ABCG2 gene polymorphism and its association with milk traits in Dangi cattle

Introduction

Livestock production constitutes a cornerstone of the Indian agrarian economy, with the dairy sector contributing substantially to the national Gross Value Added (GVA). India is the world's largest producer of milk; however, the average daily yield of indigenous cattle breeds remains considerably lower than that of exotic Bos taurus breeds and their crossbreds. In this context, the genetic improvement of native breeds through modern molecular tools, particularly Marker Assisted Selection (MAS), represents a promising avenue to enhance productivity without compromising breed integrity or environmental adaptability. The Dangi breed locally known as "Kandadi" is one of the 55 officially recognized indigenous cattle breeds of India. Native to the hilly tracts of the Western Ghats and regions of high rainfall in Maharashtra, Dangi cattle exhibit remarkable adaptive traits, including a distinctive oily skin secretion that protects against heavy precipitation and humid conditions. Despite these ecological advantages, Dangi cows demonstrate modest milk production, with an average lactation yield ranging from 175 to 800 kg. The ABCG2 gene (ATP-binding cassette subfamily G member 2), located on bovine chromosome 6, encodes a transporter protein involved in the efflux of diverse substrates across cellular membranes. It has been identified as a candidate gene for milk yield and composition, primarily due to its role in the secretory pathway of the mammary gland. A functional missense mutation in exon-14 (Y581S; c.A>C), previously reported in exotic breeds, has been associated with increased milk yield but reduced fat and protein percentages. The ABCG2 gene is one of the promising genes influencing milk production and its components. The ABCG2 gene variants were reported to be associated with milk production traits and the composition of bovine milk (Bouwman et al., 2011; Leonard et al., 2005). It also affects absorption and distribution, changing the toxicity and efficacy of medications in humans and animals (Sarkadi et al., 2004; Jonker et al., 2005) [4,11,17,8] . Given the potential of ABCG2 as a molecular marker for selective breeding, this investigation was undertaken to characterize allelic variation at key regions of the gene in Dangi cattle and to evaluate any associations with phenotypic milk production traits.

Materials and Methods: Experimental Animals and Sample Collection:

The study was conducted on 60 lactating Dangi cows maintained under farmer herd conditions in the Ahilyanagar district of Maharashtra, India. Animals were selected based on confirmed purebred status, active lactation, and the absence of any known clinical conditions. All sample collection procedures were carried out in accordance with institutional animal ethics guidelines. Blood samples (5 ml) were collected aseptically from the jugular vein into EDTA-coated vacutainers and stored at -20°C until DNA extraction. Milk samples (10 ml) were collected fortnightly over a period of six months and subjected to compositional analysis for fat, protein, solidsnot-fat (SNF), and lactose content using a calibrated LACTOSCAN milk analyzer.

DNA Extraction and Quality Assessment

Genomic DNA was isolated from whole blood using the phenol: chloroform:isoamyl alcohol (PCI; 25:24:1) method following standard protocols. DNA concentration and purity were assessed by spectrophotometric measurement at 260 nm and 280 nm using a NanoDrop ND-2000 spectrophotometer, and only samples with an A260/A280 ratio between 1.8 and 2.0 were selected for downstream molecular analyses. The integrity of the isolated DNA was further verified by electrophoresis on 0.8 percent agarose gel, which showed intact genomic DNA suitable for PCR amplification.

PCR Amplification

Four genomic regions of the bovine ABCG2 gene were amplified at exon-14 (207 bp), intron-3 (207 bp), exon-7 with a partial intronic region (393 bp), and intron-15 (323 bp). The amplification was carried out in a 25 ul reaction mixture containing template DNA, gene-specific forward and reverse primers, 2X PCR master mix, and nuclease-free water. The amplified PCR products were confirmed by electrophoresis on a 1.5 to 2 percent agarose gel stained with ethidium bromide and visualized under UV transillumination. The primer sequences, expected amplicon sizes, are presented in Table 1, while the PCR reaction composition and thermal cycling parameters are given in Tables 2 and3, respectively. Amplified products were checked on 2 percent agarose gels and showed clear fragments of the expected sizes.

Genotyping by PCR-RFLP and PCR-SSCP

PCR-RFLP analysis was employed for genotyping of exon-14 and intron-3. The PCR amplicon of exon-14 was digested with HinfI, while the intron-3 amplicon was digested with Hin1II (NlaIII). The digested products were separated on 3% agarose gel, stained with ethidium bromide (EtBr), and visualized under UV light, and the banding patterns were recorded for genotype identification. PCR-SSCP analysis was performed for exon-7 and intron-15.

The PCR products were denatured to obtain single-stranded DNA and separated on non-denaturing polyacrylamide gels. The gels were stained with 1% ethidium bromide (EtBr), and the banding patterns were compared among all samples to detect conformational variants associated with sequence polymorphism.

Result and Discussion

Molecular analysis of all four ABCG2 gene regions revealed a complete absence of genetic polymorphism in the study population. [9,10,15,3,24,12,16,13,21,25,6] .

Intron-3 (PCR-RFLP with Hin1II)

Restriction digestion of the intron-3 amplicon with Hin1II yielded a single, undigested 207 bp fragment across the entire sample. This pattern confirmed the presence of only the AA genotype and the absence of any restriction site variation (Figure 2). The present study revealed complete monomorphism at the ABCG2 intron-3 locus in Dangi cattle, with only the AA genotype and A allele (frequency = 1.00). Similar findings were reported by Kowalewska Łuczak et al. (2009) [10] in Jersey cattle, where only the AA genotype was observed, indicating monomorphism at this locus. Likewise, Cohen-Zinder et al. (2005) identified an A→T SNP in intron-3 in Israeli and U.S. Holstein cattle but reported no significant association of this polymorphism with milk production traits, suggesting that the exon-14 Y581S mutation is the causal quantitative trait nucleotide (QTN). In contrast, Kowalewska Łuczak et al. (2009) [6,10] also reported polymorphism in Holstein Friesian cattle, detecting AA, AT, and TT genotypes with both A and T alleles, indicating breed-specific variation in the distribution of ABCG2 intron-3 polymorphism. These findings suggest the complete fixation of the A allele observed in the present Dangi cattle is consistent with several indigenous populations but differs from certain Bos taurus breeds exhibiting intron-3 polymorphism.

Exon-7 (PCR-SSCP)

SSCP analysis of exon-7 revealed a single, uniform banding pattern (designated P1) in all 60 animals (Figure 3). The present study demonstrated complete monomorphism at exon-7 and the partial intronic region of the ABCG2 gene in Dangi cattle, with only the AA genotype and A allele (frequency = 1.00). Similar findings were reported by and partial intronic region of the ABCG2 gene is highly conserved in Dangi cattle, suggesting breed-specific genetic stability at this locus [5,29,12] .

Intron-15 (PCR-SSCP)

Similarly, intron-15 exhibited a single conformational pattern (P2) (Figure 4) with no evidence of alternative conformers. The genotypic and allelic frequencies for all four loci were AA = 1.00 and A = 1.00, respectively, confirming complete fixation of the ancestral A allele within this Dangi population. The present study revealed complete monomorphism at the intron-15 locus of the ABCG2 gene in Dangi cattle, with only the AA genotype and A allele (frequency = 1.00). In contrast, Chouhan et al. (2021) [5] reported polymorphism in Rathi cattle, identifying two SSCP patterns (P1 and P2) corresponding to AA and AB genotypes, with genotypic frequencies of 0. [20,27,18,6] emphasized that identifying such fixed loci is important for assessing genetic diversity and developing effective marker-assisted selection and conservation strategies. Owing to the limited number of published reports on ABCG2 polymorphisms in intron-3, exon-7 (with a partial intronic region), and intron-15, the present findings provide valuable baseline information for future genetic studies in indigenous cattle.

Conclusion

This study demonstrates that exon-14, intron-3, exon-7, and intron-15 of the ABCG2 gene are monomorphic in the sampled Dangi cattle population, with all animals fixed for the ancestral A allele. The absence of allelic variation at these loci precluded any association analysis with milk composition traits. These findings contribute to the foundational genomic characterization of the Dangi breed and reinforce the notion that certain ABCG2 regions subject to diversifying selection in exotic dairy breeds are highly conserved in Indian zebu cattle, likely as a consequence of evolutionary adaptation to local environments and management systems. Future research should involve larger, geographically diverse Dangi populations and extend molecular analysis to additional functional regions of the ABCG2 gene and other candidate loci. Integration of high-throughput genotyping platforms with phenotypic data on milk yield and extended composition profiles will be essential to develop evidencebased Marker Assisted Selection strategies tailored to the genetic background of Dangi and other indigenous Indian breeds.

Acknowledgement

AcknowledgementsThe authors acknowledge the support of the Mumbai Veterinary College, Department of animal genetics and breeding, and the farmers of the Ahilyanagar district, who provided access to their herds for this research. The guidance provided by the Department of Animal Genetics and Breeding is gratefully acknowledged.

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