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).
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).
F primer: 5”-CCCATTTCGCCTTCTCTGTA-3”
R primer: 5”-CAGCGCTGTGAGAAAGATGA-3”
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.
4.1. 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).
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.
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.
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.
We declare no conflict of interest in this study.