Introduction

Milk protein such as casein and whey proteins are associated with milk production performance. Among variety of casein, Kappa-casein protein (about 15% of the total caseins) is encoded by Kappa-casein (k-CN) gene and Beta-lactoglobulin (β-LG), a globular protein member of the lipocalin family, is the main whey protein of ruminant milk; around 5% of the total whey proteins (Selvaggi et al., 2014). Several single nucleotide polymorphisms and their association with milk production traits have been reported in the bovine milk protein gene (Caroli et al., 2004, Satyanarayana et al., 2006). Till now very limited studies are available on milk proteins in buffalo (Patel et al., 2007, Meignanalakshmi and Nainer 2009, Gouda et al., 2013 and Cinar et al., 2016). Many genes are found to be associated with mastitis such as, Major Histocompatibility Complex (MHC), β-defensin etc. (Gulhane and Sangwan, 2012). Among them, BoLA-DRB3 exon 2 alleles in cattle have been found to be associated with resistance or susceptibility to various diseases. DRB3.2 alleles have found to be associated with somatic cell count (SCC) and mastitis resistance in cattle (Baltian et al., 2012). The MHC of buffalo is called BuLA, i.e. buffalo lymphocyte antigen (Kumar et al., 1993). There is a paucity of information on MHC polymorphism in buffaloes.

Considering the above, the present investigation was undertaken to study the polymorphism of k-CN, β-LG and DRB3 genes in Indian Murrah buffaloes using PCR-RFLP and its association with milk production traits.

Materials and Methods

Animals’ Source, DNA Isolation and Amplification

A total of 45 females of Murrah buffalo maintained at Instructional Livestock Farm Complex (ILFC), Mathura (Uttar Pradesh), were utilized in the present investigation. The animals were kept in identical environmental conditions and were fed a standard diet. Genomic DNA was extracted from venous blood using the standard protocol of Sambrook and Russel (1991). The following primers pair (‘F’: 5′- ATC ATT TAT GGC CAT TCC ACC AAA G – 3′ & ‘R’: 5′- GGC CAT TTC GCC TTC TCT GTA ACA GA -3′) and (’F’: 5′- TGT GCT GGA CAC CGA CTA CAA AAA – 3′ & ‘R’: 5′- GCT CCC GGT ATA TGA CCA CCC TCT -3′) were used for amplification of k-CN and β-LG gene, respectively as per method described by Patel et al. (2007). The 304 bp PCR product of exon 2 of DRB3 gene was amplified by using the primer (Siguardardtottir et al., 1991) (‘F’: 5′- GAT GGA TCC TCT CTC TGC AGC ACA TTT CCT – 3′ & ‘R’: 5′- CTT GAA TTC GCG CTC ACC TCG CCG CTG -3′).

PCR-RFLP Assay and Gene and Genotypic Frequency

The restriction digestion was carried out at 37^oC for 14-16 h in a total volume of 15μl containing 10.0 µl of PCR product, 1.5 µl of 10X RE buffer and 10 Units of HinfI, HaeIII and HaeIII (New England Biolab, USA) to digest k-CN, β-LG and DRB3 amplified products, respectively.

Statistical Analysis

The data was generated by estimating the frequency of different digested products. The allelic and genotypic frequencies of k-CN, β-LG and DRB3 genes were estimated by standard procedure (Falconer and Mackay, 1996). The chi square (χ²) test (P ≤ 0.05) was also performed to test whether the distribution of the genotype frequencies was in the Hardy-Weinberg equilibrium (Snedecor and Cochran, 1989).

Association Study

The association study of different genotypes with the following milk production traits- Lactation period (LP = date of drying –date of calving), Total milk yield (TMY = calculated by totaling of daily milk records of individual cow after completion of their lactation.), Milk yield in 300 days (MY300 = calculated by totaling of daily milk records of individual cows up to 300 days of lactation), Dry period (DP = date of calving – date of drying), Calving interval (CI = difference between two successive calving) and SCC (somatic cell count) was performed. Somatic cell count was performed by direct method as per method described by Singh (2013). Statistical analysis of milk production traits and SCC in relation to different genotypes was carried out using the General Linear Model (GLM) using SPSS software. The following linear model was applied-

Y^ij = μ + Gⁱ + eij

Where, Y^ij– observed trait value in animal; μ – mean trait value; Gⁱ – effect of genotype; eij– random error.

Significant differences among least square means of different genotypes were calculated using Duncan’s multiple-range test, and P values of 0.05 were considered statistically significant.

Results and Discussion

The amplified products of k-CN and β-LG gene were 350 and 247 bp, respectively. The k-CN/HinfI PCR-RFLP assay revealed only one type of banding pattern (BB genotype) of 266 and 84 bp in all the screened buffaloes. We could not identify any animal with AA homozygote (134, 132 and 84 bp) and AB (134, 132 and 84 bp); heterozygote genotypes. In the present study, all the screened buffalo were found monomorphic in nature with only B allele (1.00) for k-CN/HinfI polymorphism (Fig. 1). The β-LG/HaeIII PCR-RFLP assay also revealed only one type of banding pattern of 148, 99 and 74 bp (AB genotypes) in all the screened Murrah buffalo.

Fig.1: k-CN/HinfI PCR-RFLP assay showing genotype pattern in 2.0% agarose gel; Lane 1-4:BB genotype (266 and 84 bp); M: Marker (50bp) and PCR: Undigested PCR product (350bp)

We could not identify any animal with AA (148 and 99 bp) and BB (99 and 74 bp); heterozygote genotype. This revealed that the screened buffalo population used in the present study was monomorphic in nature for β-LG SNP with both types of allele A (0.50) and B (0.50) (Fig. 2).

Fig.2: β-LG/HaeIII PCR-RFLP assay showing genotype pattern in 2.0% agarose gel; Lane 1-6: AB genotype (148, 99 and 74bp); M: Marker (50bp) and PCR: Undigested PCR product (247bp)

DRB3/HaeIII PCR-RFLP assay revealed five types of genotypes, viz., AA (170, 82 and 52 bp); BB (222 and 82 bp), EE (170 & 134 bp) genotypes and heterozygous pattern have AB (222, 170, 82 and 52 bp) and BD (222, 193, 82 & 29 bp) genotype (Fig. 3). AB genotype was the most frequent (34.68%) in all the screened buffalo samples, followed by the AA, EE, BB and BD genotype.

Fig 3: The HaeIII/PCR-RFLP assay showing five types of banding pattern in 1.5% agarose gel; Lane: AA (170, 82, 52 bp); BB ( 222 & 82 bp); EE (170 & 134 bp) and heterozygous pattern, AB (222, 170, 82 & 52 bp bands) & BD (222, 193, 82 & 29 bp) M1=100 bp ladder, M2=50 bp ladder

The frequency of DRB3/HaeIII A, B, D and E alleles were 0.417, 0.411, 0.055 and 0.111, respectively (Table 1). The χ² calculated value for DRB3/HaeIII gene was 115.6, while χ² table values were 10.64 and 16.81 at 5% and 1% level of significance, respectively for degree of freedom 6. These results revealed that χ²_(cal) > χ²_(tab) at 1% level of significance i.e. screened population of Murrah buffalo was not found in Hardy-Weinberg equilibrium.

Table 1: Genotypic and allelic frequencies of DRB3/HaeIII gene in Murrah buffalo

Association study revealed that there was significant (P = 0.003) difference observed for MY300 and TMY (P = 0.013) among AA, AB, BB, BD and EE genotypes in the first lactation of screened buffalo. The MY300 of BD genotype (2330.0 ± 63.00 liter) was higher than AA (1580.0 ± 97.17 liter), AB (1880.0 ± 76.46 liter), BB (1490.0 ± 112.00 liter), EE (2080.0 ± 12.50 liter) genotypes. No significant difference was observed for SCC among AA, AB, BB, BD and EE genotypes in the screened buffalo population (Table 2).

The k-CN/HinfI PCR-RFLP assay showed only BB genotypes in all the screened buffaloes which was similar to the reports of Pipalia et al. (2001) in different Indian buffalo, Riaz et al. (2008) in Nili Ravi buffalo, Nair et al. (2011) in Nagpuri buffalo and Cinar et al. (2016) in Anatolian water buffalo. However, Singh et al. (2005) found two alleles A and B for k-CN locus in Murrah and Bhadawari breeds but they have reported monomorphism in Surti and Mehsana breeds of buffalo.

Table 2: Association of DRB3/ HaeIII genotypes with milk production trait in Murrah buffalo different letters in columns differ significantly (P<0.05)

Where; N = Sample size, n = Number of animals in particular genotype, χ2 test = test of Hardy -Weinberg equilibrium, ⃰⃰ ⃰ = highly significant (P< 0.01) for degree of freedom =6

Similarly, two types of alleles were found in Murrah, Surti and Pandharpuri breeds of buffalo (Patel et al., 2007) and in Egyptian buffalo (Gouda et al., 2013). β-LG/HaeIII PCR-RFLP showed only AB genotype in all the screened buffalo which was in accordance with the reports of Meignanalakshmi and Nainar (2009) in Murrah, Nair et al. (2011) in Nagpuri, Nualchuen et al.(2011) in Swamp and Murrah buffalo of Thailand. However, Ren et al. (2011) observed only BB genotype in Water buffalo. In contrast, Patel et al. (2007) and Cinar et al. (2016) observed all the three genotypes of β-LG in various breed of Indian buffalo and Anatolian water buffalo, respectively. All the screened animals were found monomorphic for both k-CN and β-LG genes, consequently we could not perform association study with milk production traits. However, previous studies reported that B allele of both genes had a favourable and significant effect on both milk and milk protein yield (Mao et al., 1992).

DRB3/HaeIII allelic frequency pattern in Murrah buffalo was observed as A (0.422), B (0.411), D (0.055) and E (0.111) in the present study. Allele A had higher frequency among all other alleles found in screened Murrah buffalo. The various DRB3/HaeIII allelic frequency pattern had been observed in different buffalo breeds by various authors (Table 3).

Table 3: Allelic frequencies of DRB3/HaeIII gene in different buffalo breeds observed by other authors

*The most frequent alleles in each breed shown in bold

Arvindakshan et al. (2000) found five different types of alleles with higher frequency of allele B than A in Indian Murrah buffalo which were not in accordance with the present findings. While Stafuzza et al. (2015) observed four types of allele with higher frequency of B allele among all other alleles in brazilian Murrah buffalo. The variation in the frequency of alleles might be due to the geographical variations and sampling.

Conclusion

In the present study, all the screened animals were found monomorphic for k-CN and β-LG SNPs, consequently we could not perform association study with milk production traits. While, the status of DRB3 gene polymorphisms revealed four types of allele viz., A, B, D and E. Significant difference was observed for TMY and MY300 in first lactation of screened murrah buffalo. It would be interesting to further investigate this polymorphism with association of these SNPs with milk production and disease resistance traits in other breeds of Indian buffalo.

Acknowledgement

The Instructional Livestock Farm Complex, DUVASU, Mathura (U.P.) is gratefully acknowledged for providing help during sample and data collection. The authors are thankful to Vice Chancellor, DUVASU, Mathura, (U.P) for providing necessary facilities and funding during entire research work at this esteemed university.

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