Detection of A1 and A2 Alleles at Beta-casein Locus in Bargur and Umblachery (Indian Zebu) Cattle Breeds by Allele-specific PCR

Caseins and whey proteins are the major proteins of bovine milk. Among these, caseins account for 80% of milk proteins which is secreted by cells of the mammary glands (Sulimova et al., 2007). Among casein proteins, beta-casein constitutes about 25 – 30% of total milk proteins and comprises of 209 amino acids with a molecular weight of 24 kD (Keating et al., 2008). Bovine casein milk proteins are synthesized by four genes, alpha s1, beta, alpha s2 and kappa (CSN1S1, CSN2, CSN1S2 and CSN3 respectively) (Farrell et al., 2004). The four casein genes were mapped to bovine chromosome 6 in the order of CSN1S1-CSN2-CSN1S2-CSN3 (Ferretti et al., 1990; Threadgill and Womack, 1990).

Many genetic variants of beta-casein in different breeds of cattle have been reported including A1, A2, A3, B, C, D, E, F, G, H1, H2 and I. The A1 and A2 are the most common variants, B variant is less common and A3 and C variants are rare (Jaiswal et al., 2014; Sharma et al., 2013). Each variant is differing from other variants in amino acid substitution at a fixed position. In polypeptide chain position 67, His is substituted by Pro, for A1 and A2, respectively.

It was shown that A1 beta casein produces betacasomorphin-7 or BMC-7 during the digestion of raw or processed milk or cheese in the human gut and BMC-7 may be a significant riskfactor in human ischemic heart disease, arteriosclerosis, type I diabetes and sudden infant death syndrome (Elliot et al., 1999; Thorsdottir et al., 2000; McLachlan, 2001; Sun et al., 2003; Tailford, 2003).

Lipkin et al., (2008) observed negative influences of A1 variant on production traits. Instead; it was shown that A2 variant has positive relationship with milk performance traits such as milk protein and milk yield apart from health promoting properties (Ikonen et al., 1999; Ikonen et al., 2001; Olenski et al., 2010). Moreover, the level of BCM-7 in beta-casein A1 hydrolysed milk is reported to be four times higher than the A2 milk (Cieslinska et al., 2007). However, the overall evidence for gastrointestinal effects from A1 and BCM-7 in animal and in vitro studies is conclusive, but the evidence from human studies is still limited and the clinical implication of A1 milk on human health is still under discussion.

An attempt was made to study the frequency of A1 and A2 alleles at beta-casein locus in Bargur and Umblachery cattle of Tamil Nadu, India. This is the first report on the status of the beta-casein alleles in these breeds. Bargur cattle are medium sized animals; they are usually red coloured with white patches. These are bred in the Bargur hills of Western Ghats in Erode district of Tamil Nadu (Ganapathi et al., 2009). Umblachery animals are short and grey coloured. These are bred in Thiruvarur and Nagapattinam districts of Tamil Nadu (Rajendran et al., 2008).

This study was carried out on Bargur and Umblachery breeds of cattle of Tamil Nadu, India. The blood samples were collected from the randomly selected animals in the home tract of respective breeds of cattle. In the present study, a total of 48 Bargur and 42 Umblachery animals were screened for A1 and A2 alleleic variants in bovine beta–casein (CSN2) gene.

Sample collection and DNA extraction

Blood sample (5-10 ml) was aseptically collected from each animal in an EDTA containing vacutainer tube by jugular vein puncture and transported to laboratory at 4°C. Genomic DNA was isolated from the blood using standard phenol chloroform extraction method. Nanodrop (Thermoscientific) was used to assess the quality and quantity of DNA. Samples showing an optical density ratio (260 nm / 280 nm) of between 1.8-2.0 were diluted to 50 ng / µl and stored at -20°C.

Allele specific PCR

Allele specific PCR (AS-PCR) was carried out as reported by Ganguly et al., (2013). AS-PCR was carried by using the forward primers, IGBhF (5'-CTTCCCTGGGCCCATCCA-3'), IGBpF (5'-CTTCCCTGGGCCGATCCC-3') and common reverse primer, IGBR (5'-AGACTGGAGCAGAGGCAGAG-3'). For each sample, two separate reactions were set with primer pairs, IGBhF and IGBR to amplify the 244 bp A1 variant (CCA-Histidine) and primer pairs IGBpF and IGBR were used to amplify the 244 bp A2 variant (CCC – Proline). AS-PCR was carried out in a total volume of 20 µl contained 50 ng of DNA template, 10 µl Taq DNA polymerase 2x master mix (Ampliqon), 1µl each of forward and reverse primers (10 pmoles each) and nuclease free water to final volume of 20 µl. PCR was carried out in a T100 thermal cycler (BioRad) with cyclic conditions of initial denaturation at 94°C for 5 min, followed by 5 cycles of 94°C for 30 sec, 64°C for 30 sec and 72°C for 30 sec; thereafter 30 cycles of  94°C for 30 sec, 62°C for 30 sec and 72°C for 30 sec and a final extension of 72°C for 5 min. PCR products were visualized after electrophoresis  in a 2% agarose gel (Fig 1).

Fig 1: Agarose gel electrophoresis of AS-PCR products of beta-casein genotypes with 244 bp. Lane M: Marker (100 bp DNA ladder). Lane 1, 3, 5, 7-A1 specific PCR. Lane 2, 4, 6, 8-A2 specific PCR. Lanes 1 and 2 demonstrate A2A2 genotype; lanes 3 and 4 indicate A2A2 genotype; lanes 5 and 6 show A1A2 genotype; lanes 7 and 8 shows A2A2 genotype.

The gene and genotype frequencies were calculated as per Falconer (1991). Effectiveness of allele incidence was evaluated using theoretical heterozygosity (Heexp) as per Nei (1973), experimental heterozygosity (Heobs), expected homozygosity (E), effective number of alleles (ENA), level of possible variability realization (V%). as per Crow and Kimura (1970). Polymorphism information content value (PIC) was estimated according to Botstein et al., (1980).

The genotype and gene frequencies of beta - casein A1A2 in Bargur and Umblachery cattle are presented in Table 1. Only two types of genotypes i.e. A1A2 and A2A2 were observed in both the breeds (Fig 1). We detected a predominance of the A2A2 genotype; a lower frequency of the A1A2 genotype and absence of the A1A1 genotype.

Table 1: Genotype and gene frequencies of beta-casein (CSN2) gene in Bargur and Umblachery cattle.

Among the 48 Bargur animals screened, 42 animals were of A2A2 genotype and the 6 animals were of  A1A2 genotype. The genotype frequencies of A1A1, A1A2 and A2A2 were 0.000, 0.125 and 0.875 respectively in Bargur breed. The frequency of allele A2 was very high in the population and amounted to 0.937. The frequency of allele A1 was 0.063 in Bargur breed.

Out of 42 Umblachery cattle, 40 animals were of A2A2 genotype and 2 animals were having A1A2 genotype (Fig 1). The genotype frequencies of A1A2 and A2A2 were 0.048 and 0.952 respectively in Umblachery breed. The frequencies of A1 and A2 alleles were 0.024 and 0.976 respectively in Umblachery breed. Hence, this study revealed the presence of A1 allele in low frequency in purebred Umblachery and Bargur cattle. The present results showed predominance of A2A2 genotype and A2 allele in both the breeds. The results of our study are compatible with the results of Mishra et al., (2009) who studied the b- casein variants among 15 Indian cattle breeds and reported the frequencies of A2 allele as 0.904 in Malnad Gidda, 0.891 in Kherigarh and fixation of allele in Kangayam, Nimari, Red Kandhari, Malvi, Amritmahal, Kankrej, Gir, Sahiwal, Hariana, Tharparkar, Rathi, Mewathi and Red Sindhi. They reported the absence of A1A1 genotype among the investigated breeds and also near fixation of A2 allele in Indian cattle breeds except for very low frequency of A1A2 in Malnad Gidda and Kherigarh cattle. They have further estimated the mean frequencies of A1 and A2 alleles among these 15 breeds as 0.013 and 0.987 respectively. The predominance of A2 allele had also been reported in Sahiwal, i.e., 0.93 in Sahiwal (Mir et al., 2014) and 0.94 in Ongole (Ganguly et al., 2013). Malarmathi et al., (2014) did not find any A1 allele and reported that A2 allele had been fixed in Kangayam breed of cattle, one of the four recognised breeds of cattle of Tamil Nadu state of India.  Ramesha et al., (2016) screened the various breeds of cattle of India and reported the frequencies of A2 allele as 0.986 in Malnad Gidda, 0.958 in Kasargod cattle and 1 (fixed) in Deoni and Khillar cattle breeds. Kumar et al., (2018) also reported the abundancy of A2 allele in Sahiwal cattle and the frequencies of A1 and A2 alleles were 0.06 and 0.94 respectively with absence of A1A1 genotype in the population they studied. Genotype frequencies for A1A2 and A2A2 genotypes were 0.13 and 0.87 in Sahiwal cattle. The frequency of A1 allele in Bargur and Umblachery breeds is in contrast to the allelic frequency of A1 allele, ranging from 0.01 to 0.72 throughout the world (Kaminsky et al., 2006). If the beneficial property of the A2 milk is proved beyond the doubt, it can be commercially exploited and utilised for conservation of Bargur cattle, which has become endangered (Ganapathi et al., 2009) and Umblachery cattle, the population size of which is declining at a faster rate.

Effectiveness of alleles

The values of experimental heterozygosity (Hobs), theoretical heterozygosity (Hexp), polymorphism information content (PIC), expected homozygosity (E), effective number of alleles (ENA), level of possible variability realization (V%) were 0.125, 0.1163, 0.1095,0.8837, 1.131 and 11.88 respectively in Bargur population. These values were 0.048, 0.0468, 0.0458, 0.9532, 1.049 and 4.79 respectively in Umblachery population (Table 2). In the Bargur cattle, the bovine CSN2 gene showed a high proportion of A2A2 ho­mozygosity (87.5%), as described with the high value of the coefficient of homozygosity (0.8837). In the Umblachery cattle, the same locus showed a highest proportion of A2A2 homozygosity (95.2%) as described with the highest value of coefficient of homozygosity (0.9532). Effectiveness of alleles in a population can be described with the effective number of alleles. In a bi-allelic system, a limit of 2.0 indicates that both alleles are effectively involved in the development of genotypes. These results revealed that both the breeds have lower amount of heterozygosity. In this study, the ENA decreased to 1.131 in Bargur and 1.049 in Umblachery cattle, showing that the effect of alleles A1 and A2 is not balanced in both the breeds. Polymorphism information content, an indicative of degree of informativeness of a marker and genetic diversity ranges from 0 to 1. In present investigation, the PIC values (0.1095 and 0.0458) were substantially lower than a threshold value (0.5), also indicating a low polymorphic level and thereby low genetic diversity. The low level of polymorphism caused a decrease in a level of possible variability realization (11.88 and 4.79%).

Table 2: Effectiveness of alleles for beta - casein (CSN2) gene in Bargur and Umblachery cattle.