Characterization of β-casein Gene Sequence Variants in Cholistani Cattle

Ruqayya Bint Khalid1, Asif Nadeem1,2*, Maryam Javed1, Muhammad Zubair Shabbir3 and Masroor Ellahi Babar4 1Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan. 2Department of Biotechnology, Virtual University of Pakistan, Lahore, Pakistan 3Department of Microbiology, University of Veterinary and Animal Sciences, Lahore, Pakistan; 4The University of Agriculture, Dera Ismail Khan, Khyber Pakhtunkhwa, Pakistan Article Information Received 10 June 2020 Revised 03 July 2020 Accepted 07 July 2020 Available online 06 November 2020


INTRODUCTION
A griculture is considered as an important sector of Pakistan's economy. Livestock species play a vital role in country's economic, social and cultural values. It contributes about 58.3% to the agriculture sector and 11.4% to the overall GDP of Pakistan. Within livestock sector, milk is the major commodity. Annual milk production of Pakistan is over 50 billion liters (GOP, 2018). Milk is considered as a complete diet as it is composed of essential micro and macronutrients. Milk is an essential source of nutrients like proteins, carbohydrates and particular micronutrients. It is rich in vitamin B12, vitamin A, riboflavin, folate, and calcium but iron occurs in less amount (Bermudez et al., 2010).
Proteins are one of the most important component of milk, out of which caseins have gained most of the attention due to their recognized association with health related O n l i n e

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peptide, betacasomorphin-7 (BCM-7) De Noni, 2008). Several studies have reported adverse effect of BCM-7 on human health like diabetes type I (Elliott et al., 1999;Laugesen and Elliott, 2003), heart diseases (Laugesen and Elliott, 2003;McLachlan, 2001), sudden death syndrome in infants (SIDS) (Sun et al., 2003), atherosclerosis (Tailford et al., 2003) and autism (Cieślińska et al., 2015). Due to association with several human diseases, A1 has been titled as "Devil in the milk". Prevalence of A1 and A2 variants differ among cattle species. A1 type is common in European cattle milk while A2 variant is secreted in milk of Asian and African cattle (Woodford, 2009). Sequence of animals other than cattle and human β-casein gene shows the presence of A2 variant (Ng-Kwai-Hang and Grosclaude, 2003). Due to adverse effect of A1 variant on human health, screening of β-casein gene status in cattle has gained importance from breeding point of view. While no significant evidence has been reported regarding association of A2 allele with certain diseases, therefore, A2A2 genotype gained importance in breeding programs. Polymorphism in β-casein gene is highly associated with milk performance traits. These variations might help in selection of cattle with better milk quality and yield. Keeping in view the importance of β-casein gene, current study was conducted to molecular characterize the exonic regions of β-casein gene and to explore the status of A1/A2 β-casein type in Cholistani cattle breed of Pakistan.

Sampling
Blood samples of Cholistani cattle breed (n=20), from Government Livestock Farm, Jugait Peer, Bahawalpur, were collected in EDTA vacutainers. Samples were transported in laboratory in ice box. Samples were stored at 4°C to avoid any degradation.

PCR amplification of β-casein
Genomic DNA was extracted from whole blood using standard phenol chloroform extraction protocol (Maniatis, 1989).
Specific primers were designed against exonic regions β-casein gene for PCR, by using Primer3 (version 0.4.0) software. Reference sequence of Bos taurus (NC_037333.1) reported in NCBI database was used for primer designing. For further confirmation and optimization PCR Primer stats (Stothard, 2000), OligoCalc (Kibbe, 2007) were also used. UCSC genome browser (Kent et al., 2002)  . These products were sequenced by using the method of Sanger sequencing. Sequenced data was analyzed with the help of BioEdit software (Hall, 1999). To identify the conserved regions of β-casein protein among all these species, multiple sequence alignment of protein sequences was performed with the help of PRALINE tool (Simossis and Heringa, 2005) by using BLOSUM62 exchange weights matrix, associated gap open penalty of 12 and associated gap extension penalty of 1, respectively. PSI-BLAST iterations were set at 3 at an e-value cut off of 0.01.

Phylogenetic tree construction
NJ (Neighbor-Joining) tree was constructed by using MEGA 6.0 software (Tamura et al., 2013). Model selected for NJ tree construction was maximum composite likelihood model and pair wise deletion was selected for gap/missing data treatment. Phylogenetic relationships among the nucleotide sequences of β-casein of species under investigation were based on genetic distances. Total number of 1000 bootstrap replications were selected to calculate the reliability of tree by bootstrap confidence values, respectively. All of the nucleotide sequences were trimmed to equal length, so that, they all correspond to same regions before the generation of tree.

Estimation of evolutionary divergence between sequences
Analysis for the estimation of evolutionary divergence between sequences were conducted by selecting the maximum composite likelihood model and 1000 bootstrap iterations were selected to get the standard error estimates O n l i n e F i r s t A r t i c l e (Tamura et al., 2004). This analysis involved nine nucleotide sequences. This analysis was performed with the help of MEGA6 software (Tamura et al., 2013).
Protein structure prediction SWISS-MODEL was used for the prediction of 3D structure of β-casein protein. Both wild and mutated protein structures were predicted in order to identify the change that occurred in structure of protein due to amino acid change (Waterhouse et al., 2018).

Physical and chemical properties identification
Physical and chemical parameters of β-casein protein of Cholistani cattle were identified by using ProtParam tool available on ExPASy proteomics tools (Gasteiger et al., 2005).

Statistical data analysis
POPGENE tool (Yeh et al., 1997) was used for calculation and identification of allelic and genotypic frequencies as well as for the calculation of Hardy-Weinberg equilibrium. Allelic and genotypic frequencies significance verification was done with the help of Chi-square test.

RESULTS
Purpose of this study was to molecular characterize the exonic regions of β-casein gene and to explore the status of A1/A2 β-casein type in Cholistani cattle breed of Pakistan. BioEdit software was used for identification of variations in the sequence. Total 9 SNPs were identified including 1 in exonic and 8 in intronic regions of β-casein gene ( Table I). The exonic SNP was non-synonymous as arginine changed to serine (Table II). In this study, A1/A2 allelic polymorphism in cholistani cattle was also being explored. Our results represent the presence of β-casein A2 allele in milk of Cholistani Cattle of Pakistan ( Fig. 1).  Multiple sequence alignment of nine protein sequences was done using PRALINE tool. MKVLILACLVALALARE and QRAVPVQALLLNQE were found to be highly conserved regions among various species (Fig. 2). Phylogenetic tree was constructed with the help of MEGA6 software to find out the evolutionary relationships among species. Figure 3 represents the optimal tree with the sum of branch length 1.90855609. The percentage of replicate trees are shown next to the branches (associated taxa are clustered together in bootstrap test). NJ tree was drawn to scale, having similar units of branch lengths as those of evolutionary distances utilized to deduce the phylogenetic tree. This tree represents that Cholistani cattle is closely related to Bos indicus and Bos taurus.
Pairwise distances were measured by using MEGA6 software. Total number of base substitutions per site from between sequences and standard error estimate(s) above the diagonal have been shown in Table III. Here the lower diagonal represents the average genetic distances between species and upper diagonal shows the standard error estimate(s). Evolutionary divergence estimation indicates that the Cholistani cattle is highly similar to Bos taurus and Bos indicus and it is highly divergent from Mus musculus and Homo sapiens, respectively. As non-synonymous mutations lead to change in amino acid residue. SWISS MODEL was utilized for indication of change in structure of β-casein protein due to amino acid change (Fig. 4).
Physical and chemical parameters of β-casein protein were calculated with the help of ProtParam tool.  (Table V). Wild and mutant genotypic frequencies were calculated and has been shown in Table VI. POPGENE tool was utilized for the calculation of genic variation (Table VII) and heterozygosity of all loci (Table VIII).

DISCUSSION
Milk is a complete food as it contains all the basic nutrients for consumption. However, milk proteins remain the center of controversy in regard their association with human health issues. As several studies have reported the association of BCM-7, bioactive peptide released after proteolytic digestion of A1 milk, with several diseases (Laugesen and Elliott, 2003;McLachlan, 2001;Sun et al., 2003;Tailford et al., 2003). β-casein gene is highly polymorphic and A1/A2 allelic variance of β-casein gene is considered as most important polymorphic variants. This study was conducted to molecular characterize the exonic regions of β-casein gene and to explore the status of A1/A2 β-casein type in Cholistani cattle breed of Pakistan. A total of nine single nucleotide polymorphisms have been identified in β-casein gene of Cholistani cattle breed of Pakistan. Eight identified SNPs were found in intronic region while one was present in exonic region. C allele serves as a genetic marker for A2 variant of β-casein (Caroli et al., 2009). It has been reported that SNP at 85451132 (CSN 3) position, β-casein C allele helps in discrimination of B and C allele from A1 variant (Cecchinato et al., 2014). It was reported that β-casein 85451132 C allele was positively associated with curd firming (Cecchinato et al., 2015). C > G polymorphism has also been found O n l i n e

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at 85451132 (CSN 3) chromosomal position of Cholistani cattle. 85450410 (C>A) have also been reported in Holstein crossbred while studying association between protein composition traits and polymorphisms in milk protein genes and SNPs were significantly associated with milk protein composition traits (Huang et al., 2012).  [Kimura and Crow (1964)]; * I, Shannon's Information index [Lewontin (1972)].  Single amino acid substitution in A2 allele of β-casein gene is one of the most common and abundant form of genetic polymorphism that results in the production of A1 allele. These variations can be helpful for germplasm fingerprinting and population studies. Insilico analysis of β-casein gene indicated that A1 and A2 allele differ only at 67 th position, where histidine amino acid is substituted instead of proline (Jaiswal and Sarsavan, 2013).
Presence of A2 allele in Cholistani cattle of Pakistan can be reported in this study. Similarly, it was reported that local Ladakh cattle in India have higher A2 allelic frequency and exhibit A2A2 genotype which is the source A2 milk (Sharma et al., 2018). A relatively high allelic frequency of A1 β-casein variant was identified in Iranian naive cattle breed (Gholami et al., 2016). Another study on Bangladesh bovine reported high A2A2 genotype, whereas, no A1A1 genotype was found. Heterozygous A1A2 genotype was also present in some breeds. So, it was concluded that A2 β-casein variants were dominant in Bangladesh.
Major milk proteins, including casein, have been found to perform important biological activities. These proteins are involved in the regulation of various health conditions of human beings as well as involved in provision of immunological protection and important nutritive components. Presence of A2 allele was reported in Sahiwal cattle breed, responsible for the prevention of harmful BCM-7 opioid production after enzymatic digestion of milk in stomach. It was also reported that crossbred cattle breed composed of A1 genetic variant while indigenous cattle produce A2 β-casein variant milk (Kumar et al., 2019). A2 β-casein gene variant has also been found in this study.
Various studies have reported that consumption A1 β-casein gene variant milk was correlated with high mortality rate. A2 β-casein variant is good for health as compared to A1 variant (Laugesen and Elliott, 2003). In a study conducted on sahiwal cattle breed of Pakistan population, A2 allelic variance was reported, similar to results of this study (Mir et al., 2014). Whereas, A1A1 and A1A2 genotypes of β-casein gene were detected in Holstein and native cattle breeds (Firouzamandi et al., 2018).
Association of genetic variants of casein and milk serum proteins with milk, protein, and fat production was studied in dairy cattle. Strong association of B allele of αsl-casein gene and A allele of β-casein gene with high protein, fat and milk yields was reported. β-casein variants were found to be correlated with fat percentage as well as fat yield. It was also reported that A3 allele was highly associated with milk production trait (Ng-Kwai-Hang et al., 1984). Polymorphisms in β-casein gene are highly associated with performance traits. It was observed that β-casein gene (as a member of casein cluster) influence the traits regarding milk performance (Singh et al., 2015). A study was designed to find out the effects of genetic polymorphisms of caseins on Swedish O n l i n e

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Red, Danish Holstein, and Danish Jersey cattle breeds. Milk composition differ in each breed significantly. These variations might help in selection of cattle with better milk quality and yield. These might also help in processing of cheese (Gustavsson et al., 2014). Multiple sequence alignment of protein sequences represented highly conserved region MKVLILACLVALALARE. Similar region has been found conserved in another study (Pareek et al., 2012). Phylogenetic analysis revealed the evolutionary relationship of β-casein among Cholistani Cattle_Pakistan, Bos indicus and Bos taurus. Similarly, cattle, goat and sheep formed a clade, as they all belong to same bovidae family. Rabbit and horse gene sequences were somewhat related to this clade but maintained at a distant relationship. These findings are similar with some other studies (Pareek et al., 2012;Vincent et al., 2014).
Physical and chemical analysis of Cholistani cattle protein indicated that this protein is acidic in nature. Aliphatic index represented the stability of proteins at high temperature. Grand average hydropathicity (GRAVY) value of this protein indicated the hydrophilic nature of proteins. This might be helpful to increase the oligomerization as well as functional properties of proteins. Similarly, in another study aliphatic index, GRAVY, and hydrophobicity all suggested αs1-CN as a moderately hydrophobic protein (Huppertz et al., 2018).
Genotypic frequencies calculated in Cholistani Cattle for homozygous A2A2 waere 0.62 and for heterozygous A1A2 was 0.37, respectively. Genetic polymorphisms of β-casein gene were studied in various breeds of cattle. β-casein have been found to constitute approximately 45 percent of bovine milk caseins. A1 and A2 are the most common whereas, B is the less common allele of β-casein gene in dairy cattle. A1 was found to be associated with autism, type I diabetes mellitus and coronary heart diseases whereas, A2 variant was found to be associated with reduction of serum cholesterol. Genotypic frequencies detected for homozygous A1A1 genotype was 0.1261, for A2A2 homozygous 0.5405. For heterozygous A1A2 genotype frequency in Simmental breed frequency was found to be 0.3333. Similarly, 0.1379, 0.4023 and 0.4598 genotypic frequencies for A1A1, A2A2 and A1A2 were detected for Holstein breed and 0.3034, 0.1798 and 0.5168 for Pinzgau breed, respectively. A2 allelic frequency was higher in Holstein and Simmental breeds respectively whereas, with 0.5618 A1 allelic frequency was higher in Pinzgau breed (Miluchová et al., 2014). A2A2 and A2B genotypic frequencies of Sahiwal cattle were 0.14 and 0.86, respectively (Mir et al., 2014).
Heterozygosity statistics for all loci of Cholistani cattle were calculated using Levene's heterozygosity and homozygosity and Nei's (1973) expected heterozygosity. Mean value for Nei's calculation was 0.4430. In another study, genetic variation of β-casein gene was find out. Heterozygosity statistics revealed that Holstein population had 0.4983 Nei's (1973) expected heterozygosity. Sarabi population had 0.4990 and Gaja population had 0.5000 Nei's expected heterozygosity value (Firouzamandi et al. 2018). Genic variation statistics for all loci of Cholistani cattle was calculated using Shannon's information index. Similar method was also utilized in another study finding genetic diversity of β-casein gene in Indian goats (Rout et al., 2010).

CONCLUSION
Hence, sequence characterization has shown that β-casein gene is highly polymorphic with the identification of exonic and intronic single nucleotide polymorphisms. Present study results indicated that A2 β-casein variant is present in Cholistani cattle breed of Pakistan. Phylogenetic analysis revealed the species relatedness that is consistent with well-established evolutionary history. Dairy cattle breeders should start genotyping of all cattle for A2A2 β-casein variants. Selective breeding programs should be improved to gain good quality and quantity milk.

Statement of conflict of interest
The authors have declared no conflict of interest.

O n l i n e F i r s t A r t i c l e O n l i n e F i r s t A r t i c l e
Molecular Characterization of Bovine β-casein Gene