International Journal of Biotechnology and Bioengineering Research. ISSN 2231-1238, Volume 4, Number 3 (2013), pp. 249-256 © Research India Publications http://www.ripublication.com/ ijbbr.htm

Status of β-casein (CSN2) Polymorphism in Frieswal (HF X Sahiwal Crossbred) Cattle

Indrajit Ganguly*1, Sushil Kumar2, G K Gaur3, U Singh4, Arun Kumar5,

Sunil Kumar6, Sandeep Mann7 and Arjava Sharma8

1,8National Bureau of Animal Genetic Resources, Karnal, Haryana-132001, India.

2,4,5,6,7Project Directorate on Cattle, Meerut Cantt., Meerut, Uttar Pradesh–250 001, India.

3Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh-243122, India.

Abstract Cow milk is a rich source of protein and selected micronutrients such as calcium. Recently, a relationship between disease risk and consumption of a specific bovine β-casein fraction either A1 or A2 genetic variants has been identified. Polymorphism at codon 67 of the beta-casein gene: CCT→CAT causes a substitution of amino acid proline (in A2 variant) by histidine (in A1 variant). The presence of histidine residue at codon 67 makes A1 variants exclusively vulnerable to gastrointestinal proteolysis digestion to release bioactive peptide, beta-casomorphin-7 (BCM-7) which has been implicated in potential risk of human cardiovascular disease, type 1 diabetes, atherosclerosis and sudden infant death syndrome. Furthermore, consumption of milk with A2 variant may be associated with less severe symptoms of autism and schizophrenia. Besides, A2 variant has also been reported to have positive relationship with milk production traits especially protein and milk yield. Considering the importance of CSN2 polymorphism with health promoting properties and milk production traits, the present investigation was undertaken to explore A1/A2 polymorphism in crossbred, Frieswal (HF X Sahiwal; N=172) cattle population using allele-specific PCR. All the three possible genotypes (viz., A1A1, A1A2, and A2A2) were identified in Frieswal population. The sequences, specific to A1 and A2 allele, were submitted to GenBank (JQ408387 and JQ408386). The distribution of three genotypes was in Hardy-Weinberg equilibrium [χ2 test (P>0.05)].

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Overall frequency of A2 allele in the population observed to be 0.65. Gene heterozygosity, gene homozygosity, fixation index, polymorphism information content (PIC) were calculated. The observed heterozygosity (0.41) and PIC value (0.351) pointed towards the existence of medium genetic variability in the tested population. Present value of fixation index (0.110) revealed that random mating is being practiced in the population. In view of healthfulness of A2 milk as well as positive relationship of A2 allelic variant with milk performance traits in different cattle breeds, the existing variability of β-casein (A1/A2) locus in Frieswal population may be exploited near future for genetic selection once detail associations study of A1/A2 polymorphism with milk production traits in large number of Frieswal cattle is available. Keywords: Allele-specific PCR; CASB; Milk protein; Polymorphism; Bos taurus x Bos indicus.

1. Introduction Beta-casein is the most polymorphic bovine milk protein gene with 13 known protein variants (Roginski, 2003). The most common forms of beta-casein in dairy cattle breeds are A1 and A2, while B is less common, and A3 and C are rare (Farrell et al. 2004). The nucleotide changes, encoding the amino acid differences between A1, A2, A3 and B variants are located in exon VII region that encodes the major part of the mature protein (Bonsing et al.1988). Polymorphism at codon 67 of the beta-casein gene: CCT→CAT causes a substitution of amino acid proline (A2) by histidine (A1). This difference in amino acid sequence might generate conformational changes in the secondary protein structure and therefore affect physical properties of casein micelles. The presence of histidine residue at codon 67 makes A1 and B variants exclusively vulnerable to gastrointestinal proteolysis digestion to release bioactive peptide, beta-casomorphin-7 (BCM-7) (Cieslinska et al. 2007; De Noni 2008). Bovine BCM-7 has been implicated in potential risk of human ischemic heart diseases (McLachlan, 2001; Laugesen and Elliott, 2003), insulin-dependent diabetes (Elliott et al. 1999; Laugesen and Elliott, 2003) atherosclerosis (Tailford et al. 2003) and sudden infant death syndrome (Sun et al. 2003). In New Zealand, A2 Corporation Ltd. (A2C) has been established in early 2000 to exploit the commercial potential of A2 milk. The company has started large scale marketing of A2 milk as a premium brand (a2TM) and related dairy products in New Zealand, Australia, Asia and United States (http://www.a2corporation.com). Besides health promoting properties, the A2 variant has also been reported to have positive relationship with milk production traits especially protein and milk yield (Winkelman and Wickham, 1997; Ikonen et al. 1999; Ikonen et al. 2001; Olenski et al. 2010), whereas A1 variant shows the opposite influence (Freyer et al. 1999; Lipkin et al. 2008). In India, predominance of A2 allelic

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variant has been observed in 15 zebu cattle breeds and 8 breeds of riverine buffaloes (Mishra et al. 2009). All the breeds were reported to carry fixed A2 allele, except two cattle breeds (Malnad Gidda and Kherigarh) were found to carry A1 allele, mostly in A1/A2 heterozygous condition, with a frequency of 0.096 and 0.109, respectively. The mean frequency of A2A2 and A1A2 genotypes for all Bos indicus cattle breeds was 0.974 and 0.026, respectively while the mean A1 and A2 allele frequency was 0.013 and 0.987 (Mishra et al. 2009). India is the largest milk producer (112 MMT, year 2009-10; http://dahd.nic.in) in the world where cattle contributes to about 43% of total milk; out of which 20% is produced by indigenous cows and 23% by crossbreds and exotic cows. Although the entire crossbred populations (33 million) is only 16.5% of the total cattle population (199 million) in India, however, their contribution is appreciable in terms of milk production. Moreover, Country’s crossbred population is increasing very rapidly (18th Livestock census 2007, http://dahd.nic.in). Since crossbred’s milk constitutes a major part of total milk produced in India, understanding the status of A1/A2 CSN2 polymorphism, association of said polymorphism with other milk production traits as well as screening of crossbred AI bulls is of paramount importance to minimize the risk of A1 milk. Frieswal is a crossbred population which is being developed by crossing Holstein Friesian and Sahiwal breeds at Military Dairy Farms of India in order to evolve a National dairy breed capable of producing 4000 kg milk in a mature cow with a lactation of 300 days with 4% butter fat. The population has undergone seven generations of interbreeding and reached to the level of 3550 kg in 4th lactation. Currently, there are more than 17,000 Frieswal females at all the Military dairy farms. The present study was undertaken to explore the status of CSN2-A1/A2 polymorphism in crossbred, Frieswal cattle and to assess its association with milk production traits. 2. Materials and Methods A total of 172 Frieswal animals consisting of 124 Frieswal heifers and 48 Frieswal bulls were included in this study. Genomic DNA was isolated from the venous blood using standard phenol chloroform extraction method (Sambrook et al. 1989). Primers were designed using Primer 3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). The genomic specificity of the primers was tested using the Primer-BLAST program (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). Allele-specific PCR was carried out using forward primers with either A (IGBhF 5’ CTT CCC TGG GCC CAT CCA 3’) or C (IGBpF 5’ CTT CCC TGG GCC CAT CCC 3’) at the 3′ end a common reverse primer (IGBR 5’ AGA CTG GAG CAG AGG CAG AG 3’) to amplify a 244 bp fragment (Figure 1a) (Ganguly et al., 2013). Primer pairs IGBhF-IGBR and IGBpF-IGBR were intended to give rise to Histidine (A1) and Proline (A2) specific amplicon, respectively. PCR was carried out from a starting template of approximately 50 ng of genomic DNA in a final reaction volume of 25 μl containing 1X Taq DNA polymerase buffer (Sigma), 1.5 mM MgCl2 (Sigma), 200 μM dNTPs (Sigma), 0.5 μM of each primer and 1U Taq polymerase (Sigma). PCR

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conditions were: initial denaturation at 94 0C for 5 min; followed by 5 cycles of 94 0C for 30 s, 66 0C for 30 s and 72 0C for 30 s; thereafter 30 cycles of 94 0C for 30 s, 64 0C for 30 s and 72 0C for 30 s and a final extension 72 0C for 5 min. PCR products were visualized in 1.5% agarose gel. Further, to validate genotypes, a pair of primer (A1 A2 Seq F: 5’ CCA GGA TAA AAT CCA CCC CT 3’; A1 A2 Seq R: 5’ AGG GAA GGG CAT TTC TTT GT 3’) was designed to amplify a 202 bp fragment of exon VII encompassing the polymorphic site (Ganguly et al., 2013). Representative amplicons were sequenced directly using automated DNA sequencer. 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. The population genetic indices i.e., gene heterozygosity (He), gene homozygosity (HO), and fixation index (FIS) were calculated by Popgene 32 software version 1.32 (Yeh et al. 2000). Polymorphism information content (PIC) was calculated according to Botstein et al. (1980). For associations between genotypes and milk production traits, all the heifers are being followed for recording of fat%, protein % and 305 days milk yields.

3. Results and Discussion The role of milk proteins in relation to human health remains controversial. However, it is evident from the previous studies that the A1 beta-casein is associated with a range of illnesses by preferentially releasing an opioid peptide called beta-casomorphin-7 (BCM-7) upon digestion (McLachlan, 2001; Laugesen and Elliott, 2003; Tailford et al .2003; Sun et al. 2003). In the present investigation an allele-specific PCR (AS-PCR) was utilized to explore A1/A2 polymorphism in crossbred Frieswal (HF X Sahiwal) cattle. All the three genoypes viz., A1A1, A1A2 and A2A2 were identified in Frieswal population by this technique (Figure. 1b). The genotypes were further confirmed by direct nucleotide sequencing (Figure. 1c).

The sequences, specific to A1 and A2 allele, were submitted to GenBank (JQ408387 and JQ408386). In Frieswal cattle population overall genotype frequencies were found to be 0.15 (A1A1), 0.41 (A1A2) and 0.44 (A2A2). The frequency of A2 and A1 allele was 0.65 and 0.35, respectively. The distribution of the genotypes was within Hardy–Weinberg equilibrium in the tested population (P>0.05). The value of gene homozygosity (Ho), gene heterozygosity (He), polymorphism information content (PIC) and fixation index (FIS) were found to be 0.59, 0.41, 0.351 and 0.110, respectively. The results revealed that Frieswal population harbour intermediate homozygosity and PIC values. Polymorphism information content, an indicative 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 0.351 indicating medium genetic diversity. Fixation index (FIS), a measure of deviation of genotypic frequencies from panmictic frequencies in terms of heterozygous deficiency or excess. Negative FIS values indicate heterozygote excess in out breeding and positive values indicate heterozygote deficiency in inbreeding compared with Hardy–Weinberg

Status of β-casein (CSN2) Polymorphism in Frieswal (HF X Sahiwal Crossbred) 253

equilibrium expectations. The present value of FIS in Frieswal (0.110) indicates that random mating is being practiced in the population. The frequency of A1 allele in different breeds varies 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) as reviewed by Kaminski et al. (2007). Recently, the frequencies of A1 (0.35) and A2 (0.65) alleles from Polish Holstein–Friesian bulls (Olenski et al., 2010) reported to be very similar to what we observed in Frieswal population. At present, the number of heifers, completed first lactation milk yield, is too small to be able to find beta-casein allele association with production traits. Ongoing research is continued to have sufficient records for association study shortly. Positive relationship of A2 variant with milk production traits especially with protein and milk yield has been observed in various cattle breeds (Ikonen et al. 1999; Ikonen et al. 2001; Winkelman and Wickham, 1997; Olenski et al. 2010). Alternatively, Lipkin et al. (2008) observed negative influences of A1 variant on production traits. 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).

Figure 1: Results of allele-specific PCR and sequencing confirmation of genotypes a.

Schematic representation of amplification of A1 and A2 beta-casein (CSN2) allelic variants, b. Three animals with genotypes A2A2, A1A2 and A1A1 are shown in the

photograph. Lane 1,8: Marker (M); Lane 2, 4, 6: A1-specific PCR product; Lane 3, 5, 7: A2-specific PCR product, c. Chromatograms of beta-casein (CSN2) genotypes

A2A2, A1A2, and A1A1. Presence of two peaks at SNP site (arrow head) indicates hetrozygosity of the sample.

4. Conclusion Frieswal cattle were genotyped for the β-casein locus by allele specific PCR. Distribution of all the three genotypes (viz., A1A1, A1A2, and A2A2) was in Hardy-Weinberg Equilibrium. A medium genetic diversity and the practice of random mating

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were recorded in the population under study. The existing genetic variability in β-casein (A1/A2) locus of Frieswal population can be exploited for genetic selection to minimize the negative effect of A1 milk. However, detail association study of A1/A2 polymorphism with milk production traits in large number of Frieswal cattle is underway prior to reach some meaningful conclusion.

5. Acknowledgement The authors are grateful to Director, Project Directorate on Cattle and Director, Frieswal Project, Meerut for providing necessary facilities to carry out the present investigation.

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