β casein Polymorphism in Indigenous and Exotic Cattle Breeds of Pakistan

Shakila Mumtaz1, Khalid Javed1, Muhammad Dawood1*, Muhammad Imran2, Asad Ali1 and Nazia Ramzan1 1Department of Livestock Production, University of Veterinary and Animal Sciences, Ravi Campus Pattoki, Pakistan 2Institute of Biochemistry and Biotechnology, University of Veterinary and Animal Sciences, Lahore, Pakistan Article Information Received 07 March 2019 Revised 21 July 2019 Accepted 15 December 2020 Available online 31 May 2021

Milk composition depends largely on the breed's genetics and ration fed. Different proteins can be found in milk. Beta-caseins are thought to be more important because some serious health-related issues in humans have been reported with the consumption of A1 milk (mutated casein variant). This study was planned to investigate the polymorphism in the beta-casein gene (CSN2) in Sahiwal (40), American Holstein Friesian (40) and the crossbred (Sahiwal × HF) (50). PCR-RFLP and conformational sequencing were performed to investigate the beta-casein polymorphism. Results of the present study showed that there was not any mutated genotype (A1A1) available in all of the three breeds. All three breeds possess dominant genotype A2A2 with genotypic frequency (0.925, 0.4, 0.64) respectively and we have also found some heterozygous genotypes A1A2 in all breeds with genotypic frequency (0.075, 0.6, 0.36) respectively. Findings of the present study revealed that A2 (the dominant allele) is present with a pretty much higher frequency (0.9625, 0.7, 0.82) in all the three studied breeds and the mutated allele A1 is present with very low allelic frequency (0.037, 0.3, 0.18). M ilk proteins constitute 3% of the total chemical components of milk. Caseins constitute 80% of total milk proteins (Ho et al., 2014). Three major types of caseins are found in milk which include alpha, beta and kappa casein. Beta-caseins are the most important proteins found in milk and share the major part about 30% of the milk protein (Ho et al., 2014;Malarmathi et al., 2014;Boro et al., 2016). There are twelve different genetic forms of beta-casein available, among those two A1 and A2 are more common and have an important role regarding milk production and the human health aspect (Farrell et al., 2004). In cattle, the dominant variant for beta-casein protein was A2 beta-casein. Many years ago, a natural mutation appeared in cows from European breeds which have resulted in another casein variant A1 beta-casein and producing A1 milk (Boro et al., 2016). Casein protein is composed of 209 amino acids and a change at 67 th amino acid (proline to histidine) can cause A2 milk to A1 milk. Proline (CCT) is responsible for A2 milk and histidine (CAT) is responsible for A1 milk Ho et al., 2014;Singh et al., 2015). Some other variants like B, C, D, E, F, G, H1, H2, and I are also found but are not as importance as A1 and A2 (Farrell et al., 2004).
Beta-casein variants A1 is responsible for causing serious health problems for human such as ischemic heart disease, arteriosclerosis, type 1 diabetes (DM-I) and unexpected infant death syndrome (Laugesen and Elliott, 2003;Birgisdottir et al., 2006;Caroli et al., 2009;Massella et al., 2017). Currently increased consumption of dairy products has been considered to be associated with serious health risks and worst symptoms of some disorders like gastrointestinal malfunction, immunity related issues and heart diseases ( Barnett et al., 2014;. Some of these effects are linked with beta-casomorphin-7, a peptide associated with A1 milk consumption. Betacasomorphin-7(BCM-7) adversely affects the digestibility, reduces the production of lymphocytes and ultimately affects the immunity (Trompette et al., 2003;Zoghbi et al., 2005). The present study aims at investigating the betacasein polymorphism in indigenous cattle Sahiwal, exotic breed American Holstein Friesian (HF) and crossbred (Sahiwal × HF). This study will help in selection of superior milk and breed improvement programs.

O n l i n e F i r s t A r t i c l e
Materials and methods Whole blood samples (5 ml each) were collected from three cattle breeds-Sahiwal (n=40), HF (n=40) and Crossbred (Sahiwal x HF) (n=50). Sampling from Sahiwal cattle and Holstein Friesian (HF) was done at two Research and Training Farms at the University of Veterinary and Animal Sciences, Ravi Campus, whereas sampling from crossbred was done at Livestock Experiment Station, Qadirabad, Sahiwal. All unrelated animals were used for this study. Bovine gDNA was extracted using a standard protocol (Grimberg et al., 1989). PCR product was amplified using the following reported pair of primers (Mclachlan, 2006).
For PCR-RFLP analysis, the PCR product (121 bp) was digested with 5 units of the DdeI enzyme overnight at 37 ο C. Agarose gel (4%) was used to visualize the digested fragments. Conformational sequencing was also performed to validate the RFLP results. Gene and genotypic frequencies were calculated using the pop gene 32 software (Yeh et al., 2000). Table I shows genotype frequencies of three breeds. It was observed that most of the animals in Sahiwal breed have A2A2 genotype but a few of these showed heterozygous genotype A1A2. We have not found any A1A1 genotype in the Sahiwal population. In the present study, the frequency of the A2A2 genotype was observed as 0.925, while A1A2 come up with a frequency of 0.075 in Sahiwal cattle. Whereas the allelic frequencies were 0.9625 for the A2 allele and 0.0375 for the A1 allele ( Table I). The sequence result revealed that the selected herd of Sahiwal breed had no A1A1 genotype (Fig. 1). Some studies on their native breeds like. Kangeyam breed carried only A2 allele (Malarmathi et al., 2014), Zebu cattle carried A2 allele with frequency 0.987 (Mishra et al., 2009), Slovak spotted breed carried A2 with frequency 0.7072 (Miluchova et al., 2013).

Results and discussion
RFLP analysis of HF shows that all the individuals possess two genotypes, dominant genotype A2A2 and heterozygous genotype A1A2 with the genotypic frequency of 0.4 (A2A2) and 0.6 (A1A2) respectively. We have not found any mutated A1A1 genotype for beta-casein protein

O n l i n e F i r s t A r t i c l e
in the selected herd (Table II). Allelic frequency for betacasein in Friesian cattle was observed as 0.3 (A1) and 0.7 (A2) ( Table I). DNA sequence also revealed the absence of the A1A1 genotype in the selected herd of HF but the other two genotypes are available (A2A2, A1A2) (Fig.  1). Our results are partially supported by the findings of some researchers who reported that HF carried A2 as the abundant allele with frequency of 0.6322 but they also reported that HF carried all the three genotypes for beta-casein (A2A2, A1A2, and A1A1) with frequency of 0.4023, 0.4598, 0.1379 respectively (Miluchova et al., 2014). A study by Malamarthi et al. (2014) reported that HF carried a high frequency of A2 allele (0.595) and low frequency of A1 allele (0.405). A study on German Friesian and Hungary Friesian partially supported the findings of the present investigation reported that German Friesian and Hungary Friesian carried allele A2 with a frequency of 0.496 and 0.470 (Mishra et al., 2009). A study also reported that Italian Friesians carried the highest frequency of A2 followed by A1. Dutch HF carried the highest frequency of A2 (0.692) followed by A1 (0.285), A3 (0.001) and B (0.022). In Swedish HF A2 (0.60) was predominant but A1 and B present in the lowest frequencies (0.34, 0.06) (Caroli et al., 2016). A study on Chinese Holstein revealed that these cattle have the highest frequency of A1A2 (0.353) genotype and the lowest frequency of A1 (0.030). The allele A1 and A2 present with frequencies 0.432 and 0.459 (Dai et al., 2016).
Our results showed that most of the Crossbred animals possess dominant beta-casein genotype A2A2. No animal comes up with mutated casein genotype A1A1 (Table II), a few heterozygous animals (A1A2) are present in selected crossbred herd. RFLP analysis of crossbred cattle showed that in cross-bred genotypic frequency of A2A2 type was 0.64 and for A1A2 type was 0.36, whereas the allelic frequencies for A2 allele was observed 0.82 and for allele, A1 was 0.18 (Table I). Conformational DNA Sequence results revealed that A1A1 genotype is not present in the selected herd of crossbred, but the other two genotypes are available (A2A2, A1A2) (Fig. 1). These results are partially supported by the findings of some researchers who reported that crossbred (Sahiwal × HF) possesses high frequency of A2 allele (0.65) and A1 present in low frequency (0.35).  showed that Frieswal had three genotypes A2A2 (0.44), A1A2 (0.41) and A1A1 (0.15). Another study reported that in HF crossbred there was the superiority of A1 allele (0.6383) and A2 allele was present in lower frequency (0.3617). This crossbred carried two genotypes A1A1 and A1A2 with frequencies 0.28 and 0.72 (Shende et al., 2017). Frieswal heifers carried high frequency of A2 (0.68) and had all three genotypes A2A2, A1A2, A1A1 with frequencies 0.48, 0.40, 0.12 . Another study revealed that Brazil breeds Gir and Guzerá carried 0.98 and 0.97 A2 allele, 0.96 and 0.93 A2A2 genotype (Rangel et al., 2017). HF and Braunvieh maintained at Northern Italy (Emilia Romagna) carried an abundance of A2 allele (0.546) and A1A2 genotype (0.403) (Massella et al., 2017).

Conclusion
Beta casein is one of the major proteins found in milk and it has two important variants A1 and A2. The results from the present study showed that A2 was the dominant allele found in the studied population and we did not find any mutated genotype (A1A1). The majority of the studied animals from all three breeds possessed dominant allele A2 and dominant genotype A2A2.