Identification of Novel Milk Protein Gene Variants in Sahiwal Cattle Breed of Pakistan

This novel study was aimed at identification of new genetic variants in Sahiwal cattle breed of Pakistan and determined the effects of these variants on milk yield. Five major milk protein genes in Sahiwal cattle were analyzed and two single nucleotide polymorphisms identified through bi-directional sequencing. These include A to T in exon XI at position 11462 of the alpha s1 casein gene; resulting in a Glutamic Acid (GAA) to Aspartic acid (GAU) substitution at position 84 of alpha s1 casein protein and T to C change at position 8491 of the exon VII in beta-casein gene resulting in a Valine to Alanine substitution at position 197 of beta casein protein. Amplification Refractory Mutation System (ARMS) and SNaPshot genotyping protocols were optimized for genotyping new genetic variants. The genotypes in both the alpha-s1 casein and beta casein genes were found associated with milk yield but their influence was not statistically significant. However, the least square means of milk yield for TT genotypes of alpha s1 casein and of beta casein genes were higher compared to other genotypes.


INTRODUCTION
Top 10 dairy companies were turning over $114 billion in 2009 in worldwide agriculture and 84% of it was shared by dairy cows as a result there is great importance of the production potential of these animals (Whitley, 2010).Sahiwal cattle breed is one of the best dairy breeds of Pakistan and India due to its qualities of being heat tolerant and resistant to internal as well as external parasites.Cows produce 2270 kg of milk on average during one lactation period while also suckling a calf (Khan et al., 1999).
Milk protein genetic polymorphism provides the opportunity to estimate the genetic merit of sires and cows and practice selection independent of sex and age of animal; as a result many populations of dairy cattle were screened to determine the differences in various breeds or group of breeds (Gurcan, 2011;Bonfatti et al., 2010), calculate frequencies of these variants in different cattle breeds (Hamza et al., 2010) and investigate relationships between genetic variants and productive traits (Hristov et al., 2012).
With newly developed genotyping techniques based on DNA analysis, which include Amplification Refractory Mutation System (ARMS) and SNaPshot SNP genotyping it is now possible to include information on milk protein genotypes into selection programs.It could provide more accurate predictions of breeding values of animals to be selected and thus improve response to selection (Lara et al., 2002).
Five major milk protein genes including αs1casein, β-casein, κ-casein, α-lactalbumin and βlactoglobulin were screened through sequencing which resulted in identification of two new genetic variants in αs1-casein and β-casein genes reported here.Two genotyping techniques; amplification refractory mutation system and SNaPshot were optimized for large sets of sample size.

MATERIALS AND METHODS
Genomic DNA samples: Blood samples of 150 Sahiwal cattle breed were collected from Research Centre for Conservation of Sahiwal cattle (RCCSC, Khanewal) and 120 samples from Livestock Production Research Institute (LPRI, Okara).DNA was extracted using a standard protocol (Sambrook and Russell, 2001).Lactation records obtained from dairy farms were adjusted, those greater than 150 and less than 305 days were fixed to 305 days values using dairy farms correction factors.

Phenotypic data and statistical analysis:
Primers design: Primers were designed using the bovine αs1-casein and β-casein gene sequences available at NCBI website.The exons XI and VII of αs1-casein and β-casein were amplified by PCR using primers X111S1F, X111S1R and X7.2BcnF X7.2BcnR for αs1-casein and β-casein respectively (Table 1).The tetra-primer ARMS-PCR procedure required the design of two outer primers to amplify a 200-500 bp fragment and two allele-specific inner primers (Ye et al., 2001).The inner primers were designed as described by Ye et al. (2001) following the rules to introduce a second deliberate mismatch at position -2 from the 3' end utilizing the web-based program accessible from http://www.cedar.genetics.soton.ac.uk/ public_html/pr mer1.html(Ye et al., 2001).Primers for SNaPshot SNP genotyping were designed using the website http://www.basic.northwestern.edu/biotools/oligocalc.ht ml.The primers were tailored to immediately end 5' site before the target SNP (Table 1).α-S1cn and 67°C for β-casein) and 30 sec extension (72°C) and an additional 5-min extension at 72°C at the end of the 30 cycles.Amplification products were resolved by electrophoresis in 3% agarose gels with ethidium-bromide.

PCR amplification for SNaPshot genotyping:
The SNaPshot® Multiplex Kit a single-tube was used to interrogate SNPs at known locations using recommended protocol from Applied Biosystems, CA.

RESULTS AND DISCUSSION
Nineteen exons of αs1-casein gene were sequenced, bi-directionally.Sequencing of exon XI using primer X111S1 (Table 1) revealed a point mutation from A to T in exon XI (Fig. 1) at position 11462 of the sequence published by Koczan et al. (1991) (GenBank accession no.X59856).All three genotypes AA, AT and TT were found in the samples studied.A change of amino acid from Glutamic acid (E) to Aspartic acid (D) at position 84 of the protein was recorded by comparison of this amino acid sequence with that reported by Treweek et al. (2011).Total of 9 exons of β-casein gene were sequenced and sequencing of PCR product from exon VII with primer X7.2BCN (Table 1) showed a T to C point mutation (Fig. 2) at position 8491 of the sequence published by Mackinlay et al. (1965) (Acc. No. X14711).This mutation causes a Valine (GTA) to Alanine (GCA) substitution at position 197 of the protein.All three genotypes TT, CT and CC were found in samples screened.
ARMS SNP genotyping and SNaPshot genotyping assays were successfully applied to type two different SNPs (Fig. 3 to 6) respectively.SNaPshot SNP genotyping is generally considered quite reliable for biallelic discrimination (Syvanen, 1999).Multiplexing can also be done that makes it cost efficient and high throughput.Tetra-primer ARMS-PCR technique does not involve post-PCR manipulations so it is a more rapid and lower cost technique that carries the potential of being a reproducible higher throughput method for SNP genotyping.In addition to that these techniques can be used not only for the selection of sires for artificial insemination but also for selection of immature cows and for pre-implantation embryos for embryo transfer thus saving the cost of raring an animal.The gene counting method was used to calculate allele frequencies for the total number of animals included in this study at the αs1-casein and β-casein.It was noted that new DNA variant at the αs1-casein locus (GenBank accession no.EF538766) (Shahla et al., 2007a) occurred at higher frequency 35% in Sahiwal cattle.The new allelic variant of β-casein (GenBank accession no.EF628290) (Shahla et al., 2007b) also had high frequency in Sahiwal cattle 13%.
Additionally, we identified two single base substitutions in exon VII of β casein (A to G variation at position 8093 and T to C change at 8261) but did not cause amino acid change.
Data on milk protein genotypes was statistically analyzed to find out association with milk yield.Chi square test was applied for association of genotypes with milk yield which showed quite significant association of genotypes with milk yield (Table 2).Although the results were not significant (p>0.05),animals with genotype TT of αs1-casein had higher least square means of milk yield than genotypes AA and AT.The least square means of milk yield for new β-casein TT genotype were higher than either CC or CT (Table 3).
Genetic polymorphism of milk proteins has great interest in animal breeding, due to its relationship with production traits, milk composition and milk quality (Treweek et al., 2011).However, before the genetic variability of milk proteins can be used in selection programs, it needs to be established that no unfavorable associations exist between the economically interesting milk protein alleles and other traits of importance for milk production.Also, the gene frequencies in the population of interest should be investigated because the prospects of improving a given trait depend on the frequency of the favorable allele (Lunden et al., 1997).
Two genotyping techniques were optimized for fast and accurate futuristic genotyping studies.Studies on large population size on new variants should be conducted to observe the positive influence of TT genotypes of αs1-casein and β-casein on milk yield as Least Square Means were high for these genotypes so that it can be incorporated in genetic improvement programs of Sahiwal cattle.Likewise, the negative influence of β-casein CC genotype should also be studied and animals carrying this genotype should be eliminated from breeding stock upon validation of studies.

Fig. 1 :
Fig. 1: Results of direct sequencing of a 345-bp fragment of the alpha s1-casein gene new variant, showing polymorphic site at position 11462 and sequence changes (indicated by arrows)

Table 2 :
Chi square test for association of genotypes with milk yield