Beta-casein constitutes up to 45% of the casein, the most abundant protein in cow's milk, and it is reported to be the most polymorphic milk protein gene. There are 12 variants present in bovine milk including A1, A2, A3, B, C, D, E, F, H1,H2, I and G, of which A1 and A2 are the most common variants (Farrell et al. Reference Farrell, Jimenez-Flores, Bleck, Brown, Butler, Creamer, Hicks, Hollar, Ng-Kwai-Hang and Swaisgood2004; Oleński et al. Reference Oleński, Cieślińska, Suchocki, Szyda and Kamiński2012; Singh et al. Reference Singh, Jayakumar, Sharma, Gupta, Dixit, Gupta and Gupta2015). It's reported that β-casein not only provides a group of active peptides, mainly opioids, which are known to play an important role in the response to stress and pain (Bell et al. Reference Bell, Grochoski and Clarke2006; Givens et al. Reference Givens, Aikman, Gibson and Brown2013; Nguyena et al. Reference Nguyena, Johnsona, Busettib and Solaha2015), but also has an influence on milk protein composition and milk-production traits according to its genetic polymorphism (Boettcher et al. Reference Boettcher, Caroli, Stella, Chessa, Budelli, Canavesi, Ghiroldi and Pagnacco2004; Oleński et al. Reference Oleński, Kamiński, Szyda and Cieslinska2010; Visker et al. Reference Visker, Dibbits, Kinders, Van Valenberg, Van Arendonk and Bovenhuis2011). Therefore, β-casein plays an essential role both for nutrition and for its impact on the technological properties of the dairy products. It has already been investigated intensively at the protein as well as at the DNA level, especially in dairy breeds (Barroso et al. Reference Barroso, Dunner and Cañón1999; Freyer et al. Reference Freyer, Liu, Erhardt and Panicke1999; Lipkin et al. Reference Lipkin, Tal-Stein, Friedmann and Soller2008).
Currently, the Chinese Holstein cow is the main dairy cow breed in China, characterised by its very distinctive markings and outstanding milk production. Few studies have been performed in the Chinese Holstein and no genetic variants have been reported so far for β-casein, although several molecular diagnostic techniques have been developed as dairy cow breeding in China is still evolving (Sun et al. Reference Sun, Fan, Xie, Chu, Sun, Zhang, Zhang, Gong, Chen, Li, Shi and Zhang2011).
The technique of sequence analysis was successfully applied for the detection of different point mutations of bovine β-casein gene in genomic DNA from 133 Holstein cows and we screened the full coding region of the β-casein gene which includes exons 4, 5, 6 and 7. The aims of this study were to analyse the occurrence of different polymorphic variants of bovine β-casein, as well as their frequencies, and the proportions of the key β-casein variants in Southern Chinese Holstein. Furthermore, all additional β-casein sequence variants – and, if possible, intragenic haplotypes – were recorded.
Material and methods
Animal welfare and experimental procedures were carried out strictly in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of Institute of Shanghai Jiaotong University.
Animals
133 Holstein cows were randomly selected from Xinghuo farm (Shanghai, China) for experiment. Hair follicle samples from the tails of these cows were collected into dry, sterile tubes, stored in a −20 °C freezer and prepared for analysis.
DNA extraction and PCR amplification
Bovine genomic DNA was extracted from somatic cells in hair follicles using the direct lysis method (TransDirect Animal Tissue PCR Kit, Trangen Biotech, BeiJing) and used to estimate β-casein genotypes by means of sequence analysis.
For the β-casein polymorphic sites considered, a set of primers were designed on the basis of the genomic GenBank sequence NM_181008 of bovine β-casein using Primer3 software to amplify parts of the gene containing the polymorphic sites for almost all known alleles. The genomic specificity of the primers was tested using the BLAST programme. The details of primers along with expected product size for different regions of β-casein gene and the melting temperatures (Tm) have been summarised in Supplementary Table S1.
The PCR amplifications with their respective primer pairs were performed using 4 µl tissue extract in a reaction volume of 20 µl containing 10 µl 2 × TransDirectTM PCR SuperMix(+dye), 10 µm forward primer and reverse primer. PCR was carried out in a thermal cycler using the following cycling programme: 95 °C for 5 min, followed by 35 cycles at 95 °C for 30 s, 60–66 °C for 30 s and 72 °C for 1 min with a final extension at 72 °C for 7 min. The amplified products were analysed by electrophoresis on 1·5% agarose gel at 120 V for 20 min using ethidium bromide staining. The purification was carried out using AxyPrepTM DNA Gel Extraction kit (Corning, NY, USA).
DNA sequencing
After purification of the PCR products, sequencing was carried out using a BigDye Terminator cycle sequencing kit (Applied Biosystems, CA, USA) on an automated Genetic Analyzer ABI 3730 (Applied Biosystems, CA, USA). The obtained sequences were analysed and compared with the genomic GenBank sequence NM_181008 using the software Seqman (DNASTAR, Inc., WI, USA).
Results and discussion
Figure 1 shows the five β-casein variants found in this study and corresponding amino acid differences deduced from the sequence analysis. β-casein has 209 amino acids and there are at least 12 variants of this protein that differ at different amino acid positions (Gallinat et al. Reference Gallinat, Qanbari, Drögemüller, Pimentel, Thaller and Tetens2013; Haq et al. Reference Haq, Kapila, Shandilya and Kapila2014; Singh et al. Reference Singh, Jayakumar, Sharma, Gupta, Dixit, Gupta and Gupta2015). For instance, the A2 allele differs from A1 and A3 alleles by only one amino acid change at positions 67 (His/Pro) and 106(Gln/His), respectively (Barroso et al. Reference Barroso, Dunner and Cañón1999; Kamiński et al. Reference Kamiński, Cieoelińska and Kostyra2007; Cieślińska et al. Reference Cieślińska, Kostyra, Kostyra, Oleński, Fiedorowicz and Kamiński2012). The difference between A1 and B variants involves only one amino acid substitution (Ser for Arg) at position122 of the β-casein. B variant differs from A2 in having a proline in position 67 and an arginine in place of a serine at position 122 (Clemens, Reference Clemens2011; Vallas et al. Reference Vallas, Kaart, Varv, Parna, Joudu, Viinalass and Parna2012).
Fig. 1. Nucleotides present at polymorphic and corresponding amino acid changes in five β-casein variants observed.
The natural mutations that give rise to this difference are a result of a single nucleotide polymorphism of the β-casein gene. The β-casein A1 allele differs from A2 allele by a A → C substitution at position 304 of the cow β-casein reference sequence (GenBank, NM_181008). The B allele differs by 2 nonsynonymous mutations from the A2 allele. The variant is characterised by a A → C transition at nucleotide position 304, resulting in the amino acid exchange Pro → His, and a C → G transition at position 470, resulting in a Ser → Arg substitution. This difference in the amino acid sequence suggests a conformational difference in the secondary structure of the expressed protein (Elliott et al. Reference Elliott, Harris, Hill, Bibby and Wasmuth1999; McLachlan, Reference McLachlan2001).
Frequencies for all observed variants and genotypes of the β-casein gene were calculated by direct counting for all the examined population. Figure 2 shows the gene and genotype frequencies of variants in the investigated cows. Genotype A1A3 appeared only once and was therefore excluded from further statistical analyses.
Fig. 2. Frequency of genotypes and alleles within β-casein gene in a population of Holstein cows.
In the population included in the study we detected eight genotypes: Two homozygote genotype A1A1 (27 animals) and A2A2 (30 animals) with percentages of 0·203 and 0·226, respectively; heterozygote genotype A1A2 (47 animals) with prevalence greater than 35%, and the others with percentages less than 7%. In the total population of cow heterozygote genotype A1A2–0·353 were the most frequent, while A1I –0·030 were the least frequent one.
The heterozygote A1A2 genotype was more common (0·353), while A1A1 and A2A2 genotype was at relatively low frequency among the cows (0·203 and 0·226). The heterozygote genotype frequency is similar to those reported for Estonian Holstein cows (Vallas et al. Reference Vallas, Kaart, Varv, Parna, Joudu, Viinalass and Parna2012). However, the expected homozygosity is rather less; the genotypes A1B, A2I, A2B, A1I and A1A3 are found in Chinese Holstein with low frequencies.
The most common forms of β-casein detected in this population are A1 and A2 with relative frequencies of 0·432 and 0·459, respectively, while B and I are less common (0·060 and 0·045), and A3 is rare (0·004). The relatively higher prevalence of desirable A2 allele observed across all the population corresponded to the existence of high frequencies of heterozygous genotype A1A2 and homozygote genotype A2A2. In addition, in the population of Chinese Holstein studied, the β-casein C, D, E, F, G and H allele could not be detected. These alleles were previously reported to occur at very low frequencies (<0·1) and sometimes they were even absent.
Frequencies of β-casein A1/A2 alleles in our population were similar to some early studies. For instance, a study in Denmark indicated a slight superiority of A1 β-casein for Black-and-White breed (0·550 of total β-casein; Bech & Kristiansen, Reference Bech and Kristiansen1990) and Ehrmann (Reference Ehrmann, Bartenschlager and Geldermann1997) reported that the situation in Germany was similar (A1 0·573 of total β-casein for Red-and-White). More recently a study from Holstein-Friesian cows in India showed that A2 β-casein was the main β-casein variant (A2 0·559, A1 0·441 of total β-casein; Sodhi et al. Reference Sodhi, Mukesh, Mishra, Kishore, Prakash, Kapil, Khate, Kataria and Joshi2012) and this was also the case in a study in the Czech Republic (A2 0·550, A1 0·450; Manga & Dvořák, Reference Manga and Dvořák2010). Compared with these studies, the frequency of β-casein A1/A2 alleles in the Chinese Holstein is relatively low, perhaps as a result of natural evolution and crossbreeding. The predominance of β-casein A2 allele (0·880–0·970) was detected by Ehrmann et al. (Reference Ehrmann, Bartenschlager and Geldermann1997) in population of Guernsey cow. The higher frequency of allele A1 was reported by Bech & Kristiansen (Reference Bech and Kristiansen1990) (A1 0·710) for Red breed and Swaissgood (Reference Swaissgood and Fox1992) for Ayrshire breed in the US (A1 0·720).
With the exception of the familiar A1/A2 allele, β-casein protein variant A3 was also found in our study with much lower frequency. However, A3 variant has been observed in the Holstein-Friesian population before, while its frequency of 0·100 in Estonian Holstein cows (Vallas et al. Reference Vallas, Kaart, Varv, Parna, Joudu, Viinalass and Parna2012) shows that it is actually one of the common variants. In most cows, the β-casein alleles present are either A1, A2, or B, with the other alleles being relatively rare and the most common variants are always A1 and A2 (Kamiński et al. Reference Kamiński, Cieoelińska and Kostyra2007).
In conclusion, in this paper we demonstrate genetic variability in the β-casein by analysing genomic DNA sequences that span the bovine β-casein gene. The most frequent β-casein genotype in Chinese Holstein cow was A1A2, with prevalence greater than 35%. Moreover, as a result of DNA-sequence analysis four nucleotide substitutions and restriction-site polymorphisms are identified. Among the five genetic variants characterised, the A2 and A1 variants were found to be predominant over other alleles with a slight superiority of A2 allele. Whether there are definite health effects to milk containing the A1/A2 genetic variants is unknown and requires further investigation. However, the β-casein gene can still be assumed to be a perspective marker for cow breeding and screening of all breeding cows for their A1/A2 variant status would be a promising way to check the flow of undesirable alleles in our breeds. Therefore, our study can promote a better understanding of the genetic background of β-casein in milk and based on these findings it will be essential for us to carry out screening of dairy breeds being used in large scale crossbreeding programme for desirable A2 allele and better milk quality in China.
We thank Dr Yan Zhang for valuable comments to this manuscript.
Supplementary material
The supplementary material for this article can be found at http://dx.doi.org/10.1017/S0022029916000303.
