WO2007107862A2 - Method for the identification of bovine milk protein genetic polymorphisms - Google Patents

Abstract

A method to determine milk protein polymorphisms, e.g. in genes CSNlSl, CSN2, CSN3, LGB & Bov-A2, in cattle based on PCR amplification of genomic regions and subsequent ligation of probes complementary to these regions, wherein the primers and probes are designed to allow the simultaneous determination of polymorphisms at different gene regions. The method and reagents allow bovine milk protein genotyping in order to improve milk quality and to increase its production.

Description

METHOD FOR THE IDENTIFICATION OF BOVINE MILK PROTEIN GENETIC POLYMORPHISMS

The present invention relates to a method for the determination of milk protein polymorphisms in cattle based on PCR amplification of specific genomic regions and subsequent ligation of probes complementary to these regions, the probes are suitably designed to exactly determine the characteristic polymorphisms of the individual under study. More precisely, the invention provides primers and probes to be used in amplification and ligation reactions, and a support or microarray to visualize the genotypes corresponding to the examined samples. The PCR primers and the probes were weredesigned to allow the simultaneous determination of polymorphisms at different gene regions. The method and reagents of the invention provide an accurate, inexpensive and high-throughput tool for bovine typing useful for improving milk quality and increasing its production. Background of the invention Milk production has considerable research interest because of its nutritional properties associated to fresh consumption, as well as for dairy production. Furthermore, the "interprofessional agreements for a differentiated milk payment based on quality" have become effective with the enforcement of Law no. 88/1988. According to such agreements, the breeder is induced to improve the quality of milk produced based on precise and measurable parameters. The casein protein fraction has a central role for a qualitative evaluation of milk, both for nutritional and dairy transformation purposes.

Milk proteins are a difficult research subject because of their complexity.New analysis methods and advancements in the fields of molecular biology, genetics, and biochemistry in the latest years have given new impulse to research, and remarkably widened the knowledge in this field. The simultaneous presence of quali-quantitative differences at loci of calcium- sensitive caseins required , occasionally, to perform a molecular approach to milk protein investigation. In some cases, it is considerably difficult to trackprotein phenotypes back to genotypes of the tested animals. Furthermore, molecular technologies allow the typization of animals in the absence of a gene product, independent of age and sex. This means that on the one hand it is nowadays possible to recognize with certainty the gene mutations responsible for the differential synthesis of a given protein, on the other hand to identify an increasing number of polymorphisms. State of the art and technical problem

Caseins amount to 78-80% of total milk proteins (Renner, 1982), and together with seroproteins represent more than 95% of milk proteins.

Caseins (CN), α_Si-CN, α_S2-CN, /3-CN₅ and K-CN₅ are coded by four autosomic genes (CSNlSl, CSNl S2, CSN2, and CSN3, respectively), closely linked and clustered in a 250 Kb DNA stretch (Figure 1). In cattle, these genes were mapped to chromosome 6 (Threadgill & Womack, 1990; Hayes et al., 1993).

Another important milk protein gene codes for /3-lactoglobulin (LGB). This protein, together with α-lactoaϊbumin (α-LA), constituites the main fraction of milk seroproteins. LGB gene was mapped to chromosome 11 (Vaiman et al., 1994), outside the casein cluster. Polymorphisms at this locus also determine considerable effects on milk characteristics and cheese-making properties (Creamer and Harris, 1997; Lunden et al., 1997; Di Stasio and Mariani, 2000). A recent review of bovine milk protein nomenclature (Farrell et al., 2004) indicates 8 α_srCN, 4 α_S2-CN, 12 β-CN, 11 K-CN, and 11 /3-LG variants.

Furthermore, several studies of non-coding regions of milk protein genes highlighted a moderate polymorphism, in particular in 5' flanking regions, with important effects on milk characteristics and composition (Prinzenberg et al., 2003; Ibeagha-Awemu et al., 2005). As anexample, Damiani and coll. (2000b) described a polymorphism within the SINE (Short Interspersed Nucleotide Element) element Bov-A2 in the second intron of CSN3 gene. Statistically significant associations with somemilk-production characteristics were found (Damiani et al., 2000a) also for such polymorphism. More recently, polymorphisms of CSNlSl gene promoter region have been described and foundsignificantly associated with milk protein content (Prinzenberg et al., 2003). Thus, casein cluster polymorphisms should be considered as a single complex which determines diverse effects depending on all the mutations contained. This view is summarized in the concept of casein haplotype.

Unfortunately, the high degree of polymorphism and structural complexity of milk protein genes makes a complete analysis of an individual's milk protein variation difficult. At the moment, for each of these polymorphic regions different tests exist to directly analyze DNA by PCR . However, the use of such methods takes a great deal of time and money, so rarely all genes involved are investigated. Furthermore, restricting the study to these analyses, the effect of a given gene allele is often estimated without an adequate correction for the allelic effect of other flanking polymorphisms.

In addition, it is objectively difficult to compare the results obtained with different races as the haplotype pattern is breed-specific (Boettcher et al., 2004; Caroli et al., 2004), and it is therefore necessary to expand research to a sufficiently high number of animals for each haplotype to provide information on its actual effect on productions.

The microarray technique approach to bovine milk protein genotyping is a suitable research tool to conduct investigations in this field. It enables to include a high number of polymorphisms at milk protein genes, and other polymorphisms at non-coding sequences of milk protein cluster, in a single analysis with clear applicative consequences. Indeed, microarrays allow breeders to know much of milk proteins' genetic variation, and choose their own animals for selection accounting for such variation.

Description of the invention

According to a first aspect, the invention relates to a method for the simultaneous determination of polymorphisms at genes or gene elements which control the synthesis of milk proteins in cattle, these polymorphisms being selected from:

Gene Nucleotides Protein Ammo acid involved involved

CSNlSl AJG α_sl-casein GIu₁₉₂ZGIy₁₉₂

CSN2 GZA β -casein Glu₃₇/Lys₃₇

A/C cc Metp₃ZLeu₉₃

C/G Ser₁₂₂ZArgi₂₂

CSNS G/A κ-casein Arg₉₇/His₉₇

C/T ThT₁₃₅ZIIe₁₃₅

C/T ThT₁₃₆ZIIe₁₃₆

• A/C Asp₁₄₈ZAla₁₄₈

A/G Ser₁₅₅ZGly₁₅₅

A/G AIa₁₆₈ZAIa₁₆₈

LGB TGA/CGG β-lactoglobulin ASp₆₄ZGIy₆₄

G/T VaI₁₀₅ZVaI₁₀₅

GGT/ GGC VaI₁₁₈ZAIa₁₁₈ or TGC Gene Nucleotides Position Reference element involved sequence

Bov-A2 A/G 107 SEQ ID element NO: 88

CSNlSl A/C 289 SEQ ID promoter NO: 89

TT Del. 395-396

GTTT Del. 397-400

A/G 444

G/C 484 wherein the same polymorphisms can be referred to the deposited gene sequences, as follows:

- CSNlSl gene, polymorphism at nucleotide position 17807 of sequence deposited with GenBank Accession No. X59856; - CSN2 gene, polymorphisms at nucleotide positions 6690, 8101, 8178,

8219, and 8267 of sequence deposited with GenBank Accession No. X14711;

- CSN3 gene, polymorphisms at nucleotide positions 12951, 13065, 13068, 13104, 13124, and 13165 of sequence deposited with GenBank Accession No. AY380228; - LGB gene, polymorphisms at nucleotide positions 3982 and 5174 of sequence deposited with GenBank Accession No. X14710; or at nucleotide positions 5223 and 5261 of sequence deposited with GenBank Accession No. NM_173929;

- CSN3 gene, SINE (Short Interspersed Nucleotide Element) element Bov-A2, polymorphism at nucleotide position 8371 of sequence deposited with GenBank Accession No. AY380228;

- CSNlSl gene promoter region, polymorphisms at nucleotide positions 289, 395-396, 397-400, 444, and 484 of sequence deposited with GenBank Accession No. AF549502; this method comprising:

1) amplification, in the same reaction medium, of gene fragments containing the above cited polymorphisms by multiplex PCR using primer pairs selected from:

2) addition of a pair of "discriminating" probes having sequences complementary to the regions containing each of the two polymorphic variants, respectively , and a "common" probe for each pair of discriminating probes, wherein this common probe has a sequence complementary to the region contiguous to the first one in the same amplified fragment, such discriminating and common probes being selected from:

or, in case the searched polymorphism is a deletion in CSNlSl gene promoter, addition of a pair of discriminating probes and a pair of common probes selected from:

wherein these discriminating probes are labelled at their 5' ends with a different fluorophore for each of the two discriminating probes necessary to identify a given polymorphism, while these common probes are bound at their 3 ' ends to a different random nucleotide sequence (ZipCode) for each polymorphism examined; 3) ligation of discriminating and common probes by adding a ligase enzyme to the reaction media followed by incubation;

4) hybridization of the probes obtained in step 3 on a support on the surface of which polynucleotide sequences complementary to the random ZipCode sequences used in steps 2 and 3 have been previously adsorbed or bound, directly or by a spacer sequence, in separate areas ("spots"), so that polynucleotide molecules complementary to a unique ZipCode sequence are bound in each separate area;

5) sample analysis by reading the fluorescence emitted from the fluorescent label bound to the 5' end of the discriminating probe.

The method herein described can be applied to a simultaneous analysis of different polymorphisms, preferably at least 14, more preferably at least20, even more preferably all the 22 polymorphisms above described.

At their 5' ends, the "discriminating" probes bear a fluorophore, which is preferably Cyanine 3 and Cyanine 5, or is selected from 5-FAM, Alexa 488, Alexa 532, Alexa 546, Alexa 405, Alexa 568, Alexa 594, Alexa 647, Alexa 660, Allophycocyanin (APC), BODIPY 530, BODIPY 558, BODIPY 564, BODIPY 630, BODIPY TMR, Calcein, Calcium Crimson, Calcium Green- 1, Calcium Orange, Cyanine 2, Cyanine 3.5, Cyanine 5.5, DiD DilC(5), DiI, dsRed, Ethidium Bromide, FAM, FITC, FluorX, GFP, GFP Red Shifted (rsGFP), JOE, JOE-514, Magnesium Green, Magnesium Orange, Nile Red, Oregon Green 488, Oregon Green 500, PBXL-I, PBXL-3, Phycoerythrin R&B, Pyronin Y, Red Reflect, Rhodamine 110, Rhodamine 123, Rhodamine B, Rhodamine Green, Rhodamine Phalloidin, Rhodamine Red, Ribo Green, ROX, R-Phycocyanin (R-PC), R-Phycocerithrin (R-PE), SYBR Green, Sypro Ruby, TAMRA, Texas Red, Thiadicarbocyanine, TO-PRO-3, TO-PRO-I, TOTO-3, TOTO-I, TRITC, YO-PRO-I, YOYO-I, while the common probes are bound to a random nucleotide sequence (ZipCode) at their 3' ends. Once discriminating and common probes have bound to one another by the action of a ligase, the ligation product binds to a support by means of the ZipCode sequences. These sequences are expressly designed not to have any naturally occurring complementary sequence, and can be selected from those available in the literature (Chen et al., 2000; Consolandi et al., 2004).

In one preferred embodiment, hybridization of probes (step 4 of the method) occurs on a microarray-type support made of a slide whose surface is divided into discrete areas on which ZipCode sequences are attached according to a prefixed matrix scheme, so that a specific area corresponds to a single sequence.

In another aspect, the invention refers to a combination of at least 4, preferably 8 pairs of (forward and reverse) primers to simultaneously amplificate, by a multiplex PCR reaction, gene fragments containing the above indicated polymorphisms, these primers being selected from the group comprising:

Forward primer Reverse primer SEQ ID NO: SEQ ID NO:

1 2

3 4

5 6

7 8

9 10

11 12

13 14

15 16

In a further aspect, the invention provides a combination of probes to be used in a LDR/UA (Ligation Detection Reaction/Universal Array) reaction, such probes being capable of base pairing to a gene fragment containing an above-indicated polymorphism, wherein this combination, specific for a given polymorphism, consists of two discriminating probes and at least one common probe, and is selected from the group comprising:

The combination of multiplex PCR reactions, LDR (Ligase Detection

Reaction), and (micro)array analyses is the method of choice to identify milk protein polymorphisms according to the present invention. Nevertheless, both the primers and oligonucleotide probes having sequences SEQ ID No. 1-16 and 17-87, respectively, can be used in other techniques such as hybridization,

PCR, RT-PCR, for genotyping, minisequencing, and high-throughput systems based on magnetic microbeads (Yingyongnarongkul et al., 2003). In a further aspect the invention refers to a kit for carrying out the method disclosed herein, this kit containing different combinations of primers and probes, and optionally Taq polymerase and ligase enzymes.

According to a further aspect, the invention relates to a support consisting of a slide whose surface is divided into discrete areas on which polynucleotide molecules complementary to ZipCode sequences are attached according to a prefixed matrix scheme, so that a single ZipCode sequence corresponds to a given area. In one preferred embodiment, this support is in a microarray format.

Furthermore, the invention relates to the use of primers and probes in different combinations to detect insertion or deletion of single nucleotide polymorphisms, as indicated above. Polymorphism determination according to the present invention is particularly useful for bovine milk protein genotyping.

Description of the figures

Figure 1: Genomic organization of bovine casein locus (Martin et al., 2002). Figure 2: Schematic representation of a ligation reaction.

Figure 3: Schematic representation of a universal array hybridization. Figure 4: Example of the DNA amplification from 12 animals by the multiplex PCR herein described. Concentration of each DNA fragment was calculated with Bioanalyzer 2100 (Agilent Technologies). Figure 5: Universal array deposition scheme. Among the ZipCodes placed on the slide, 22 correspond to the considered SNPs, as described in Table 4. ZipCodes 63 and 66 correspond to ligation and hybridization controls, respectively, while ZipCode 35 corresponds to the internal reference for fluorescent signal normalization processes within genotype calculations. Figure 6: Example of some results obtained by using probes which are the subject of the instant invention with the LDR/universal array method. Images were obtained by overlapping fluorescent signal emitted from Cy3 with that from Cy 5. Spots indicate the analyzed SNPs and controls. Depending on spot color, it is possible to determine whether the animal under study is homozygous for one of the two alleles or heterozygous at each of the investigated polymorphisms, being it consequently possible to obtain its genotype. Figure 7: Determination of the genotype obtained by using probes which are the subject of the instant invention with the LDR/universal array method. The image shows the assignment of each of 22 SNPs to one of three genotypic classes (homozygous for one allele or the other, or heterozygous) for 80 analyzed samples. Detailed description of the invention

The invention provides an effective, rapid, and high-throughput method for the simultaneous detection of single nucleotide polymorphisms (SNPs), as well as insertion or deletion polymorphisms in genes involved in milk protein synthesis. The method includes an initial step wherein regions of interest in target genes are simultaneously amplified, a subsequent step wherein labeled probes specific for each polymorphism and complementary to these regions are used, and a final step wherein polymorphisms are visualized on suitable supports or microarrays. Polymorphism-bearing genes that can be analyzed according to the invention are the following: CSNlSl, CSNl S2, CSN2, and CSN3, coding for α_sl-CN, CIs₂-CN, and /3-CN caseins, respectively, and K-CN; the β-lactoglobulin (LGB) gene, mapped to chromosome 11 (Vaiman et al., 1994); the non-coding regions of milk protein genes, in particular within SINE (Short Interspersed Nucleotide Element) element Bov-A2 in the second intron of CSN3 gene, and in the sequence of CSNlSl gene promoter. The list of alleles traceable to milk protein loci according to the method of the invention is set forth in the following Table 1 :

Multiplex PCR Reaction

For the amplification reaction, DNA fragments containing polymorphisms and having such a size that they can be distinguished on agarose gels, enabling at the same time to obtain satisfactory amplification efficiencies, were selected. More single or multiple nucleotide polymorphisms are located in each selected fragment, as shown in the following Table 2:

Locus Polymorphism Nucleotides Alleles Reference Amino acid position involved sequence^Λ involved

CSNlSJ 17807 A B X59856 GlUi92

G C GIy₁₉₂

CSN2 6690 G A1-A2-A3-B-I X14711 GIu₃₇

A C Lys₃₇

8101 C A2-A3-I PrO₆₇

A Al-B-C His₆7

8178 A A1-A2-A3-B- Met93

C

C I Leu₉₃

8219 C A1-A2-B-C-I HiS₁₀₆

A A3 Glnioδ

8267 C A1-A2-A3-C-I Ser₁₂₂

G B Arg₁₂₂

CSN3 12951 G A-B-E-H AY380228 Arg₉₇

A C HiS₉₇ 13065 C A-B-C-E Thr_]35 T H Hens 13068 C A-C-E-H ThT₁₃₆ T B He₁₃₁; 13104 A A-C-E-H Asp_14S C B AIa₁₄₈

(continued) 13124 A A-B-C-H Ser₁₅₅ G E Glyiss 13165 A A-C-E-H AIa₁₆₈

CGG B GIy₆₄

5174 C A Asn₈₈

T B Asn₈₈

T B VaI₁₀₅

5261 GGT A VaI₁₁₈

GGC or TGC B AIa₁₁₈

Bov-A2 8371 A A AY380228 -

Element

G B -

CSNlSl 289 A 2-3-4 AF549502 - promoter

C 1 -

Del*395-396 TT 1 -

2-3-4 -

Del*397-400 GTTT 3-4 -

1-2 -

444 A 2-3-4 -

G 1 -

484 G 1-2-3 -

C 4 -

Fragments of polymorphism-bearing genes can be amplified in a single tube ("one pot") by a multiplex PCR using the primer pairs set forth in the following Table 3 :

Name Sequence 5' -> 3' Gene involved Tm Amplicon CQ size

CSNlSl TGGATGCCTATCCATCTGGT CSNlSl exon 60.30 333 bp exl7F 17

CSNlSl CACTGCTCCACATGTTCCTG CSNlSl exon 60.31 exl7R 17

CSN2 CATCAATAAGGTAAAACCCCTCA CSN2 exon 6 59.28 275 bp ex6F

CSN2 TTTGTCAAAGTTTTTATTTCTTGCAC CSN2 exon 6 59.98 ex6R

CSN2 TCCAGGATGAACTCCAGGAT CSN2 exon 7 59.46 545 bp ex7F

CSN2 CATCAGAAGTTAAACAGGCACAG CSN2 exon 7 58.96 ex7R

(continued) CSN3 TAGGTCACCTGCCCAAATTC CSN3 exon 4 59.93 462 bp ex4F

CSN3 ex ATTAGCCCATTTCGCCTTCT CSN3 exon 4 60.06 4R

LGB ex3F TGGCTCCATCTGACTTCTCC LGB exon 3 60.35 404 bp

LGB CACGGCAGTGTCTTCATCAC LGB exon 3 60.32 ex3R

LGB ex4F GAGTTGGGCTTCCAGAGTGA LGB exon 4 60.39 494 bp

LGB CCGGTATATGACCACCCTCT LGB exon 4 58.74 ex4R

Bov-A2F AGCTACAAGCCCCATGGATA CWi intron 2 59.55 423 bp Bov-A2R TGCTGCTGCTGCTAAGTCAC CSN5 intron 2 60.50

CSNlSl TTTGCAGGAAAAAGATTAGACCA CSNlSl 60.11 518 bp PromF promoter

CSNlSl GGTGATGGCAGACTTTTGCT CSNlSl 60.26 PromR promoter

Multiple amplification of these fragments is performed in a 50 μL solution containing 15 μL (100 to 150 ng) of DΝA under study, IX AccuPrime™ PCR Buffer II, 1 μL of AccuPrime™ Taq DΝA Polymerase enzyme (Invitrogen, Milan, Italy), 10 pmol of primers for CSNlSl gene exon 17, LGB gene exon 3, Bov-A2 element, and CSNlSl promoter, and 16 pmol of primers for CSN2 gene exons 6 and 7, CSN3 gene exon 4, and LGB gene exon 4.

Amplification conditions are as follows: a first denaturation step at

94° C for 5', followed by 35 cycles consisting of a denaturation step at 94° C for 30", an annealing step at 56°C for 30", and an extension step at 68⁰C for 1 '. The final extension was carried out at a temperature of 68°C for T using a

PTC-0200 DΝA (MJ Research Inc., Waltham, MA) thermal cycler.

Then, PCR products are purified by using a GFX PCR DΝA purification kit (Amersham Pharmacia Biotech Inc, Piscataway-ΝJ) according to manufacturer's instructions. They are eluted in 40 μL of bidistilled water, and quantified using a Bioanalyzer 2100 and a DΝA 7500 kit (Agilent

Technologies, Milan, Italy).

As it can be seen from Figure 4, multiplex PCR amplification gives a DΝA concentration per amplified fragment which varies depending on the sample, but is anyway in the range between 5 and 50 femtomoles per fragment. Therefore, it is sufficient for subsequent analyses.

Oligonucleotide Probes to Detect Polymorphisms at Milk Protein Loci The probes used in the next step (LDR ligation or Ligation Detection Reaction) were directly designed on gene sequences by moving upstream and downstream of each polymorphic site, so that all the probes shared similar thermodynamical characteristics, and temperature values for hybridization to template DNA sequences which were between 67°C and 69⁰C.

Two discriminating probes (allele-specific probes designed so that they have their 3' ends on the nucleotide responsible for the polymorphism) and at least one common probe were designed for each polymorphism. The two discriminating probes were labeled at their 5' ends with two different fluorophores (Cy3 or Cy5) while the common probes have a phosphate group at this position. The sequences used as a reference to design probes are available from

GenBank under the following Accession Nos.: X59856 for CSNlSl, M94327 for CSN1S2, X14711 for CSN2, AY380228 for CSN3, AF549499-AF549500- AF549501-AF549502 for the four variants found in the promoter region of CSNlSl, and X14908 for Bov-A2. A pair of discriminating probes (GS¹Ni-UNI) hybridizing with CSN2 gene sequence, so that this region is heterozygous in all analyzed samples, have also been designed. This pair of probes is used as an internal reference ("IR") for fluorescent signal normalization within genotype calculations.

Probe sequences are set forth in Table 4. When a probe sequence had a degenerate base, this base was replaced with Inosine during oligonucleotide synthesis. Modificat Tm

SNP Probe sequences ion (°C)

CSNlSl ^'-17807-A Cy3 TCTGACATCCCTAATCCCATTGGCTCTGAGAACAGTGA 67

CSNlSl-mO7-G Cy5 TCTGACATCCCTAATCCCATTGGCTCTGAGAACAGTGG 68

AAAGACTACTATGCCACTGTGGTGGTAAGTTCATTTAA

Commonprobe-17807 Ziρ20 A 67

TGACTGCATATTG

CSN2-6690-G Cy3 TTTTAACCAGAAAATTGAGAAGTTTCAGAGTGAGG 69

CSN2-6690-A Cy5 TTTTAACCAGAAAATTGAGAAGTTTCAGAGTGAGA 68

Common probe-6690 Zip38 AACAGCAGCAAACAGAGGTAATTTGTTCACTA 68

CW2-8101-C Cy3 AGTCTCTAGTCTATCCCTTCCCTGGICCCATCCC 67

GW2-8101-A Cy5 AGTCTCTAGTCTATCCCTTCCCTGGICCCATCCA 67

Common ρrobe-8101 Zip40 TAACAGCCTCCCACAAAACATCCCTCCTCTTACTC 67

CS7V2-8178-A Cy3 GTGGTGGTGCCGCCTTTCCTTCAGCCTGAAGTAA 68

CW2-8178-C Cy5 GTGGTGGTGCCGCCTTTCCTTCAGCCTGAAGTAC 68

Common probe-8178 Zip41 TGGGAGTCTCCAAAGTGAAGGAGGCTATGGCTCCTA 68

GS7V2-8219-C Cy3 GTCTCCAAAGTGAAGGAGGCTATGGCTCCTAAGCAC 68

CW2-8219-A Cy5 GTCTCCAAAGTGAAGGAGGCTATGGCTCCTAAGCAA 68

Common ρrobe-8219 Zip42 AAIGAAATGCCCTTCCCTAAATATCCAGTTGAGCCCTTT 67

GW2-8267-C Cy3 CCTTCCCTAAATATCCAGTTGAGCCCTTTACTGAAAGC 67

GW2-8267-G Cy5 CCTTCCCΓAAATATCCAGTTGAGCCCTTTACTGAAAGG 67

Common probe-8267 Zip44 CAGAGCCTGACTCTCACTGATGTTGAAAATCTGCACC 67

GW5-12951-G Cy3 CCCAGCCAACTACCATGGCACG 68

GWJ-12951-A Cy5 GCCCAGCCAACTACCATGGCACA 68

Common probe-12951 Zipl TCACCCACACCCACATTTATCATTTATGGC 69

GW3-13065-C Cy3 TAGTGGTGAGCCTACAAGTACACCTAC 68

GW5-13065-T Cy5 CTAGTGGTGAGCCTACAAGTACACCTAT 68

Common ρrobe-13065 Zip2 CACCGAAGCAGTAGAGAGCACTGT 67

GSM-13068-C Cy3 GTGAGCCTACAAGTACACCTACCAC 67

CS/V3-13068-T Cy5 TGGTGAGCCTACAAGTACACCTACCAT 68

Common probe-13068 Zip3 CGAAGCAGTAGAGAGCACTGTAGCTA 68

GW3-13104-A Cy3 GTAGAGAGCACTGTAGCTACTCTAGAAGA 69

CW5-13104-C Cy5 GAGAGCACTGTAGCTACTCTAGAAGC 68

Common probe-13104 Zip4 TTCTCCAGAAGTTATTGAGAGCCCACC 68

GW5-13124-A Cy3 GCTCTAGAAGATTCTCCAGAAGTTATTGAGA 68

CW3-13124-G Cy5 CTCTAGAAGATTCTCCAGAAGTTATTGAGG 68

Common probe- 13124 Zip5 GCCCACCTGAGATCAACACAGTCC 69

GW3-13165-A Cv3 ATCAACACAGTCCAAGTTACTTCAACTGCA 68

(continued) GWJ-13165-G Cy5 CAACACAGTCCAAGTTACTTCAACTGCG 68 Common probe- 13165 Zip6 GTCTAAAAACTCTAAGGAGACATCAAAGAAGAC 69

Modificat Tm

SNP ion Probe sequences (°C)

LGβ-3982-TGA Cy3 CGTGGCTCAGAAAGCAGCTGTCTTTCAGGGAGAATGA 68

Z,G£-3982-CGG Cy5 TGGCTCAGAAAGCAGCTGTCTTTCAGGGAGAACGG 68 TGAGTGTGCTCAGAAGAAGATCATTGCAGAAAAAACCA

Common probe-3982 Zip^' AGATCCC

LGB-5174-C Cy3 TCACTTTCCTCCCGTCTTGATCTCTTCCAGCCTTGAAC 68

LGBSIlA-T Cy5 TCACTTTCCTCCCGTCTTGATCTCTTCCAGCCTTGAAT 67

GAGAACAAAGTCCTTGTGCTGGACACCGACTACAAAAA ,_e

Common probe-5174 Zip 16

GTACC ⁶⁸

LGB-5223-T Cy3 CCTTGTGCTGGACACCGACTACAAAAAGTACCTGCTCT 68

LGB-5223-G Cy5 CTTGTGCTGGACACCGACTACAAAAAGTACCTGCTCG 68

Common ρrobe-5223 Zip 18 TCTGCATGGAGAACAGTGCTGAGCCCGAGCAAAG 68

Z6S-5261-GGT Cy3 AGAACAGTGCTGAGCCCGAGCAAAGCCTGGT 67

Z,G5-5261-KGC Cy5 AGAACAGTGCTGAGCCCGAGCAAAGCCTKGC 68

Common probe-5261 Zipl9 CTGCCAGTGCCTGGGTGGGTGCCAAC 67

Bov-A2-8371-A Cy3 TAGACTGCAGTCCATGGGGTCACTAA 68

BOV-A2-8371-G Cy5 GACTGCAGTCCATGGGGTCACTAG 69

Common probe-8371 Zipl4 GAGTCAGACACGACTGTGTAACTTTCAAG 69

PtomCSNlSl-289-A Cy3 TTGAAGACCCCATTTTGTCCCAAGAATTTCA 68 PxomCSNlSl-289-C Cy5 GAAGACCCCATTTTGTCCCAAGAATTTCC 69 Common probe-289 Zip7 TTTACAGGTATTGAATTTTTCAAAGGTTACAAAGGAAA 68

PromGS'M^-Del-395-

Cy5 CATAAATCTAGGGTTTTGTTGGGGTTTTTTG 67 396

PromCSM>S7-395-396 Cy3 CCATAAATCTAGGGTTTTGTTGG^GTTTTTT 67 Common probe-Del- 395 Zip8 TTTGTTAATTTAGAACAATGCCATTCCATTTCCT 61

Common ρrobe-395 Zip8 TTGTTAATTTAGAACAATGCCATTCCATTTCCT 61

VromCSNlSl-Oel-391- _ ,

400 °y⁵ CTAGGGTTTTGTTGGGGTTTTTTITGTTTG 67

ΕmmCSNlSl-391-400 Cy3 ATAAATCTAGGGTTTTGTTGGGGTTTTTTTTGTT 67

Common probe-Del- „. ~

TTAATTTAGAACAATGCCATTCCATTTCCTGTATA 67

397 ^Z*⁹

Common ρrobe-397 Zip9 AATTTAGAACAATGCCATTCCATTTCCTGTATAAT 67

PromGS7W6'7-444-A Cy3 CAATGCCATTCCATTTCCTGTATAATGAGTCA 69

PromGW;S7-444-G Cy5 ATGCCATTCCATTTCCTGTATAATGAGTCG 68

Common probe-444 Zip 10 CITCΓTΓGTTGTAAACTCTCCTTAGAATTTCTTG 68

PromC5M,S';-484-G Cy3 GTAAACTCTCCTTAGAATTTCTTGGGAGAG 68

PromGS'M5i-484-C Cy5 GTAAACTCTCCTTAGAATTTCTTGGGAGAC 68

Common probe-484 Zipl2 GAACTGAACAGAACATTGATTTCCTATGTGAG 69 GHV2-UNI-Cy3 Cy3 GAAAGCAGTGCCCTATCCCCAGAGAGATATGCCCA 68

GSM-UM-CyS Cy5 GAAAGCAGTGCCCTATCCCCAGAGAGATATGCCCA 68

Common probe-UNI Zip35 TTCAGGCCTTTCTGCTGTACCAGGAGCCTGTACTC 68

Table 4: List of discriminating and common probes, and their melting temperature (T_m) for each considered SNP. The fluorophore used to label each discriminating probe, as well as the cZipCode (Zip) attached to each common probe are indicated. Specificity of probe sequences was verified in GenBank database

(www.ncbi.n1m.nih.gov) by using BLAST software. In this way, absence of homology with other sequences was ascertained.

A ZipCode sequence selected from among those available in the literature (Chen et al., 2000; Consolandi et al., 2004) was randomly assigned to every polymorphic site considered, and a sequence complementary to each ZipCode (cZipCode) was synthetized at the 3' end of the common probe corresponding to the specific polymorphic site. The possibility that common probe sequences formed any secondary structures with cZipCode sequences was tested using dedicated analysis software available on-line (www.bioin.fo.rmati.cs.vg/index.shtml).

Finally, a pair of discriminating probes annealing to a synthetic template of 50 bp in length (5'-

AGCCGCGAACACCACGATCGACCGGCGCGCGCAGCTGCAGCTTGCT CATG-3'), which is used as the ligation reaction control, were designed. A sequence was also synthetized which is labeled both with Cy3 and

Cy5 fluorophores. This sequence is complementary to ZipCode 66 sequence

(5 '-GTTACCGCTGGTGCTGCCGCCGGTA-S '), and is used as the hybridization reaction control.

All oligonucleotide probes were synthetized by Thermo-Hybaid GmbH (Germany). Probes were purified by HPLC and checked by MALDI-MS. LDR (Ligation Detection Reaction)

The ligation reaction is performed in a 20 μL final volume of a solution containing 20 niM Tris-HCl (pH 7.5), 20 niM KCl, 10 mM MgCl₂, 0.1% NP₄O, 0.01 mM ATP, 1 mM DTT, 2 picomoles of each discriminating probe, 2 picomoles of each common probe, 5-50 femtomoles of PCR product, 2 μL of 1OX Pfu ligase Buffer (Stratagene, La Jolla, California), 1 μL of Pfu DNA ligase (4U/μL), (Stratagene, La Jolla, California), and bidistilled water. The ligation reaction is performed in a GeneAmp 2700 (Applied Biosystem, Applera Italy, Monza, Italy) thermal cycler, and comprises the following steps: a first denaturation step at 94⁰C for 2', followed by 30 cycles consisting of a 30" denaturation at 94⁰C, a 4' ligation at 65⁰C, and a 10' final step at

4°C. Then, ligation products are stored at -2O⁰C until hybridization on a slide.

Preparation of Micro array Support according to the "Universal Array "

Procedure Microarray "Universal Array" slides (Gerry et al., 1999) are prepared using "CodeLink™ Activated" slides (Amersham Biosciences Europe GmbH, Milan, Italy), and a MicroGrid II Compact pin contact arrayer (BioRobotics Ltd, Cambridge, UK). ZipCode sequences are synthetized with an amino group at their 5' ends. Furthermore, they have an additional poly(dA)₁₀ tail at the same end, which serves as a spacer so that the ZipCode sequences bound to the activated surface of the slide are rendered sterically more accessible to hybridization. ZipCode probes are resuspended in 100 mM phosphate buffer (pH 8.5) at a final concentration of 50 μM, and deposited on the slides in four repetitions. Following ZipCode deposition, the slides are processed to inactivate all free reactive sites according to manufacturer's instructions.

As shown in Figure 5, each microarray slide consists of eight equivalent subarrays, which permits to simultaneously test eight different samples. Each subarray is formed by a matrix where four repetitions of the same ZipCode are present. Each ZipCode is indicated by a number and is selected from those described by Chen et al., 2000.

To ascertain the quality of microarray slides after ZipCode deposition, a

5X SSC solution containing 1 pmol/μL poli(dT)io oligonucleotides labeled at their 5' ends with Cy3, and 0.1 mg/ml salmon sperm DNA (Sigma- Aldrich), is hybridized on one of the microarray slides produced in the same series. The hybridization reaction is performed at room temperature for 30 minutes.

Following incubation, the slides are washed for 5 minutes in a 2X SSC solution containing 0.1% SDS. Fluorescent signals are acquired by reading with ScanArray^® Lite laser and ScanArray 3.1 software (Perkin Elmer Life

Sciences, Boston, MA).

Hybridization of Ligation Products on a Slide

The hybridization solution consists of 20 μL of the above-performed ligation mix, SSC 5X buffer, 0.1 mg/ml salmon sperm DNA (Sigma- Aldrich), 100 picomoles each of the two probes used to check the hybridization reaction, which are labeled with Cy3 and Cy5 respectively, and sterile bidistilled water in a final volume of 65 μL. The solution is denatured at 94°C for 2 minutes and immediatley cooled on ice. At this point it is ready to be appied to a slide by a suitable hybridization chamber (Press-to-seal Silicon isolator, eight wells, Schleicher&Schuell, USA), so that each slide is used to analyze 8 different samples in parallel, as shown in Figure 5.

The hybridization reaction is performed in the dark, at 65°C for approximately two hours in a hybridization oven and at a controlled temperature. The slide is then washed in a IX SSC-0.1% SDS solution prewarmed for 15 minutes at 65°C.

Signal Detection and Statistical Analysis of Data

The fluorescent signal is detected at a resolution of 5 μm. with a ScanArray^® Lite microarray scanner and ScanArray 3.1 dedicated software (Perkin Elmer Life Sciences, Boston, USA) for acquisition of images by using the laser for Cy3 fluorophore (λ_eX 543 nm/λ_em 570 nm) and Cy5 (X_6x 633 nm/λ_em 670 nm). Power of both laser and photomultiplier (PMT) can be set in the range between 50% and 95%. Images generated by the reader represent fluorescence intensity for every spot of each matrix. Some examples of the images obtained following hybridization are shown in Figure 6.

For image quantitification, ScanArray Quantitative Microarray Analysis quantification software (Perkin Elmer Life Sciences, Boston, USA) is used, which enables to store data in an electronic sheet in Excel format. For quantification, an array is placed on the acquired image after slide hybridization. At the edge of all matrices eight spots are present which hybridize with the probes used as controls for the hybridization reaction (Figure 5), so that boundaries of the matrix are marked and positioning of the array to quantitate fluorescence is facilitated. The quantification method chosen is referred to as "fixed circle". Fluorescence intensities of spots are calculated by using the mean intensity option.

Analysis of data from each experiment is carried out as follows:

- Local background noise is subtracted from the intensity of each spot;

- Mean fluorescence (IF) of four replicates for each ZipCode in each one of two analyzed channels (Cy3 and Cy5) and the coefficient of variation

(CV) from four replicates are calculated; only signals having a CV < 5% are considerated as the optimum;

- The ratio (Rm) between the signal emitted from fluorochrome Cy3 and that from Cy5 for the internal reference sequence (IFCy3/IFCy5 = R_IR) is calculated; this R_JR value is used to balance the two fluorophores one with respect to the other, and to compare results obtained from different experiments; each mean intensity from IFCy3 fluorescence is divided by the R_IR value, so that a corrected fluorescent intensity is obtained for Cy3 fhioroptiore (cIFCy3).

Genotype Determination

To define the genotype, allelic fractions for each analyzed SNP are calculated. These estimate the relative quantity of allele 1 (labeled with Cy3) compared to allele 2 (labeled with Cy5) in the analyzed sample.

The allelic fraction allows the transformation of fluorescence values in a value close to 1 when the individual is homozygous for allele 1 and a value close to 0 when is homozygous for allele 2 at a given SNP₅ and a value close to 0.5 when the individual is heterozygous for alleles 1 and 2 in that mutation site.

The formula to obtain the allelic fraction is as follows:

AF = cIFCy3/(cIFCy3 + IFCy5), wherein AF is the allelic fraction, cIFCy3 is the corrected Cy3 fluorescence intensity, and IFCy5 is Cy5 fluorescence intensity. Obtained allelic fraction values enable to assign one of three possible genotypes (homozygous for allele 1 or for allele 2, or heterozygous) to the sample under study for each SNP.

The file obtained from reading the slide is used by a SAS software expressly generated for this application that outputs the animal's genotype with an assignment rule weighted for each analyzed SNP. In general, it can be stated that homozygotes for allele 1 have allelic fraction values in the range between 0.87 and 1.00, homozygotes for allele 2 have allelic fraction values in the range between 0.01 and 0.21, and heterozygotes have values in the range between 0.36 and 0.65. In Figure 7 some of these genotypic assignments are shown.

Results

To validate the above described primers, probes and methods applicative tests were performed on 145 samples of DNA extracted from blood or milk from Friesian (120), Somba, Zebu, and Reggiana (25) cattle breeds. Some of the results obtained from a multiplex PCR reaction are shown in Figure 4. Subsequently, the ability of probes to discriminate polymorphisms of interest was tested, when they are used all together in a single mix undergoing a LDR ligation reaction.

The PCR primers and probes used in this method are an accurate, cheap, and high-throughput tool whose results do not generate interpretative errors due to false-positive and/or false-negative signals. Thus, the developed primers and probes are particularly suitable tools for animal genotyping. All oligonucleotide probes were designed so that their melting temperatures are in the range between 67 and 69°C, thus preventing problems owing to formation of secondary structures during reactions of molecular recognition of target DNA.

The oligonucleotide probe mix is used to recognize eight different amplicons at the same time and in the same reaction. Development of a multiplex PCR reaction to simultaneously amplify the eight fragments of interest enables to increase productivity while reducing costs and working time.

The good quality of the obtained PCR products in terms of lenght and concentration ensures the successful outcome of subsequent analyses aimed at detection of polymorphisms.

An important aspect of applicability of the probes here described is that they can be successfully used for detection of heterozygotes. If two different alleles are located at the same site, the experiments have shown that good signals are obtained for both the bases involved. This means that genotype determination is reliable also in this case.

The experiments have shown that, with the probes herein provided, it is also possible to obtain the genotypes of polymorphisms due to insertions or deletions. In such cases, two common probes were designed which differ at their 3' ends in order to define with certainty the two allelic variants.

Furthermore, to normalize performance differences from the whole process, which consists of multiplex PCR, LDR, and hybridization on microarray, every hybridization signal obtained was corrected by using an internal control located within CSN2 gene and corresponding to a region designed to be heterozygous in all analyzed individuals.

Some examples of results obtained after hybridization on a slide are illustrated in Figure 6. A diagram showing allelic fractions for genotype assignment of the 22

SNPs considered for 80 out of 145 samples analyzed is set forth in Figure 7.

All SNPs are unambigously clustered in three genotypic classes.

In conclusion, the great potential for applying primers and probes to a method such as a LDR reaction in association with slide microarray to rapidly and reliably identify milk protein genetic polymorphisms in bovines was established.

Primers and probes have proven themselves a useful tool enabling the simultaneous genotyping of different genomic regions that are of interest for breeders for the productive implications they have displayed. Some of these polymorphisms are used for breeding plans. Furthermore, the probes can be used for relationship diagnosis. In the latter field, SNP usage is attractive compared to currently used molecular markers (microsatellites) since SNPs are genetically stable polymorphisms which can be easily employed for automated processes such as that based on LDR and microarrays but also, for example, for hybridization, PCR, RT-PCR genotyping, and minisequencing techniques, and systems based on the use of magnetic microbeads. Bibliography

Boettcher PJ., A. Caroli, A. Stella, S. Chessa, E. Budelli, F. Canavesi, S. Ghiroldi, G. Pagnacco. (2004). Effects of casein haplotypes on production traits in Italian Holstein and Brow Cattle. J. Dairy Sci. 87: 4311-4317.

Caroli A., S. Chessa, P. Bolla, E. Budelli, G.C. Gandini (2004). Genetic structure of milk protein polymorphisms and effects on milk production traits in a local dairy cattle. J. Anim. Breed. Genet. 121: 119-127. Chen J., M.A. Iannone, M. S. Li, J.D. Taylor, P. Rivers, AJ. Nelsen, K.A. Slentz-Kesler, A. Roses, M.P. Weiner (2000). A microsphere-based assay for multiplexed single nucleotide polymorphism analysis using single base chain extension. Genome Res. 10: 549-557.

Consolandi C, A. Frosini, C. Pera, G.B. Ferrara, R. Bordoni, B. Castiglioni, E. Rizzi, A. Mezzelani, L. R. Bernardi, G. De Bellis, and C. Battaglia (2004). Polymorphism analysis within the HLA-A locus by universal oligonucleotide array. Hum Mutat. 24: 428-434.

Creamer L.K., D.P. Harris (1997). Relationship between milk protein polymorphism and physico-chemical properties. Proc. Inter. Dairy Federation Seminar, IDF, Brussels, B: 110-123. Damiani G., E. Budelli, S. Florio, A. Caroli, G. Pagnacco (2000a). Polymorphism of κ-casein SINE Bov-A2 and CYP21 -hydroxylase in some bovine breeds. Zoot. Nutriz. Anim. 3: 145-148.

Damiani G., S. Florio, E. Budelli, P. Bolla, A. Caroli (2000b). Single nucleotide polymorphisms (SNPs) within Bov-A2 SINE in the second intron of bovine and buffalo K -casein (CSN3) gene. Anim. Genet. 31 : 277-279.

Di Stasio L., P. Mariani (2000). The role of protein polymorphism in the genetic improvement of milk production. Zoot. Nutriz. Anim. 26: 69-90. Farrell H.M. Jr., R. Jimenez-Flores, G. T. Bleck, E.M. Brown, J.E. Butler, L.K. Creamer, CL. Hicks, CM. Hollar, K.F. Ng-Kwai-Hang, H.E. Swaisgood

(2004). Nomenclature of the Proteins of Cows' Milk: Sixth Revision. J. Dairy

Sci. 87: 1641-1674.

Hadjipanayiotou, M. (1995). Composition of ewe, goat and cow milk and of colostrum of ewes and goats. Small Ruminant Research, 18: 255-262.

Hayes H., E. Petit, C. Bouniol, P. Popescu (1993). Localisation of the alpha- S2-casein gene (CASA2) to the homeologus cattle, sheep and goat chromosomes 4 by in situ hybridisation. Cytogenet. Cell Genet. 64: 282-285.

Ibeagha-Awemu E.M., Prinzenberg E. M., Erhardt G (2005). High variability of milk protein genes in Bos indicus cattle breeds of Cameroon and Nigeria and characterization of a new alphas 1 -casein promoter allele. J Dairy Res. 72:

1-9.

Lunden A., M. Nilsson, L. Janson (1997). Marked effect of polymorphism on the ratio of casin to total protein in milk. J. Dairy Sci. 80: 2996-3005. Martin P., M. Szymanowska, L. Zwierzchowski, C. Leroux (2002). The impact of genetic polymorphisms on the protein composition of ruminants milks. Reprod. Nutr. Dev. 42:433-459.

Prinzenberg E.M., C Weimann, H. Brandt, J. Bennewitz, E. KaIm,

M. Schwerin, and G. Erhardt (2003). Polymorphism of the bovine CSNlSl promoter: Linkage mapping, intragenic haplotypes, and effect on milk production traits. J. Dairy Sci. 86: 2696-2705.

Renner, E. (1982). Milk and Milk Products in Human Nutrition. Volkswirtsch.

Verlag, Munich: 467.

Rook, J.A.F. (1961). Variations in the chemical composition of the milk of the cow-Part 1. Dairy Science Abstract. 23: 251-258.

Salvadori Del Prato, O. (1998). Trattato di tecnologia casearia. Edagricole,

Bologna.

Threadgill D.W., J.E. Womack (1990). Genomic analysis of the major bovine milk protein genes. Nucleic Acid Res. 18: 6935-6942.

Vaiman D., D. Mercier, A. Eggen, I. Bahri-Darwich, C. Grohs, E. P. Cribiu, G. DoIf, A. Oustry, G. Guerin, H. Leveziel (1994). A genetic and physical map of bovine chromosome 11. Mamm Genome. 5: 553-556. Yingyongnarongkul B.E., S. E. How, JJ. Diaz-Mochon, M. Muzerelle, M. Bradley (2003). Parallel and multiplexed bead-based assays and encoding strategies. Comb. Chem. High Throughput Screen. 6: 577-587. Review.

Claims

1. A method for the simultaneous determination of single nucleotide polymorphisms or polymorphisms by insertion or deletion at genes or gene elements involved in the synthesis of milk proteins in cattle, these polymorphisms being selected from:

Gene Nucleotides Protein Amino acid involved involved

CSNlSl AlG ocgi-casein Glu₁₉₂/Glyi₉₂

CSN2 G/A β-casein Glu₃₇/Lys₃₇

C/A Hisioδ/Glnioe

CSN3 G/A κ-casein ATg₉₇ZHiS₉₇

C/T Thr₁₃₅/ne₁₃₅

PJG AlaWAlaies

LGB TGA/CGG β-lactoglobulin ASp₆₄AjIy₆₄

G/T Valio5/Valio5

GGT/ GGC or Valiig/Alane TGC

Gene Nucleotides Position Reference element involved sequence

Bov-A2 PJG 107 SEQ ID NO: element 88

CSNlSl A/C 289 SEQ ID NO: promoter 89

TT Del. 395-396

GTTT Del. 397-400

PJG 444

G/C 484 said method comprising: 1) amplification, in the same reaction medium, of gene fragments containing the above cited polymorphisms by multiplex PCR using primer pairs selected from:

Amplified gene or gene Forward primer Reverse primer element region SEQ ID NO: SEQ ID NO:

CSNlSl exon 17 1 2

CSN2 exon 6 3 4

CSN2 exon 7 5 6

CSN3 exon 4 7 8

LGB exon 3 9 10

LGB exon 4 11 12

SINE BovA2 13 14

CSNlSl Promoter 15 16

2) addition of a pair of "discriminating" probes having sequences complementary to the regions containing each of the two polymorphic variants respectively, and a "common" probe for each pair of discriminating probes, wherein said common probe has a sequence complementary to the region contiguous to the first one in the same amplified fragment, such di s criminating and common probes being selected from:

(continued)

or, in case the searched polymorphism is a deletion in CSNlSl gene promoter, addition of a pair of discriminating probes and a pair of common probes selected from:

wherein said discriminating probes are labelled at their 5' ends with a different fluorophore for each of the two discriminating probes necessary to identify a given polymorphism, and said common probes are bound at their 3 ' ends to a different random nucleotide sequence (ZipCode) for each polymorphism studied; 3) ligation of discriminating and common probes by adding a ligase enzyme to the reaction media followed by incubation;

4) hybridization of the probes obtained in step 3 on a support on the surface of which polynucleotide sequences complementary to the random ZipCode sequences used in steps 2 and 3 have been previously adsorbed or bound, directly or by a spacer sequence, in separate areas (spots), so that polynucleotide molecules complementary to a unique ZipCode sequence are bound in each separate area;

5) sample analysis by reading the fluorescence emitted from the fluorescent label bound to the 5' end of the discriminating probe.

2. A method according to claim 1 wherein at least 14, preferably at least 20 polymorphisms are simultaneously determined.

3. A method according to claim 1 wherein all indicated polymorphisms are simultaneously determined.

4. A method according to claim 1 wherein in step 4 probes are hybridized on a support consisting of a slide whose surface is divided into discrete areas on which polynucleotide molecules complementary to ZipCode sequences are attached according to a prefixed scheme, so that a single sequence corresponds to a given area.

5. A method according to claim 1 for bovine milk protein genotyping.

6. A combination of forward and reverse primers to simultaneously amplify, by a multiplex PCR reaction, gene fragments containing the polymorphisms indicated in claim 1, said primers being selected from the group comprising:

Forward primer Reverse primer SEQ ID NO: SEQ ID NO:

1 2

3 4

5 6

7 8

9 10

11 12

13 14

15 16

7. A combination of probes to be used in a LDR/UA (Ligation Detection Reaction/Universal Array), wherein these probes are capable of annealing to a gene fragment containing a polymorphism according to claim 1, and each of said combinations, which is specific for a given polymorphism according to claim 1 , consists of two discriminating probes and at least a common probe, and is selected from the group comprising:

8. A combination of probes according to claim 7 wherein said probes bear a fluorophore group at their 5' ends.

9. A combination of probes according to claim 8 wherein a random nucleotide sequence (ZipCode), which is not naturally occurring, is bound at the 3 ' end of each common probe.

10. A Kit for carrying out a method according to claims 1-5, containing different combinations of primers and probes according to claim 6 and claims 7-9, respectively, and optionally Taq polymerase and ligase enzymes.

11. The use of primers and probes in different combinations according to claims 6 and claims 7-9, respectively, to determine single nucleotide polymorphisms or polymorphisms by insertion or deletion according to claim 1.

12. The use according to claim 11 for bovine milk protein genotyping.