Polymorphism of κ-Casein Gene in Iranian Holsteins

Document Type: Research Paper


1 Research and Technology Deputy, University of Shahrekord Medical Sciences, Shahrekord, I.R. Iran

2 Department of Basic Sciences, School of Veterinary Medicine, University of Shahrekord, Shahrekord, I.R. Iran

3 Department of Food Hygiene and Quality Control, School of Veterinary Medicine, University of Shahrekord, Shahrekord, I.R. Iran


Background: Genetic polymorphism of milk proteins has been associated with composition, manufacture, and traits of milk. Caseins are the most important milk proteins whose genes are strongly linked and inherited as a raceme. κ-casein which is a quantitatively minor constituent of bovine milk is thought to play a critical role in organization, fixation and aggregation of casein micelles and firmness of curd during cheese making. Objectives: In this study, we considered polymorphism of κ-casein gene in Iranian Holstein cows. Materials and Method: In this study, κ-casein gene polymorphism among 50 DNA samples of Iranian Holstein cows via Polymerase Chain Reaction sequence analysis (PCR-Sequencing) was considered. For data analysis SPSS 11.5 (ANOVA test) was used. Results: Four polymorphic sites that created 4 variants and seven different genotypes of κ-casein gene were identified. In this population the frequencies of A, B, C, and E alleles were estimated as 0.391, 0.413, 0.087, and 0.109 respectively. Conclusion: We suggested that the B allele of κ-casein gene frequency should be increased in Iranian Holsteins because it has an essential factor in marker-assisted selection for milk traits.


1. Background

The most important milk proteins are caseins which are secreted from mammary gland cells. They constitute about 80% of bovine milk proteins and are divided into four principal classes: αs1, αs2, β, and κ-casein (1). About 95% of the caseins exist in milk as large colloidal particles known as micelles, on a dry matter basis. Casein micelles contain 94% protein and 6% low weight molecular species referred to as colloidal calcium phosphate (2). These micelles increase the solubility of minerals and facilitate the transfer of nutrients from mother to offspring (3). Caseins and their genetic variants have been widely studied and reported as an essential factor associated with milk protein content and cheese yield (4-6). The κ-casein which represents about 15% of the total milk casein (2) has a crucial role in the formation, stabilization and aggregation of casein micelles and thus effects the technological (7) and nutritional properties of milk (8, 9). The mature κ-casein protein has a peptide bond that is cleaved in the gut by the action of renine to produce an insoluble peptide (para κ-casein or PKC) as well as a soluble hydrophilic glycopeptide (caseinomacropeptide or CMP) (10). The caseins are encoded by single copy genes clustered in a region about 200 kb on chromosome 6 in bovine (11-13), 4 in goat, human, and sheep, 5 in mouse, and 12 in rabbit (14) arranged in the following order: αs1, αs2, β, and κ-casein (15). Specifically the κ-casein gene comprehends a 13kb sequence subdivided into 5 exons and 4 introns (16) and sequences of this gene constitute about 25% of the casein gene cluster (15). κ-casein gene 5’ regions’ is organized differently from that of other casein genes, showing that its expression is independent from other casein genes (16). Polymorphisms of milk proteins are important because genetic selection and genetic characterization of bovine breeds are related to promotion of positive properties of milk and cheese yield (17, 18). Different breeds of cattle have various allelic variants for κ-casein gene, including A, B, C, E, F, G, H, I, and A1 (19, 20).

2. Objectives

In this study polymorphism of κ-casein gene in Iranian Holstein dairy cows was considered.

3. Materials and Methods

Sampling of the animals consisted of 50 Holstein cattle randomly selected from the second parity cows in Ghiam Dairy Co. (Iran). Peripheral whole blood was collected from jugular veins into tubes containing citrate as an anticoagulant. High molecular weight DNA was extracted by a modified salting-out method (21). Primer pairs targeting all coding regions of the κ-casein gene were designed based on the reference GenBank sequence NC_007304 using Vector NTI software v10.1 (Invitrogen). An annealing temperature 60 °C and concentrations of MgCl2 (1–4 mM) was applied to optimize the PCR, which consisted of template DNA (50 ng), primers (16 pmol for each), dNTPs (0.2 mM), 1X buffer, and 1 U Taq polymerase in a 25 μl reaction. The DNA was denatured at 95°C for 5 min. Reactions were cycled 30 times through 95 °C30s-1, annealing  temperature 60 °C.30s-1, extension 72 °C.30s-1, and finally incubated at 72 °C for 5 min. All of the PCR products were electrophoresed at 150 V for 40 min through a 2% agarose gel containing 1X TBE buffer and 0.14 mg.ml-1 ethidium bromide to check whether amplification had been successful or not. The purified PCR products were sequenced (Macrogen, Korea) in both directions using the appropriate PCR primers (22, 23).



3.1. Statistical Analysis

Statistical analysis was done by SPSS software (version 11.5) and comparision between frequency of genotypes and alleles was done by ANOVA.

4. Results

4.1. Observation of gel Electrophoresis Bond

Electrophoresis of PCR production with ethidium bromide staining showed that only one bond with 13kb weight for κ-casein gene existed in the samples of study (Figure 1).

4.2. Sequencing

Polymorphic sites in exon 4 and different genotypes in Holstein κ-casein gene were identified, as follows:

1- Sequencing analysis of 28 samples indicated that in the positions of 2523, 10711, 10731, 10825, 10828, 10863, and 10884 of κ-casein gene the nucleotides of G, G, T, C, C, A and A were presented and created A variant. The frequency of this variant in this study was 0.391.

2- At positions of 10828 and 10863 region of κ-casein gene, two mutations of C→T (Transition) and A→C (Transversion) occurred and put Isoleucine136 instead of Threonine 136 and Alanine148 instead of Aspartate148 amino acids in protein and created B variant of κ-casein. Frequency of B allele in this population was estimated 0.413 (P < 0.05).

3- At position of 10711 region of this gene, G→A (Transition) mutation was presented and put Histidine97 instead of Arginine97 amino acid in protein. This mutation created C variant. Also in this variant 2 previous mutations were presented. Frequency of C allele was estimated 0.087 (P < 0.05).

4- At position of 10884 region of κ-casein gene A→G (Transition) mutation occurred, and put Glycine155 instead of Serine155 amino acid and created E variant of this gene. Frequency of E allele was estimated as 0.109 (P < 0.05) (Table 1).

Frequency of 7 different genotypes and 4 different alleles of κ-casein gene in Holstein cows has been summarized in Tables 2, 3. A and B alleles’ frequency in this breed and other breeds has been summarized in Table 4.

5. Discussion

Cattle breeds’ allelic variants have demonstrated that the B allele of κ-casein may allow betterment in the milk quality for manufacturing processes, being excellent for cheese making due to faster coagulation and firmer curd (24, 25), unlike the E variant of κ-casein which has been found related to inferior coagulation feature (26, 27). In this study four polymorphic sites in exon 4 and 7 different genotypes in Holstein κ-casein gene were identified and B allele frequency was higher (0.413) compared to other alleles (A, C, and E). Genetic improvement of breeds or herds could be associated with B allele and therefore we should try to select the cows with this allele of κ-casein for production purpose.

A study of alleles frequency consideration of caseins in the Finnish Ayrish breed showed that A and E alleles’ frequency of κ-casein was 0.612 and 0.307, respectively (28). In another study, alleles’ frequency of κ-casein gene was checked in 1316 cattles from the Brazilian Bos Indicus breed and the highest frequency of the B allele was 0.30. On the other hand, in different breeds, the frequencies of this allele ranged from 0.01 to 0.18 (29). Due to a single base mutation in the κ-casein locus in B variant, Isoleucine substituted by Threonine and Aspartic acid replaced by Alanine (30) were found to be related to different sizes of  micelles,  thermal resistance, shorter coagulation, and better curdles, which are dominant in cheese making and cheese yield (31, 32). In a study in China the A allele of κ-casein gene was dominant in chinese Holstein cattle and its frequency was estimated 0.73 (33) that is similar to the results on Indian goat (34) and Russian cattle breeds (35). In casein molecule, the variants that named as post transcriptional variants are also seen (18, 36). Polymorphisms of κ-casein gene has been reported in different cattle breeds and frequencies of all alleles of this gene were estimated (17, 18, 37). In view of existing evidence on the whole casein group, casein haplotype effects on productive traits have been examined and confirmed. Moreover, non coding sequences mutations could affect specific protein expression, milk composition, and cheese making. Milk protein variants are also useful tools for breed characterization, variety, and phylogenetic research. Improvement of human nutrition quality is dependent on beneficial allele selection in food animal for milk proteins and should be tested to produce the specific milk, e.g. hypoallergenic milk (38). Recently, researchers found 16 polymorphic sites at the κ-casein (CSN3) gene in domesticated dairy goat (Capra hircus). 13 mutation sites were created protein variants and 3 of them were silent mutations in exon 4 (39). In a sample of 540 dairy goats, 67 different haplotypes with frequency of 0.01 and 27 with frequency of 0.03 were reported. Analysis of 41 White Shorthaired (WSH) trio families and 44 Brown Shorthaired (BSH) trio families in 2 dairy goat breeds showed that respectively 14 and 20 haplotypes were present. Various genomic techniques were used to type the casein loci. 22 different combinations of κ-casein alleles have been found (40). Study of casein complex by milk isoelectrofocusing and analyses at the DNA level in three goat breeds from northern Italy showed that the majority of all known polymorphisms were present and a new allele of β-casein was identified which seemed to be specific to the Frisa breed. It was named β-casein*E and characterized by a transversion mutation (TCT→TAT) responsible for amino acid replacement Ser166→Tyr166 in this protein (41). In Iran there has been no selection for specific protein variants in breeding programs and by this study the allele frequency of κ-casein gene was determined in this study, probably being helpful for selection of cows’ breeding.

Polymorphisms of κ-casein gene have not been previously reported for Holstein via PCR-Sequencing method. Genetic variants detection by means of PCR-RFLP method was limited to the mutation identification in gene and we suggest PCR-Sequencing method for discovering the new κ-casein gene mutations. The polymorphism of milk proteins affects the milk composition and cheese quality and the mutations should be used as a molecular marker-assisted selection. In this study four polymorphic sites in exon 4 and 7 different genotypes in Holstein κ-casein gene were identified and B allele frequency was higher (0.413) compared to other alleles (A, C, and E).

In the present study, we reported mutations of κ-casein gene in Iranian Holstein cows via PCR-Sequencing. The B allele frequency of this gene was higher than that of others. Use of this allele as a genetic marker in Holstein cows around the world may increase milk protein and cheese yield, hence we suggest using this allele to improve milk quality.


We thanks Ghiam Dairy Co. and Research and Technology Deputy of University of Shahrekord Medical Sciences for their cooperation.

Author's contribution

Homayon Reza Shahbazkia suggested the primary idea and protocol, abstracted the research work, analyzed the data, and wrote the manuscript. Zahra Molavi Choobini recorded samples, designed and did the laboratory research, analyzed the data, wrote the manuscript and submitted it.  Mohammad Shadkhast, Hamdollah Moshtaghi and Said Habibian Dehkordi supervised the laboratory research.


This research work with grant number 285 was jointly supported by Department of Basic Sciences and Faculty of Veterinary Medicine, University of Shahrekord, Shahrekord, Iran.

Financial Disclosure

We declare no conflict of interest in this study.

1.            Farrell Jr HM, Jimenez-Flores R, Bleck GT, Brown EM, Butler JE, Creamer LK, et al. Nomenclature of the Proteins of Cows’ Milk—Sixth Revision. Journal of Dairy Science. 2004;87(6):1641-74.

2.            Fox PF, McSweeney PLH. Dairy Chemistry and Biochemistry. Springer; 1998.

3.            Dev BC, Sood SM, DeWind S, Slattery CW. Kappa-casein and beta-caseins in human milk micelles: structural studies. Arch Biochem Biophys. 1994;314(2):329-36.

4.            Bobe G, Beitz DC, Freeman AE, Lindberg GL. Effect of Milk Protein Genotypes on Milk Protein Composition and Its Genetic Parameter Estimates. Journal of Dairy Science. 1999;82(12):2797-804.

5.            Hallen E, Wedholm A, Andren A, Lunden A. Effect of beta-casein, kappa-casein and beta-lactoglobulin genotypes on concentration of milk protein variants. J Anim Breed Genet. 2008;125(2):119-29.

6.            Celik S. β-Lactoglobulin genetic variants in Brown Swiss breed and its association with compositional properties and rennet clotting time of milk. International Dairy Journal. 2003;13(9):727-31.

7.            Wedholm A, Larsen LB, Lindmark-Månsson H, Karlsson AH, Andrén A. Effect of Protein Composition on the Cheese-Making Properties of Milk from Individual Dairy Cows. Journal of Dairy Science. 2006;89(9):3296-305.

8.            Mercier JC, Brignon G, Ribadeau-Dumas B. [Primary structure of bovine kappa B casein. Complete sequence]. Eur J Biochem. 1973;35(2):222-35.

9.            Mercier J-C, Chobert J-M, Addeo F. Comparative study of the amino acid sequences of the caseinomacropeptides from seven species. FEBS Letters. 1976;72(2):208-14.

10.          Qian Z-Y, Jollès P, Migliore-Samour D, Schoentgen F, Fiat A-M. Sheep κ-casein peptides inhibit platelet aggregation. Biochimica et Biophysica Acta (BBA) - General Subjects. 1995;1244(2–3):411-7.

11.          Ferretti L, Leone P, Sgaramella V. Long range restriction analysis of the bovine casein genes. Nucleic Acids Res. 1990;18(23):6829-33.

12.          Threadgill DW, Womack JE. Genomic analysis of the major bovine milk protein genes. Nucleic Acids Research. 1990;18(23):6935-42.

13.          Hayes H, Petit E, Bouniol C, Popescu P. Localization of the alpha-S2-casein gene (CASAS2) to the homoeologous cattle, sheep, and goat chromosomes 4 by in situ hybridization. Cytogenet Cell Genet. 1993;64(3-4):281-5.

14.          Eggen A, Fries R. An integrated cytogenetic and meiotic map of the bovine genome. Animal Genetics. 1995;26(4):215-36.

15.          Lien S, Rogne S. Bovine casein haplotypes: number, frequencies and applicability as genetic markers. Anim Genet. 1993;24(5):373-6.

16.          Alexander LJ, Stewart AF, Mackinlay AG, Kapelinskaya TV, Tkach TM, Gorodetsky SI. Isolation and characterization of the bovine kappa-casein gene. Eur J Biochem. 1988;178(2):395-401.

17.          DelLama S, Zago M. Identification of the kappa-casein and beta-lactoglobulin genotypes in Brazilian Bos indicus and Bubalus bubalis populations. Brazilian J Genet. 1996;19(1):73-7.

18.          Kemenes PA, Regitano LCdA, Rosa AJdM, Packer IU, Razook AG, Figueiredo LAd, et al. k-Casein, b-lactoglobulin and growth hormone allele frequencies and genetic distances in Nelore, Gyr, Guzerá, Caracu, Charolais, Canchim and Santa Gertrudis cattle. Genetics and Molecular Biology. 1999;22:539-41.

19.          Soria LA, Iglesias GM, Huguet MJ, Mirande SL. A PCR-RFLP test to detect allelic variants of the bovine kappa-casein gene. Anim Biotechnol. 2003;14(1):1-5.

20.          Prinzenberg EM, Krause I, Erhardt G. SSCP analysis at the bovine CSN3 locus discriminates six alleles corresponding to known protein variants (A, B, C, E, F, G) and three new DNA polymorphisms (H, I, A1). Anim Biotechnol. 1999;10(1-2):49-62.

21.          Shahbazkia HR, Aminlari M, Tavasoli A, Mohamadnia AR, Cravador A. Polymorphisms of the β-1,4 galactosyltransferase-I gene in Holsteins. Livestock Science. 2010;131(2):297-300.

22.          Shahbazkia HR, Aminlari M, Cravador A. Association of polymorphism of the beta(1, 4)-galactosyltransferase-I gene with milk production traits in Holsteins. Mol Biol Rep. 2012;39(6):6715-21.

23.          Heck JM, Schennink A, van Valenberg HJ, Bovenhuis H, Visker MH, van Arendonk JA, et al. Effects of milk protein variants on the protein composition of bovine milk. J Dairy Sci. 2009;92(3):1192-202.

24.          Ren DX, Miao SY, Chen YL, Zou CX, Liang XW, Liu JX. Genotyping of the k-casein and beta-lactoglobulin genes in Chinese Holstein, Jersey and water buffalo by PCR-RFLP. J Genet. 2011;90(1):e1-5.

25.          Ikonen T, Ahlfors K, Kempe R, Ojala M, Ruottinen O. Genetic Parameters for the Milk Coagulation Properties and Prevalence of Noncoagulating Milk in Finnish Dairy Cows. Journal of Dairy Science. 1999;82(1):205-14.

26.          Hallén E, Allmere T, Näslund J, Andrén A, Lundén A. Effect of genetic polymorphism of milk proteins on rheology of chymosin-induced milk gels. International Dairy Journal. 2007;17(7):791-9.

27.          Ikonen T, Ruottinen O, Erhardt G, Ojala M. Allele frequencies of the major milk proteins in the Finnish Ayrshire and detection of a new kappa-casein variant. Anim Genet. 1996;27(3):179-81.

28.          Azevedo AL, Nascimento CS, Steinberg RS, Carvalho MR, Peixoto MG, Teodoro RL, et al. Genetic polymorphism of the kappa-casein gene in Brazilian cattle. Genet Mol Res. 2008;7(3):623-30.

29.          Pinder SJ, Perry BN, Skidmore CJ, Savva D. Analysis of polymorphism in the bovine casein genes by use of the polymerase chain reaction. Anim Genet. 1991;22(1):11-20.

30.          McLean DM, Schaar J. Effects of β-lactoglobulin and κ-casein genetic variants and concentrations on syneresis of gels from renneted heated milk. Journal of Dairy Research. 1989;56(02):297-301.

31.          Verdier-Metz I, Coulon J-B, Pradel P. Relationship between milk fat and protein contents and cheese yield. Anim Res. 2001;50(5):365-71.

32.          Jum Z, Huang J, Li Q, Wang H, Zhong J, Wang C. The polymorphisms of k-casein gene and their associations with milk production traits and expression analysis in Chinese Holstein Cattle. African J Biotechnol. 2011;10(62):13368-75.

33.          Kumar A, Rout P, Mandal A, Roy R. Kappa-Casein gene polymorphism in indian goats. Indian J Biotechnol. 2009;8:214-17.

34.          Sulimova GE, Azari MA, Rostamzadeh J, Mohammad Abadi MR, Lazebny OE. κ-casein gene (CSN3) allelic polymorphism in Russian cattle breeds and its information value as a genetic marker. Russian Journal of Genetics. 2007;43(1):73-9.

35.          Sedlmeyer F, Daimer K, Rademacher B, Kulozik U. Influence of the composition of milk-protein κ/ι-hybrid-carrageenan gels on product properties. Colloids and Surfaces B: Biointerfaces. 2003;31(1–4):13-20.

36.          Golijow C, Giovambattista G, Poli M, Dulout F, Lojo M. kappa-casein gene frequencies support subdivision and historical origin of Argentine Creole cattle. Bra J Genet. 1996;19(4):583-6.

37.          Caroli AM, Chessa S, Erhardt GJ. Invited review: milk protein polymorphisms in cattle: effect on animal breeding and human nutrition. J Dairy Sci. 2009;92(11):5335-52.

38.          Prinzenberg EM, Gutscher K, Chessa S, Caroli A, Erhardt G. Caprine κ-Casein (CSN3) Polymorphism: New Developments in Molecular Knowledge. Journal of Dairy Science. 2005;88(4):1490-8.

39.          Sztankoova Z, Matlova V, Kysel'ova J, Jandurova OM, Riha J, Senese C. Short communication: Polymorphism of casein cluster genes in Czech local goat breeds. J Dairy Sci. 2009;92(12):6197-201.

40.          Caroli A, Chiatti F, Chessa S, Rignanese D, Bolla P, Pagnacco G. Focusing on the Goat Casein Complex. Journal of Dairy Science. 2006;89(8):3178-87.

41.          Golijow CD, Giovambattista G, Rípoli MV, Dulout FN, Lojo MM. Genetic variability and population structure in loci related to milk production traits in native Argentine Creole and commercial Argentine Holstein cattle. Genetics and Molecular Biology. 1999;22:395-8.