Identification of Point Mutation in Exon 3 of Leptin Gene in Munjal Sheep

Sheep, with its multi-faceted utility for meat, milk, skin, wool and manure, form a vital part of rural economy in most areas of the country particularly in the arid, semi-arid and mountainous areas. India is rich source of sheep genetic resources having 43 breeds. As per 20^th  Livestock Census (2019), India stands at number three in world sheep population with 74.26 million sheep. Munjal sheep is large in size, tall and rectangular in shape. The origin of Munjal sheep are not known exactly but it is supposed to have originated in India through the sheep breeders of Rajasthan, Punjab and Haryana breeding Nali with Lohi sheep (Arora et al., 1986; Mason, 1988). Munjal sheep is very popular among farmers of Haryana, Punjab and Rajasthan for their height and heavy weight.

Leptin, a diverse hormone which plays vital role in body growth by regulating the balance between food intake and energy expenditure by signaling to the brain. Leptin has diverse effect on controlling appetite, energy metabolism, growth, reproduction, body composition and immunity. Leptin is produced by white adipocytes and also from the tissues of stomach, skeleton, muscle and placenta (Friedman and Halaas, 1998). Leptin has a huge effect in controlling whole body energy metabolism and can be regarded as a “metabolism modifier.” Leptin is encoded of three exons and two introns and is found on fifth chromosome in sheep (Pomp et al., 1997). Leptin gene polymorphism has drawn much attention in biomedical research particularly from animal scientists for their possible role in economically important production and reproduction traits. Zhou et al., (2009) found four SNPs in the exon 3 of Leptin gene of which three were non-synonymous and resulted in amino acid changes.  Boucher et al., (2006) conducted a study to identify the SNPs in leptin gene in sheep and their associations with growth traits in a population set of Dorset and Suffolk lambs. Barzehkar et al., (2009) demonstrated the polymorphism of the Leptin gene and its association with growth and carcass traits in three Iranian sheep breeds. Shojaei et al., (2010) also reported association of leptin gene polymorphism and growth traits in Kermani sheep and found that the growth traits were significantly affected by the genotypes. Cauveri et al., (2014) reported two SNPs (PCR-RFLP), 16973 G>A (SNP-L1) and 17476 C>T (SNP-L2) in the Exon 3 in Nilagiri sheep. Animals of SNP-L1 were homozygous to the A allele. Bakhtiar et al., (2017) also explored exon 3 region of leptin gene to explore g.332G>A in Sanjabi sheep. Similarly, many researchers targeted exon 3 of Leptin gene to screen candidate marker associated with various economic traits in sheep (Meena et al., 2017; Kaplan et al., 2018; Saleem et al., 2018). Taking into the consideration these facts, the study was carried with the objective to explore candidate point mutation in exon 3 of Leptin Gene in Munjal Sheep.

The study was carried on randomly selected Munjal sheep maintained at Sheep Breeding Farm, Department of Animal Genetics and Breeding, Lala Lajpat Rai University of Veterinary and Animal sciences, Hisar, India. A total of 50 animals were taken to detect polymorphism in genomic region of Leptin gene. 5 ml of blood was aseptically collected from the jugular vein in vacutainer tube containing EDTA (2.7%). DNA was extracted in Automated Maxell RSC DNA/RNA purification system (Promega) by using Maxwell RSC whole blood DNA kit and quality of DNA was also assesed (Fig 1). DNA samples were dissolved in 0.1X TE buffer (pH 8.0).


 
DNA amplification and genotyping
 
Reported set of primers was used to amplify exon 3 coding sequence of the Leptin gene (Bakhtiar et al., 2017) (Table 1). PCR amplification was carried out in a total volume of 25 ml with 100 ng DNA template, Dream Taq Green PCR Master Mix (Promega). Polymerase chain reaction (PCR) was carried out in thermal cycler (T-100 BIO-RAD) as per the conditions given in Table 2. The PCR product was checked on 2.5% agarose gel. PCR-RFLP was also performed to genotype animal for reported 332G>A candidate SNP (Bakhtiar et al., 2017), amplified PCR products (10 μl) of all animals were digested with 2 U Cail 1 restriction enzyme (Thermo Scientific) at 37°C for 10 h and were subsequently resolved in 2.5% agarose gel stained with ethidium bromide.

In the present study, the PCR product (463 bp) of the Leptin gene was amplified encompassing exon 3 in Munjal sheep (Fig 2). The PCR product was digested with Cail 1 restriction enzyme and digested products were resolved on 2.5% agarose gel, revealed uncut single band (Fig 3). The PCR- RFLP demonstrated the existence of only one allele A, showing single band consisting of 463 bp, was assigned as the AA genotype corresponding to Glutamin homozygote for Munjal sheep. Thus, the animals under study were found to be monomorphic, which was reported first time in Munjal Sheep. Contrary to our study, Bakhtiar et al., (2017) observed all the three possible genotyope at targeted loci with predominance of G allele and reported that polymorphism in 332G>A locus had a significant effect on viability trait, water test and scrotal circumference as GA genotypes had the highest amounts for these traits compared with GG genotypes (P<0.05) in Sanjabi rams. Our observations are in agreement with those of Gregorio et al., (2014) who observed uniform pattern in the partial genomic sequence of part of intron-1 to part of exon-3 of ovine leptin gene. The results are also in agreement with Cauveri et al., (2014) that studied polymorphism in exon 3 of leptin gene in Nilagiri sheep. Qureshi et al., (2015) reported monomorphic pattern of leptin gene in three sheep breeds of Pakistan using PCR RFLP technique. However, they observed polymorphism at position C1467T and A3050- in intron 2 of Ovine leptin gene. In an earlier attempt, Li et al., (2008) also did not find polymorphism in exon 2 using PCR-SSCP protocol in 358 sheep including Poll Dorset, Suffolk, Texel and Tan sheep. Study of polymorphism in exon 2 of LEP gene have been done in Dorset and Suffolk breeds (Boucher et al., 2006) and Shal, Zandi and Zel breeds (Barzhekar et al., 2009) but no polymorphism was detected by them in this region. However, leptin gene polymorphism in sheep were reported by many scientists like Zhou et al., (2009), Shojaei et al., (2010), Hajihosseinlo et al., (2012), Bahrami et al., (2013), Mahmoud et al., (2014), Meena et al., (2016), Maitra et al., (2014), Quirino et al., (2016) in different sheep breeds.

This study insight an evidence that Munjal sheep have no variability in targeted leptin locus. All animals were found to be monomorphic with respect to g.332G>A SNP. As a result, the targeted loci in Munjal sheep is largely conserved. It is possible that this monomorphism is a breed-specific trait. Therefore, reported SNPs were not considered to be a universal marker for a specific feature across all breeds. As a result, there is a need to investigate before implementing selection criteria.

The authors are indebted to the Director Research, LUVAS, Hisar, Haryana and Rastrtriya Krishi Vikas Yojna for funding and facility that enabled successful completion of the research.