Analysis of beta-casein gene (CSN2) polymorphism in Tharparkar and Frieswal cattle

Cow’s milk plays an important role in human health. In milk, total proteins content is around 3.5% (Miller et al., 2007) and the major protein groups are represented through casein and whey proteins (ß-lactoglobulin and alpha-lactalbumin), accounting for about 80% and 20% respectively (Hoffman and Falvo, 2004).
       
The casein is a family of phosphoprotein synthesized in the mammary glands in response to lactogenic hormone and various other stimuli. These are secreted as large colloidal aggregates termed as micelles, which are responsible for the physical property of the milk (Swiasgood, 1992). Bovine milk contains four major casein groups, namely, alpha s1-, alpha s2, beta- and kappa-casein accounting for 38, 10, 36 and 13% of total proteins in milk, respectively (Sulimova et al., 2007). Bovine beta casein (CASB) gene is located on chromosome number 6 (Roginsky, 2003). Among caseins, the beta-casein (CSN2) is the most polymorphic milk protein gene and consists of 209 amino acids (Keating et al., 2008). There are 13 different types of genetic variants of beta-casein in different breeds of cattle including A1, A2, A3, A4, B, C, D, E, F, H1, H2, I and G. The A1 and A2 variants are the most common forms, the B variant is less common and the A3 and C variants are rare (Farrell et al., 2004). These changes in nucleotide, encoding the amino acid differences between A1, A2, A3 and B variants are situated in exon VII region that codes the major part of the protein (Bonsing et al., 1988). The difference between A1 and A2 beta casein variants is replacement of  a cytosine (C) nucleotide with an adenine (A) nucleotide in the beta casein gene results in replacement of histidine in A1 variant with proline amino acid at position 67^th of the beta-casein chain in A2 variant (Jaiswal et al., 2014). These changes in amino acid sequence produced conformational changes in the secondary protein structure and therefore affect the physical properties of casein micelles. Histidine found in A1 milk has a very weak bond, which is easily broken during enzymatic hydrolysis in gastrointestinal tract, to release bioactive peptide beta-casomorphin-7 (BCM7), an important bioactive peptide with strong opioid (Morphine) like activity; while prolein found in A2 milk has very strong bond, which prevents the hydrolysis of peptide bond between residues 66th and 67th in the β-casein and hence, inhibits production of BCM-7 (Sharma et al., 2013). The presence of BCM-7 in milk is associated with many diseases including human ischemic heart disease, sudden infant death syndrome, schizophrenia, autism, type 1 diabetes and milk intolerance (Kamiñski et al., 2006).
       
Due to contradictory role of A1 and A2 beta-casein variants on human health, the present study was undertaken to determine the frequency of these variants in Tharparkar and Frieswal (HF X Sahiwal) cattle.

Experimental animals
 
A total of 181 lactating cattle were screened for genetic variants A1 and A2 of beta-casein gene (CSN2). Tharparkar (N=81) cattle, maintained at Cattle and Buffalo Farm, IVRI, Izatnagar and Frieswal (N=100) cattle, maintained at Military Farm, Bareilly, were utilized for the present study.
 
Sample collection and DNA isolation
 
8-10 ml of blood sample was collected aseptically from jugular vein in a vacutainer tube containing 0.5 per cent EDTA. The samples were stored at 4°C prior to DNA isolation. The genomic DNA was extracted from whole blood within 24 hours of collection of blood sample using phenol chloroform extraction method as described by Sambrook and Russel (2001).
 
Evaluation of quality and concentration of DNA
 
Genomic DNA quality was checked to ensure the presence of intact DNA by running the DNA on 0.8% agarose gel. The purity of the genomic DNA was assessed by UV spectrophotometer by checking the optical density (OD) value at 260 and 280 nm. The samples having OD ratio (260 nm/ 280 nm) ranging from 1.8 to 2.0 were used for further experiment. The concentration of DNA was calculated by using the following formula: DNA concentration (µg/µl) = OD260 x (Dilution factor) x 50/ 1000. Finally, DNA was diluted in distilled water at the concentration of 100 ng / µL make working solution for the PCR reaction 2 µl diluted DNA (approximately 200 ng) was used.
 
PCR-RFLP Technique
 
The technique involved in study was Polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP). The created restriction site in PCR-RFLP technique as described by McLachlan (2006) was used for screening. The forward primer 5'- CCT TCT TTC CAG GAT GAA CTC CAG G-3' and reverse primer 5'-GAG TAA GAG GAG GGA TGT TTT GTG GGA GGC TCT-3', was used to amplify a 121 bp fragments in beta casein gene. Briefly, PCR amplification was performed in a reaction volume of 25 μl containing 0.20µl Taq polymerase (Fermentas), 2.5 μl 10X PCR buffer, 0.5 μl dNTP, 1µl of each primers, 2 µl DNA and 17.8 µl nuclease Free Water. The following PCR cycle condition were applied: an initial denaturation at 95°C for 5 minutes, 35 cycles of denaturation at 95°C for 30 seconds, annealing at 58°C for 45 seconds, elongation at 72°C for 45 second and a final extension at 72°C for 4 minutes. After PCR, the amplified products were analyzed by loading on 1.5% agarose gel containing Sybr Safe DNA gel staining (Thermo Fisher Scientific, USA) and running in 1 X TBE buffer at 100 V for 30 minutes along with a 50 bp DNA marker. The gel was visualized and documented using gel documentation system (Gel doc 1000, Bio-Red, USA). Further the PCR products were digested using 2 units of Dde I restriction enzyme (Fermentas) at 37°C for 16 hours in the incubator to liberate the restriction fragments. After incubation the digested products were loaded on 3% agarose gel (Invitrogen) containing 1 × TBE buffer along with 50 bp DNA marker at 120 V for 5 min, followed by 90 V for 2 hours. Then gel was examined for different band pattern under UV trans-illuminator and documented using gel documentation system to record the result.
 
Statistical analysis
 
The gene (allele) and genotype frequencies were calculated by simple frequency calculations (Falconer and Mackay, 1996). The deviation of genotypic frequencies from expectations (Hardy–Weinberg equilibrium) was analyzed using Chi-square test of significance as described by Snedecor and Cochran (1994) considering the allelic frequencies in a 2×2 table. The population genetic indices were evaluated by following parameters: theoretical gene heterozygosity (He exp), experimental gene homozygosity (HO obs), fixation index (FIS), polymorphism information content (PIC), expected homozygosity (E), effective number of alleles (ENA) and level of possible variability realisation (V %).
 
Effective number of alleles (ENA) (Kimura and Crow, 1964)
The effective number of alleles (ne) is the reciprocal of the sum of the square of allele frequencies.

                                                               
Where Pi²=p²+q²
 
Experimental heterozygosity (He) (Nei, 1978)
Heterozygosity is the state of possessing different alleles at a given locus in regard to a given character. It is a measure of heterozygosity or genetic variation in a population. The population heterozygosity at a locus is given by formula:

H=1-ΣPi²

 
Expected heterozygosity
 
The expected heterozygosity per locus (E) is defined as the mean of heterozygosity over all structural loci in the genome.

 
                                E = ΣPi²

 
However, the unbiased estimate of expected heterozygosity at a locus is (if N <50): N is the sample size

 
 

 
Polymorphism information content (PIC)
 
PIC is the measure of in formativeness of marker and the PIC value for each locus was calculated according to Boltstein et al., (1980): using the formula:

 

 
Level of possible variability realization (V %) (Crow and Kimura., 1970)

                                                                               
F-statistics and Gene flow (Slatkin and Barton, 1989)
F-statistic can be determined from the ratio of observed to expected heterozygosity

                               

 
Where Hs is the average expected heterozygosity estimated from each subpopulation

 
                                HS = 1-ΣPi²

 
And Hi is the average observed heterozygosity
 
 
For K sub populations.
Negative FIS values indicate heterozygote excess in out breeding and positive values indicate heterozygote deficiency in inbreeding compared with Hardy–Weinberg equilibrium expectations.

Two cattle breeds (i.e. Tharparkar and Frieswal) were screened for beta casein gene variants, A1 and A2. A2A2 genotype showed the product size of 86 bp and 35 bp while A1A2 genotype showed the product size of 121 bp, 86 bp and 35 bp. A1A1 genotype, which is expected to show the product size of 121 bp. Among 81 Tharparkar cattle screened, 74 animals were of A2A2 and 7 animals were of A1A2 genotype, while none of the animal was of A1A1 genotype. In 100 Frieswal cattle all 3 types of genotypes were observed, 16 animals were of A1A1, 35 animals were of A1A2 and 49 animals were of A2A2 genotype, respectively. Genetic variants of beta casein observed in cattle breeds are shown in Fig 1. The genotypic frequencies and allele frequencies of A1 A2 variants in both the cattle breeds are shown in Table 1.
 

       
It was observed that A2 allele is prominent in Tharparkar cattle with very low frequency of A1 allele.In the present study, Tharparkar cattle showed A1 allele frequency as 0.04, indicating very high frequency of A2 variants.  Earlier reports of screening in Tharparkar cattle also showed similar allelic frequency of 0.05 and 0.95 for A1 and A2 allele respectively (Kumar et al., 2018). The study on other indigenous breeds of cattle showed similar frequency of A2 allele viz., 0.94 in Ongole), 0.93 in Sahiwal and 0.94 in Gir cattle breeds, (Ganguly et al., 2013, Mir et al., 2014 and Kumar et al., 2018). Consistent with the present finding, Ramesha et al., (2016) had also reported very low frequency of A1 allele in Malnad Gidda (0.014) and Kasargode (0.042) cattle. Various study on Tharparkar, Kangayam, Deoni and Khillari cattle showed fixation of A2 allele (Mishra et al., 2009; Malarmathi et al., 2014 and Ramesha et al., 2016). In contrast Muhammed et al., (2012) observed high frequency of A1 allele in indigenous Vechur (0.34) and Kasargode (0.79) cattle.
       
In Frieswal cattle, the genotype frequencies of A1A1, A1A2 and A2A2 was found to be 0.17, 0.39 and 0.44, respectively. The frequency of A2 and A1 allele was 0.63 and 0.37, respectively. The frequency of A1 allele (0.37) observed in the present study was in accordance with Ganguly et al., (2013), who reported frequency of 0.32 and 0.44 in Frieswal heifers and bulls respectively. Similar to the results observed in this study Malarmathi et al., (2014) and Muhammed et al., (2012) showed allele frequency of 0.40 and 0.46 respectively in Indian crossbred cattle. However, Jaiswal et al., (2014) reported comparatively low frequency of A1 variants (0.17) in Karan fries cross bred cattle. Olenski et al., (2010) reported frequencies of A1 (0.35) and A2 (0.65) alleles in Polish Holstein–Friesian bulls and similar to what we observed in Frieswal population. The A1 allele frequency in different breeds of exotic cattle had been reported between 0.01-0.06 (Guernsey), 0.09-0.22 (Jersey), 0.31–0.66 (Holstein), 0.43-0.72 (Ayrshire) and 0.71 (Danish Red) (Kaminski et al., 2007).
       
The distribution of the genotypes was within Hardy-Weinberg equilibrium in the tested Tharparkar population (P>0.05). The value of theoretical gene heterozygosity (He exp), experimental gene homozygosity (HO obs), fixation index (FIS), polymorphism information content (PIC), expected homozygosity (E), effective number of alleles (ENA) and level of possible variability realisation (V %) are shown in Table 2. The results revealed that Tharparkar cattle population harbor low homozygosity and PIC values while Frieswal population harbour intermediate values. Polymorphism information content, an indicator of degree of informativeness of a marker and genetic diversity ranges from 0 to 1. In present investigation, the value of PIC was found to be 0.0739 in Tharparkar cattle indicating low genetic diversity while PIC value in Frieswal cattle (0.357) indicating medium genetic diversity. The present value of FIS in Tharparkar (0.9885) and Frieswal (0.991) cattle population indicates that random mating is being practiced in both the population.
 

From this investigation, we can infer that the nearly fixed A2 allele in Tharparkar indigenous cattle population (0.96) can be associated with better human health in the country. The moderate A1 allele frequency (0.37) in Frieswal cattle can be reduced with proper breeding policy. If a cow is of A2A2 genotype, she/he is guaranteed to pass on the A2 allele to their progeny. Similarly, an A1A1 cow/bull is guaranteed to pass on the A1 allele.