India is a rich reservoir of genetic diversity in cattle with 43 recognized cattle breeds. The total number of cattle populations was 190.90 million, out of which indigenous cattle population comprised of 151.17 million which is 37.28% of the total livestock population (19th Livestock Census, 2012). India is the largest producer of milk in the world with 176.3 million tonnes of total milk production during 2017-18 (BAHS, 2018). The share of indigenous cattle in milk production is around 20% of the total milk produced in the country. Sahiwal is one of the best milch breed of cattle in India known for its higher milk production, disease resistance ability, remarkable power of endurance for hot climate of tropics, higher feed conversion efficiency and low cost of maintenance (Nivsarkar et al., 2000). Selection of superior phenotypes and their selective breeding is a conventional tool which is used for the genetic improvement of livestock. Molecular genetics tools can be used as an aid to conventional breeding programmes for accurate and effective selection of animals having high production potential.
The key objective of dairy cattle genomics is to identify genes underlying genetic variability of milk production and composition traits that could be implemented in breeding programs (Szyda and Komisarek, 2007). There are number of candidate genes like prolactin, leptin, diacylglycerol acyl-transferase (DGAT1), β-lactoglobulin, ATP-binding cassette subfamily G member 2 (ABCG2), signal transducer and activator of transcription (STAT5A), growth hormone releasing hormone (GHRH), Fibroblast Growth Factor 2 (FGF2) etc. which are associated with milk production traits. (Grisart et al., 2002; Blott et al., 2003; Liefers et al., 2005; He et al., 2006; Olsen et al., 2007; Khatib et al., 2008; Ganai et al., 2009 ; Singh et al., 2019). ABCG2 a member of the ATP binding cassette (ABC) super-family, is a “half-transporter”, with only one ATP binding cassette in the N terminus and one C-terminal transmembrane domain (Ejendal and Hrycyna 2002; Gottesman et al., 2002). ABCG2 gene expression is significantly enhanced during lactation and is accountable for the secretion of vitamin K3 or cholesterol into milk (Van Herwaarden et al., 2007; Farke et al., 2008). The ABCG2 gene in cattle is located in the narrow region of chromosome 6 (BTA 6) and spans over 117474 base pairs (bp) long consisting of fifteen introns and sixteen exons encoding 658 amino acids (aa), harbouring the QTL with a large impact on milk production traits (Ron et al., 2006; Olsen et al., 2005). Cohen –Zinder et al. (2005) identified a single nucleotide polymorphism (SNP) A to C resulting from translocation of adenine/cytosine on exon 14 of bovine ABCG2 which was a missense mutation named Y581S and it leads to alteration in milk yield and proportion of milk fat and protein. Polymorphism of ABCG2 gene have been studied in different exotic (Bos taurus) cattle breeds (Cohen-Zinder et al., 2005; Ron et al., 2006; Soltani-Ghombavani et al., 2016; Fontanesi et al., 2015) while limited work has been done in Bos indicus cattle (Tantia et al., 2006; Sharma et al., 2016). The present investigation was undertaken to examine the polymorphism and allele frequency of ABCG2 gene in Sahiwal cattle.
Materials and Methods
Location and Climatic Conditions of the Study Area
The present study was conducted at Livestock Research Centre and Dairy Cattle Breeding Division, ICAR-NDRI, Karnal situated at an altitude of 235 to 252 meters above the mean sea level at 29.68°N latitude and 76.98°E longitude in eastern zone of Haryana which comes under the Trans-Gangetic plain agro-climatic zone of India.
A total of 130 animals of Sahiwal cattle maintained at Livestock Research Centre (LRC) were used for the present study. The selection of animals was done randomly and all animals were maintained under proper and uniform managemental conditions.
Isolation and Extraction of DNA from Blood
Blood samples were collected from randomly selected Sahiwal cattle after obtaining permission from Institute Animal Ethics Committee. Genomic DNA was isolated by Phenol-Chloroform method (Sambrook and Russel, 2001) with minor modifications. The quality and quantity of DNA was checked by agarose gel electrophoresis and UV spectrophotometer. The stock solutions were store at -20°C and used for further analysis. The working solution was prepared by diluting the stock to 100ng/µL for utilizing as DNA template in PCR.
Determination of ABCG2/PstI Polymorphism
For the amplification of 292 bp fragment of exon 14 in PCR the following forward and reverse primers were used; Forward Primer: 5′-AACAGCCTCAGCTCCAGAGAGATAT-3′ and Reverse Primer: 5′- CGGTGACAGATAAGGAGAACATACT-3′ (Cohen-Zinder et al., 2005). PCR reaction was performed in a final volume of 25 µl containing 100 ng of template DNA, 10 pmol of each primer, 10X PCR buffer (20mM Tris-HCL pH 8.4, 50mM KCl), 1mM MgCl2, 2.0 mM of dNTPs and 1 ul of Taq DNA polymerase (M/s Genetix Biotech Asia Pvt. Ltd). This solution was initially denatured at 95°C for 5 min, followed by 35 cycles of denaturation (95°C for 1 min), annealing (58°C for 1 min), elongation (72°C for 1 min) and a final extension at 72°C for 5 min. The amplified products were detected in 1.5% agarose gel electrophoresis. Aliquots of 5 µl of PCR products were applied to the gel. About 10 µl of amplified product was digested with 10 units of Pst I enzyme overnight at 37°C in water bath. The amplified product was digested at 37°C for 14 hours. The digested products were detected by electrophoresis in 2% agarose gel in 1X TBE buffer and ethidium bromide (10 mg/µl).
The allelic frequencies of ABCG2 gene were estimated by simple allele counting according to Hardy-Weinberg equilibrium (Falconer and Mackay, 1996).
Results and Discussion
In the present study a 292 bp fragment of genomic DNA was amplified (Fig. 1). PCR-RFLP analysis of exon 14 was carried out using PstI. PstI–RFLP for exon 14 revealed monomorphic pattern (Fig. 2) showing only one type of genotype, AA (292 bp) with genotypic frequencie of 1.0. AC and CC genotype were not observed in the studied Sahiwal population. Similar findings were reported by Sharma et al. (2016) in Sahiwal and Hariana cattle and Tantia et al. (2006) in different Bos indicus breeds. The allelic frequencies for ABCG2-A and ABCG2-C allele were 1.0 and 0, respectively. This is in agreement with the results presented by Soltani-Ghombavani et al. (2016) in Iranian Holstein cows (0.97 and 0.03), Fontanesi et al. (2015) in Reggiana cattle breed of Italy (1.0 and 0), Hosseinpour-Mashhadi et al. (2012) in Holstein cows (0.98 and 0.02), Kowalehiska-luczak et al. (2007) in Jersey cows (0.80 and 0.20) and Cohen-Zinder et al. (2005) in Holstein bulls (0.99 and 0.01).
Fig. 1: Resolution of PCR products of ABCG2 gene in Sahiwal cattle
Lane 1-12: PCR product (292 bp)
Lane M: Marker (100 bp ladder)
Fig. 2: PstI PCR-RFLP pattern of ABCG2 gene showing genotype pattern.
Lane 1-12: AA genotype (292 bp)
Lane M: Marker (50 bp ladder)
Atila et al. (2014) carried out a study to determine polymorphisms present in ABCG2 gene in South Anatolian Red (SAR) and East Anatolian Red (EAR) indigenous cattle breeds in Turkey. They amplified target region of ABCG2 (SNP. Y581S) and found that genotype AA (SAR: 0.50; EAR: 0.62) allele A (SAR: 0.63; EAR: 0.64) were high in both SAR and EAR cattle breeds. Ron et al. (2006) studied the polymorphism in ABCG2 gene in 32 Bos taurus and 3 Bos indicus breeds and observed that allele – A was most frequent in all the populations and is responsible for less milk yield and more fat and protein concentration. The ABCG2- C allele was present only in Bos taurus breed suggesting that allele – A is the ancestral allele. Milk composition traits are significantly influenced by the non-conservative Y518S mutation in ABCG2 gene as reported by Cohen-Zinder et al. (2005) in Israeli Holstein cattle and Olsen et al. (2007) in Norwegian Red cattle breed. There is increase in frequency of ABCG2-A allele if selection is done for higher milk fat and protein percentage as reported by Cohen-Zinder et al. (2005). Nahas et al. (2018) reported higher frequency of AA genotype which is associated with higher lactose percentage and low milk yield in less selected population of Baladi cattle breed of Egypt. These are several findings which indicate that ABCG2 is a strong candidate gene which significantly influence milk composition traits due to its physical role in mammary system and chromosomal positioning.
In the present study, the selected Sahiwal population was found to be monomorphic for ABCG2 gene as only single genotype (AA) was found in all the animals which were screened for studying polymorphism in ABCG2 gene and hence no association could be established between genotype and milk composition traits. The absence of ABCG2-C allele also indicates that ABCG2-A is the ancestral allele, and that the Y518S substitution occurred after the separation of Bos indicus and Bos taurus lineages. Further investigation on larger population of this breed is required in order to explore the polymorphism (A/C: Y518S) in ABCG2 gene and to exploit it for marker assisted selection for milk composition traits.
The authors are thankful to Director ICAR-National Dairy Research Institute, Karnal, Head Dairy Cattle Breeding Division and Head Livestock Research Complex for providing all the necessary facilities required for successful completion of research. Financial assistance in the form of institute fellowship is deeply acknowledged.
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