WO2010050829A1

WO2010050829A1 - Marker assisted selection of a mammalian subject for desired immunoglobulin phenotype

Info

Publication number: WO2010050829A1
Application number: PCT/NZ2009/000233
Authority: WO
Inventors: Natalie Louise Thomas; Alayna Katie Burrett; Sarah Dianne Berry; Stephen Richard Davis; Elizabeth Marie Beattie; Klaus Lehnert; Russell Grant Snell
Original assignee: Vialactia Biosciences (Nz) Limited
Priority date: 2008-10-31
Filing date: 2009-10-30
Publication date: 2010-05-06

Abstract

The present invention provides methods of identifying or selecting mammalian subjects having one or more desired immunoglobulin phenotype(s) by determining a PIGR genotype of the subject. Particularly contemplated are methods of identifying or selecting bovine wherein the presence or absence of the G allele or the C allele at the C903G polymorphism in the bovine PIGR gene, or the presence or absence of the G allele or the A allele at the A3938G polymorphism in the bovine PIGR gene, or the presence or absence of the G allele or the A allele at the A4029G polymorphism in the bovine PIGR gene, or the presence or absence of the G allele or the A allele at the G5183A polymorphism in the bovine PIGR gene, is associated with variation in milk or colostrum immunoglobulin content.

Description

MARKER ASSISTED SELECTION OF A MAMMALIAN SUBJECT FOR DESIRED

IMMUNOGLOBULIN PHENOTYPE

FIELD OF THE INVENTION

This invention relates to an application of marker assisted selection of mammalian subjects for quantitative trait loci (QTL) associated with immunoglobulin (Ig) uptake, production, regulation, or secretion, including for example milk or colostrum Ig content, particularly by assaying for the presence of polymorphisms associated with the QTL. BACKGROUND

The genetic basis of mammalian milk and colostrum production is of great economic, veterinary, and medical significance. Genetic bases for variations in the composition of milk, such as the relative amounts of major milk proteins, and the effect of these variations on milk production characteristics and milk processing properties, has been the subject of considerable research, debate, and review. An ability to modulate milk or colostrum composition has the potential to alter farming practices, improve animal and human health, and to produce products which are tailored to meet a range of requirements. In particular, a method of genetically evaluating mammals to select those which express desirable phenotypes, such as desirable milk or colostrum immunoglobulin content, would be useful.

Colostrum plays a critical role in the transmission of maternal immunoglobulins to offspring, which is important for the growth and health of newborn mammals. In humans, maternal immunoglobulins with specific antimicrobial activity are transferred via the placenta to the newborn infant, and are reported to confer a degree of passive immunity (see Hannu Korhonen, et al., 2000). However, in other mammalian species such as pigs, horses, sheep and cows, it has been reported that Igs are transferred postnatally via colostrum as the placenta does not allow significant transfer of macromolecules. In a newborn calf, for example, the Igs are reportedly absorbed from the colostrum into the circulation within 24-36 hours after birth (see Hannu Korhonen, et al., 2000). This Ig complement system provides the newborn mammal with protection against microbial pathogens and their toxins. This major antimicrobial effect confers passive immunity until the newborn's own immune system has matured.

Bovine serum and lacteal secretions (which are representative of these secretions in mammals) contain three major classes of immunoglobulins: immunoglobulin G (IgG), immunoglobulin M (IgM), and immunoglobulin A (IgA). These immunoglobulins are selectively transported from the serum into the mammary gland. Indeed, the first colostrum contains very high concentrations of immunoglobulins (40-200 mg/ml). IgGl accounts for over 75 % of the immunoglobulins in colostral whey, followed by IgM, IgA and IgG2. Total immunoglobulin concentration decreases within a few days to approximately 0.7-1.0 mg/ml, with IgGl remaining the major Ig class in milk throughout lactation (see Hannu Korhonen, et al., 2000).

IgA is a major immunoglobulin in mammalian secretions and provides protection against pathogens, binding to disease-causing viruses, bacteria, fungi and their toxins. IgA is produced by plasma cells and is frequently present as polymeric IgA (plgA). IgA is transcytosed across secretory epithelia following binding of plgA dimers to the polymeric IgA receptor (PIGR) at the basolateral membrane. PIGR, the extracellular ligand-binding region of which is also known as secretory component (SC), is a glycoprotein reportedly expressed at the basolateral surface of epithelial cells. At the apical membrane, SC is cleaved and released either in free form, or bound to plgA. The complex of SC and plgA is known as secretory IgA (slgA). PIGR is believed to also transport IgM, although at a lesser extent than IgA. See Kaetzel 2005, Wiburg et al, 2006.

Strategies to increase or decrease milk or colostrum immunoglobulin content could provide health benefits and are expected to be economically valuable. IgA is normally present in cows' milk at low levels, and commercial scale purification of IgA typically requires a method to increase the concentration and yield of IgA. The established method to increase the yield of IgA on a commercial scale is by immunisation regimes to boost IgA levels in the milk. It will be appreciated, however, that these methods and products prepared using these methods are undesirable in many markets or for particular applications.

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

It is an object of the present invention to provide methods for marker assisted identification or selection of mammalian subjects with a desired immunoglobulin uptake, production, regulation or secretion phenotype, including production of milk or colostrum with a desired immunoglobulin content, to provide subjects identified or selected using the method of the invention as well as milk or colostrum produced by the selected subject, uses for such milk or colostrum, or to provide the public with a useful choice. SUMMARY OF THE INVENTION

This invention relates to the elucidation of the role of the gene encoding Polymeric immunoglobulin receptor (PIGR) in milk or colostrum immunoglobulin content, particularly milk or colostrum IgA content, milk or colostrum IgM content, milk or colostrum SC content, and milk or colostrum slgA content. In particular, the invention relates to the identification for the first time of the C903G polymorphism in the bovine PIGR gene, of the A3938G polymorphism in the bovine PIGR gene, of the A4029G polymorphism in the bovine PIGR gene, and of the G5183A polymorphism in the bovine PIGR gene. The invention further relates to the association for the first time of any of the C903G C allele, the A3938G A allele, the A4029G A allele, or the G5183A G allele with production of milk and colostrum with increased IgA content, and of any of the C903G G allele, the A3938G G allele, the A4029G G allele, or the G5183A A allele with production of milk and colostrum with decreased IgA content.

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

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

In one embodiment, the mammalian subject is bovine.

In various embodiments, milk or colostrum immunoglobulin content is one or more of milk or colostrum IgA content, milk or colostrum IgM content, milk or colostrum SC content, or milk or colostrum slgA content. In one embodiment, milk or colostrum immunoglobulin content is one or more of colostrum IgA content, colostrum IgM content, colostrum SC content, or colostrum slgA content.

In various embodiments, the genetic status with respect to milk or colostrum immunoglobulin content is production of milk or colostrum with one or more of increased milk or colostrum IgA content, increased milk or colostrum IgM content, increased milk or colostrum SC content, or increased milk or colostrum slgA content. Accordingly, in one embodiment the invention provides a method for identifying or selecting a bovine that produces milk, colostrum, blood, serum, mucosal secretions, or has mucosal surfaces with one or more of increased IgA content, increased IgM content, increased SC content, or increased slgA content, or a bovine capable of producing progeny that produce milk, colostrum, blood, serum, mucosal secretions, or having mucosal surfaces with one or more of increased IgA content, increased IgM content, increased SC content, or increased slgA content, the method comprising determining a PIGR allelic profile of the bovine, and identifying or selecting the bovine on the basis of the determination.

In various embodiments, the genetic status with respect to milk or colostrum immunoglobulin content is production of milk or colostrum with one or more of decreased milk or colostrum IgA content, decreased milk or colostrum IgM content, decreased milk or colostrum SC content, or decreased milk or colostrum slgA content.

Accordingly, in one embodiment the invention provides a method for identifying or selecting a bovine that produces milk, colostrum, blood, serum, mucosal secretions, or has mucosal surfaces with one or more of decreased IgA content, decreased IgM content, decreased SC content, or decreased slgA content, or a bovine capable of producing progeny that produce milk, colostrum, blood, serum, mucosal secretions, or having mucosal surfaces with one or more of decreased IgA content, decreased IgM content, decreased SC content, or decreased slgA content, the method comprising determining a PIGR allelic profile of the bovine, and identifying or selecting the bovine on the basis of the determination.

In various embodiments, increased immunoglobulin content is increased total immunoglobulin concentration, such as for example, increased [total Igjmol.L^"1 milk, or increased [total Igjmol.L^"1 colostrum. Similarly, in various embodiments decreased immunoglobulin content is decreased total immunoglobulin concentration, such as for example, decreased [total Igjmol.L^"1 milk, or decreased [total Igjmol.L^"1 colostrum.

In various embodiments, increased IgA content is increased IgA concentration, or increased IgA concentration relative to a second class of immunoglobulin, such as for example, increased IgA concentration relative to IgG concentration, for example in milk or colostrum. Similarly, in various embodiments decreased IgA content is decreased IgA concentration, or decreased IgA concentration relative to a second class of immunoglobulin, such as for example, decreased IgA concentration relative to IgG concentration, for example in milk or colostrum. In various embodiments, increased IgM content is increased IgM concentration, or increased IgM concentration relative to a second class of immunoglobulin, such as for example, increased IgM concentration relative to IgG concentration, for example in milk or colostrum. Similarly, in various embodiments decreased IgM content is decreased IgM concentration, or decreased IgM concentration relative to a second class of immunoglobulin, such as for example, decreased IgM concentration relative to IgG concentration, for example in milk or colostrum.

In various embodiments, increased SC content is increased SC concentration, or increased SC concentration relative to a second class of immunoglobulin, such as for example, increased SC concentration relative to IgG concentration, for example in milk or colostrum. Similarly, in various embodiments decreased SC content is decreased SC concentration, or decreased SC concentration relative to a second class of immunoglobulin, such as for example, decreased SC concentration relative to IgG concentration, for example in milk or colostrum.

In various embodiments, increased slgA content is increased slgA concentration, or increased slgA concentration relative to a second class of immunoglobulin, such as for example, increased slgA concentration relative to IgG concentration, for example in milk or colostrum. Similarly, in various embodiments decreased slgA content is decreased slgA concentration, or decreased slgA concentration relative to a second class of immunoglobulin, such as for example, decreased slgA concentration relative to IgG concentration, for example in milk or colostrum.

In one embodiment the PIGR allelic profile is a PIGR nucleic acid profile. In another embodiment, the PIGR allelic profile is a PIGR protein profile, such as a profile comprising data about the expression or activity of a PIGR gene product, such as PIGR.

As used herein, the phrase "PIGR allelic profile" contemplates data indicative of the presence or absence of one or more alleles at one or more polymorphisms in the PIGR gene or which affect expression from the PIGR gene or the expression or activity of a PIGR gene product or which are associated with variation in the expression from the PIGR gene or in the expression or activity of a PIGR gene product. The PIGR allelic profile may be a PIGR nucleic acid profile, a PIGR protein profile, or a combination thereof. In preferred embodiments, the PIGR allelic profile is a PIGR nucleic acid profile comprising data indicative of the presence or absence of one or more alleles at one or more polymorphisms associated with increased or decreased milk or colostrum immunoglobulin content. For example, in various preferred embodiments the PIGR allelic profile of bovine comprises data indicative of a) the presence or absence of the C allele at the C903G (S29T) polymorphism in the PIGR gene; or b) the presence or absence of the G allele at the C903G (S29T) polymorphism in the PIGR gene; or c) the presence or absence of the A allele at the A3938G (M404I) polymorphism in the PIGR gene; or d) the presence or absence of the G allele, at the A3938G (M404I) polymorphism in the PIGR gene; or e) the presence or absence of the A allele at the A4029G (A435T) polymorphism in the PIGR gene; or f) the presence or absence of the G allele at the A4029G (A435T) polymorphism in the PIGR gene; or g) the presence or absence of the G allele at the G5183A (A504T) polymorphism in the PIGR gene, or h) the presence or absence of the A allele at the G5183A (A504T) polymorphism in the PIGR gene, or i) the presence or absence of a polymorphism in linkage disequilibrium (LD) with any one or more of a)-h) above, or j) any combination of any two or more of a)-i).

It will be appreciated that methods comprising determining the expression or activity of a PIGR gene product encompass determining expression from or of a PIGR gene. In one embodiment, expression or activity of a PIGR gene product is determined using PIGR mRNA, for example by determining the presence or amount of PIGR mRNA. In other embodiments, expression or activity of the PIGR gene product is determined using PIGR protein, preferably by determining the amount of PIGR protein, or by determining the activity of PIGR protein, for example the activity of PIGR protein present in a sample obtained from the subject, or by determining the amount of a PIGR protein variant, derivative or fragment, for example the amount of SC present in a sample obtained from the subject.

In still other embodiments, the expression or activity of the PIGR gene product is determined using PIGR DNA, preferably by determining the presence or absence of one or more polymorphisms associated with decreased or increased PIGR expression or activity, for example one or more or the polymorphisms associated with increased or decreased expression or activity as described herein.

In another embodiment, the PIGR allelic profile of the subject is determined together with the allelic profile of the subject at one or more genetic loci associated with milk or colostrum immunoglobulin content.

In one embodiment, the one or more genetic loci are one or more polymorphisms in one or more genes associated with milk or colostrum immunoglobulin content.

The one or more polymorphisms can be detected directly or by detection of one or more polymorphisms which are in linkage disequilibrium with said one or more polymorphisms.

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

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

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

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

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

Conveniently, in said method the presence or absence of DNA encoding wild type PIGR in said subject is determined, directly or indirectly, for example using an expressed gene product. For example, the presence or absence of at least one nucleotide difference from the nucleotide sequence encoding wild type PIGR in said subject is determined, directly or indirectly.

In various embodiments, the method comprises determining the PIGR C903G allelic profile of the bovine, or determining the PIGR A3938G allelic profile of the bovine, or determining the PIGR A4029G allelic profile of the bovine, or determining the PIGR G5183A allelic profile of the bovine. More preferably, the method comprises determining the PIGR allelic profile at two or more of these polymorphisms, three or more of these polymorphisms, or all four of these polymorphisms.

More specifically, in said method the presence or absence of one or more of the G allele or C allele at the C903G polymorphism in the PIGR gene is determined, directly or indirectly. For example, the presence of the G allele or C allele at the C903G polymorphism in the PIGR gene may be determined using a polymorphism in linkage disequilibrium with the G allele or with the C allele at the C903G polymorphism.

Similarly, the presence or absence of a particular allele at any one or more of the A3938G polymorphism, the A4029G polymorphism, or the G5183A polymorphism, may be determined directly or indirectly, for example by analysis of a polymorphism in LD with one or more of these polymorphisms.

In one embodiment, the method includes ascertaining, from a sample of material containing DNA obtained from the subject, whether a sequence of the DNA encoding a protein "(A)" having biological activity of wild type PIGR is present, or whether a sequence of the DNA encoding an allelic protein "(B)" at least partially lacking the activity of (A) is present, or whether a sequence of the DNA encoding (A) and a sequence of the DNA encoding (B) are both present. The absence of the DNA encoding (A) and the presence of the DNA encoding (B) indicates an association with low relative milk, colostrum, blood, serum, mucosal secretions, or mucosal surface Ig content, particularly with the production of milk or colostrum with, inter alia, decreased IgA content. The reverse association holds true, where the presence of the DNA encoding (A) and the absence of the DNA encoding (B) indicates an association with high relative milk, colostrum, blood, serum, mucosal secretions, or mucosal surface Ig content, particularly with the production of milk or colostrum with, inter alia, increased IgA content. The presence of both the DNA encoding (A) and the DNA encoding (B) indicates an association with intermediate relative Ig content, particularly with the production of milk or colostrum with, inter alia, intermediate IgA content.

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

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

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

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

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

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

In another aspect directed to veterinary or agricultural applications, the invention is a method for determining the PIGR genotype of a mammalian subject, as may be desirable to know for breeding purposes.

In one embodiment, the method includes ascertaining, with reference to a sample of material containing nucleic acid obtained from the subject and uncontaminated by heterologous nucleic acid, whether the sample contains (i) nucleic acid molecule encoding a protein having biological activity of wild type PIGR and optionally ascertaining whether the sample contains an (ii) allelic nucleic acid molecule encoding a protein lacking biological activity of wild type PIGR.

In another embodiment, the method includes ascertaining, with reference to a sample of material containing protein obtained from the subject and uncontaminated by heterologous protein, whether the sample contains (i) a protein having biological activity of wild type PIGR and optionally ascertaining whether the sample contains (iii) a protein lacking biological activity of wild type PIGR.

In a further embodiment, the invention provides a method of determining genetic status of a subject with respect to milk or colostrum IgA content which comprises determining the PIGR allelic profile of the subject, together with determining the allelic profile of the subject at one or more genetic loci associated with milk or colostrum IgA content, with milk or colostrum IgM content, with milk or colostrum SC content, or with milk or colostrum slgA content.

In one embodiment, the one or more genetic loci is one or more polymorphisms in one or more genes associated with milk or colostrum IgA content.

In a further aspect, the invention includes a probe containing a nucleic acid molecule sufficiently complementary with a nucleic acid sequence present in SEQ ID NO:1 or a nucleotide sequence encoding a wild type bovine PIGR, or its complement, so as to bind thereto under stringent conditions, as well as a diagnostic kit containing such a probe. Particularly contemplated are probes that comprise one or other of the alleles at one or more of the C903G polymorphism, the A3938G polymorphism, the A4029G polymorphism, or the G5183A polymorphism, for example a probe that includes an adenosine at the site corresponding to the A3938G polymorphism.

The invention also includes a primer composition useful for detection of the presence or absence of DNA encoding wild type PIGR and/or the presence of the DNA encoding a variant protein at least partially lacking wild type activity. In one form, the composition can include a nucleic acid primer substantially complementary to a nucleic acid sequence comprising at least about 12 contiguous nucleotides of a nucleic acid sequence encoding wild type PIGR, or its complement. The nucleic acid sequence can in whole or in part be identified in SEQ ID NO: 1 or SEQ ID NO:2. Diagnostic kits including such a composition are also included.

Particularly contemplated are primers comprising or substantially complementary to a nucleic acid sequence comprising at least about 12 contiguous nucleotides of a nucleic acid sequence present in SEQ ID NO:1 and within approximately 1 to about 2000 bp of one of the C903G polymorphism, the A3938G polymorphism, the A4029G polymorphism, or the G5183A polymorphism, more preferably within approximately 1 to about 1000 bp, or within approximately 1 to about 500 bp, approximately 1 to about 400 bp, approximately 1 to about 300 bp, approximately 1 to about 200 bp, approximately 1 to about 100 bp, approximately 1 to about 50 bp, or within approximately 1 to about 20 bp of one of the C903G polymorphism, the A3938G polymorphism, the A4029G polymorphism, or the G5183A polymorphism.

Examples of such primers are presented herein as SEQ ID NOs: 4 to 51.

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

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

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

Thus, in another aspect the invention provides a method of assessing the genetic status of a bovine with respect to milk or colostrum immunoglobulin content which comprises the step of determining the presence or absence of one or more polymorphisms selected from the group comprising: the C903G (S29T) polymorphism in the PIGR gene, or the A3938G (M404T) polymorphism in the PIGR gene, or the A4029G (A435T) polymorphism in the PIGR gene, or the G5183A (A504T) polymorphism in the PIGR gene, or one or more polymorphisms in linkage disequilibrium with one or more of these polymorphisms.

In one embodiment the method comprises the step of determining the presence or absence of one or more of the C allele or the G allele at the C903G (S29T) polymorphism in the PIGR gene; the A allele or the G allele at the A3938G (M404T) polymorphism in the PIGR gene; the A allele or the G allele at the A4029G (A435T) polymorphism in the PIGR gene; or the G allele or the A allele at the G5183A (A504T) polymorphism in the PIGR gene. Again, the one or more polymorphisms can be detected directly or by detection of one or more polymorphisms which are in linkage disequilibrium with the one or more polymorphisms.

In another aspect, the present invention provides a method for identifying or selecting a mammalian subject with one or more desired immunoglobulin uptake, production, regulation, or secretion phenotypes, the method comprising determining a PIGR allelic profile of said subject, and identifying or selecting the subject on the basis of the determination.

In one embodiment, the PIGR allelic profile is determined by providing the results of an analysis of a sample from said subject for the presence or absence of one or more polymorphisms in the PIGR gene associated with one or more of:

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

(b) increased or decreased immunoglobulin uptake, or (c) increased or decreased immunoglobulin secretion, or

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

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

In one embodiment, the subject is bovine.

In one embodiment, the immunoglobulin content is IgA content, IgM content, SC content, or slgA content.

In one embodiment, the phenotype is desired milk or colostrum immunoglobulin content such as desired milk or colostrum IgA content, desired milk or colostrum IgM content, desired milk or colostrum SC content, or desired milk or colostrum slgA content.

In one embodiment, the invention provides a method for identifying or selecting a bovine with a PIGR allelic profile indicative of increased milk or colostrum immunoglobulin content, preferably of increased milk or colostrum IgA content, increased milk or colostrum content, increased milk or colostrum SC content, or increased milk or colostrum slgA content.

Preferably the method comprises determining the presence of one or more of the C allele at the C903G polymorphism, the A allele at the A3938G polymorphism, the A allele at the A4029G polymorphism, or the G allele at the G5183A polymorphism in the PIGR gene, and selecting the bovine on the basis of the determination. Alternatively or additionally, the method comprises determining the absence of one or more of the G allele at the C903G polymorphism, the G allele at the A3938G polymorphism, the G allele at the A4029G polymorphism, or the A allele at the G5183A polymorphism in the PIGR gene, and identifying or selecting the bovine on the basis of the determination.

Preferably, the method comprises determining the presence of one or more of the CC genotype at the C903G polymorphism, the AA genotype at the A3938G polymorphism, the AA genotype at the A4029G polymorphism, or the GG genotype at the G5183A polymorphism in the PIGR gene, and identifying or selecting the bovine on the basis of the determination.

In various embodiments, the invention provides a method for identifying or selecting a bovine with a PIGR allelic profile indicative of one or more of increased IgM content, of increased slgA content, or of increased SC content. In a further embodiment the invention provides a method for identifying or selecting a bovine with a PIGR allelic profile indicative of decreased milk or colostrum immunoglobulin content, preferably of decreased milk or colostrum IgA content.

Preferably the method comprises determining the absence of one or more of the C allele at the C903G polymorphism, the A allele at the A3938G polymorphism, the A allele at the A4029G polymorphism, or the G allele at the G5183A polymorphism in the PIGR gene, and selecting the bovine on the basis of the determination. Alternatively or additionally, the method comprises determining the presence of one or more of the G allele at the C903G polymorphism, the G allele at the A3938G polymorphism, the G allele at the A4029G polymorphism, or the A allele at the G5183A polymorphism in the PIGR gene, and identifying or selecting the bovine on the basis of the determination.

Preferably, the method comprises determining the presence of one or more of the GG genotype at the C903G polymorphism, the GG genotype at the A3938G polymorphism, the GG genotype at the A4029G polymorphism, or the AA genotype at the G5183A polymorphism in the PIGR gene, and identifying or selecting the bovine on the basis of the determination.

In a further embodiment the invention provides a method for identifying or selecting a bovine with a PIGR allelic profile indicative of intermediate milk or colostrum immunoglobulin content, preferably of intermediate milk or colostrum IgA content.

Preferably, the method comprises determining the presence of one or more of the GC genotype at the C903G polymorphism, the AG genotype at the A3938G polymorphism, the AG genotype at the A4029G polymorphism, or the GA genotype at the G5183A polymorphism in the PIGR gene, and identifying or selecting the bovine on the basis of the determination.

In various embodiments, the invention provides a method for identifying or selecting a bovine with a PIGR allelic profile indicative of one or more of decreased IgM content, of decreased slgA content, or of decreased SC content.

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

In one embodiment, the presence of an allele is determined by sequencing a PIGR polynucleotide obtained from the bovine. In a further embodiment the determination comprises the step of amplifying a PIGR polynucleotide sequence from genomic DNA, mRNA or cDNA produced from mRNA derived from said bovine, for example by PCR.

Preferably the determination is by use of primers which comprise a nucleotide sequence having at least about 12 contiguous bases of or complementary to the sequence of SEQ ID NO:1 or SEQ ID NO:2 or a naturally occurring flanking sequence.

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

In an alternative embodiment, the method comprises restriction enzyme digestion of a nucleotide derived from the bovine. Such digestion may also be performed on a product of the PCR amplification described above.

In a further embodiment, the presence of an allele is determined by mass spectrometric analysis of a PIGR polynucleotide obtained from the bovine.

In an alternative embodiment, the presence of an allele is determined by hybridisation of a probe or probes comprising a nucleotide sequence of or complementary to the sequence of SEQ ID NO: 1 or SEQ ID NO:2.

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

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

In an alternative embodiment, the presence of an allele is determined by analysis of a PIGR polypeptide obtained from the bovine.

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

In still a further aspect the invention provides a method of selecting a herd of bovine, comprising selecting individuals by a method of the present invention, and segregating and collecting the selected individuals to form the herd. The invention further provides a herd of bovine so selected, as well as a herd comprising bovine produced by bovine selected by the methods described herein.

In a still further aspect, the invention provides a method of determining genetic status of a bovine with respect to one or more milk or colostrum immunoglobulin content phenotypes, or with respect to capability of producing progeny predisposed to or with one or more milk or colostrum immunoglobulin content phenotypes, the method comprising providing data about the PIGR allelic profile of said bovine, and determining the genetic status of the bovine on the basis of the data.

Preferably, the data about the PIGR allelic profile comprises data representative of the presence or absence of one or more of the C allele or the G allele at the C903G polymorphism, the A allele or the G allele at the A3938G polymorphism, the A allele or the G allele at the A4029G polymorphism, or the G allele or the A allele at the G5183A polymorphism, in the PIGR gene.

Preferably, the method additionally comprises providing data comprising the result of at least one analysis of one or more genetic loci associated with one or more milk or colostrum immunoglobulin content phenotypes, wherein the data is representative of the genetic status of the bovine.

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

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

In one embodiment, the one or more milk or colostrum immunoglobulin content phenotypes is selected from the group comprising production of or capability of producing milk with increased immunoglobulin content, production of or capability of producing colostrum with increased immunoglobulin content, production of or capability of producing milk with increased IgA content, production of or capability of producing colostrum with increased IgA content, production of or capability of producing milk with increased IgM content, or production of or capability of producing colostrum with increased IgM content, production of or capability of producing milk with increased SC content, or production of or capability of producing colostrum with increased slgA content.

In another embodiment, the one or more milk or colostrum immunoglobulin content phenotypes is selected from the group comprising production of or capability of producing milk with decreased immunoglobulin content, production of or capability of producing colostrum with decreased immunoglobulin content, production of or capability of producing milk with decreased IgA content, production of or capability of producing colostrum with decreased IgA content, production of or capability of producing milk with decreased IgM content, or production of or capability of producing colostrum with decreased IgM content, production of or capability of producing milk with increased SC content, or production of or capability of producing colostrum with increased slgA content.

In a further aspect the invention provides a method for identifying or selecting a mammalian subject with respect to one or more desired immunoglobulin uptake, production, regulation, or secretion phenotypes, the method comprising providing the result of one or more genetic tests of a sample from the subject, and analysing the result for the presence or absence of one or more polymorphisms selected from the group comprising:

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

(b) one or more polymorphisms in the PIGR gene associated with increased or decreased immunoglobulin uptake, or

(c) one or more polymorphisms in the PIGR gene associated with increased or decreased immunoglobulin secretion, or

(d) one or more polymorphisms in the PIGR gene associated with production of milk, colostrum, blood, serum, mucosal secretions, or having one or more mucosal surfaces with increased or decreased immunoglobulin content, or

(e) one or more polymorphisms in linkage disequilibrium with one or more polymorphisms in the PIGR gene associated with one or more of (a) to (d) above, wherein a result indicative of the presence or absence of one or more of said polymorphisms is indicative of a subjectwith one or more desired immunoglobulin uptake, production, regulation, or secretion phenotypes; and identifying or selecting the subject on the basis of the result.

Preferably, the one or more polymorphisms associated with increased or decreased expression or activity of PIGR gene product is one or more polymorphisms in the PIGR gene.

In one embodiment, the one or more immunoglobulin uptake, production, regulation, or secretion phenotypes is one or more milk or colostrum immunoglobulin content phenotypes, including increased milk or colostrum immunoglobulin content, or increased milk or colostrum IgA content, increased milk or colostrum SC content, or increased milk or colostrum slgA content.

In a further aspect the invention provides a method for identifying or selecting a bovine with one or more desired milk or colostrum immunoglobulin content phenotypes, the method comprising a) providing the result of one or more genetic tests of a sample from the bovine, and b) analysing the result for the presence or absence of one or more polymorphisms selected from the group comprising: the C903G polymorphism in the PIGR gene, or the A3938G polymorphism in the PIGR gene, or the A4029G polymorphism in the PIGR gene, or the G5183 A polymorphism, in the PIGR gene, or one or more polymorphisms in linkage disequilibrium with the C903G polymorphism, the A3938G polymorphism, the A4029G polymorphism, or the

G5183A polymorphism, in the PIGR gene, wherein a result indicative of the presence or absence of one or more of said polymorphisms is indicative of a bovine with one or more desired milk or colostrum immunoglobulin content phenotypes.

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

Preferably, said system is accessible via the internet or by personal computer. Preferably, said reference genetic database comprises or includes the results of one or more analyses of one or more genetic loci associated with one or more milk or colostrum immunoglobulin content phenotypes, more preferably the one or more genetic loci are one or more polymorphisms in one or more genes associated with one or more milk or colostrum immunoglobulin content phenotypes. In yet a further aspect, the invention provides a computer program suitable for use in a system as defined above comprising a computer usable medium having program code embodied in the medium for causing the computer program to process received data consisting of or including the result of at least one genetic analysis of one or more genetic loci associated with one or more milk or colostrum immunoglobulin content phenotypes in the context of both a reference genetic database of the results of said at least one genetic analysis and optionally a reference database of non-genetic factors associated with one or more subject milk or colostrum immunoglobulin content phenotypes.

Preferably, the one or more genetic loci are one or more polymorphisms in one or more genes associated with one or more milk or colostrum immunoglobulin content phenotypes.

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

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

In still another aspect, the invention provides a method of determining genetic status of a bovine with respect to milk or colostrum immunoglobulin content, or with respect to capability of producing progeny that will have increased or decreased milk or colostrum immunoglobulin content, the method comprising determining milk or colostrum immunoglobulin content of the bovine, determining the PIGR allelic profile of the bovine, comparing the PIGR allelic profile of the bovine or the milk or colostrum immunoglobulin content of the bovine with that of a bovine having a known PIGR allelic profile; determining the genetic status of the bovine on the basis of the comparison. It will be appreciated that for the purposes of the comparison, the milk or colostrum immunoglobulin content associated with the known PIGR allelic profile is known. It will further be appreciated that the association of milk or colostrum immunoglobulin content with a particular PIGR allelic profile may be established by the methods described herein.

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

(a) at least 12 contiguous nucleotides of SEQ ID NO:1 and comprising one or more of the C903G polymorphism, or the A3938G polymorphism, or the A4029G polymorphism, or the G5183A polymorphism; or (b) at least 12 contiguous nucleotides of SEQ ID NO:2 and comprising one or more of the C903G polymorphism, or the A3938G polymorphism, or the A4029G polymorphism, or the G5183A polymorphism; or

(c) at least 12 contiguous nucleotides of a variant of SEQ ID NO:1 ; or

(d) at least 12 contiguous nucleotides of a variant of SEQ ID NO:2; or

(e) any one or more of SEQ ID NOs:4 - 51 ; or

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

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

In one embodiment, the nucleic acid molecule comprises nucleotide sequence selected from

(a) at least 12 contiguous nucleotides of SEQ ID NO:1 and comprising a guanine at the C903G polymorphism in the PIGR gene; or

(b) at least 12 contiguous nucleotides of SEQ ID NO:2 and comprising a guanine at the C903G polymorphism; or

(c) at least 12 contiguous nucleotides of SEQ ID NO: 1 and comprising a cytosine at the C903G polymorphism in the PIGR gene; or

(d) at least 12 contiguous nucleotides of SEQ ID NO:2 and comprising a cytosine at the C903G polymorphism; or

(e) at least 12 contiguous nucleotides of SEQ ID NO:1 and comprising a guanine at the A3938G polymorphism in the PIGR gene; or

(f) at least 12 contiguous nucleotides of SEQ ID NO:2 and comprising a guanine at the A3938G polymorphism; or

(g) at least 12 contiguous nucleotides of SEQ ID NO:1 and comprising an adenine at the A3938G polymorphism in the PIGR gene; or

(h) at least 12 contiguous nucleotides of SEQ ID NO:2 and comprising an adenine at the A3938G polymorphism; or (i) at least 12 contiguous nucleotides of SEQ ID NO:1 and comprising a guanine at the A4029G polymorphism in the PIGR gene; or (j) at least 12 contiguous nucleotides of SEQ ID NO:2 and comprising a guanine at the A4029G polymorphism; or (k) at least 12 contiguous nucleotides of SEQ ID NO:1 and comprising an adenine at the A4029G polymorphism in the PIGR gene; or (1) at least 12 contiguous nucleotides of SEQ ID NO:2 and comprising an adenine at the A4029G polymorphism; or (m) at least 12 contiguous nucleotides of SEQ ID NO: 1 and comprising an adenosine at the G5183 A polymorphism in the PIGR gene; or (n) at least 12 contiguous nucleotides of SEQ ID NO:2 and comprising an adenosine at the G5183 A polymorphism; or (o) at least 12 contiguous nucleotides of SEQ ID NO:1 and comprising a guanine at the G5183A polymorphism in the PIGR gene; or (p) at least 12 contiguous nucleotides of SEQ ID NO: 2 and comprising a guanine at the G5183 A polymorphism; or

(q) at least 12 contiguous nucleotides of a variant of SEQ ID NO:2; or (r) any one or more of SEQ ID NOs:4 - 51 ; or (s) a complement of any one of (a) to (r); or (t) a sequence of at least 12 contiguous nucleotides and capable of hybridising to the nucleotide sequence of any one of (a) to (s) under stringent conditions. In one embodiment, the PIGR nucleic acid molecule is a PIGR fragment as defined herein, wherein the PIGR fragment comprises one or more of the C903G polymorphism, or the A3938G polymorphism, or the A4029G polymorphism, or the G5183A polymorphism, or a combination of any two or more thereof.

The invention also provides a genetic construct comprising a PIGR nucleic acid molecule of the invention, a vector comprising the genetic construct or a nucleic acid sequence as described above, a host cell comprising the genetic construct or vector, a polypeptide encoded by a PIGR nucleic acid molecule of the invention, an antibody which selectively binds a polypeptide of the invention, and a method for recombinantly producing a polypeptide of the invention.

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

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

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

In various embodiments the one or more conditions associated with immunoglobulin uptake, production, regulation, or secretion, is one or more conditions associated with decreased or increased IgA secretion or uptake, or with decreased or increased IgM secretion or uptake, or with decreased or increased SC secretion or uptake, or with decreased or increased IgA secretion or uptake.

In one embodiment the one or more conditions is associated with decreased immunoglobulin uptake.

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

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

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

In one embodiment, the infectious disease is mastitis.

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

Examples of conditions associated with immunoglobulin uptake, production, regulation, or secretion include IgA nephropathy, autoimmune diseases including rheumatoid arthritis, multiple sclerosis, psoriasis, systemic lupus erythematosus, and inflammatory bowel disease, dermatitis, eczema, and the like.

In one embodiment wherein the subject is bovine, the one or more polymorphisms are selected from the group consisting of: the C903G polymorphism in the PIGR gene, or the A3938G polymorphism in the PIGR gene, or the A4029G polymorphism in the PIGR gene, or the G5183 A polymorphism, in the PIGR gene, or one or more polymorphisms in linkage disequilibrium with one or more of the C903G polymorphism, or the A3938G polymorphism, or the A4029G polymorphism, or the G5183A polymorphism, in the PIGR gene, wherein the presence or absence of one or more of said polymorphisms is indicative of the subject's risk of developing one or more conditions associated with decreased or increased PIGR expression or activity.

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

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

In another aspect there is provided a method of identifying or selecting a subject that would benefit from increased immunoglobulin intake, the method comprising providing the results of an analysis of a sample from said subject for the presence or absence of one or more polymorphisms in the PIGR gene associated with one or more of:

(a) increased or decreased expression or activity of a PIGR gene product, or (b) increased or decreased immunoglobulin uptake, or

(c) increased or decreased immunoglobulin secretion, or

(d) production of milk, colostrum, blood, serum, mucosal secretions, or mucosal surfaces with increased or decreased immunoglobulin content, or

(e) one or more polymorphisms in linkage disequilibrium with one or more polymorphisms in the PIGR gene associated with one or more of (a) to (d) above, and identifying or selecting the subject on the basis of the results.

In one embodiment, a result indicative of the presence of one or more polymorphisms in the PIGR gene associated with one or more of decreased expression or activity of a PIGR gene product, of decreased immunoglobulin uptake, of decreased immunoglobulin secretion, or of production of milk, colostrum, blood, serum, mucosal secretions, or mucosal surfaces with decreased immunoglobulin content, is indicative of a subject that would benefit from increased immunoglobulin intake.

In another aspect there is provided a method of identifying or selecting a nursing subject that would benefit from increased immunoglobulin intake, the method comprising providing the results of an analysis of a sample from said subject's mother for the presence or absence of one or more polymorphisms in the PIGR gene associated with one or more of:

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

(b) increased or decreased immunoglobulin secretion, or

(c) production of milk, colostrum, blood, serum, mucosal secretions, or mucosal surfaces with increased or decreased immunoglobulin content, or

(d) one or more polymorphisms in linkage disequilibrium with one or more polymorphisms in the PIGR gene associated with one or more of (a) to (c) above, and identifying or selecting the subject on the basis of the results.

In one embodiment, a result indicative of the presence of one or more polymorphisms in the PIGR gene associated with one or more of decreased expression or activity of a PIGR gene product, of decreased immunoglobulin secretion, or of production of milk, colostrum, blood, serum, mucosal secretions, or mucosal surfaces with decreased immunoglobulin content, is indicative of a subject that would benefit from increased immunoglobulin intake.

In various embodiments, the nursing subject is a nursing neonatal subject, including but not limited to a breastfeeding neonatal human or a nursing neonatal calf, or a nursing infant subject, such as a breastfeeding infant human or a nursing infant calf. In another aspect there is provided a method of treating a subject with or at risk of developing one or more conditions associated with immunoglobulin uptake, production, regulation, or secretion, the method comprising administering to the subject a milk or colostrum product or composition as herein described.

In one embodiment, the milk or colostrum composition comprises or is derived from milk or colostrum with increased IgA content, with increased IgM content, with increased SC content, or with increased slgA content.

In another embodiment, the milk or colostrum composition comprises or is derived from milk or colostrum with decreased IgA content, with decreased IgM content, with decreased SC content, or with decreased slgA content.

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

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

In one embodiment, the method additionally comprises determining the PIGR allelic profile of the subject.

In another aspect, the invention relates to a method of using milk or colostrum as provided herein for maintaining or raising immunoglobulin levels in a foetal subject, the method comprising providing a pregnant mother with a composition comprising or derived from milk or colostrum as provided herein and informing the mother that the composition will maintain or increase immunoglobulin levels in the foetal subject.

In various embodiments, the immunoglobulin levels are one or more of IgA content, or IgM content, or SC content, or slgA content, including increased or decreased IgA concentration relative to the concentration of another class of immunoglobulin, or increased or decreased IgM concentration relative to the concentration of another class of immunoglobulin, or increased or decreased SC concentration relative to the concentration of another class of immunoglobulin, or increased or decreased slgA concentration relative to the concentration of another class of immunoglobulin.

It will be appreciated that the invention also contemplates use of milk or colostrum of the invention in the manufacture of a composition for maintaining or raising immunoglobulin levels in a foetal, infant, child or adult subject.

In embodiments relating to maintaining or raising immunoglobulin levels in a foetal subject, the composition is suitable for oral administration to a mother during gestation. In certain embodiments the composition is a maternal formula, infant formula, follow- on formula, growing-up formula or dietetic product. In other embodiments the composition is a nutraceutical.

In another aspect the invention relates to an isolated, purified or recombinant protein comprising at least about 10 contiguous amino acids of SEQ ID NO:3 and comprising one or more of the following:

(i) threonine at the position corresponding to amino acid 29 of pro-PIGR (PIGR including the signal peptide) which corresponds to amino acid position 11 of SEQ ID NO:3;

(ii) isoleucine at the position corresponding to amino acid 404 of pro-PIGR (PIGR including the signal peptide) which corresponds to amino acid position 386 of SEQ ID NO:3;

(iii) threonine at the position corresponding to amino acid 435 of pro-PIGR (PIGR including the signal peptide) which corresponds to amino acid position 417 of SEQ ID NO:3;

(iv) threonine at the position corresponding to amino acid 504 of pro-PIGR (PIGR including the signal peptide) which corresponds to amino acid position 486 of SEQ ID NO:3; or

(v) any combination of two or more of (i)-(iv).

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

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

It is intended that reference to a range of numbers disclosed herein (for example, 1 to 10) also incorporates reference to all rational numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.

The term "comprising" as used in this specification means "consisting at least in part of. When interpreting each statement in this specification that includes the term "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises" are to be interpreted in the same manner.

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

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a graph of the range of colostrum IgA concentration determined as described in the Example. A) IgA range at the second milking and B) IgA range at the eighth milking. Animals were milked twice daily thus the second milking occurred on day one of lactation, and the eighth milking occurred on day four of lactation. Note the difference in scale between A and B.

Figure 2 shows a graph of the range of colostrum IgA, determined as described in the example, for each of the six Fl sire families within the trial population. Data are from the eighth milking.

Figure 3 shows a graph depicting the detection of major QTL for colostrum IgA content on bovine chromosome 16. The maximum F-value (y-axis) for the QTL was 1 1.8, and the most likely position was estimated at 7 cM. Bootstrap analysis (n = 1000 iterations) showed that the 95% confidence interval for the QTL was 7 - lO cM.

Figure 4 is a schematic showing the polymorphisms identified within the bovine PIGR gene. A: Graphical representation of the predicted gene structure of bovine PIGR where the unfilled blocks represent 5' and 3' untranslated regions, and the filled blocks represent exons. B. graphical representation of the C903G polymorphism (see SEQ ID NO:1), the A3938G polymorphism (see SEQ ID NO.l), the A4029G polymorphism (see SEQ ID NO: 1), and the G5183A polymorphism (see SEQ ID NO:1) in bovine PIGR.

Figure 5 is four graphs showing the effect of the PIGR alleles on colostrum IgA concentration at the C903G polymorphism, the A3938G polymorphism, the A4029G polymorphism, and the G5183 A polymorphism. The main effect for genotype on IgA content was significant at P < 0.0001, for IgA both at the second and the eighth milkings. Samples were taken at the eighth milking.

Figure 6 is a graph showing the adjusted statistical effect of one of the PIGR polymorphisms on the bovine chromosome 16 colostrum IgA QTL dark grey line: original IgA QTL; light grey line: QTL after IgA phenotype was adjusted for C903G polymorphism. The decrease in QTL significance (F-value) shows a significant association of the PIGR genotypes with the colostrum IgA QTL variation. Similar effects were seen for each of the PIGR polymorphisms. DETAILED DESCRIPTION OF THE INVENTION

The present invention recognises for the first time that polymorphisms in the PIGR gene in bovine are associated with a QTL for variations in one or more immunoglobulin phenotypes, and particularly variations in milk or colostrum immunoglobulin content.

For the sake of clarity, the phrase "milk or colostrum immunoglobulin content" is to be read as referring to milk immunoglobulin content or colostrum immunoglobulin content. Grammatical equivalents or components thereof are to be read likewise, such that the phrase "milk or colostrum IgA content" is to be read as "milk IgA content or colostrum IgA content".

It will be apparent to those skilled in the art that milk or colostrum immunoglobulin content can readily be determined qualitatively or quantitatively. In some applications, a qualitative comparison of a sample of milk or colostrum relative to another sample (preferably from an individual of known PIGR allelic profile) may be sufficient to correlate association with a particular genotype. Methods for quantitative determination of milk or colostrum immunoglobulin content are also known in the art, and examples are provided herein.

The invention provides methods for identifying or selecting a mammalian subject with a genotype indicative of one or more desired immunoglobulin uptake, production, regulation, or secretion phenotypes, including desired milk or colostrum immunoglobulin content, particularly desired milk or colostrum IgA content. One of the major applications of the present invention is in the identification or selection of bovine having one or more of the C allele at the C903G polymorphism, the A allele at the A3938G polymorphism, the A allele at the A4029G polymorphism, or the G allele at the G5183A polymorphism, in the PIGR gene, which are each independently associated with increased milk or colostrum IgA content, or of bovine having one or more of the G allele at the C903G polymorphism, the G allele at the A3938G polymorphism, the G allele at the A4029G polymorphism, or the A allele at the G5183A polymorphism, in the PIGR gene which are each independently associated with decreased milk or colostrum IgA content. Accordingly, one method comprises determining the presence or absence of one or more of the C allele or the G allele at the C903G polymorphism, the A allele or the G allele at the A3938G polymorphism, the A allele or the G allele at the A4029G polymorphism, or the G allele or the A allele at the G5183A polymorphism, of the PIGR gene, and selecting the bovine on the basis of the determination. The invention also provides methods of assessing the genetic status of a bovine with respect to milk or colostrum immunoglobulin content, more particularly milk or colostrum IgA content. One such method comprises the step of determining the PIGR allelic profile of said bovine. Another such method comprises the step of determining the level of the PIGR gene product of said bovine.

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

The production of a wide variety of dairy products is well known in the art, and dairy products contemplated herein include ice creams, yoghurts and cheeses, dairy based drinks (such as milk drinks including milk shakes, and yogurt drinks), milk powders, maternal formulas, infant formulas, follow-on formulas and growing up formulas, formulas such as dairy based sports supplements, food additives such as protein sprinkles and dietary supplement products including daily supplement tablets.

Similarly, the production of a wide range of colostrum products, such as but not limited to nutraceutical supplements and the like, is well known in the art. Particularly contemplated herein are colostrum compositions having increased IgA content or produced from colostrum having increased IgA content. Methods to prepare compositions enriched in IgA and PIGR (secretory component) are also known in the art, as exemplified by those discussed in European Patent EP0446876, incorporated herein by reference in its entirety.

The present invention recognises that mutations in the gene encoding PIGR, as well as PIGR levels or activity, may be used as a selection tool to breed animals with higher or lower milk concentrations of immunoglobulin (and particularly IgA content). This in turn may allow the production of milk products more suitable to particular markets, such as the production of foods high in immunoglobulin.

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

A "subject" is an animal, preferably a mammal, more preferably a mammalian companion animal, a mammalian agricultural animal, or a human. Preferred companion animals include feline, equine, and canine. Preferred agricultural animals include bovine, ovine, cervine, and porcine. 1 PIGR

Polymeric immunoglobulin receptor (PIGR) binds its ligand, polymeric IgA and mediates the transfer of IgA across epithelial cell membranes. A reference genomic sequence for the bovine PIGR gene (GeneID:281401) is available at NC_007314.3. See also US Patent Nos 6,020,161 and 6,232,441, incorporated herein in their entirety.

As described herein, the present invention relates to the identification that mutations in the PIGR gene lead to variation in milk or colostrum immunoglobulin content, particularly variations in milk or colostrum IgA content. The PIGR polymorphisms described herein were each closely associated with colostrum IgA concentration phenotype. Animals homozygous for the C allele at the C903G polymorphism, or for the A allele at the A3938G polymorphism, or for the A allele at the A4029G polymorphism, or for the G allele at the G5183A polymorphism produced milk with approximately 340% more IgA than animals homozygous for the G allele at the C903G polymorphism, or for the G allele at the A3938G polymorphism, or for the G allele at the A4029G polymorphism, or for the A allele at the G5183A polymorphism. See Table 2 herein.

The GG genotype at the C903G polymorphism, the GG genotype at the A3938G polymorphism, the GG genotype at the A4029G polymorphism, or the AA genotype at the G5183A polymorphism, were each present in approximately 3.4% in the Holstein-Friesian x Jersey crossbred trial. Removal of these animals from the herd would increase the herd average immunoglobulin concentration by approximately 5.3%. Removal of both homozygous recessive and heterozygous animals at any polymorphic site would increase the herd average immunoglobulin concentration by approximately 16.3%. Conversely, removal of homozygote wild-type animals would decrease herd average immunoglobulin concentration by approximately 20.7%, and removal of the homozygous wildtype and heterozygous animals would decrease herd average immunoglobulin concentration by approximately 67%. IgA antibodies are effective when taken orally because they are resistant to degradation by enzymes in the gut. As a result, they are ideally suited to use as nutraceuticals or food supplements. IgA may be combined with probiotics to inhibit or reduce adverse effects due to pathogens. Applications include use of IgA as a nutraceutical ingredient to target pathogens which cause infections of mucosal surfaces such as in the nose, eyes, ears, lungs, breast and vagina. Moreover, IgA-containing products are suitable for gut and oral health applications.

A reference bovine PIGR nucleotide sequence referred to as "wild type PIGR" by the applicants is presented as SEQ ID NO. 1 , and the compiled coding sequence is presented as SEQ ID NO. 2, with the corresponding amino acid sequence is presented as SEQ ID NO: 3. Accordingly, as used herein the term "wild type" recognizes the characteristics of the PIGR nucleotide sequences presented as SEQ ID NOS: 1 and 2, and of the protein product encoded thereby. For example, when used with reference to activity, the term "wild type" denotes activity associated with the wild type PIGR protein. Similarly, when used with reference to expression level, the term "wild type" denotes a level of expression associated with the wild type PIGR promoter or of the wild type PIGR gene.

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

As used herein, such as when used in reference to an allelic protein lacking the activity of wild type PIGR, the phrase "lacking the activity of (A)" contemplates activity both greater than that of (A) and less than that of (A). For example, an allelic protein lacking the activity of wild type PIGR may be a variant PIGR protein of greater or lesser enzymatic activity than that of wild type PIGR.

The activity of PIGR protein, such as that present in a sample obtained from the subject, may be determined by methods well known in the art, for example by determining the PIGR- mediated transport of IgA (and particularly polymeric IgA) or another PIGR substrate across secretory epithelia, or by determining the presence or absence of slgA or SC in, for example, cell secretions or in culture media. Methods to assay the expression or activity of PIGR are well known in the art. For example, one such method utilises Northern analysis or immunostaining for PIGR as described in Johansen et al., 1999. Another exemplary method described in Kaetzel et al., 1991, assays transcytosis across epithelial cells, where the appearance of labeled immunoglobulin in cell culture supernatant correlates with PIGR activity. Similarly, methods to indirectly measure the activity of PIGR are available, and include a determination of the presence, absence or concentration of IgA, such as that present in a sample obtained from bovine.

The genetic polymorphism identified in the bovine PIGR gene is identified as a variant in SEQ ID NO.l and 2, and is reported in Figure 3. The nucleic acid and protein sequences of the PIGR polymorphisms described herein are shown in Figure 3B. 2 Identification and analysis of polymorphisms

The polymorphisms described herein are numbered according to their position in the genomic nucleotide sequence relative to the +1 translation start site of the bovine PIGR gene (i.e., the PIGR protein inclusive of the signal peptide, also referred to herein as pro-PIGR). Those skilled in the art will recognise that these positions can readily be expressed relative to the coding sequence, or relative to their position in the mature PIGR polypeptide lacking the signal peptide. It will be apparent to those skilled in the field that the convention of identifying polymorphisms effecting an amino acid substitution by their codon position in the gene in which they occur and the amino acid substitution effected thereby is also contemplated herein. Accordingly, the C903G polymorphism, the A3938G polymorphism, the A4029G polymorphism, and the G5183A polymorphism described herein may be referred to by reference to the codon of the PIGR gene within which they are located and the amino acid substitution effected, namely the S29T polymorphism, the M404I polymorphism, the A435T polymorphism, and the A504T polymorphism, respectively.

Each of the C903G polymorphism, the A3938G polymorphism, the A4029G polymorphism, or the G5183 A polymorphism, can be detected directly or by detection of one or more polymorphisms which are in linkage disequilibrium with one or more of these polymorphisms. Linkage disequilibrium (LD) is a phenomenon in genetics whereby two or more mutations or polymorphisms are in such close genetic proximity that they are co- inherited. This means that in genotyping, detection of one polymorphism as present implies the presence of the other. (Reich DE et al; Linkage disequilibrium in the human genome, Nature 2001, 41 1 :199-204.)

Various degrees of linkage disequilibrium are possible. Preferably, the one or more polymorphisms in linkage disequilibrium with one or more of the polymorphisms specified herein are in greater than about 60% linkage disequilibrium, are in about 70% linkage disequilibrium, about 75%, about 80%, about 85%, about 90%, about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100% linkage disequilibrium with the C903G polymorphism, the A3938G polymorphism, the A4029G polymorphism, or the G5183A polymorphism, of the PIGR gene. (Devlin and Risch 1995; A comparison of linkage disequilibrium measures for fine-scale mapping, Genomics 29: 311-322).

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

In another aspect, the present invention provides a method for determining the genetic status of a bovine with respect to milk or colostrum IgA content, and particularly with respect to milk or colostrum IgA content. The method includes ascertaining, from a sample of material containing DNA obtained from the bovine, whether a sequence of the DNA encoding (a) a protein having biological activity of wild type PIGR is present, and whether a sequence of the DNA encoding (b) an allelic protein lacking the activity of (a) is present.

An example of another art standard method known for determining whether a particular DNA sequence is present in a sample is the Polymerase Chain Reaction (PCR). A preferred aspect of the invention thus includes a step in which ascertaining whether a sequence of the DNA encoding (a) is present, and whether a sequence of the DNA encoding (b) is present includes amplifying the DNA in the presence of primers based on a nucleotide sequence encoding a protein having biological activity of wild type PIGR, and/or in the presence of a primer containing at least a portion of a polymorphism known to naturally occur and which when present results in high relative immunoglobulin levels, and particularly in milk or colostrum having inter alia a higher IgA content, and/or in the presence of a primer containing at least a portion of a polymorphism known to naturally occur and which when present results in low relative immunoglobulin levels, and particularly in milk or colostrum having inter alia a lower IgA content.

A primer of the present invention, used in PCR for example, is a nucleic acid molecule sufficiently complementary to the sequence on which it is based and of sufficient length to selectively hybridise to the corresponding portion of a nucleic acid molecule intended to be amplified and to prime synthesis thereof under in vitro conditions commonly used in PCR. Likewise, a probe of the present invention, is a molecule, for example a nucleic acid molecule of sufficient length and sufficiently complementary to the nucleic acid molecule of interest, which selectively binds under high or low stringency conditions with the nucleic acid sequence of interest for detection in the presence of nucleic acid molecules having differing sequences.

Accordingly, a preferred embodiment of the invention thus includes the step of amplifying a PIGR polynucleotide in the presence of at least one primer comprising a nucleotide sequence of or complementary to, the PIGR gene (SEQ ID NO:1 and SEQ ID NO:2) or flanking sequence thereof, and/or in the presence of a such a primer comprising sequence corresponding to or flanking the allele-specific nucleotides described herein. PCR methods are well known by those skilled in the art (Mullis et al., 1994.) The template for amplification may be selected from genomic DNA, mRNA or first strand cDNA derived from a sample obtained from the bovine under test (Sambrook et al., 1989).

Primers suitable for use in PCR based methods of the invention should be sufficiently complementary to the PIGR gene sequence, such as SEQ ID NO:1 or SEQ ID NO:2 or flanking sequence thereof, and of sufficient length to selectively hybridise to the corresponding portion of a nucleic acid molecule intended to be amplified and to prime synthesis thereof under in vitro conditions commonly used in PCR. Such primers should comprise at least about 12 contiguous bases of or complementary to SEQ ID NO: 1 or SEQ ID NO:2, or naturally occurring flanking sequences thereof. Examples of such PCR primers are presented herein as SEQ ID NOs:4-51.

Suitable PCR primers may include sequence corresponding to the C903G C allele- specific, the C903G G allele-specific, the A3938G A allele-specific, the A3938G G allele- specific, the A4029G A allele-specific, the A4029G G allele-specific, the G5183A G allele- specific, or the G5183A A allele-specific, nucleotides described herein. Generation of a corresponding PCR product, or the lack of product, may constitute a test for the presence or absence of the specified nucleotides in the PIGR gene of the test bovine.

Other methods for determining whether a particular nucleotide sequence is present in a sample may include the step of restriction enzyme digestion of nucleotide sample. Separation and visualisation of the digested restriction fragments by methods well known in the art, may form a diagnostic test for the presence of a particular nucleotide sequence. The nucleotide sequence digested may be a PCR product amplified as described above. Still other methods for determining whether a particular nucleotide sequence is present in a sample include a step of hybridisation of a probe to a sample nucleotide sequence. Thus, methods for detecting one or more of the allele-specific nucleotides described herein may comprise the additional steps of hybridisation of a probe derived from the PIGR sequence of SEQ ID NO:1 or SEQ ID NO:2.

Such probes should comprise a nucleic acid molecule of sufficient length and sufficiently complementary to the PIGR gene sequence, to selectively bind under high or low stringency conditions with the nucleic acid sequence of a sample to facilitate detection of the presence or absence of the allele-specific nucleotides described herein.

With respect to polynucleotide molecules greater than about 100 bases in length, typical stringent hybridization conditions are no more than 25 to 30° C (for example, 10° C) below the melting temperature (Tm) of the native duplex (see generally, Sambrook et al., 1989; Ausubel et al., 1987). Tm for polynucleotide molecules greater than about 100 bases can be calculated by the formula Tm = 81. 5 + 0. 41% (G + C-log (Na+).

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

Such a probe may be hybridised with genomic DNA, mRNA, or cDNA produced form mRNA, derived from a sample taken from a bovine under test.

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

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

In another aspect, the invention provides a method for determining the genetic status of bovine with respect to milk or colostrum IgA content with reference to a sample of material containing mRNA obtained from the bovine. In one embodiment this method includes ascertaining whether a sequence of the mRNA encoding (A) a protein having biological activity of a wild type PIGR is present, and whether a sequence of the mRNA encoding (B) a protein at least partially lacking the activity of (A) is present, and may include determining the amount of mRNA. The absence of the mRNA encoding (A) and the presence of the mRNA encoding (B) indicates an association with low relative milk or colostrum immunoglobulin levels, particularly with the production of milk or colostrum with, inter alia, decreased IgA content. The reverse association again holds true.

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

Other methods to quantitate or otherwise assess the amount of nucleic acid, particularly the amount of mRNA are well known in the art. These include Northern analysis using probes able to hybridise to the target PIGR mRNA. Such probes should comprise a nucleic acid molecule of sufficient length and sufficiently complementary to the PIGR coding sequence to selectively bind under high or low stringency conditions with the nucleic acid sequence of a sample to facilitate detection and assessment of the amount of PIGR mRNA present. As is evident to the person skilled in the art, such quantitative methods generally utilise an internal control, for example in the case of Northern analysis quantitation may be done with reference to, for example, rRNA present in the sample.

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

In one embodiment, said genetic loci is a polymorphism in a gene associated with milk or colostrum IgA content, preferably a polymorphism in a gene involved in immunoglobulin production or secretion.

Exemplary methods of the invention are reliant on genetic information such as that derived from methods suitable to the detection and identification of polymorphisms, particularly single nucleotide polymorphisms (SNPs) associated with the qualitative trait for which an assessment is desired. For the sake of convenience the following discussion refers particularly to SNPs, yet the art-skilled worker will appreciate that the methods discussed are amenable to the detection and identification of other genetic polymorphisms, such as triplet repeats or microsatellites. A SNP is a single base change or point mutation resulting in genetic variation between individuals that can occur in both coding or non-coding regions.

A number of databases have been constructed of known SNPs, and for some such SNPs, the biological effect associated with a SNP. For example, the NCBI SNP database "dbSNP" is incorporated into NCBI' s Entrez system and has records for over 4 million SNPs mapped onto the human genome sequence. Similar databases exist for other mammalian genomes.

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

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

A number of sequencing methods and platforms are particularly suited to large-scale implementation, and are amenable to use in the methods of the invention. These include pyrosequencing methods, such as that utilised in the GS FLX pyrosequencing platform available from 454 Life Sciences (Branford, CT) which can generate 100 million nucleotide data in a 7.5 hour run with a single machine, and solid-state sequencing methods, such as that utilised in the SOLiD sequencing platform (Applied Biosystems, Foster City, CA).

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

The majority of methods to detect or identify SNPs by site-specific hybridisation require target amplification by methods such as PCR to increase sensitivity and specificity (see, for example U.S. Pat. No. 5,679,524, PCT publication WO 98/59066, PCT publication WO 95/12607). US Patent Application Publication No. US 20050059030 (incorporated herein by reference in its entirety) describes a method for detecting a SNP in total human DNA without prior amplification or complexity reduction to selectively enrich for the target sequence, and without the aid of any enzymatic reaction. The method utilises a single-step hybridization involving a hybridization of a first portion of the target sequence to a capture probe, and hybridization of a second portion of said target sequence to a detection probe.

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

The technique of Lynx Therapeutics (Hayward, Calif.) using MEGATYPE technology can genotype very large numbers of SNPs simultaneously from small or large pools of genomic material. Two populations are compared using fluorescently labelled probes, enabling detection and recovery of SNPs that distinguish the two populations.

Other methods for detecting and identifying SNPs include mass spectrometry. A preferred example is the use of mass spectrometric determination of a nucleic acid sequence which comprises the polymorphisms of the invention (whether the coding sequence or a complementary sequence). Such mass spectrometric methods are known to those skilled in the art, and the genotyping methods of the invention are amenable to adaptation for the mass spectrometric detection of the polymorphisms of the invention.

SNPs can also be determined by ligation-bit analysis. A further discussion of these methods can be found in U.S. Pat. Nos. 5,919,626; 5,945,283; 5,242,794; and 5,952,174. A large number of methods reliant on the conformational variability of nucleic acids have been developed to detect SNPs. For example, Single Strand Conformational Polymorphism (SSCP, Orita et al, PNAS 1989 86:2766-2770), and various modifications of SSCP as are well known in the art. These include the use of differing gel running conditions, such as for example differing temperature, or the addition of additives, and different gel matrices, RNA-SSCP, restriction endonuclease fingerprinting-SSCP, dideoxy fingerprinting (a hybrid between dideoxy sequencing and SSCP), bi-directional dideoxy fingerprinting (in which the dideoxy termination reaction is performed simultaneously with two opposing primers), and Fluorescent PCR-SSCP (in which PCR products are internally labelled with multiple fluorescent dyes, may be digested with restriction enzymes, followed by SSCP, and analysed on an automated DNA sequencer able to detect the fluorescent dyes).

Other methods which utilise the varying mobility of different nucleic acid structures include Denaturing Gradient Gel Electrophoresis (DGGE), Temperature Gradient Gel Electrophoresis (TGGE), Heteroduplex Analysis (HET), and capillary electrophoresis. Denaturing High Pressure Liquid Chromatography (HPLC) is yet a further method utilised to detect SNPs, using HPLC methods to detect, for example, homoduplexes and heteroduplexes which elute from the HPLC column at different rates, thereby enabling detection of mismatch nucleotides and thus SNPs.

Yet further methods to detect SNPs rely on the differing susceptibility of single stranded and double stranded nucleic acids to cleavage by various agents, including chemical cleavage agents and nucleolytic enzymes. Further examples include the Protein Translation Test (PTT), used to resolve stop codons generated by variations which lead to a premature termination of translation and to protein products of reduced size, and the use of mismatch binding proteins. US Patent 6,821,733 (incorporated herein in its entirety) describes methods to detect differences in the sequence of two nucleic acid molecules.

Protein- and proteomics-based approaches are also suitable for polymorphism detection and analysis. These approaches typically require separation of the various proteins within a sample, by, for example, gel electrophoresis or HPLC, and identification of said proteins or peptides derived therefrom, for example by NMR or protein sequencing such as chemical sequencing or more prevalently mass spectrometry. Proteomic methodologies are well known in the art, and have great potential for automation. For example, integrated systems, such as the ProteomlQ™ system from Proteome Systems, provide high throughput platforms for proteome analysis combining sample preparation, protein separation, image acquisition and analysis, protein processing, mass spectrometry and bioinformatics technologies.

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

Associated technologies are also well known, and include, for example, protein processing devices such as the "Chemical InkJet Printer" comprising piezoelectric printing technology that allows in situ enzymatic or chemical digestion of protein samples electroblotted from 2-D PAGE gels to membranes by jetting the enzyme or chemical directly onto the selected protein spots. After in-situ digestion and incubation of the proteins, the membrane can be placed directly into the mass spectrometer for peptide analysis.

Suitable polypeptide-based analyses include those able to discriminate between full- length and truncated protein products, and may include but are not limited to, the following: Native polyacrylamide gel electrophoresis (PAGE), isoelectric focussing, 2D PAGE, or Western blotting with specific antibodies. Mass spectroscopy, immunoprecipitation, and peptide fingerprinting are also suitable.

Those skilled in the art will know that a particular SNP, particularly when it occurs in a regulatory region of a gene such as a promoter, can be associated with altered expression of a gene. Altered expression of a gene can also result when the SNP is located in the coding region of a protein-encoding gene. Such altered expression can be determined by methods well known in the art, such as quantitative PCR, RT-PCR, quantative Northern analysis, and can thereby be employed to detect such SNPs. Similarly, where a SNP occurs in the coding region of a gene and results in a non-synonomous amino acid substitution, such substitution can result in a change in the function of the gene product. Similarly, in cases where the gene product is an RNA, such SNPs can result in a change of function in the RNA gene product. Any such change in function, for example as assessed in an activity or functionality assay, can be employed to detect such SNPs. The above methods of detecting and identifying SNPs are amenable to use in the methods of the invention. 3 Polynucleotide and polypeptide variants

The term "polynucleotide(s)," as used herein, means a single or double-stranded deoxyribonucleotide or ribonucleotide polymer of any length but preferably at least 15 nucleotides, and include as non-limiting examples, coding and non-coding sequences of a gene, sense and antisense sequences complements, exons, introns, genomic DNA, cDNA, pre- mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polypeptides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers and fragments. A number of nucleic acid analogues are well known in the art and are also contemplated.

A "fragment" of a polynucleotide sequence provided herein is a subsequence of contiguous nucleotides that is preferably at least 15 nucleotides in length. The fragments of the invention preferably comprises at least 20 nucleotides, more preferably at least 30 nucleotides, more preferably at least 40 nucleotides, more preferably at least 50 nucleotides and most preferably at least 60 contiguous nucleotides of a polynucleotide of the invention. A fragment of a polynucleotide sequence can be used in antisense, gene silencing, triple helix or ribozyme technology, or as a primer, a probe, included in a microarray, or used in polynucleotide-based selection methods.

The term "fragment" in relation to promoter polynucleotide sequences is intended to include sequences comprising cis-elements and regions of the promoter polynucleotide sequence capable of regulating expression of a polynucleotide sequence to which the fragment is operably linked.

Preferably fragments of polynucleotide sequences of the invention comprise at least 20, more preferably at least 30, more preferably at least 40, more preferably at least 50, more preferably at least 100, more preferably at least 200, more preferably at least 300, more preferably at least 400, more preferably at least 500, more preferably at least 600, more preferably at least 700, more preferably at least 800, more preferably at least 900 and most preferably at least 1000 contiguous nucleotides of a polynucleotide of the invention.

The term "primer" refers to a short polynucleotide, usually having a free 3 'OH group, that is hybridized to a template and used for priming polymerization of a polynucleotide complementary to the template. Such a primer is preferably at least 5, more preferably at least 6, more preferably at least 7, more preferably at least 9, more preferably at least 10, more preferably at least 1 1, more preferably at least 12, more preferably at least 13, more preferably at least 14, more preferably at least 15, more preferably at least 16, more preferably at least 17, more preferably at least 18, more preferably at least 19, more preferably at least 20 nucleotides in length.

The term "probe" refers to a short polynucleotide that is used to detect a polynucleotide sequence that is complementary to the probe, in a hybridization-based assay. The probe may consist of a "fragment" of a polynucleotide as defined herein. Preferably such a probe is at least 5, more preferably at least 10, more preferably at least 20, more preferably at least 30, more preferably at least 40, more preferably at least 50, more preferably at least 100, more preferably at least 200, more preferably at least 300, more preferably at least 400 and most preferably at least 500 nucleotides in length.

The term "variant" as used herein refers to polynucleotide or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the polynucleotides and polypeptides possess biological activities that are the same or similar to those of the wild type polynucleotides or polypeptides. The term "variant" with reference to polynucleotides and polypeptides encompasses all forms of polynucleotides and polypeptides as defined herein. 3.1 Polynucleotide variants

Variant polynucleotide sequences preferably exhibit at least 50%, more preferably at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least %, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a specified polynucleotide sequence. Identity is found over a comparison window of at least 20 nucleotide positions, preferably at least 50 nucleotide positions, at least 100 nucleotide positions, or over the entire length of the specified polynucleotide sequence. Polynucleotide sequence identity can be determined in the following manner. The subject polynucleotide sequence is compared to a candidate polynucleotide sequence using BLASTN (from the BLAST suite of programs, version 2.2.10 [Oct 2004]) in bl2seq (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250), which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The default parameters of bl2seq are utilized except that filtering of low complexity parts should be turned off.

The identity of polynucleotide sequences may be examined using the following unix command line parameters: bl2seq -i nucleotideseql -j nucleotideseq2 -F F -p blastn

The parameter -F F turns off filtering of low complexity sections. The parameter — p selects the appropriate algorithm for the pair of sequences. The bl2seq program reports sequence identity as both the number and percentage of identical nucleotides in a line "Identities = ".

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

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

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

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

The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. The size of this database is set by default in the bl2seq program. For small E values, much less than one, the E value is approximately the probability of such a random match.

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

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

The term "hybridize under stringent conditions", and grammatical equivalents thereof, refers to the ability of a polynucleotide molecule to hybridize to a target polynucleotide molecule (such as a target polynucleotide molecule immobilized on a DNA or RNA blot, such as a Southern blot or Northern blot) under defined conditions of temperature and salt concentration. The ability to hybridize under stringent hybridization conditions can be determined by initially hybridizing under less stringent conditions then increasing the stringency to the desired stringency.

With respect to polynucleotide molecules greater than about 100 bases in length, typical stringent hybridization conditions are no more than 25 to 30⁰C (for example, 10°C) below the melting temperature (Tm) of the native duplex (see generally, Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Ausubel et al., 1987, Current Protocols in Molecular Biology, Greene Publishing,). Tm for polynucleotide molecules greater than about 100 bases can be calculated by the formula Tm = 81. 5 + 0. 41% (G + C-log (Na+). (Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Bolton and McCarthy, 1962, PNAS 84:1390). Typical stringent conditions for polynucleotide of greater than 100 bases in length would be hybridization conditions such as prewashing in a solution of 6X SSC, 0.2% SDS; hybridizing at 65°C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in IX SSC, 0.1% SDS at 65°C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65°C.

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

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

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

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

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

Polypeptide Variants

The term "variant" with reference to polypeptides encompasses naturally occurring, recombinantly and synthetically produced polypeptides. Variant polypeptide sequences preferably exhibit at least 50%, more preferably at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least %, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a sequences of the present invention. Identity is found over a comparison window of at least 20 amino acid positions, preferably at least 50 amino acid positions, at least 100 amino acid positions, or over the entire length of a polypeptide of the invention.

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

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

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

Variant polypeptide sequences preferably exhibit an E value of less than 1 x 10^"10, more preferably less than 1 x 10^"20, less than 1 x 10^"30, less than 1 x 10^"40, less than 1 x 10^"50, less than 1 x 10^"60, less than 1 x 10^"70, less than 1 x 10^"80, less than 1 x 10^"90, less than 1 xlO^"100, less than 1 x 10^{"1 10}, less than 1 x 10^"120 or less than 1 x 10^"123 when compared with any one of the specifically identified sequences.

The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. For small E values, much less than one, this is approximately the probability of such a random match.

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

A polypeptide variant of the present invention also encompasses that which is produced from the nucleic acid encoding a polypeptide, but differs from the wild type polypeptide in that it is processed differently such that it has an altered amino acid sequence. For example a variant may be produced by an alternative splicing pattern of the primary RNA transcript to that which produces a wild type polypeptide. 4 Diagnostic kits

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

Accordingly, in one embodiment the invention provides a diagnostic kit which can be used to determine the PIGR genotype of bovine genetic material. One kit includes a set of primers used for amplifying the genetic material. A kit can contain a primer including a nucleotide sequence for amplifying a region of the genetic material containing one of the naturally occurring mutations described herein. Such a kit could also include a primer for amplifying the corresponding region of the normal gene that produces a functionally wild type PIGR. Usually, such a kit would also include another primer upstream or downstream of the region of the gene. These primers are used to amplify the segment containing the mutation of interest. The actual genotyping is carried out using primers that target specific mutations described herein and that could function as allele-specific oligonucleotides in conventional hybridisation, Taqman assays, OLE assays, etc. Alternatively, primers can be designed to permit genotyping by microsequencing. One kit of primers can include first, second and third primers, (a), (b) and (c), respectively. Primer (a) is based on a region containing a PIGR mutation such as described above. Primer (b) encodes a region upstream or downstream of the region to be amplified by a primer (a) so that genetic material containing the mutation is amplified, by PCR, for example, in the presence of the two primers. Primer (c) is based on the region corresponding to that on which primer (a) is based, but lacking the mutation. Thus, genetic material containing the non-mutated region will be amplified in the presence of primers (b) and (c). Genetic material homozygous for the wild type gene will thus provide amplified products in the presence of primers (b) and (c). Genetic material homozygous for the mutated gene will thus provide amplified products in the presence of primers (a) and (b). Heterozygous genetic material will provide amplified products in both cases.

For example, the kit may include a primer comprising a guanine at the position corresponding to the C903G promoter polymorphism in the PIGR gene, or comprising a nucleotide capable of hybridising to a nucleotide capable of hybridising to a guanine at the position corresponding to the C903G promoter polymorphism in the PIGR gene. Those skilled in the art will recognise that in such a primer, the guanine, or the nucleotide capable of hybridising to a nucleotide capable of hybridising to a guanine, as applicable, may be substituted for a nucleotide analogue having the same discriminatory base-pairing as the substituted nucleotide. Preferably, the primer comprises at least 12 contiguous nucleotides of SEQ ID NO: 1 or 2.

In another example, the kit may include a primer comprising at least 12 contiguous nucleotides of the complement of SEQ ID NO: 1 or the complement of SEQ ID NO:2, and a cytosine at the position corresponding to the C903G promoter polymorphism in the PIGR gene, or a nucleotide capable of hybridising to a nucleotide capable of hybridising to a cytosine at the position corresponding to the C903G promoter polymorphism in the PIGR gene. Those skilled in the art will recognise that in such a primer, the cytosine, or the nucleotide capable of hybridising to a nucleotide capable of hybridising to a cytosine, as applicable, may be substituted for a nucleotide analogue having the same discriminatory base- pairing as the substituted nucleotide.

Those skilled in the art will appreciate that the invention provides kits comprising primers similarly directed to one or more of the A3938G polymorphism, the A4029G polymorphism, or the G5183A polymorphism. In one embodiment, the diagnostic kit is useful in detecting DNA comprising a variant PIGR gene or encoding a variant PIGR polypeptide at least partially lacking wild type activity in a bovine which includes first and second primers for amplifying the DNA, the primers being complementary to nucleotide sequences of the DNA upstream and downstream, respectively, of a polymorphism in the PIGR gene which results in increased or decreased immunoglobulin levels (particularly increased or decreased IgA content in milk or colostrum). In one embodiment, at least one of the nucleotide sequences is selected to be from a coding region of the PIGR gene. In another embodiment, at least one of the nucleotide sequences is selected to be from a non-coding region of the PIGR gene. The kit can also include a third primer complementary to a naturally occurring mutation of a coding portion of the wild type PIGR gene. Preferably the kit includes instructions for use, for example in accordance with a method of the invention.

In one embodiment, the diagnostic kit comprises a nucleotide probe complementary to the sequence shown in SEQ ID NO: 1 or SEQ ID NO:2 or a fragment thereof, for example, for hybridisation with mRNA from a sample of cells; means for detecting the nucleotide probe bound to mRNA in the sample with a standard. In a particular aspect, the kit of this aspect of the invention includes a probe having a nucleic acid molecule sufficiently complementary with a sequence presented in SEQ ID NO: lor SEQ ID NO:2 or complements thereof, so as to bind thereto under stringent conditions. "Stringent hybridisation conditions" takes on its common meaning to a person skilled in the art. Appropriate stringency conditions which promote nucleic acid hybridisation, for example, 6x sodium chloride/sodium citrate (SSC) at about 45°C are known to those skilled in the art, including in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989). Appropriate wash stringency depends on degree of homology and length of probe. If homology is 100%, a high temperature (65°C to 75°C) may be used. However, if the probe is very short (<100bp), lower temperatures must be used even with 100% homology. In general, one starts washing at low temperatures (37°C to 4O⁰C), and raises the temperature by 3-5°C intervals until background is low enough to be a major factor in autoradiography. The diagnostic kit can also contain an instruction manual for use of the kit.

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

5 Sample preparation

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

Conveniently, samples may be taken from milk, colostrum, tissues including blood, serum, and plasma, cerebrospinal fluid, urine, semen or saliva. Tissue samples may be obtained using standard techniques such as cell scrapings or biopsy techniques. For example, the cell or tissue samples may be obtained by using an ear punch to collect ear tissue from bovine. Similarly, blood sampling is routinely performed, for example for pathogen testing, and methods for taking blood samples are well known in the art. Likewise, methods for storing and processing biological samples are well known in the art. For example, tissue samples may be frozen until tested if required. In addition, one of skill in the art would realize that some test samples would be more readily analyzed following a fractionation or purification procedure, for example, separation of whole blood into serum or plasma components.

6 Computer-Related Embodiments

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

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

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

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

A variety of data storage structures are available to a skilled artisan for creating a computer readable medium having recorded thereon SNP information of the present invention. The choice of the data storage structure will generally be based on the means chosen to access the stored information. In addition, a variety of data processor programs and formats can be used to store the SNP information of the present invention on computer readable medium. For example, sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, represented in the form of an ASCII file, or stored in a database application, such as OB2, Sybase, Oracle, or the like. A skilled artisan can readily adapt any number of data processor structuring formats (e.g., text file or database) in order to obtain computer readable medium having recorded thereon the SNP information of the present invention. By providing the SNPs and/or the results of an analysis of the SNPs utilised in the present invention in computer readable form, a skilled artisan can routinely access the SNP information for a variety of purposes. Computer software is publicly available which allows a skilled artisan to access sequence information provided in a computer readable medium. Examples of publicly available computer software include BLAST (Altschul et at, J. MoI. Biol. 215:403-410 (1990)) and BLAZE (Brutlag et at, Comp. Chem. 17:203-207 (1993)) search algorithms.

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

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

As stated above, the computer-based systems of the present invention comprise data storage having stored therein SNP information, such as SNPs and/or the results of an analysis of the SNPs utilised in the present invention, and the necessary hardware and software for supporting and implementing one or more programs or algorithms. As used herein, "data storage" refers to memory which can store SNP information of the present invention, or a memory access facility which can access manufactures having recorded thereon the SNP information of the present invention. The one or more programs or algorithms are implemented on the computer-based system to identify or analyze the SNP information stored within the data storage. For example, such programs or algorithms can be used to determine which nucleotide is present at a particular SNP position in a target sequence, or to analyse the results of a genetic analysis of the SNPs described herein. As used herein, a "target sequence" can be any DNA sequence containing the SNP position(s) to be analysed, searched or queried.

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

One exemplary embodiment of a computer-based system comprising SNP information of the present invention that can be used to implement the present invention includes a processor connected to a bus. Also connected to the bus are a main memory (preferably implemented as random access memory, RAM) and a variety of secondary storage devices, such as a hard drive and a removable medium storage device. The removable medium storage device may represent, for example, a floppy disc drive, a CD-ROM drive, a magnetic tape drive, etc. A removable storage medium (such as a floppy disc, a compact disc, a magnetic tape, etc.) containing control logic and/or data recorded therein may be inserted into the removable medium storage device. The computer system includes appropriate software for reading the control logic and/or the data from the removable storage medium once inserted in the removable medium storage device. The SNP information of the present invention may be stored in a well-known manner in the main memory, any of the secondary storage devices, and/or a removable storage medium. Software for accessing and processing the SNP information (such as SNP scoring tools, search tools, comparing tools, etc.) preferably resides in main memory during execution. Accordingly, the present invention provides a system for performing one or more of the methods of the invention, said system comprising: computer processor means for receiving, processing and communicating data; storage means for storing data including a reference genetic database of the results of genetic analysis of a bovine with respect to one or more milk or colostrum immunoglobulin content phenotypes and optionally a reference milk or colostrum immunoglobulin content phenotypes database of non-genetic factors for bovine milk or colostrum immunoglobulin content phenotypes; and a computer program embedded within the computer processor which, once data consisting of or including the result of a genetic analysis for which data is included in the reference genetic database is received, processes said data in the context of said reference databases to determine, as an outcome, the genetic status of the bovine, said outcome being communicable once known, preferably to a user having input said data.

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

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

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

Preferably, the one or more genetic loci are one or more polymorphisms in one or more genes associated with one or more milk or colostrum immunoglobulin content phenotypes. 7 Compositions useful according to the invention

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

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

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

In another embodiment, compositions useful herein include dietetic products.

In preferred embodiments, the compositions useful herein are able to maintain or raise immunoglobulin levels in a subject to whom they are administered.

For example, such compositions are able to maintain or raise immunoglobulin levels in a foetal subject (by 'indirect' administration to a foetal subject by administration to the pregnant mother). The term "levels in a foetal subject" as used herein includes foetal blood levels and/or foetal lymph levels and/or foetal tissue levels.

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

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

The term "dietetic product" means a product specially processed or formulated to satisfy particular dietary requirements which exist because of a particular physical or physiological condition and/or specific diseases and disorders and which are presented as such. It will be appreciated that it is not intended to limit the invention to the above example only, many variations, which may readily occur to a person skilled in the art, being possible without departing from the scope thereof as defined in the accompanying claims.

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

The invention consists in the foregoing and also envisages constructions of which the following gives examples only. EXAMPLE ONE -Analysis of the genetic basis for IgA content of milk and colostrum

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

Materials and methods

1. Trial design

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

2. IgA measurement in colostrum and milk

All colostrum composition measurements were taken during the second lactation. Cows were milked twice daily; milk volume was recorded at each milking and samples taken for analysis at the second milking after calving (day one of lactation) and the eighth milking after calving (day four of lactation). IgA in milk was measured in samples taken from cows in the FJX trial just after peak yield was achieved in their second lactation (approximately days 70- 100 post-calving). IgA was measured using the bovine IgA ELISA quantitation kit (Bethyl Laboratories Inc, Montgomery, Texas, USA; catalogue number E 10-121), as per manufacturer's instructions. A reference standard of bovine serum, containing 0.18 mg/mL IgA was used to calibrate the assay for the colostrum samples. The working range of the assay was 1000 - 15.625 ng/niL. IgM was measured using a bovine IgM ELISA quantitation kit (Bethyl Laboratories Inc, Montgomery, Texas, USA; catalogue number ElO-101) as per manufacturers instructions. A reference standard of bovine serum, containing 2.5 mg/mL IgM was used to calibrate the assay for the colostrum samples. The working range of the assay was 1000 - 15.625 ng/mL.

The absolute IgA concentration values generated by the ELISA for the milk samples were higher than would be expected for milk by a factor of 7-10 fold because of less than optimal calibration of the reference, serum-derived standard. Irrespective of this, the ranking of values was unaffected. The milk IgA data also produced a large QTL for IgA on bovine chromosome 16 in a very similar position to that produced by the colostral IgA data. The maximum F value for the QTL was 9.1, (half-sib model) and the most likely position was estimated at 1 1 cM. Bootstrap analysis (n = 1000) showed that the 95% confidence interval for the QTL was 1 - 25 cM. 3. Secretory component measurement in milk Sample preparation

Whey was prepared from a subset of FJX mid lactation milk samples by the addition of acetic acid to a final concentration of 1% followed by centrifugation at 3,000 x g, 20 min, 8⁰C. Samples of the whey were reduced, alkylated with iodoacetamide, then digested with trypsin. Analysis of tryptic peptides by liquid chromatography-mass spectrometry

Samples of tryptic peptides of whey proteins and the synthetic peptide AAPAGAAISQR, which corresponds to a tryptic peptide of secretory component, were analysed by reverse phase high performance liquid chromatography using an Agilent Technologies 1100 series capillary liquid chromatograph fitted with an Agilent Technologies Zorbax SB-Cl 8 3OθA 0.3x150mm 3.5 μm column which was coupled to an Agilent Technologies model SL ion trap mass spectrometer with an electrospray ionisation interface. The Zorbax column was developed with a gradient of 4% to 56% acetonitrile in 0.1% formic acid/0.005% heptafluorobutyric acid, over 27 minutes at a flow rate of 4 μl/rnin. The mass spectrometer was operated in single ion monitoring mode which was set to isolate and fragment the doubly protonated ion of peptide AAP AGAAISQR observed at 506.8 m/z; fragment ions were observed by scanning the mass range from 430 m/z to 810 m/z. The predominant fragment ion observed at 435.9 m/z, corresponding to the doubly protonated y9 ion, was used for quantitation; ions observed at 623.3 m/z, corresponding to the b8 ion, and at 704.2 m/z, corresponding to the y7 ion, were used as qualifiers. The peptide AAPAGAAISQR observed in samples of trypsin-digested whey was quantitated by comparison to a standard curve constructed using the synthetic peptide AAPAGAAISQR.

4. Genotyping

Genomic DNA was prepared from whole blood from a total of 1679 animals within the trial pedigree (846 F2 daughters, six Fl sires, 796 Fl dams, and 13 selected FO sires). An initial whole genome scan was conducted by genotyping each animal for 285 microsatellite markers, obtained primarily from published marker maps. Subsequently, the pedigree was genotyped using the Affymetrix Bovine 1OK SNP GeneChip. A total of 6634 informative SNP markers were placed on the map.

5. Candidate gene sequencing

Polymeric immunoglobulin receptor (PIGR; GenelD: 281401, NC_007314.3) was identified as a candidate gene for IgA content of colostrum and milk. The coding region of the gene (1 1 exons) as well as 3 kb of 5' sequence was sequenced. Intron/exon boundaries were determined. Exons were amplified using the primers presented as SEQ ID NOs: 4-51 and sequenced in both directions. The determined wild-type coding sequence for the bovine PIGR gene is shown in SEQ ID NO:2.

6. Statistical analysis

The dataset consisted of IgA phenotypes (taken at two time points: the 2^nd and 8^th milkings) collected during the colostrum period for two cohorts of F2 animals. Data manipulation was performed using SAS (version 9.1). These data were matched with the following covariates: cohort (cohort 1 or cohort 2), sire (sires 1 - 6), milk volume (milking 2 and milking 8) and calving week (0-14). Five PIGR genotypes thought to be associated with IgA were matched with the data. Animals with missing data points for any of the measurements were excluded from the final datasets. The final dataset included 624 animals.

Statistical analyses were conducted using raw phenotypic data. ANOVA was conducted for IgA phenotypes to determine whether the PIGR polymorphisms were significantly associated with colostrum IgA content. The final ANOVA models for each of the time points were produced using a backward elimination process; all the covariates were included in the model at the first stage of the modeling process and the least significant covariates removed at each subsequent stage until all the remaining covariates were found to be significant (sig. level set at 0.1). The final models were as follows: IgA at second milking (IgA 2^nd): sire, cohort, milk volume at second milking, calving week; IgA at eighth milking IgA (8^th): sire, cohort, milk volume at eighth milking, calving week. For the IgM and SC datasets for milk concentrations, an ANOVA was conducted on the raw data using genotype as a fixed effect in the model. 7. QTL detection

The data used for QTL detection were the residuals from the statistical model. The raw phenotype data (no covariates or modeling) was also used to detect QTLs. QTL detection was conducted using a line of descent model and a half-sib model. Subsequently, the PIGR genotypes were included separately as a covariate into the models for IgA (2^nd) and IgA (8¹ ), to determine whether these polymorphisms explained the QTL variation.

Results

1. Colostrum IgA

The IgA content of colostrum differed at the second milking (IgA 2^nd) and at the eighth milking (IgA 8^th). The mean IgA content at the second milking was 1.66 mg/mL, and at the eighth milking, was 0.43 mg/mL (Figure 1). IgA content of colostrum also differed according to sire (Figure 2).

2. Detection of a QTL for colostrum IgA on bovine chromosome 16

Analysis of the IgA within the line-of-descent and the half-sib models of QTL analysis showed the presence of a significant QTL on bovine chromosome 16 (Figure 3). The maximum F value for the QTL was 1 1.8, and the most likely position was estimated at 7 cM. Bootstrap analysis (n = 1000) showed that the 95% confidence interval for the QTL was 7 - 10 cM. There were a total of 258 markers (19 microsatellite markers and 239 single nucleotide polymorphisms) used for this analysis on chromosome 16.

3. Identification of PIGR as a candidate gene and detection of polymorphisms affecting IgA content of colostrum

Polymeric immunoglobulin receptor (PIGR) binds its ligand, IgA and mediates the transfer of IgA across epithelial cell membranes. PIGR was identified as a candidate gene for the chromosome 16 IgA QTL effect. To determine whether this gene explained the observed variation in colostrum IgA content, the PIGR coding region in the six Fl sires was sequenced to identify any genetic polymorphisms that could potentially alter the function of this protein. Intron/exon boundaries were determined using the reference sequence NC 007314.3. Primers were designed within introns so that complete sequence was obtained from each exon. An additional 3kb of sequence was obtained to cover the 5' UTR.

Several polymorphisms were discovered (Figure 4). To determine whether these were associated with the QTL effect, the polymorphisms in the remainder of the FJXB trial pedigree was genotyped. The frequency of each PIGR genotype is shown in Table 1 below.

Table 1: Genotype frequencies of F 2 FJXB population

Polymorphism* fjN umber Frequency

C903G C 523 64.01

CG 266 32.56 G 28 3.43 Total 817 100.00

A3938G A 524 64.22

GA 264 32.35 G 28 3.43 Total 816 100.00

A4029G A 526 64.78

AG 262 32.27 G 24 2.96 Total 812 100.00

G5183A G 524 64.85

GA 257 31.81 A 27 3.34 Total 808 100.00

Note: Data based on successfully genotyped animals in F2 population 4. PIGR polymorphisms have a significant effect on IgA concentration in colostrum

The effect of the PIGR polymorphisms on IgA concentration is shown in Figure 5 and Table 2 below. The main effect for genotype on IgA content was significant at P < 0.0001, for both IgA (2^nd), and IgA (8^th). Addition of the PIGR genotypes as covariates into the ANOVA completely abolished the observed QTL effect on chromosome 16 (Figure 6).

Table 2: Summary statistics of PIGR polymorphisms on IgA content of colostrum, as measured at the eighth milking (lactation day four).

sd = standard deviation; med = median (mg/mL); min = minimum (mg/mL); max = maximum (mg/mL); n = number of observations (subset of F2 population which were phenotyped).

As can be seen in Table 2 above, an approximately three-fold increase in IgA content of colostrum was seen in bovine homozygous for the C allele at the C903G polymorphism compared to those homozygous for the G allele. Likewise, an approximately three-fold increase in IgA content of colostrum was seen in bovine homozygous for the A allele at the A3938G polymorphism, the A allele at the A4029G polymorphism, and the G allele at the G5183 A polymorphism, compared to bovine homozygous for the G allele, the G allele, or the A allele, respectively.

5. PIGR polymorphisms have a significant effect on IgA concentration in milk The effect of the PIGR polymorphisms on IgA concentration is shown below in Table 3. Table 3: IgA in milk ipllslSMs 1 PIGRT4029 IPIGR 5183 IPIGR903J fclAΪ M ¥Ai mm 93' fMmGΑ «} ^■ §EGA ξ&

Mean 0.79 0.55 0.30 0.79 0.56 0.29 0.29 0.56 0.78 0.79 0.56 0.30 sd 0.35 0.28 0.20 0.35 0.28 0.19 0.19 0.28 0.35 0.35 0.28 0.20 med 0.72 0.50 0.23 0.72 0.51 0.26 0.22 0.51 0.71 0.72 0.51 0.23

Min 0.15 0.07 0.10 0.15 0.07 0.10 0.10 0.07 0.15 0.15 0.07 0.10

Max 2.08 1.96 0.83 2.08 1.96 0.83 0.83 1.96 2.08 2.08 1.96 0.83

402 214 25 403 215 19 24 213 415 401 218 25 sd = standard deviation; med = median (mg/mL); min = minimum (mg/mL); max = maximum (mg/mL); n = number of observations (subset of F2 population which were phenotyped).

As can be seen in Table 3 above, an approximately 2.5-fold increase in IgA content of milk was seen in bovine homozygous for the C allele at the C903G polymorphism compared to those homozygous for the G allele. Likewise, an approximately 2.5-fold increase in IgA content of milk was seen in bovine homozygous for the A allele at the A3938G polymorphism, the A allele at the A4029G polymorphism, and the G allele at the G5183A polymorphism, compared to bovine homozygous for the G allele, the G allele, or the A allele, respectively. The main effect for genotype on IgA concentration was significant at P <0.001 by ANOVA for all genotypes). 6. PIGR polymorphisms have a significant effect on IgM concentration in milk

The effect of the PIGR polymorphisms on IgM concentration is shown below in Table 4. Table 4. IgM in milk

(mg/mL); n = number of observations (subset of F2 population which were phenotyped).

As can be seen in Table 4 above, an approximately 1.5-fold increase in IgM content of milk was seen in bovine homozygous for the C allele at the C903G polymorphism compared to those homozygous for the G allele. Likewise, an approximately 1.5-fold increase in IgA content of milk was seen in bovine homozygous for the A allele at the A3938G polymorphism, the A allele at the A4029G polymorphism, and the G allele at the G5183A polymorphism, compared to bovine homozygous for the G allele, the G allele, or the A allele, respectively. The main effect for genotype on IgM concentration was significant at P <0.01 for all markers except PIGR 4029 where PO.02..

7. PIGR polymorphisms have a significant effect on SC concentration in milk The effect of the PIGR polymorphisms on secretory component concentration is shown below in Table 5. Table 5: Secretory component in milk

sd = standard deviation; med = median (pmol/mL); min = minimum (pmol/mL); max = maximum (pmol/mL); n = number of observations (subset of F2 population which were phenotyped).

As can be seen in Table 5 above, an approximately 1.8-fold increase in SC content of milk was seen in bovine homozygous for the C allele at the C903G polymorphism compared to those homozygous for the G allele. Likewise, an approximately 1.8-fold increase in SC content of milk was seen in bovine homozygous for the A allele at the A3938G polymorphism, the A allele at the A4029G polymorphism, and the G allele at the G5183A polymorphism, compared to bovine homozygous for the G allele, the G allele, or the A allele, respectively. The main effect for genotype on SC concentration was significant at P < 0.05 for all markers Discussion

The results presented above show that polymorphisms in the PIGR gene, such as the PIGR polymorphisms described above, are associated with increased or decreased immunoglobulin production and secretion, and are useful in the identification of subjects with particular immunoglobulin uptake, production, regulation, or secretion phenotypes. The use of these polymorphisms provides a selection tool to breed agricultural animals with higher or lower colostrum or milk concentrations of immunoglobulins, including IgA, IgM and secretory component. Publications

Hannu Korhonen, P. Marnila and H. S. Gill, Milk immunoglobulins and complement factors. British

Journal of Nutrition (2000), 84, Suppl. 1, S75-S80. Wiburg, O.L.C, T.K. Uren, K. Simpfendorfer, F.-E. Johansen, P. Brandtzaeg and R.A. Strugnell,

Innate secretory antibodies protect against natural Salmonella typhimurium infection. Journal of

Experimental Medicine 2006, 203:21-26.

Reich DE et al; Linkage disequilibrium in the human genome, Nature 2001 , 41 1 : 199-204. Mullis et al., Eds. 1994 The Polymerase Chain Reaction, Birkhauser. Tatiana A. Tatusova, Thomas L. Madden, Blast 2 sequences - a new tool for comparing protein and nucleotide sequences, FEMS Microbiol Lett. 1999, 174:247-250. Needleman, S. B. and Wunsch, C. D., J. MoI. Biol. 1970, 48, 443-453. 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. Huang, X., On Global Sequence Alignment. Computer Applications in the Biosciences 1994, 10, 227-

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

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Ausubel et al., 1987, Current Protocols in Molecular Biology, Greene Publishing. Bolton and McCarthy, PNAS 1962, 84: 1390. Nielsen et al., Science 1991, Dec 6;254(5037): 1497-500. Giesen et al., Nucleic Acids Res. 1998, Nov l ;26(21 ):5004-6. Bowie et al., Science 1990, 247, 1306.

Current Protocols in Molecular Biology, John Wiley & Sons, NY ( 1989). Orita et al., PNAS 1989, 86:2766-2770. Devlin and Risch 1995; A comparison of linkage disequilibrium measures for fine-scale mapping,

Genomics 29: 31 1 -322. Johansen FE, Pekna M, Norderhaug IN, Haneberg B, Hietala MA, Krajci P, Betsholtz C, Brandtzaeg

P.; Absence of epithelial immunoglobulin A transport, with increased mucosal leakiness, in polymeric immunoglobulin receptor/secretory component-deficient mice. J Exp Med. 1999 Oct

4;190(7):915-22.

INDUSTRIAL APPLICATION

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

Claims

1. A method for identifying or selecting a mammalian subject with one or more desired immunoglobulin uptake, production, regulation, or secretion phenotypes, the method comprising determining a PIGR allelic profile of the subject, and identifying or selecting the subject on the basis of the determination.

2. The method as claimed in claim 1, wherein the one or more desired immunoglobulin uptake, production, regulation, or secretion phenotypes is increased milk or colostrum IgA content, increased milk or colostrum IgM content, increased milk or colostrum plgA content, increased milk or colostrum secretory component content, or any combination of two or more thereof.

3. The method as claimed in claim 1 or claim 2 wherein the PIGR allelic profile is determined by providing the results of an analysis of a sample from said subject for the presence or absence of one or more polymorphisms in the PIGR gene associated with one or more of:

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

(b) increased or decreased immunoglobulin uptake, or

(c) increased or decreased immunoglobulin secretion, or

4. The method of any one of claims 1 to 3 wherein the mammalian subject is bovine.

5. The method as claimed in claim 4, wherein the PIGR allelic profile is determined by determining the presence or absence of any one or more of the following: the G allele at the C903G polymorphism in the PIGR gene, or the C allele at the C903G polymorphism in the PIGR gene, or the G allele at the A3938G polymorphism in the PIGR gene, or the A allele at the A3938G polymorphism in the PIGR gene, or the G allele at the A4029G polymorphism in the PIGR gene, or the A allele at the A4029G polymorphism in the PIGR gene, or the G allele at the G5183 A polymorphism in the PIGR gene, or the A allele at the G5183A polymorphism in the PIGR gene.

6. The method as claimed in claim 5 wherein the PIGR allelic profile is determined by determining the presence of one or more polymorphisms in linkage disequilibrium with any one or more of the following: the G allele at the C903G polymorphism in the PIGR gene, or the C allele at the C903G polymorphism in the PIGR gene, or the G allele at the A3938G polymorphism in the PIGR gene, or the A allele at the A3938G polymorphism in the PIGR gene, or the G allele at the A4029G polymorphism in the PIGR gene, or the A allele at the A4029G polymorphism in the PIGR gene, or the G allele at the G5183A polymorphism in the PIGR gene, or the A allele at the G5183A polymorphism in the PIGR gene.

7. The method as claimed in any one of claims 1 to 6 wherein the PIGR allelic profile is determined by providing the results of an analysis of a sample from said subject for the expression or activity of a PIGR gene or gene product.

8. A method for identifying or selecting a bovine with respect to milk or colostrum immunoglobulin content, or with respect to capability of producing progeny that will have one or more desired milk or colostrum immunoglobulin content phenotypes, the method comprising providing data about a PIGR allelic profile of the bovine, and identifying or selecting the bovine on the basis of the data.

9. The method as claimed in claim 8, wherein the data about a PIGR allelic profile comprises a) data indicative of the presence or absence of one or more alleles at one or more polymorphisms which affect expression from the PIGR gene or the expression or activity of a PIGR gene product, or b) data indicative of the presence or absence of one or more alleles at one or more polymorphisms which are associated with increased or decreased expression from the PIGR gene or with increased or decreased expression or activity of a PIGR gene product, or c) data indicative of the presence or absence of one or more alleles at one or more polymorphisms in linkage disequilibrium with one or more of the polymorphisms of (a) or (b).

10. The method as claimed in claim 9, wherein the one or more polymorphisms is in the PIGR gene.

1 1. The method as claimed in claim 10, wherein the one or more polymorphisms is in the coding sequence of the PIGR gene.

12. The method as claimed in any one of claims 8 to 11, wherein the data about a PIGR allelic profile comprises data indicative of the presence or absence of one or more alleles at one or more polymorphisms selected from the group comprising the C903G polymorphism in the PIGR gene, the A3938G polymorphism in the PIGR gene, the A4029G polymorphism in the PIGR gene, the G5183 A polymorphism in the PIGR gene, or one or more polymorphisms which are in linkage disequilibrium with one or more polymorphisms selected from the group comprising the C903G polymorphism in the PIGR gene, the A3938G polymorphism in the PIGR gene, the A4029G polymorphism in the PIGR gene, or the G5183A polymorphism in the PIGR gene.

13. The method as claimed in any one of claims 9 to 12, wherein the provision of data comprises the step of amplifying at least a fragment of the bovine PIGR gene sequence to determine the presence or absence of the one or more alleles.

14. The method as claimed in claim 13, wherein the step of amplifying uses one or more primers selected from the group consisting of SEQ ID NOs: 4 to 51.

15. The method as claimed in any one of claims 9 to 12, wherein the presence or absence of the one or more alleles is determined by determining the expression or activity of a PIGR gene or gene product.

16. A probe or primer comprising a nucleotide sequence having about at least 12 contiguous bases of SEQ ID NO: 1 or SEQ ID NO: 2 or a complement thereof.

17. The probe or primer of claim 16 comprising a nucleotide sequence having about at least 12 contiguous bases of SEQ ID NO: 1 or SEQ ID NO: 2 and wherein said probe or primer comprises one or more of the following: a) a guanine at the position corresponding to the C903G polymorphism in the PIGR gene, or a nucleotide capable of hybridising to a nucleotide capable of hybridising to a guanine at the position corresponding to the C903G polymorphism in the PIGR gene, b) a guanine at the position corresponding to the A3938G polymorphism in the PIGR gene, or a nucleotide capable of hybridising to a nucleotide capable of hybridising to a guanine at the position corresponding to the A3938G polymorphism in the PIGR gene, c) a guanine at the position corresponding to the A4029G polymorphism in the PIGR gene, or a nucleotide capable of hybridising to a nucleotide capable of hybridising to a guanine at the position corresponding to the A4029G polymorphism in the PIGR gene, d) an adenosine at the position corresponding to the G5183 A polymorphism in the PIGR gene, or a nucleotide capable of hybridising to a nucleotide capable of hybridising to an adenosine at the position corresponding to the G5183 A polymorphism in the PIGR gene.

18. The probe or primer of claim 16 comprising a nucleotide sequence having about at least 12 contiguous bases of the complement of SEQ ID NO: 1 or the complement of SEQ ID NO: 2 and wherein said probe or primer comprises one or more of the following: a) a cytosine at the position corresponding to the C903G polymorphism in the PIGR gene, or a nucleotide capable of hybridising to a nucleotide capable of hybridising to a cytosine at the position corresponding to the C903G polymorphism in the PIGR gene, b) a cytosine at the position corresponding to the A3938G polymorphism in the PIGR gene, or a nucleotide capable of hybridising to a nucleotide capable of hybridising to a cytosine at the position corresponding to the A3938G polymorphism in the PIGR gene, c) a cytosine at the position corresponding to the A4029G polymorphism in the PIGR gene, or a nucleotide capable of hybridising to a nucleotide capable of hybridising to a cytosine at the position corresponding to the A4029G polymorphism in the PIGR gene, d) a thymine at the position corresponding to the G5183 A polymorphism in the PIGR gene, or comprising a nucleotide capable of hybridising to a nucleotide capable of hybridising to a thymine at the position corresponding to the

G5183 A polymorphism in the PIGR gene.

19. A probe or primer having about at least 12 contiguous bases of one of SEQ ID NO: 4 to 51 or a complement thereof.

20. The probe or primer as claimed in claim 16 comprising a nucleotide sequence having at least about 12 contiguous bases of SEQ ID NO: 1 or SEQ ID NO: 2 or a complement thereof wherein the about 12 contiguous bases comprise or are within about 1 to about 2000 nucleotides of the C903G polymorphism in the PIGR gene.

21. A probe or primer as claimed in claim 16 comprising a nucleotide sequence having at least about 12 contiguous bases of SEQ ID NO: 1 or SEQ ID NO: 2 or a complement thereof wherein the about 12 contiguous bases comprise or are within about 1 to about 2000 nucleotides of the A3938G polymorphism in the PIGR gene.

22. A probe or primer as claimed in claim 16 comprising a nucleotide sequence having at least about 12 contiguous bases of SEQ ID NO: 1 or SEQ ID NO: 2 or a complement thereof wherein the about 12 contiguous bases comprise or are within about 1 to about 2000 nucleotides of the A4029G polymorphism in the PIGR gene.

23. A probe or primer as claimed in claim 16 comprising a nucleotide sequence having at least about 12 contiguous bases of SEQ ID NO: 1 or SEQ ID NO: 2 or a complement thereof wherein the about 12 contiguous bases comprise or are within about 1 to about 2000 nucleotides of the G5183 A polymorphism in the PIGR gene.

24. A pair of primers comprising two primers as claimed in any one of claims 16 to 23.

25. A bovine identified or selected by the method of any one of claims 4 to 15.

26. The bovine as claimed in claim 25, wherein the bovine is a bull.

27. Collected semen produced by a bovine as claimed in claim 26.

28. The bovine as claimed in claim 25, wherein the bovine is a cow.

29. A method of selecting a herd of bovine, comprising selecting individuals by the method of any one of claims 4 to 15, and segregating and collecting the selected individuals to form the herd.

30. A herd of bovine selected by the method of claim 29.

31. A herd of bovine comprising two or more bovine, wherein the bovine are the progeny of one or more bovine selected by the method of any one of claims 4 to 15.

32. Collected or pooled milk produced by bovine as claimed in claim 28.

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

34. Collected or pooled milk as claimed in claim 32 or 33 having increased or decreased IgA content when compared to milk produced by a bovine having a PIGR gene comprising the nucleotide sequence of SEQ ID NO: 2 or a functional variant thereof or capable of expressing a functional equivalent of the PIGR gene product of SEQ ID NO:2 or SEQ ID NO:3.

35. Collected or pooled colostrum produced by bovine as claimed in claim 28.

36. Collected or pooled colostrum produced by a herd of bovine as claimed in claim 30.

37. Collected or pooled colostrum as claimed in claim 35 or 36 having increased or decreased immunoglobulin content when compared to milk produced by a bovine having a PIGR gene comprising the nucleotide sequence of SEQ ID NO: 2 or a functional variant thereof or capable of expressing a functional equivalent of the PIGR gene product of SEQ ID NO:2 or SEQ ID NO:3.

38. A dairy product made from the milk as claimed in any one of claims 32 to 34.

39. A composition comprising colostrum as claimed in claim 35 to 37.

40. A kit for genotyping a bovine with respect to one or more milk or colostrum immunoglobulin content phenotypes, comprising a probe or primer as defined in any one of claims 16 to 23 or a pair of primers as defined in claim 24.

41. An isolated, purified or recombinant nucleic acid molecule comprising at least 12 contiguous nucleotides of SEQ ID NO: 1 or SEQ ID NO:2.

42. The isolated, purified or recombinant nucleic acid molecule of claim 41 comprising a nucleotide sequence selected from

(a) at least 12 contiguous nucleotides of SEQ ID NO: 1 and comprising a guanine at the C903G polymorphism in the PIGR gene; or

(c) at least 12 contiguous nucleotides of SEQ ID NO: 1 and comprising a guanine at the A3938G polymorphism in the PIGR gene; or

(d) at least 12 contiguous nucleotides of SEQ ID NO:2 and comprising a guanine at the A3938G polymorphism; or

(e) at least 12 contiguous nucleotides of SEQ ID NO:1 and comprising a guanine at the A4029G polymorphism in the PIGR gene; or

(f) at least 12 contiguous nucleotides of SEQ ID NO:2 and comprising a guanine at the A4029G polymorphism; or

(g) at least 12 contiguous nucleotides of SEQ ID NO:1 and comprising an adenosine at the G5183A polymorphism in the PIGR gene; or (h) at least 12 contiguous nucleotides of SEQ ID NO:2 and comprising an adenosine at the G5183A polymorphism; or

(i) at least 12 contiguous nucleotides of a variant of SEQ ID NO:2; or Q) any one or more of SEQ ID NOs:4 - 51 ; or (k) a complement of any one of (a) to (j); or (1) a sequence of at least 12 contiguous nucleotides and capable of hybridising to the nucleotide sequence of any one of (a) to (k) under stringent conditions.

43. A vector comprising the nucleic acid of claim 41-42.

44. A method of determining genetic status of a bovine with respect to milk or colostrum immunoglobulin content, or with respect to capability of producing progeny that will have increased or decreased milk or colostrum immunoglobulin content, the method comprising determining milk or colostrum immunoglobulin content of the bovine, determining the PIGR allelic profile of the bovine, comparing the PIGR allelic profile of the bovine or the milk or colostrum immunoglobulin content of the bovine with that of a bovine having a known PIGR allelic profile; determining the genetic status of the bovine on the basis of the comparison.

45. A method for identifying or selecting a mammalian subject with respect to one or more desired immunoglobulin uptake, production, regulation, or secretion phenotypes, the method comprising providing the result of one or more genetic tests of a sample from the subject, and analysing the result for the presence or absence of one or more polymorphisms selected from the group comprising:

(d) one or more polymorphisms in the PIGR gene associated with production of milk, colostrum, blood, serum, mucosal secretions, or having one or more mucosal surfaces with increased or decreased immunoglobulin content, or (e) one or more polymorphisms in linkage disequilibrium with one or more polymorphisms in the PIGR gene associated with one or more of (a) to (d) above, wherein a result indicative of the presence or absence of one or more of said polymorphisms is indicative of a subjectwith one or more desired immunoglobulin uptake, production, regulation, or secretion phenotypes; and identifying or selecting the subject on the basis of the result.

46. A method for identifying or selecting a subject that would benefit from increased immunoglobulin intake, the method comprising providing the results of an analysis of a sample from said subject for the presence or absence of one or more polymorphisms in the PIGR gene associated with one or more of:

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

(b) increased or decreased immunoglobulin uptake, or

(c) increased or decreased immunoglobulin secretion, or