversibly combines a Fe with high-affinity³⁺Ability (the Moore SA of ion, Anderson BF, Groom CR, the A resolution.J Mol Biol. (1997) 274 (2) of Haridas M, Baker EN.Three-dimensional structure of diferric bovine lactoferrin at 2.8：222-36).Lactoferrin in cow's milk is natively about 12% to 18% iron saturation, and has reported that the lactoferrin of more subway saturation is beneficial as supplements-iron or the part (PCT International Publication No.s WO 2006/054908) as modality of cancer treatment.Ox Lf genes (GeneID：280846) reference gene group sequence can be obtained in Genbank accession number NC_007320.3, and can be obtained with reference to mRNA sequence in Genbank accession number NM_180998, and the reference amino acid sequence of ox Lf albumen can be obtained in Genbank accession number NP_851341.

As described herein, the present invention relates to such identification：Lf gene mutations cause Lf contents, the change for producing or secreting --- the particularly change of breast or colostrum Lf contents.Lf polymorphisms described herein and breast and colostrum Lf concentration phenotypes are closely related.The breast with many about 2.5 to 4 times Lf is produced compared with the animal of the C allele homozygosis in 30126T/C polymorphisms in the animal of the T allele homozygosis of 30126T/C polymorphisms, this depends on the stage of lactation.Also observation is led depending on the similar differences in 7447A/G polymorphisms He the Lf content phenotypes of the genotype of -7G/C polymorphisms.Referring to this paper table 2.

It is present in the CC genotype of 30126T/C polymorphisms with about 2% in Holstein-Friesian xJersey cross experiments described herein.It is present in the GG genotype of 7447A/G polymorphisms with about 6% in Holstein-Friesianx Jersey cross experiments, and is present in the CC genotype of -7G/C polymorphisms with about 10% in Holstein-Friesian x Jersey cross experiments described herein.For example, the animal removed from group behind these will increase group mean Lf concentration about 7%.Group mean Lf concentration about 25% will be increased in the homozygous recessive of any polymorphic site and the animal of heterozygosis by removing.On the contrary, group mean Lf concentration about 21% will be reduced by removing homozygote wild type animal, the animal of the wild type and heterozygosis that remove homozygosis will reduce group mean Lf concentration about 61%.

Applicant is referred to as that " wild type Lf " reference ox Lf nucleotide sequences are presented in NC_007320.3, and corresponding amino acid sequence is presented in NM_180998.Therefore, as used by herein when mentioning Lf, term " wild type " identification is presented on the Lf nucleotide sequences in NC_007320.3 and NM_180998, and the protein thus encoded characteristic.For example, working as on activity in use, term " wild type " refers to the activity related to wild type Lf albumen.Similarly, when on expression in use, term " wild type " refers to the expression with wild type Lf promoters or wild type Lf gene-correlations.

It should be appreciated that term " activity " can refer to the intrinsic activity of single Lf molecules, it can be wild-type activity or be smaller than or more than wild-type activity, such as may depend on, for example amino acid sequence, the presence of any 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, the availability of co-factor etc.；And refer to the gross activity of the Lf molecular populations of presence (for example, in ox or in the sample of ox is derived from), such as the activity and the level (for example, exist how many molecule) of expression of each molecule for may depend on presence.

As used herein, the allelic protein of shortage wild type Lf activity is mentioned as worked as in use, phrase " lacking (A) activity " consideration is more than the activity of (A) and less than the activity of (A).For example, the allelic protein for lacking wild type Lf activity can be the Lf protein variants with the enzymatic activity greater or lesser compared with wild type Lf activity.

Determine Lf expression or the method for activity is well known in the art.For example, using RNA analysis, RT-PCR or Lf immunostaining.Another illustrative methods utilizes reversed-phase HPLC (Palmano KP after the isoelectric precipitation of milk casein, Elgar DF, Detection and quantitation of lactoferrin in bovine whey samples by reversed-phase high-performance liquid chromatography on polystyrene-divinylbenzene.J Chromatogr A. (2002) Feb 22；947(2)：307-11).Similarly, using the method for measurement Lf activity indirectly, it includes determining Fe or Fe ions, such as Fe³⁺--- as being present in from the sample that ox obtains --- presence, shortage or concentration.

In various embodiments, the method for the present invention is used together with the method for other known increase Lf breasts or concentration or yield, described in the NZ patent applications NZ550859 disclosed in 30 days October in 2009 method.

In various embodiments, using period of not milking, those embodiments milked once a day one to two days after twice daily milking, the method of the present invention is used to produce breast and milk product rich in lactoferrin, it includes at least about 600mg/l, more preferably at least 800mg/l, more preferably at least 1000mg/l, more preferably at least 1500mg/l, more preferably at least 2000mg/l, more preferably at least 2500mg/l, more preferably at least 3000mg/l, more preferably at least 3500mg/l, more preferably at least 4000mg/l, more preferably at least 4500mg/l, more preferably at least 5000mg/l, most preferably at least 5000mg/l.

For example, the present invention provides the method for producing breast or milk product rich in lactoferrin, comprise the following steps：

Ox as described herein is recognized or selects,

The milk sample product collected from ox are provided, and optionally

Modified milk sample is to produce milk product.

In another example, the present invention provides the method for producing breast or milk product rich in lactoferrin, comprises the following steps：

Ox as described herein is recognized or selects,

The milk sample product prepared by following steps are provided,

A) period milked to ox more than 24 hours is stopped；

B) milk from ox is collected in after completion a) in the period of up to 7 days；Optionally

Modified milk sample is to produce milk product.

The identification and analysis of polymorphism

By polymorphism described herein according to they in genome nucleotide sequence relative to ox Lf genes+1 translation initiation site Position Number.It would be recognized by those skilled in the art that these positions are relative to coded sequence, or can easily it be expressed relative to their positions in ripe Lf polypeptides.Polymorphism and the thus routine of caused 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor that their codon position in the gene that polymorphism occurs causes 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor are identified by it is apparent to those skilled in the art that being also contemplated herein.Therefore, 7447A/G polymorphisms described herein, i.e. I145V polymorphisms can be mentioned by mentioning the codon and caused 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor of the Lf genes of polymorphic position in the inner.

It each can directly detect or be detected by the detection with one or more polymorphisms in linkage disequilibrium one or more in these polymorphisms in 30126T/C polymorphisms, 7447A/G polymorphisms or -7G/C polymorphisms.Linkage disequilibrium is the phenomenon in science of heredity, and mutation two or more whereby or polymorphism are in so close heredity in, so that they are co-inheritances.This means the presence for the other polymorphisms of polymorphic sexual cue in Genotyping, detecting a presence.(Reich DE et al；Linkage disequilibrium in the human genome, Nature2001,411：199-204.)

The linkage disequilibrium of a variety of degree is possible.Preferably, in the linkage disequilibrium that greater than about 60% with one or more polymorphisms specified herein is in one or more of one or more linkage disequilibriums of polymorphism specified herein polymorphism, in about 70% linkage disequilibrium, in about 75%, about 80%, about 85%, about 90%, about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or about 100% linkage disequilibrium.It will be understood by those skilled in the art that when a pair of alleles frequency on observing and desired frequency departure are expressed, linkage disequilibrium can also be represented by capital D.Correspondingly, phrase " two allele in LD " is often referred to D and is not equal to 0.On the contrary, " linkage equilibrium " refers to D=0 situation.When using the nomenclature, with one or more of one or more LD of polymorphism specified herein polymorphism be preferably in greater than about D '=0.6, about D '=0.7, about D '=0.75, about D '=0.8, about D '=0.85, about D '=0.9, about D '=0.91, about D '=0.92, about D '=0.93, about D '=0.94, about D '=0.95, about D '=0.96, about D '=0.97, about in D '=0.98, about D '=0.99 or the about LD of D '=1.0.(Devlin and Risch 1995；A comparison of lmkagedisequilibrium measures for fine-scale mapping, Genomics 29：311-322).

There are many standard methods known in the art to be used to determine whether that specific DNA sequence dna is present in sample, the step of many in these methods includes sequencing DNA sample.Therefore, in an embodiment of the invention, determine whether that the nucleotides specified is present in the step of step in the nucleic acid from ox is including sequencing nucleic acid.The method of nucleotide sequencing is to known to those skilled in the art.

On the other hand, the present invention, which is provided, determines ox on method of the Lf contents especially with regard to breast or the genetic state of colostrum Lf contents.This method includes determining coding (a) from the sample of the material containing the DNA obtained from ox：Protein DNA sequence with wild type Lf bioactivity whether there is, and coding (b)：The DNA sequence dna for lacking the allelic protein of (a) activity whether there is.

The example for becoming known for determining whether another technical standard method that specific dna sequence is present in sample is polymerase chain reaction (PCR).The preferred aspect of the present invention is therefore including such step, the DNA sequence dna that the DNA sequence dna of wherein determination coding (a) whether there is and encode (b) whether there is, the step is included in the case of the primer in the presence of the nucleotide sequence based on protein of the coding with wild type Lf bioactivity and/or contains at least part polymorphism existing --- and it is known naturally occurring and it causes high relative Lf levels when it is present, particularly with higher Lf contents etc. breast or colostrum in --- primer in the case of, and/or contain at least part polymorphism existing --- it is known naturally occurring and it causes low relative Lf levels when it is present, particularly with relatively low Lf contents etc. breast or colostrum in --- primer in the case of DNA amplification.

The primer --- such as PCR --- of the present invention is the nucleic acid molecules with sequence fully complementation, and the nucleic acid molecules with PCR usually used conditions in vitro optionally with the appropriate section of the nucleic acid molecules of expectation amplification hybridize and start it are synthesized based on the sequence and with enough length.Equally, probe of the invention is molecule, such as the nucleic acid molecules with sufficient length and with target nucleic acid molecules fully complementation, and it is optionally combined for detecting the presence with not homotactic nucleic acid molecules under high or low stringent condition with target nucleic acid sequence.

Correspondingly, in the case that therefore the preferred embodiment of the present invention is included in the presence of at least one primer --- nucleotide sequence including Lf genes (NC_007320.3 and NM_180998) or its flanking sequence or with Lf genes (NC_007320.3 and NM_180998) or the complementary nucleotide sequence of its flanking sequence ---, and/or the step of Lf polynucleotides are expanded in the case of there is such primer --- including corresponding to allele-specific nucleotide described herein or in sequence of allele-specific nucleotide flank described herein ---.PCR method is to (Mullis et al., 1994.) known to those skilled in the art.The template of amplification may be selected from the genomic DNA, mRNA or the first chain cDNA (Sambrook et al., 1989) of the analyte derivative obtained from ox in test.

The primer for being suitable for the method for PCR-based of the present invention should be with Lf gene orders, and such as NC_007320.3 or NM_180998 or its flanking sequence are fully complementary and are hybridized with the appropriate section of the nucleic acid molecules under the usually used conditions in vitro of PCR optionally with expecting amplification with enough length and are started it and synthesize.Such primer should include NC_007320.3 or NM_180998 or its naturally occurring flanking sequence at least about 12 continuous bases or with NC_007320.3 or NM_180998 or at least about 12 continuous bases of its naturally occurring flanking sequence complementation.The example of such PCR primer may be from herein such as SEQ ID NOs：1 to 3 sequence presented.

Appropriate PCR primer may include 30126T/C C allele-specific nucleotides, 30126T/C T allele-specific nucleotides, 7447A/G G allele-specific nucleotides, 7447A/G A allele-specific nucleotides, -7G/C C allele-specific nucleotides, or -7G/C G allele-specific nucleotides, it is as described herein.The corresponding generation of PCR primer or the shortage of product may make up the presence or the test of shortage of designated nucleotide in the ox Lf genes to test.

Determine whether that specific nucleotide sequence is present in other methods in sample and may include the step of restriction endonuclease of nucleotide sample digests.The separation and visualization of the restriction fragment of method digestion well known in the art can form the diagnostic test of specific nucleotide sequence presence.The nucleotide sequence of digestion can be the PCR primer expanded as described above.

Determine whether that specific nucleotide sequence is present in other methods in sample and may include the step of probe hybridizes with sample oligonucleotide sequence.Therefore, detect that methods one or more in allele-specific nucleotide described herein may include the other step of probe hybridization of the Lf sequences from NC_007320.3 or NM_180998.

Such probe should include sufficient length and the nucleic acid molecules with the fully complementation of Lf gene orders, optionally to be combined presence or shortage that allele-specific nucleotide described herein is detected with promotion with the nucleotide sequence of sample under high or low stringent condition.

The polynucleotide molecule of 100 bases is greater than about on length, common stringent hybridization condition is to be no more than 25 to 30 DEG C below (for example in natural double helix melting temperature (Tm), 10 DEG C) (generally referring to Sambrook et al., 1989；Ausubelet al., 1987).The Tm of the polynucleotide molecule of greater than about 100 bases can (G+C-log (Na+) be calculated by formula Tm=81.5+0.41%.

It is less than the polynucleotide molecule of 100 bases on length, exemplary stringent hybridization condition is in 5 to 10 DEG C of below Tm.On an average, the Tm of polynucleotide molecule of the length less than 100bp is lowered about (500/ oligonucleotide length) DEG C.

The cDNA that such probe can be produced with genomic DNA, mRNA or from mRNA hybridizes, and it is derived from the sample of ox in experiment.

At least 12 continuous nucleotides of the sequence complementation for generally including at least 12 continuous nucleotides of the sequence that NC_007320.3 or NM_180998 is presented or being presented with NC_007320.3 or NM_180998 may include the sequence corresponding to allele-specific nucleotide described herein by such probe.

Such probe can comprise additionally in the method for detecting the probe presence when being attached to sample oligonucleotide sequence.(see, for example, Sambrook et al., 1989) that the method for label probe such as radioactive label is well-known in the art.

On the other hand, the present invention provide determine ox on Lf contents --- on the sample of the material containing mRNA obtained from ox --- genetic state method.In one embodiment, this method includes determining coding (A)：The mRNA sequence of protein with wild type Lf bioactivity whether there is, and coding (B)：The mRNA sequence at least partly lacking the protein of (A) activity whether there is, and may include measure mRNA amount.The presence for encoding the mRNA of (A) shortage and the mRNA of coding (B) indicates that Lf levels relative to low are related, related particularly to the generation of the breast or colostrum of the Lf contents with reduction etc..Reverse correlation is applicable again.

And, if amplification method such as PCR is used to determine whether that the mRNA sequence for encoding (A) has and whether encoded the mRNA sequence presence of (B), so this method includes amplification mRNA, for example in the case where there is the pair of primers complementary with encoding the nucleotide sequence of the protein with wild type Lf bioactivity, or in the case where there is the pair of primers complementary with encoding the nucleotide sequence of variant Lf albumen.It should be appreciated that in the embodiments of the present invention for relying on the amount for evaluating the LfmRNA being present in sample, using quantitative amplification method well known in the art, such as quantitative RT-PCR, microarray analysis and other methods described herein.

Amount that is quantitative or evaluating nucleic acid in addition, what other methods of particularly mRNA amount were well-known in the art.These using can with target Lf mRNA hybridize probe RNA analysis.Such probe should include sufficient length and the nucleic acid molecules with the fully complementation of Lf coded sequences, optionally to be combined with promotion detection and evaluated the LfmRNA existed amount with the nucleotide sequence of sample under high or low stringent condition.It is apparent to those skilled in the art that such quantitative approach generally utilizes internal contrast, such as, quantitatively can be for for example, the rRNA being present in sample be carried out in the case of RNA analysis.

In a further aspect, the present invention provides the method for determining ox on breast or the genetic state of colostrum Lf contents, it includes determining the Lf allele distributions of the ox, and determines the ox in one or more allele distributions to genetic loci newborn or that colostrum Lf contents are related.

In one embodiment, the genetic loci is the polymorphism in the polymorphism in the gene related to breast or colostrum Lf contents, the gene being related in preferably Lf generations or secretion.

The exemplary method of the present invention relies on hereditary information Tathagata from the hereditary information for being adapted to detect and recognize polymorphism, the particularly method of the SNP (SNPs) related to the qualitative character of expectancy evaluation.For convenience, following discussion refers in particular to SNPs, but skilled labourer will be understood that the method for discussion is easy to modification to detect and recognize other genetic polymorphisms, and such as triplet is repeated or microsatellite.SNP is single sequence change or point mutation, and it causes can be in the hereditary variation between the individual that coding or noncoding region occur.

Many databases are by known SNPs, and --- and for some such SNPs, the biological effect related to SNP --- is constituted.For example, NCBI snp databases " dbSNP ", which are incorporated into NCBI ' s Entrez systems and had, is plotted to being recorded more than 1,000 7 million refSNPs in human genome sequence.In the presence of the similar database for other mammalian genomes.

Detection SNPs well known in the art methods of genotyping includes DNA sequencing, needs following method：The hybridization of the allele specific of primer or probe, the primer combined near or adjacent to polymorphism that is incorporated into of the allele specific of nucleotides (is often referred to as " Single base extension ", or " micro sequence "), the connection (ligation) (connection (joining)) (connection chain reaction or connection lock type probe) of the allele specific of oligonucleotides, the cutting of oligonucleotides or the allele specific of PCR primer caused by Restriction Enzyme (restriction fragment length polymorphism analysis or RFLP) or chemical agent or other doses, pass through electrophoresis or the resolution of chromatographic mobilities difference for the allele dependence that the special enzyme of structure --- including the special enzyme of invasion and attack structure --- or mass spectrography are carried out.The analysis of amino acid change is also possible, and wherein SNP is located at code area and causes amino acid change.

DNA sequencing allows directly to determine and recognize SNPs.Micro sequence includes allowing primer and the DNA sequence dna of neighbouring SNP site hybridizes on test specimen in research.

Many sequence measurements and platform are particularly suitable for performing and be easily used to the method for the present invention on a large scale.These include pyrosequencing method, are such as used for the Genome Sequencer obtained from 454 Life Sciences (Branford, CT)^TMThe pyrosequencing method of FLX pyrosequencing platforms, its available individual machine produces 400,000,000 few nucleotide evidences in being run at 10 hours；Solid-state sequence measurement, is such as used for SOLiD^TMThe solid-state sequence measurement of microarray dataset (Applied Biosystems, Foster City, CA)；Second generation synthesis sequencing technologies such as TruSeq^TMMicroarray dataset (Illumina, San Diego, CA) based on a large amount of parallel terminators；The real-time Single-molecule Sequencing Systems of PacBio RS (Pacific Biosciences, CA)；PostLight^TMMicroarray dataset (Ion Torrent, Guilford, CT) based on semiconductor；Sequencing technologies based on nano-pore include the united nano-pore sequencing of exonuclease (Oxford Nanopore Technologies, Oxford, UK)；And tSMS^TMPlatform (Helicos BioScience Corporation, Cambridge, MA) based on single-molecule sequencing flow cell.

Include site-specific and/or allele specific hybridization currently used for many methods that SNP is detected.These methods rely primarily on the differentiated combination of oligonucleotides and the target sequence containing target SNP.Illumina (SanDiego, CA), Affymetrix (Santa Clara, CA.) and Nanogen Inc. (San Diego, Calif.) technology are especially known and use the fact that：Unstable many of the duplex of the more complete base pairing of DNA duplex containing single base mismatch.The presence of the duplex of pairing is generally detected by fluorescence.The full-length genome Genotyping product and solution that many is easy to or be suitable for the present invention are obtainable now, including available from those products and solution of above company.

It is most by locus specificity hybridization check or recognize SNPs method need the targeting amplification by method such as PCR with increase sensitivity and specificity (referring to, such as U.S. Patent No. 5,679, No. 524, PCT Publication WO98/59066, PCT Publication WO 95/12607).U.S. Patent Application Publication No. US 20050059030 (is all merged in) method that description detects SNP in total human DNA with its by quoting, it need not previously expand or complexity reduction is to be optionally enriched with target sequence, and without the auxiliary of any enzymatic reaction.This method utilize single step hybridization --- the hybridization of Part I and capture probe including target sequence, and the target sequence Part II and the hybridization of detection probe.

The modification (U.S. Patent No. 5,871,918) for the electrochemical detection method that the nucleic acid that U.S. Patent Application Publication No. US 20050042608 (is all merged in) description Thorp etc. with its by quoting hybridizes.In short, the probe base sequence on the every side of capture probe and SNP bases containing different SNP bases is fixed on different electrodes.Each the degree of hybridization between capture probe and nucleic acid target spot detects in the redox reaction of each electrode to detect by using transition metal complex.

Utilize MEGATYPE^TMThe Lynx Therapeutics (Hayward, Calif.) of technology technology can be simultaneously from the small or big larger numbers of SNPs of mixture Genotyping of Genomic material.Compare Liang Ge colonies using the probe of fluorescence labeling, the SNPs of difference Liang Ge colonies detection and recovery is carried out.

Detection and identification SNPs other methods include mass spectrography.It is preferred that example be the nucleotide sequence for including the polymorphism of the present invention mass spectroscopy (whether coded sequence or complementary series) use.Such mass spectrometry method is known to the skilled person, and methods of genotyping of the invention is readily adapted to accommodate the Mass Spectrometer Method of the polymorphism of the present invention.

SNPs can also be determined by connection-position (ligation-bit) analysis.The other discussion of these methods can be in U.S. Patent No. 5,919,626；5,945,283；Found in 5,242,794 and No. 5,952,174.

The a large amount of methods for the conformation variability for relying on nucleic acid have been developed to detect SNPs.For example, single-strand conformation polymorphism (SSCP, Orita et al., PNAS 198986：2766-2770) the various modifications with SSCP are well known in the art.These methods are using different gel service conditions, such as such as different temperatures, or add additive, with different gel-type vehicles, RNA-SSCP, restriction endonuclease fingerprint analysis-SSCP, double deoxidation fingerprint analysis (hybrid between dideoxy sequencing and SSCP), (wherein PCR primer is internally marked with multiple fluorescent dyes by two-way double deoxidation fingerprint analysis (wherein double deoxidation terminating reaction is carried out simultaneously with two reverse primers) and fluorescent PCR-SSCP, restriction Enzyme digestion can be used, it is SSCP afterwards, and analyzed in the automation DNA sequencer for can detect fluorescent dye).

Other methods using the different mobilities of different nucleic acid structures include denaturing gradient gel electrophoresis (DGGE), TGGE (TGGE), heteroduple analysis (HET) and Capillary Electrophoresis.Denaturing high pressure liquid chromatography (HPLC) is the other method to detect SNPs utilized, it is detected for example using HPLC methods, the homoduplex and heteroduplex eluted with friction speed from HPLC column, detection and therefore SNPs detection thus, it is possible to carry out mismatched nucleotide.

Detect that SNPs other method relies on single-stranded and double-strandednucleic acid by various doses --- including chemical cleavage agent and nucleolytic enzyme --- the different neurological susceptibilities of cutting.Other example includes protein translation experiment (PTT), and it is used to differentiate the terminator codon that variation is produced --- and it causes the premature end of translation and causes the protein of size reduced；With the use of mispairing conjugated protein.United States Patent (USP) 6,821,733 (is all incorporated herein) method that description detects the sequence difference of two nucleic acid molecules with its by quoting.

Method based on protein and proteomics also is adapted for polymorphic detection and analysis.These methods usually require to separate various protein in sample by, for example, gel electrophoresis or HPLC, and for example by NMR or protein sequencing as described in the mass spectrography identification of chemistry sequencing or more popular protein or peptide from it.It is that proteomics method is well-known in the art and with very big automation potential.For example, integrated system, such as ProteomIQ from proteomic system^TMSystem, is Proteomic analysis, and --- separation of joint sample, Separation of Proteins, image acquisition and analysis, protein processing, mass spectrography and bioinformatics technique --- provides high flux platform.

The proteomics method of most identification of proteins utilizes mass spectrography, it includes ion trap mass spectrometry, liquid chromatography (LC) and LC/MSn mass spectrographies, gas chromatography (GC) mass spectrography, Fourier Transform Ion cyclotron Resonance mass spectrograph (FT-MS), MALDI-TOF mass spectrographies and ESI mass spectrographies, and their derivative.Mass spectrometry method is additionally operable to determine the posttranslational modification of protein, such as phosphorylation or glycosylation, and therefore has effectiveness in polymorphism --- it causes the change of the posttranslational modification of protein or related to the change of the posttranslational modification of protein --- is determined.

Related technology is also well known, and including, for example, protein processing unit is as included " the chemical ink-jet printer " of piezo printing technique, and it allows by the way that enzyme or chemicals are directly injected on the protein spots of selection and the protein example of enzymatic digestion or chemical digestion in situ from 2-D PAGE gels electroblottings to film.After the digestion in site and incubation of protein, film can directly be put into mass spectrograph is used for peptide analysis.

Analysis suitably based on polypeptide includes those analyses that can be made a distinction between total length and the protein of truncation, and may include but be not limited to following analysis：Native polyacrylamide gel electrophoresis (PAGE), isoelectric focusing, 2D PAGE or the immunoblotting with specific antibody.Mass spectrography, immunoprecipitation and peptide fingerprint analysis are also suitable.

It will be appreciated by those skilled in the art that, specific SNP --- particularly when it occurs the regulatory region such as promoter in gene --- can be related to the gene expression of change.When SNP is located at the code area of protein coding gene, the gene expression of change can be also produced.The expression of this change can be determined by methods known in the art, such as quantitative PCR, RT-PCR, quantitative RNA assay, and can be consequently for detecting such SNPs.Similarly, occur in SNP in the code area of gene and in the case of causing non-synonymous 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, this substitution can cause the change of gene outcome function.Similarly, in the case where gene outcome is RNA, such SNPs can cause the change of rna gene product function.Any such function changes, such as evaluated in activity or functional assays, available for the such SNPs of detection.

Detect above and identification SNPs method is easily used to the method for the present invention.

Polynucleotides and polypeptide variants

As used herein, term " polynucleotides (or multiple) " refers to the single-stranded or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length but preferably at least 15 nucleotides, including unrestricted example, the coding and non-coding sequence of gene, sense and antisense sequence complementary strand, extron, introne, genomic DNA, cDNA, premessenger RNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozyme, recombinant polypeptide, the naturally occurring DNA or RNA sequence of separation and purifying, the RNA of synthesis and DNA sequence dna, nucleic acid probe, primer and fragment.It is that many nucleic acid analogs are well-known in the art and contemplated.

Provided herein is polynucleotide sequence " fragment " be preferably at least 15 length of nucleotides continuous nucleotides subsequence.The fragment of the present invention preferably includes at least 20 nucleotides of the polynucleotides of the present invention, more preferably at least 30 nucleotides, more preferably at least 40 nucleotides, more preferably at least 50 nucleotides, most preferably at least 60 continuous nucleotides.The fragment of polynucleotide sequence can be used for antisense, gene silencing, triple helix or ribozyme technology, or as the primer in microarray, probe, or for the system of selection based on polynucleotides.

The term " fragment " relevant with promoter polynucleotide sequence is intended to include such sequence, and it includes the area of cis element and promoter polynucleotide sequence, and it can adjust the expression for the polynucleotide sequence that fragment is operably connected to.

The preferred fragment of the polynucleotide sequence of the present invention includes at least the 20 of the polynucleotides of the present invention, 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 continuous nucleotides.

Term " primer " refers to short polynucleotides, generally has 3 ' free OH groups, and it hybridizes with template and is used to trigger polymerizeing for the polynucleotides complementary with template.Such primer preferred length 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, the nucleotides of more preferably at least 20.

Term " probe " refers to short polynucleotides, and it is used for detection and the polynucleotide sequence of probes complementary in the measure based on hybridization.Probe can be made up of polynucleotides " fragment " as defined herein.It is preferred that such probe is length 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 the nucleotides of most preferably at least 500.

Term " variant " refers to the polynucleotides or peptide sequence of the sequence different from especially recognizing as used herein, and wherein one or more nucleotides or amino acid residue are removed, replace or added.Variant can be naturally occurring allele variant, or non-naturally occurring variant.Variant may be from identical or from other species and may include homologue, collateral homologue and ortholog thing.In some embodiments, the variant of polynucleotides and polypeptide has and wild-type polynucleotide or the same or analogous bioactivity of polypeptide.Term " variant " on polynucleotides and polypeptide includes the polynucleotides limited herein and polypeptide of form of ownership.

Phrase " functional variety " recognizes the amino acid sequence that may change protein and keeps the feature that is substantially identical.If for example, variant peptides in immunology with urporotein cross reaction, then for specific function, a kind of protein can be considered as the functional variety of another protein, and preferably have at least substantially with urporotein identical function.

Polynucleotides variant

The preferred display of variant polynucleotide sequence and specific polynucleotide sequence 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% homogeneity.Homogeneity is at least 20 nucleotide positions, preferably at least 50 nucleotide positions, is found in the comparison window of at least 100 nucleotide positions, or is found in whole length of specific polynucleotide sequence.

Polynucleotide sequence homogeneity can be determined in the following manner.Object polynucleotide sequence and candidate polynucleotide sequence are utilized can be from NCBI (ftp://ftp.ncbi.nih.gov/blast/) open bl2seq (the Tatiana A.Tatusova obtained, Thomas L. Madden (1999), " sequences-a new tool for comparing protein andnucleotide sequences ", the FEMS Microbiol Lett.174 of Blast 2：BLASTN (come from blast program group, version 2 .2.10 [in October, 2004]) in 247-250) compares.Using bl2seq default parameters, except the filtering of low-complexity part should be closed.

The homogeneity of polynucleotide sequence can be checked using following unix command line parameters：

Bl2seq-i nucleotides seq1-j nucleotides seq2-F F-p blastn

Parameter-F F close the filtering of low-complexity part.Parameter-p selects the appropriate algorithm of sequence pair.Sequence identity is reported as the row " Identities=" numbers and percentage of middle identical nucleotides by bl2seq programs.

Polynucleotide sequence homogeneity is also using overall sequence alignment programs (such as Needleman, S.B.andWunsch, C.D. (1970) J.Mol.Biol.48,443-453) calculate in whole length overlapping between candidate and object polynucleotide sequence.The complete implementation of Needleman-Wunsch global alignment algorithms is in EMBOSS program bags (Rice, P.Longden, I.and Bleasby, A.EMBOSS：The European Molecular Biology Open Software Suite, Trends in Genetics June 2000, vol 16, No 6.pp.276-277) --- it can be from http://www.hgmp.mrc.ac.uk/Software/EMBOSS/ obtain --- needle programs in find.European Bioinformatics research institute (European Bioinformatics Institute) server also provides facility with online in http:The upper EMBOSS-needle that carried out between two sequences of/www.ebi.ac.uk/emboss/align/ are totally compared.

Alternatively, using GAP programs, it calculates the optimal overall comparison of two sequences, not to end gap penalties.GAP is described in following paper：Huang, X. (1994) On Global Sequence Alignment.Computer Applications in the Biosciences 10,227-235.

The polynucleotides variant of the present invention also includes such polynucleotides variant, and its display is one or more similar to the sequence --- it may keep the functional equivalent of those sequences --- especially recognized, and it can not reasonably expect accidentally occurred.Such sequence similarity on polypeptide is available from NCBI (ftp://ftp.ncbi.nih.gov/blast/) disclose the bl2seq programs of acquisition to determine from blast program group (version 2 .2.10 [in October, 2004]).

The similitude of polynucleotide sequence can be determined using following unix command line parameters：

Bl2seq-i nucleotides seq1-j nucleotides seq2-F F-p tblastx

Parameter-F F close the filtering of low-complexity part.Parameter-p selects the appropriate algorithm of sequence pair.The program finds the similitude area between sequence and to each such area's record " E values " --- it is that a people can accidentally see the desired number of times of such matching in the database containing random sequence of fixed reference size.The size of database default setting in bl2seq programs.For small E values --- much smaller compared with one, E values are about the probability of such random fit.

When any of sequence with especially recognizing is compared, the preferred display of variant polynucleotide sequence is less than 1 × 10^-10, more preferably less than 1 × 10^-20, less than 1 × 10^-30, less than 1 × 10^-40, less than 1 × 10^-50, less than 1 × 10^-60, less than 1 × 10^-70, less than 1 × 10^-80, less than 1 × 10^-90, less than 1 × 10^-100, less than 1 × 10^-110, less than 1 × 10^-120Or less than 1 × 10^-123E values.

Alternatively, variant polynucleotides of the invention hybridize under strict conditions with specific polynucleotide sequence or its complementary strand.

Term " under strict conditions hybridize " and its grammatically equivalent refer to the ability that polynucleotide molecule hybridizes with target polynucleic acid molecules (fixed target polynucleic acid molecules on such as DNA or RNA traces, such as Southern traces or Northern traces) at the temperature and salt concentration conditions of restriction.The ability hybridized under stringent hybridization condition can increase to desired stringency to determine by the initially hybridization under the conditions of less stringent and then by stringency.

The polynucleotide molecule of 100 bases is greater than about on length, common stringent hybridization condition is to be no more than 25 to 30 DEG C below (for example in natural double helix melting temperature (Tm), 10 DEG C) (generally referring to, Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, the second edition .Cold Spring Harbor Press；Ausubel et al., 1987, Current Protocols in Molecular Biology, Greene Publishing).The Tm of the polynucleotide molecule of greater than about 100 bases can (G+C-log (Na+) be calculated by formula Tm=81.5+0.41%.(Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, second edition .Cold Spring Harbor Press；Bolton and McCarthy, 1962, PNAS 84：1390).The common stringent condition that length is more than the polynucleotides of 100 bases will be the hybridization conditions such as prewashing in 6 × SSC, 0.2%SDS solution；At 65 DEG C, 6 × SSC, 0.2%SDS hybridize a whole night；It is the cleaning of 30 minutes twice afterwards, every time at 65 DEG C in 1 × SSC, 0.1%SDS, and the cleaning of 30 minutes twice, every time at 65 DEG C in 0.2 × SSC, 0.1%SDS.

DNA analog (Nielsen et al., Science.1991 Dec6 on being referred to as peptide nucleic acid (PNAs)；254(5037)：1497-500), Tm values are higher than DNA-DNA or DNA-RNA heterocomplexs, and using Giesenet al., Nucleic Acids Res.1998Nov 1；26(21)：Formula described in 5004-6 is calculated.The Exemplary stringent hybridization condition that length is less than the DNA-PNA heterocomplexs of 100 bases is in 5 to 10 DEG C of below Tm.

The variant polynucleotides of the present invention also include such polynucleotides, and it is different from the sequence of the present invention, but as the result of degenerate, it is encoded with the active polypeptide similar to the polypeptide of the polynucleotide encoding of the present invention.The sequence change for not changing the amino acid sequence of polypeptide is " silent variant ".Except ATG (methionine) and TGG (tryptophan), other codons of same amino acid can be changed by the generally acknowledged technology in field, for example, to optimize the expression of the codon in specific host organism.

Polynucleotide sequence change of the conservative substitution of one or several amino acid in the peptide sequence of coding without significantly changing its bioactivity is caused to be also included in the present invention.Those skilled in the art will be appreciated by preparing the method for the amino acid replacement of silence in phenotype (see, e.g., Bowie et al., 1990, Science 247,1306).

In the peptide sequence of coding due to silent variant and conservative substitution variant polynucleotides using from NCBI (ftp://ftp.ncbi.nih.gov/blast/) determined from the open bl2seq programs obtained of blast program group (version 2 .2.10 [in October, 2004]) by tblastx algorithms as described earlier.

Polypeptide variants

Term includes naturally occurring, restructuring ground and the polypeptide synthetically produced on " variant " of polypeptide.The preferred display of variant polypeptide sequence and the sequence at least 50% of the present invention, 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% homogeneity.Homogeneity is at least 20 amino acid positions, preferably at least 50 amino acid positions, is found in the comparison window of at least 100 amino acid positions, or is found in whole length of the polypeptide in the present invention.

Peptide sequence homogeneity can be determined in the following manner.By subject polypeptide sequence and candidate polypeptide sequence utilize in bl2seq BLASTP (coming from blast program group, version 2 .2.10 [in October, 2004]) --- it can be from NCBI (ftp://ftp.ncbi.nih.gov/blast/) open acquisition --- to compare.Using bl2seq default parameters, except the filtering of low-complexity part should be closed.

Peptide sequence homogeneity can also be calculated using in overall sequence alignment programs whole length overlapping between candidate and object polynucleotide sequence.EMBOSS-needle as described above is (in http:/ www.ebi.ac.uk/emboss/align/ is obtained) and GAP (Huang, X. (1994) On Global Sequence Alignment.Computer Applications in the Biosciences 10,227-235.) it also is adapted for calculating the overall sequence alignment programs of peptide sequence homogeneity.

The polypeptide variants of the present invention also include such polypeptide variants, its display and one or more similitudes in the sequence --- it may keep the functional equivalent of those sequences --- especially recognized, and it can not reasonably expect accidentally occurred.Such sequence similarity on polypeptide is available from NCBI (ftp://ftp.ncbi.nih.gov/blast/) disclose the bl2seq programs of acquisition to determine from blast program group (version 2 .2.10 [in October, 2004]).The similitude of peptide sequence can be determined using following unix command line parameters：

Bl2seq-i peptide seq1-j peptide seq2-F F-p blastp

When any of sequence with especially recognizing is compared, the preferred display of variant polypeptide sequence is less than 1 × 10^-10, more preferably less than 1 × 10^-20, less than 1 × 10^-30, less than 1 × 10^-40, less than 1 × 10^-50, less than 1 × 10^-60, less than 1 × 10^-70, less than 1 × 10^-80, less than 1 × 10^-90, less than 1 × 10^-100, less than 1 × 10^-110, less than 1 × 10^-120Or less than 1 × 10^-123E values.

Parameter-F F close the filtering of low-complexity part.Parameter-p selects the appropriate algorithm of sequence pair.The program finds the similitude area between sequence and to each such area's record " E values " --- it is that a people can accidentally see the desired number of times of such matching in the database containing random sequence of fixed reference size.For small E values --- much smaller compared with one, this is about the probability of such random fit.

The conservative substitution --- not significantly changing its bioactivity --- of one or several amino acid of the peptide sequence of description is also included in the present invention.Those skilled in the art will be appreciated by preparing the method for the amino acid replacement of silence in phenotype (see, e.g., Bowie et al., 1990, Science 247,1306).

The polypeptide variants of the present invention also include such polypeptide variants, and it is produced from the nucleic acid of coded polypeptide, but it is that it is treated differently for printing so that it has the amino acid sequence changed to be different from wild type peptide.For example variant can be produced by the alternative splicing pattern for the primary RNA transcript thing for producing wild type peptide.

Diagnostic kit

The present invention further provides diagnostic kit, it is used for the Lf allele distributions for determining mammalian object, such as the method for the present invention.

Correspondingly, in one embodiment, the present invention provides diagnostic kit, and it can be used for the Lf genotype of the inhereditary material of measure object.One kit includes one group of primer for being used to expand inhereditary material.Kit can contain primer, and it includes the nucleotide sequence for being used to expand the inhereditary material region containing one of naturally occurring mutation described herein.Such kit may also include the primer of the corresponding region for expanding the normal gene for producing functional wild type Lf.Generally, such kit is also by another primer of the upstream including gene regions or downstream.These primers are used to expand the section containing being purposefully mutated.The Genotyping of reality is carried out using such primer, and the primer targets specific mutagenesis described herein, and it can serve as allele specific oligonucleotide in conventional hybridization, Taqman measure, OLE measure etc..Alternatively, primer can be designed to allow to carry out Genotyping by microsequencing.

One primer kit can include first, second, and third primer (a), (b) and (c) respectively.Primer (a) is based on the region being mutated containing Lf as described above.The inhereditary material being mutated is contained in the region in the upstream in the region expanded by primer (a) or downstream by primer (b) coding will pass through PCR amplifications, for example, in the case where there are two primers.Primer (c) be based on corresponding to primer (a) based on region, but lack mutation.Therefore, the inhereditary material containing not mutated area will be expanded when there is primer (b) and (c).The product of amplification therefore will be provided when there is primer (b) and (c) for the inhereditary material of wild type gene homozygosis.The product of amplification therefore will be provided when there is primer (a) and (b) for the inhereditary material of the gene pure of mutation.The inhereditary material of heterozygosis will provide the product of amplification in both cases.

For example, the kit may include primer, hybridization that the primer is included in corresponding to the cytimidine of the position of 30126T/C polymorphisms in Lf genes or such nucleotides, the nucleotides energy and nucleotides --- can hybridize with the cytimidine of the position of 30126T/C polymorphisms in corresponding to Lf genes ---.It would be recognized by those skilled in the art that, in such primer, cytimidine, or energy and nucleotides --- can hybridize --- nucleotides of hybridization with cytimidine, it is such as available, can be by nucleotide analog --- with the nucleotides identical distinguishing base pairing with being replaced --- displacement.

In another example, kit may include primer, hybridization that the primer is included in corresponding to the thymidine of the position of 30126 promotor polymorphisms in Lf genes or including such nucleotides, the nucleotides energy and nucleotides --- can hybridize with the thymidine of the position of 30126 promotor polymorphisms in corresponding to Lf genes ---.It would be recognized by those skilled in the art that, in such primer, thymidine, or energy and nucleotides --- can hybridize --- nucleotides of hybridization with thymidine, it is such as available, can be by nucleotide analog --- with the nucleotides identical distinguishing base pairing with being replaced --- displacement.

It will be appreciated by those skilled in the art that the present invention provides kit, it includes similarly being related to primers one or more in 7447A/G polymorphisms or -7G/C polymorphisms.

In one embodiment, diagnostic kit is used in object, as detection includes the DNA that variant Lf genes or coding at least partly lack the variant Lf polypeptides of wild-type activity in ox, the kit includes the first and second primers for DNA amplification, this be respectively with the DNA upstream of polymorphism in Lf genes --- the Lf levels (the Lf contents particularly increased or decreased in breast or colostrum) for causing to increase or decrease --- and the complementary primer of the nucleotide sequence in downstream.In one embodiment, at least one nucleotide sequence is selected as the code area from Lf genes.In another embodiment, at least one nucleotide sequence is selected as the noncoding region from Lf genes.The kit may also include three-primer, and its naturally occurring mutation with wild type Lf gene codes part is complementary.Preferably, the kit is including the use of --- such as the method according to the invention --- specification.

In one embodiment, diagnostic kit includes the nucleotide probe with the sequence shown in NC_007320.3 or NM_180998 or the complementation of its oligonucleotide fragment, for example, for hybridizing with the mRNA from cell sample；With the method for the nucleotide probe that mRNA is attached in standard detection sample.In terms of specific, the kit of this aspect of the invention includes probe, and it has the nucleic acid molecules or its complementary strand of the sequence presented with NC_007320.3 or NM_180998 fully complementation, so as in connection under strict conditions." stringent hybridization condition " its common meaning is presented to those skilled in the art.Promote the appropriate stringent condition of nucleic acid hybridization, for example, it is known to the skilled person in about 45 DEG C of 6 × sodium chloride/sodium citrates (SSC), it is included in Current Protocols in MolecularBiology, in John Wiley ＆ Sons, NY (1989).Appropriate cleaning stringency depends on the degree and probe length of homology.If homology is 100%, then high temperature (65 DEG C to 75 DEG C) can be used.If however, probe is very short (＜ 100bp), then relatively low temperature must be used, even if with 100% homology.In general, starting cleaning in low temperature (37 DEG C to 40 DEG C), and temperature is raised into 3-5 DEG C of interval, until background is sufficiently low with the principal element as autoradiograph.The specification that the diagnostic kit can also be used containing kit.

In another embodiment, diagnostic kit includes antibody or antibody compositions, presence or shortage and/or the variant proteins at least partly lacking wild-type activity for detecting wild type Lf, the particularly presence of the clipped form of Lf albumen or shortage, and use --- for example in the method for the invention --- specification.

5 sample preparations

Such as those skilled in the art will be apparent, be suitable for the sample of the method for the present invention easily can obtain from tissue or liquid, so that sample contains detected part.For example, in the case where nucleic acid will be analyzed, tissue or liquid containing nucleic acid will be used.

Easily, sample may be derived from breast, colostrum, tissue --- including blood, serum and blood plasma, cerebrospinal fluid, urine, seminal fluid or saliva.Tissue sample can be obtained using standard technique such as cell scapes or biopsy technology.For example, cell or tissue sample can gather ear tissue to obtain by using ear card punch from ox.Similarly, routinely carry out blood sampling, for example, tested for pathogen, the method for obtaining blood sample is well-known in the art.Equally, the method for storage and processing biological sample is well-known in the art.For example, if desired, tissue sample can be frozen until being examined.In addition, it would be recognized by those skilled in the art that classification or purifying procedure --- for example, whole blood is separated into serum or plasma component --- afterwards, some test samples will be easier analyze.

The related embodiment of 6 computers

It is also understood that the method for the present invention is easy to be used together with computer system, software and program and with computer system, software and program analysis result.What computer system, software and the program of identification and analysis genetic polymorphism were well-known in the art.For example, the result of one or more genetic analyses as described herein can be analyzed using computer system and handled by such system.

The SNPs and SNPs analysis result utilized in the present invention can promote it to use to be permitted medium " offer ".As used by the part, " offer " refers in addition to the nucleic acid molecules of separation, the product of the SNP information containing the present invention.Such product provides the SNP information of such form, and the form allows technical staff using being not directly used for checking SNPs or its hypotype --- in the presence of they are in the form of the natural or purifying --- method check product.The SNP information that can be provided in such form includes any SNP information of the invention provided such as, for example, polymorphic nucleic acid and/or amino acid sequence information, the information on the SNP allele observed, optional codon, population, gene frequency, protein, phenotypic effect or the joint of SNP types and/or influence, or any information that the present invention is provided in table 1 and 2 and/or serial ID list.

In an application preferably, the SNPs and SNPs analysis result utilized in the present invention is recordable on computer-readable medium.As used herein, " computer-readable medium " refers to any medium that can be directly read and access by computer.Such medium includes but is not limited to：Magnetic storage medium, such as floppy disk, hard disk storage medium and tape；Optical storage media such as CD-ROM；Electric storage medium such as RAM and ROM；With the hybrid such as magnetic optical storage medium of these classification.Technical staff can easily be understood that how any computer-readable medium being currently known can be used for generation and include the product of computer-readable medium --- the SNP information for being recorded on the present invention ---.One such medium has the present invention, i.e., the application contains computer-readable medium (floppy disk), it has the nucleotide sequence for being used for analyzing the SNPs utilized in the present invention, together with derivative amino acid sequence, these sequences are provided/recorded on medium in serial ID list with ASCII text formattings.

As used herein, " record ", which refers to, store up on a computer-readable medium stored process.Technical staff can easily use any method being currently known of record information on a computer-readable medium to produce the product of the SNP information including the present invention.

Technical staff, which can obtain many kinds of data store organisations and be used to produce the SNP information of the present invention, is recorded computer-readable medium thereon.The selection of data store organisation will be typically based on the method to access the information stored of selection.In addition, many kinds of data processor programs and form can be used for the SNP information for storing the present invention on a computer-readable medium.For example, sequence information can be represented with word processing text, formatted, represented in the form of ascii text file in commercially available software such as WordPerfect and Microsoft Word, or be stored in database application, in such as OB2, Sybase, Oracle or similar program.Technical staff can easily adapt any number of data processor architecture form (for example, text or database) to obtain the computer-readable medium of the SNP information records of the present invention thereon.

By providing the SNPs utilized in the present invention and/or SNPs analysis result with computer-reader form, technical staff can routinely access SNP information for numerous purposes.Computer software is open obtainable, and it allows the sequence information provided on technician access computer-readable medium.The example of open obtainable computer software includes BLAST (Altschul et at, J.Mol.Biol.215：403-410 (1990)) and BLAZE (Brutlag et at, Comp.Chem. 17：203-207 (1993)) searching algorithm.

The present invention also provides system, is based particularly on system for computer, it contains SNP information described herein.Such system can be designed to store and/or analyze, for example, the information in the information on many SNP positions, or the SNP genotype from many objects.The SNP information of the present invention represents valuable information source.The SNP information of the invention of storage/analysis can be used for such application such as identification or selecting object in computer based system, it is divided into haplotype except the SNP gene frequencies in the application such as measure of computer intensive or analysis colony, drafting disease gene, genotype-Phenotype correlative study, by SNPs, makes SNP haplotypes associated with certain drug response, or for various other bioinformatics, Drug Discovery, drug development, or selection or identification application.

As used herein, " computer based system " refers to hardware, software and the data storage of the SNP information for analyzing the present invention.The minimal hardware of the computer based system of the present invention generally includes central processing unit (CPU), input equipment, output equipment and data storage.Technical staff is it can be easily understood that any one currently available computer based system is suitable for the present invention.By using SNP information, on such as floppy disk provided herein is SNP information, such system can be rewritten into the system or its hypotype of the present invention, without any experiment.

As described above, the computer based system of the present invention includes data storage, it, which has, stores SNP information therein, the SNPs and/or SNPs analysis result utilized in such as present invention, and for supporting and performing the necessary hardware and software of one or more programs or algorithm.As used herein, " data storage ", which refers to, can store the memory of the SNP information of the present invention, or may have access to memory access device of the SNP information records in product thereon of the present invention.

One or more programs or algorithm perform the SNP information with identification or analyze data memory memory storage in computer based system.For example, such program or algorithm can be used for determining the specific SNP positions which nucleotides is present in target sequence, or for the result for the genetic analysis for analyzing SNPs described herein.As used herein, " target sequence " can be any DNA sequence dna containing the SNP positions (or multiple) that will be analyzed, retrieve or inquire about.

The multiple structural forms of being permitted of input equipment and output equipment can be used for input and output information in the computer based system of the present invention.The exemplary form of output equipment is display, and it describes SNP information, such as specific target SNP positions specific nucleotide (allele) presence or shortage.Such presentation can be that many SNPs or object provide quick, two-symbol points-scoring system simultaneously.It should be understood that such output equipment can, for example remotely accessed by LAN or Internet.Generally, the property of SNP information is given, this remote access of such output equipment or computer system in itself is only the available security to keep SNP information and/or computer system to the user of checking.Control accesses computer system and existed that the methods of data thereon is well-known in the art and suitable for embodiments of the present invention.

An illustrative embodiments --- it can be used for realizing the present invention --- for the computer based system of SNP information including the present invention are including the processor being connected with bus.Be connected similarly to bus is main storage (preferably as random access storage device, RAM is performed) and many kinds of secondary storage devices, such as hard disk driver and removable media storage devices.Removable media storage devices can be showed for example, floppy disk, CD-ROM drive, tape drive etc..Removable media storage device is can be plugged into containing the control logic recorded wherein and/or the removable storage medium (such as floppy disk, CD, tape) of data.Computer system, which includes appropriate software, to be used to read control logic and/or data from the removable storage medium for being once inserted into removable media storage device.The SNP information of the present invention can be stored in main storage, any secondary storage devices and/or removable storage medium in a manner known.Access and the software (such as SNP scoring instrument, gopher, compare) of processing SNP information is being preferably in main storage during performing.

Correspondingly, the present invention is provided to carry out systems one or more in the method for the present invention, the system includes：

Computer processor device for receiving, handling and transmitting data；

For the storage device of data storage, the data include reference genetic database and the optionally reference Lf content phenotype database of the non-genetic factor of mammal Lf content phenotype of the mammalian object on the genetic analysis result of one or more Lf contents phenotypes；With

It is implanted to the computer program in computer processor, once --- it is included in by genetic analysis result for its data with reference in genetic database --- and is constituted or the data including the genetic analysis result are received, the computer program handles the data to determine the genetic state of object as a result under the background of the reference database, the result has preferably inputted the user of the data once known can be sent to.

Preferably, the system may have access to by internet or by personal computer.

In a further aspect, the present invention provides the computer program for being suitable for system as defined above, the system includes the available medium of computer, it has the program code for including the data for causing computer programs process to receive in media as well, the data are made up of or included the result of at least one genetic analysis with one or more genetic locis of one or more Lf contents phenotypes correlation the result of at least one genetic analysis of one or more genetic locis related to one or more Lf contents phenotypes, the result of at least one genetic analysis reference genetic database and optionally related to mammal Lf content phenotypes non-genetic factor reference database background under.

According to the useful composition of the present invention

Composition for this paper can be configured to food, beverage, food additives, beverage additive, dietary supplements, nutrition product, dietetic food, enteral or parenteral nursing product, Meal replacements, nutraceutical, cosmeceutical (cosmeceutical) or medicine.Appropriate preparation can be prepared by skilled worker according to the teaching of the technology and this specification.

It is that dosage, Dressing date and the total application program of the composition of administration between objects can be different, depending on age, sex and/or general health of such variable such as the method for application selected, and object as will be understood.

In one embodiment, the composition for this paper includes parent formula, infant formula, larger infant formula and growth formula.Such product is formulated to nutrient targetting fetus, baby and children.It should be appreciated that the Dai-ichi Mutual Life Insurance stage (children of fetus, baby and growth) includes important growth and development.Promoting any support of development can all play an important roll to ontogeny.

In another embodiment, the composition for this paper includes diet product.

Term " parent formula " as used by this specification refers to the composition drunk for pregnant woman in gestation.Term " infant formula " as used by this specification refers to the composition of the baby for the age between 0 day and 6 months.Term " larger infant formula " as used by this specification refers to the composition of the baby for the age between 6 months and 1 years old.Term " growth formula " as used by this specification refers to the composition of baby and children for more than 1 years old.Growth formula is included such as by the growth formula milk being identified by a person skilled in the art.

Those skilled in the art will understand in addition, the range of age of different components：" infant formula ", " larger infant formula " and " growth formula " can be different between children, depending on ontogeny.

Term " diet product " refers to especially processing or prepared to meet the product of special diet requirement, and special diet requirement exists due to specific body or physiological condition and/or special disease and symptom and existed like this.

" object " is animal, preferably mammal, more preferably companion animal (companion animal), the agricultural animal of mammal or the people of mammal.It is preferred that companion animal include cat, horse and dog.It is preferred that agricultural animal include ox, sheep, deer and pig.

It should be appreciated that being not intended to limit the invention to the unique example of the above, many changes without departing from scope defined in the appended claims that those skilled in the art can be readily apparent that are possible.

The present invention can be also largo interpreted as being present in the part in present specification individually or collectively pointed out or indicate, component and feature, any two or any or all multiple combinations with the part, component or feature, wherein specific integer is mentioned herein, it has known equivalent in related field of the invention, think that such known equivalent is incorporated herein, as being individually illustrated.

Imagine the invention reside in above-mentioned content and also content construction --- embodiment is provided to it below.

Embodiment --- the analysis of the hereditary basis of the Lf contents of breast and colostrum

The embodiment describe to study to promote the result of the QTLs related with economically important newborn phenotype, gene and the Holstein-Friesian of the discovery of mutation X Jersey cross experiments using progress it was observed that colostrum Lf changes of contents hereditary basis.

Material and method

1. experimental design

Holstein-Friesian x Jersey cross experiments are carried out using the F2 experimental designs with half sibs family structure.The reciprocal cross of Holstein-Friesian and Jersey animals is carried out to produce the F1 bulls of six high genetic states.Then to be mated by high genetic state F1 cows with these F1 bulls and to form 850 basic F2 female descendants of trial flock to produce.Group is formed in two seasons；Animal in cohort 1 was born spring in 2000, and spring in 2002 into their the first lactation, and the animal in cohort 2 was born spring in 2001 and entered their the first lactation spring in 2003.724 F2 cows enter their the second lactation and the colostrum sample of the collection more than 600 from these altogether.Using the management system based on pasture come cultivated animals under New Zealand's dairy industry cultivation practice of standard.All animal work are carried out according to the Ruakura animal welfares committee.

2. Lf is measured in breast

All newborn composition measurements are carried out during the second lactation.Mother is milked twice daily；Milk every time and all record milk volume.Three point in time measurement of the Lf during lactation：Peak lactation (after calving 35 days), middle lactation (mid-term in November) and late period lactation (late period in February).In each collection day, collection sample and it is mixed from milking for the morning and afternoon to prepare the single biased sample for each animal.For the research of milking once a day carried out in the middle lactation of lactation 2, all cows are milked once a day, continue 7 days, the am/pm milk sample of mixing is in a few days ago obtaining of milking once a day and the sub-sampling again after milking once a day the 5th day of (morning).For research of milking once a day, Lf is measured using ion-exchange chromatography and in middle lactation by ELISA in peak, middle and late period lactation (the 50th day).Utilize ox Lf ELISA quantification kits (Bethyl laboratories Inc, Montgomery, Texas, USA；Catalog number E10-126) measure Lf according to the specification of producer.Normative reference containing 1mg/ml oxen Lf is used for correcting determination.The working range of measure is 0-1000 ug/ml.

3. Genotyping

1679 animals prepare genomic DNA from whole blood altogether from experiment pedigree (the F0 breeding stocks that 846 F2 filial generations, six F1 breeding stocks, 796 F1 dams and 13 select).Initial genome-wide screening carries out Genotyping to carry out by 285 microsatellite markers for mainly obtaining from disclosed mark figure to every animal.Then, Genotyping is carried out to pedigree using Affymetrix Bovine 10K SNP GeneChip.6634 informedness SNP markers are placed on figure altogether.

4. candidate gene is sequenced

To the code area (17 extrons) of Lf genes and the sequences of 0.5kb 5 '.Determine introne/exon boundary.Amplification extron and sequencing in the two directions.

5. statistical analysis

Data set is made up of the Lf phenotypes (being obtained three time points) collected from two cohort F2 animals.Data processing is carried out using SAS (version 9.1).These data are matched with following correlated variables (covariates)：Cohort (cohort 1 or cohort 2), breeding stock (breeding stock 1-6), lipid, true protein, casein, lactose, total solid, calving all (0-13), mating all (0-11), newborn volume, FFA and Somatic Cell Count (＜ 200 or ＞=200).Mating week is only in middle and late period lactation detection.Breast composition correlated variables is individually matched by lactation.Think the three Lf genotype and Data Matching related to Lf.The animal with the loss data point for any measurement is excluded from final manifold.(including correlated variables) data set finally matched includes 502 respectively for peak, middle and late period lactation, 611 and 593 animals.

Statistical analysis is carried out using unprocessed phenotypic data.ANOVA is carried out to Lf phenotypes significantly correlated to determine whether Lf polymorphisms and breast Lf contents.The last ANOVA models for each time point are produced using back elimination process (backward elimination process)；In the first stage of modeling process, all correlated variables are included in a model, and it is important (sig.level set at 0.1) to remove most unessential correlated variables until finding all remaining correlated variables in each subsequent stage.Last model is as follows：

Lf_iP=μ+breeding stock+lipid_P+ true protein_P+ lactose_P+ total solid_P+ calving week_P+ milk production_P+ remaining_iP

Lf_iM=μ+breeding stock+cohort+lipid_M+ true protein_M+ casein average_M+ lactose_M+ mating week_M+ milk production_M+FFA_M+ remaining_iM

Lf_iL=μ+breeding stock+cohort+lipid_L+ true protein_L+ casein average_L+ lactose_L+ milk production_L+FFA_L+ remaining_iL

Wherein, i is individual animals, the lactation of P=peaks, M=centre lactations and L=late period lactations

6.QTL is detected

It is the residue from statistical model for the QTL data detected.Unprocessed phenotype term (no correlated variables or modeling) is also used for detecting QTLs.QTL detections are carried out using a line blood lineage model and half sibs model.Then, Lf genotype is individually included into model determining whether these polymorphisms explain that QTL changes as correlated variables.

As a result

1. breast Lf

The Lf contents of breast observe highest concentration in peak, middle (Fig. 1) different with late period lactation, late lactation, and on peak, minimum composition is observed in lactation.Lf concentration is always according to breeding stock different (Fig. 2) in breast.

2. breast Lf QTL detections on N chromosome 22

Lf analysis (Haley et al., 1994and Baret et al., 1998) is shown on ox chromosome 22 significant QTL (Fig. 1) in the half sibs model of qtl analysis.QTL maximum F values are 10.19, and most likely location is estimated in 70cM.Bootstrap analysis (n=1000) shows that QTL 95% confidential interval is 70-77cM.There are 205 marks (7 microsatellite markers and 198 SNPs) altogether for the analysis on chromosome 22.

3. as the Lf of candidate gene identification and influence breast Lf contents polymorphism detection

Lf combines its part, Fe and Fe ions and the transport for mediating it.Lf is accredited as the candidate gene of chromosome 22 LfQTL effects.In order to determine whether that the gene explains that the Lf code areas in the change observed in breast Lf contents, six F1 breeding stocks of sequencing can potentially change any genetic polymorphism of the protein function to recognize.Introne/exon boundary is determined using reference sequences NC_007320.3.Primer is designed in introne to obtain complete sequence from each extron.Other 0.5kb sequences are obtained from 5 ' UTR.

Three polymorphisms (breeding

stock

3,4 and 6) isolated in identical breeding stock as QTL are identified and in the residue that FJXB tests pedigree by Genotyping.The position of the polymorphism of these in gene and sequence vestige are shown in Fig. 4.The frequency of each Lf genotype is shown in following table 1.

Table 1：The genotype frequency of F2FJXB populations

	Genotype	N	Frequency
				Lf_30126	CC	19	0.0232
	CT	263	0.3211
					TT	537	0.6557
Lf_-7	CC	85	0.1037
					CG	364	0.4439
	GG	371	0.4524
				Lf_7447	AA	429	0.5303
	AG	333	0.4116
					GG	47	0.0581

Note：Based on successfully data of the animal of Genotyping in F2 populations

4.Lf polymorphisms play an important roll to Lf concentration in breast

Effect of the Lf30126T/C polymorphisms to the newborn concentration of Lf is shown in Figure 5.Genotype is significant to the P ＜ 0.0000001 that act predominantly on of Lf contents.ANOVA, which is added to, as the Lf genotype of correlated variables has fully phased out effects of the QTL observed for Lf-7G/C polymorphisms to chromosome 22 (Fig. 6), and Lf30126T/C polymorphisms explain jumpbogroup but are not all of QTL.

Following table 2 is shown in the average Lf concentration of peak, centre and late period lactation for various genotype.

Table 2：The phenotype average value of F2FJXB populations

5. LfmRNA eQTL is directed on chromosome 22

Except the ox chromosome 22 QTL for newborn Lf concentration, it was further observed that for the QTLs (" eQTL ") (referring to Fig. 7) of LfmRNA amounts in the hepatic tissue sample that is obtained in early stage lactation.Compared the maximum F values of display with the QTL for newborn Lf concentration and appear in the same position being directed on the chromosome 22 of breast Lf concentration and liver expression (referring to Fig. 7).In addition, adjusting breast Lf phenotypes by the way that Lf mRNA are used as into correlated variables, cancel QTL effects, the lactoferrin that effect of the indicator genotype to milk lactoferrin also will expand in other bodily tissues is expressed (referring to Fig. 8).

6.Lf polymorphisms play an important roll to Lf concentration in colostrum

Colostrum sample is obtained when second milks after calving and by elisa assay lactoferrin.As a result it is shown in following table 3.

The concentration of the lactoferrin in biestings in Friesian-Jersey cross experiments of table 3.

Genotype	Average lactoferrin mg/l (SEM)	N
			Lf_30126C	124(39)	16
CT	140(20)	213
			T	151(7)	426
Lf_-7C	89(13)	67
			CG	147(15)	286
G	159(9)	300
			Lf_7447G	63(10)	34
GA	124(8)	254
			A	173(14)	352

SNP Lf_7447 and SNP Lf_-7 show effect (being P ＜ 0.05 and P ＜ 0.01 respectively) statistically significantly by ANOVA.

7. milk once a day with Lf polymorphisms has synergy to Lf concentration in breast

During twice daily being milked by elisa assay and be transformed into milk once a day after the lactoferrin of sample that obtains for 5 days.As a result it is shown in following table 4.

Table 4. is transformed into milk once a day before and after milk lactoferrin concentration in lactation in the middle of Friesian-Jersey crossbred cows

Milked once a day in middle lactation make in milk lactoferrin concentration increase approximately twice as.On an average, increase is from 273 ± 7mg/l to 550 ± 14mg/l.Polymorphism and effect of milking all are statistically significant (P ＜ 0.01).Such as can table 4 it is visible, advantage of the favourable SNP genotype of increase of milking once a day in terms of increased milk lactoferrin concentration.

Embodiment two --- the generation of the whey powder rich in lactoferrin

Embodiment description produces the dairy products rich in lactoferrin from the milk rich in lactoferrin of the ox collection selected using the method for the present invention.

Milk once a day and cause the generation containing the milk for having more than 600mg/l lactoferrins with combining for one or more of the TT genotype in 30126T/C polymorphisms, the AA genotype in 7447 A/G polymorphisms or the GG genotype in -7G/C polymorphisms (referring to table 4 above).

In whey fraction, this will increase lactoferrin ratio from 1-2%w/w protein until up to 5-6%w/w protein.The generation of protein portion rich in lactoferrin is milked and in cow once a day by application in addition --- wherein other main protein, such as main lactalbumin beta lactoglobulin, exist with relatively low concentration --- select favourable Lf allele and increase.For example, beta lactoglobulin typically comprises the lactalbumin of about 50-60% in batch milk.But, compared with AA polymorphisms, the BB polymorphisms of beta lactoglobulin (Prosser et al. related to the reduction of beta lactoglobulin concentration in milk about 25%, 2000.J.Dairy Research 67,287-283, Robitaille et al., Quantitative analysis of b-lactoglobulin A and B genetic variants in milk of cows b-lactoglobulin AB throughout lactationJournal of Dairy Research (2002) 69651-654).The 7-8%w/w that lactoferrin ratio reaches lactalbumin can further be increased by combining the selection of the cow of one or more of the TT genotype of 30126T/C polymorphisms, the AA genotype in 7447A/G polymorphisms or GG genotype in -7G/C polymorphisms in ox Lf genes with the BB polymorphisms for beta lactoglobulin.

Therefore, the acquisition of the lactoferrin content in whey fraction close to 20%w/w can be realized in the milk that beta lactoglobulin exists with about 1g/l.Such concentration will promote the concentration of the dairy products with lactoferrin, without extensive purifying.

Discuss

Present invention recognizes that Lf polymorphisms described above together with the polymorphism in linkage disequilibrium with specific Lf produce, regulation or secretion phenotype object identification in it is useful, for example alternatively instrument produces to breed with higher or lower Lf, such as higher or lower colostrum or the agricultural animal of breast Lf concentration.Especially, genotype and the combination milked once a day synergistically increase the hereditary effect to milk lactoferrin content, it is allowed to obtain the bigger increase of milk lactoferrin concentration.

Publication

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Commercial Application

There is desired Lf to produce, adjust or secretion phenotype to promote to identify or select the present invention relates to the method to mammalian object Genotyping, include desired breast or the object of colostrum Lf content phenotypes.Especially, these phenotypes include it is increased breast or colostrum Lf contents, reduction breast or colostrum Lf contents.Expect in agricultural application, will produce breast or colostrum with more desirable Lf contents for the ox in groups of such Phenotypic Selection, generation will be allowed to have, for example, the product of increased Lf contents, and therefore by with important social and economical benefits.

Claims

1. the method for the mammalian object of identification or selection with one or more desired lactoferrins (Lf) generations, regulation or secretion phenotype, methods described includes the Lf allele distributions for determining the object, and on the basis of the measure recognizes or select the object.

2. method as claimed in claim 1, wherein one or more of desired lactoferrins are produced, regulation or secretion phenotype are increased breast or colostrum Lf contents.

3. such as claim 1 or claim 2 method claimed, wherein the Lf allele distributions are determined by providing the analysis result for existing or lacking to one or more polymorphisms in following one or more related Lf genes of the sample from the object：

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

(b) the Lf secretions increased or decreased, or

4. such as any one of claims 1 to 3 method claimed, wherein the mammalian object is ox.

5. such as claim 4 method claimed, wherein the Lf allele distributions by determine it is following any one or more presence or shortage determine：

A) in the Lf genes 30126T/C polymorphisms C allele；Or

B) in the Lf genes 30126T/C polymorphisms T allele；Or

C) in the Lf genes 7447A/G polymorphisms G allele；Or

D) in the Lf genes 7447A/G polymorphisms A allele；Or

E) in the Lf genes -7G/C polymorphisms C allele；Or

F) in the Lf genes -7G/C polymorphisms G allele；Or

G) there is a) presence of polymorphism or shortage into the linkage disequilibrium of any one or more in f) above, particularly with a) presence of polymorphism or shortage into 100% linkage disequilibrium (D '=1.0) of any one or more in f) above.

6. such as claim 5 method claimed, wherein the Lf allele distributions are determined by determining the presence with one or more of the following linkage disequilibrium of any one or more polymorphism：

A) in the Lf genes 30126T/C polymorphisms C allele；Or

B) in the Lf genes 30126T/C polymorphisms T allele；Or

C) in the Lf genes 7447A/G polymorphisms G allele；Or

D) in the Lf genes 7447A/G polymorphisms A allele；Or

E) in the Lf genes -7G/C polymorphisms C allele；Or

F) in the Lf genes -7G/C polymorphisms G allele；Or

7. such as any one of claim 1 to 6 method claimed, wherein the Lf allele distributions are determined by providing the analysis result of the Lf genes of the sample from the object or the expression of gene outcome or activity.

8. on breast or colostrum lactoferrin content, or on producing the method for recognizing or selecting ox by the ability with one or more desired breasts or the offspring of colostrum lactoferrin content phenotype, methods described includes the data for providing the Lf allele distributions on the ox, and on the basis of the data recognizes or select the ox.

9. method as claimed in claim 8, wherein the data on Lf allele distributions include

A) presence in one or more allele of one or more polymorphisms or the data of shortage are indicated, the polymorphism influences the expression from the Lf genes or expression or the activity of Lf gene outcomes, or

B) presence in one or more allele of one or more polymorphisms or the data of shortage are indicated, the polymorphism is related to the expression increased or decreased from the Lf genes, or the expression increased or decreased or active related to Lf gene outcomes, or

C) indicate in the polymorphism with (a) or (b) in one or more linkage disequilibriums one or more polymorphisms one or more allele presence or the data of shortage.

10. method as claimed in claim 9, wherein one or more of polymorphisms are in the Lf genes.

11. method as claimed in claim 10, wherein one or more of polymorphisms are in the coded sequence of the Lf genes.

12. such as any one of claim 8 to 11 method claimed, wherein the data on Lf allele distributions include indicating the presence in one or more allele of one or more polymorphisms or the data of shortage, the polymorphism is selected from

30126T/C polymorphisms in the Lf genes；

7447A/G polymorphisms in the Lf genes；

- 7G/C polymorphisms in the Lf genes；Or

Indicate positioned at the presence with one or more one or more allele selected from one or more of the following linkage disequilibrium of polymorphism polymorphism or the data of shortage

30126T/C polymorphisms in the Lf genes；

7447A/G polymorphisms in the Lf genes；

- 7G/C polymorphisms in the Lf genes.

13. such as any one of claim 9 to 12 method claimed, wherein the step of availability of data includes expanding the fragment of at least described ox Lf gene orders is to determine presence or the shortage of one or more of allele.

14. method as claimed in claim 13, wherein the step of amplification utilizes one or more primers, the primer includes coming from SEQ ID NOs：Any of 1 to 3 at least 12 continuous nucleotides.

15. such as any one of claim 9 to 12 method claimed, wherein the presence of one or more of allele or lacking the expression by determining Lf genes or gene outcome or activity to determine.

16. probe or primer, it includes the nucleotide sequence of the continuous base of pact at least 12 with NC_007320.3 or NM_180998, wherein the probe or primer are included in the guanine corresponding to the position of 7447A/G polymorphisms in the Lf genes；Or nucleotides, its energy and nucleotides --- can hybridize --- hybridization with the guanine of the position of 7447A/G polymorphisms in corresponding to the Lf genes.

17. probe or primer, it includes the nucleotide sequence of the continuous base of pact at least 12 of the complementary strand of the complementary strand or NM_180998 with NC_007320.3, wherein the probe or primer are included in the thymidine corresponding to the position of 7447A/G polymorphisms in the Lf genes；Or nucleotides, its energy and nucleotides --- can hybridize --- hybridization with the thymidine of the position of 7447A/G polymorphisms in corresponding to the Lf genes.

18. probe or primer, it has SEQ ID NO：At least 12 continuous bases of one of 1 to 3 pact or its complementary strand.

19. pair of primers, it includes two such as any of claim 16 to 18 primers claimed.

20. N, it is recognized or selected by the method any one of claim 4 to 15.

21. ox as claimed in claim 20, wherein the ox is bull.

22. the seminal fluid collected, it is produced by ox as claimed in claim 21.

23. ox as claimed in claim 20, wherein the ox is cow.

24. the method for a herd of cattle is selected, including by the method choice individual any one of claim 4 to 15, and separate and assemble the individual of the selection to form the group.

25. a herd of cattle, it passes through the method choice described in claim 24.

26. a herd of cattle including two or bull ox, wherein the ox is the offspring by one of the method choice any one of claim 4 to 15 or bull ox.

27. the breast collected or mixed, it passes through such as claim 23 ox claimed and produced.

28. the breast collected or mixed, it passes through such as claim 25 a herd of cattle claimed and produced.

29. such as the breast of the collection claimed of claim 27 or 28 or mixing; when compared with the breast with being produced with the ox of Lf genes; it has the Lf contents increased or decreased; the Lf genes include NC_007320.3 nucleotide sequence or its functional variety, or can express the functionally equivalent of the NM_180998 or NP_851341 Lf gene outcomes.

30. such as the breast of the collection claimed of any one of claim 27 to 29 or mixing, it has at least about 150mg.L^-1, at least about 200mg.L^-1, at least about 250mg.L^-1, at least about 300mg.L^-1, at least about 400mg.L^-1, at least about 450mg.L^-1, at least about 500mg.L^-1Or at least about 600mg.L^-1Lf。

31. the colostrum collected or mixed, it passes through such as claim 23 ox claimed and produced.

32. the colostrum collected or mixed, it passes through such as claim 25 a herd of cattle claimed and produced.

33. such as the colostrum of the collection claimed of claim 30 or 31 or mixing; when compared with the breast with being produced with the ox of Lf genes; it has the lactoferrin content increased or decreased; the Lf genes include the nucleotide sequence or its functional variety of the NC_007320.3, or can express the functionally equivalent of the NM_180998 or NP_851341 Lf gene outcomes.

34. such as the colostrum of the collection claimed of any one of claim 30 to 33 or mixing, it has at least about 150mg.L^-1, at least about 200mg.L^-1, at least about 250mg.L^-1, at least about 300mg.L^-1, at least about 400mg.L^-1, at least about 450mg.L^-1, at least about 500mg.L^-1Or at least about 600mg.L^-1Lf。

35. dairy products, it is made up of such as any one of claim 27 to 30 breast claimed.

36. composition, it includes such as claim 31 to 34 colostrum claimed.

37. the kit for carrying out Genotyping to ox for one or more newborn or colostrum lactoferrin content phenotypes, it includes the probe or primer or the pair of primers such as claim 19 restriction limited such as any one of claim 16 to 18.

38. the nucleic acid molecules of separation, purifying or restructuring, it includes being selected from following nucleotide sequence

(c) at least 12 of the variant of (a) or (b) continuous nucleotides；Or

(e) any one complementary strand in (a) to (d)；Or

39. carrier, it includes the nucleic acid described in claim 38.

40. on breast or colostrum lactoferrin content, or on producing the method that the ability with the breast increased or decreased or the offspring of colostrum lactoferrin content is determined into the genetic state of ox, methods described includes

The breast or colostrum lactoferrin content of the ox are determined,

The Lf allele distributions of the ox are determined,

Compare the Lf allele distributions of the ox or the breast of the ox or colostrum lactoferrin content and the Lf allele distributions or breast or colostrum lactoferrin content of the ox with known Lf allele distributions；

The genetic state of the ox is determined on basis of the comparison.

41. the method for producing, adjust or secreting phenotype and recognizing or select mammalian object on one or more desired lactoferrins, this method includes：

The result of one or more genetic tests of sample from the object is provided, and

The result of the analysis one or more presence or shortage selected from following polymorphism：

(b) one or more of described Lf genes related to the lactoferrin intake increased or decreased polymorphism, or

(c) one or more of described Lf genes related to the lactoferrin secretion increased or decreased polymorphism, or

(d) one or more of described Lf genes polymorphism, it is related to the generation of breast, colostrum, blood, serum, Mucosal secretions, or with one or more mucomembranous surfaces with the lactoferrin content increased or decreased, or

(e) have to one or more of linkage disequilibrium of one or more polymorphisms polymorphism in one or more related Lf genes in (a) to (d) above,

The result for wherein indicating presence one or more in the polymorphism or shortage be indicate with one or more desired lactoferrins intakes, produce, the object of regulation or secretion phenotype；With

The object is recognized or selected on the basis of the result.

42. such as any one of claim 1 to 15 method claimed, wherein one or more of desired lactoferrins are produced, regulation or secretion phenotype are that have at least about 150mg.L^-1, at least about 200mg.L^-1, at least about 250mg.L^-1, at least about 300mg.L^-1, at least about 400mg.L^-1, at least about 450mg.L^-1Or at least about 500mg.L^-1Lf newborn generation.

43. such as claim 42 method claimed, wherein one or more of desired lactoferrins are produced, regulation or secretion phenotype are that have at least about 600mg.L when milking once a day^-1Lf newborn generation.