Leptin is a candidate gene for growth rate in mammals. The worldwide studies on various livestock species have identified various point mutations in Leptin gene, which are found to be influencing important economic traits including growth rate. The main objective of present study was to determine Leptin gene polymorphism and its association with body weights in Madgyal sheep using PCR-SSCP technique. PCR-SSCP is a reproducible, rapid and quite simple method for the detection of mutations through electrophoretic mobility differences in PCR amplified DNA. We have successfully amplified 180 bp and 275bp amplicons representing exon 1/2 and exon 3 of LEP gene, respectively. The PCR-SSCP analysis of these amplicons revealed a uniform band pattern in all 100 Madgyal sheep. The occurrence of monomorphic nature of Leptin gene in the present study indicates the lack of Leptin gene mutation in Madgyal sheep.
Sheep (Ovis aries) rearing is the major occupation of Dhangar (shepherd) community in draught prone western and central region of the state of Maharashtra. Deccani and Madgyal are native sheep of Maharashtra. Madgyal village in Jat tehsil of Sangali district of Maharashtra is the main habitat of Madgyal sheep. This sheep is considered to be evolved through selective breeding of Deccani (Ghanekar, 1983; Pardeshi et al., 2007). It is categorized as an ecotype of Deccani sheep, however it is morphologically quite different than other ecotyoes of Deccani. These animals are generally white with brown patches, hairy type and tall. The Madgyal sheep is superior in respect of weight gain, prolificacy, early maturity and adult weight compared to Deccani. Yadav et al. (2014) compared Deccani ecotypes and Madgyal sheep and reported that the body measurements of Madgyal sheep are higher than all other ecotypes of Deccani sheep. The overall average birth weight is 3.0 to 3.5 kg, while weight of adult female and male is 45-50 kg and 50-55 kg, respectively. This sheep is considered as meat breed having average daily weight gain of around 200-225 gms. However, there is wide variation in the growth rate of Madgyal sheep. It produces rough wool with average annual yield of 250-260gms (Waghmode et al., 2008). Leptin gene (LEP) is one of the candidate growth gene involved intricately in the metabolism and growth of animals (Ge et al., 2000; Ge et al., 2003; Supakorn, 2009). LEP is located on chromosome 4 in the ovine genome and consists of three exons and two introns. Leptin (LEP), the hormonal product of the LEP, is the 16 kd protein secreted from adipocytes, plays an important role in regulation of growth, development and feed conversion efficiency. It also plays a key role in the regulation of reproductive performance by stimulating GnRH, FSH and LH release (Yu et al., 1997). In ruminants, LEP was also shown to be expressed in fetal tissues, mammary gland, rumen, abomasums, duodenum and pituitary gland (Yuen et al., 2002; Ehrhardt et al., 2002 and Bartha et al., 2005). The possible effects of LEP and its polymorphism on growth traits of sheep are unknown and little research has been conducted involving this hormone in sheep. Only a few studies have been reported on polymorphism in the Ovine LEP. Therefore, present research was carried out with the objective to find out potential variation in the ovine LEP using Polymerase Chain Reaction–Single-Strand Conformational Polymorphism (PCR–SSCP) analysis and to investigate their associations with growth rate in Madgyal sheep.
Materials and Methods
Experimental Animals and DNA Extraction
The experimental material comprised blood samples (n=100) from Madgyal sheep of either sex maintained at various Sheep and Goat farms, of Punyashlok Aahilyadevi Maharashtra Sheep and Goat Development Corporation Ltd., Pune. The data regarding birth weight, three month body weight and 6 month body weight was also collected during sample collection. The genomic DNA was isolated from blood samples using traditional Phenol: Chloroform: Isoamylalcohol method of DNA isolation as described by Sambrook and Russel, 2006 with minor modifications. The quantity and quality of DNA was checked by photo- spectrometer (NanoDrop ND-2000, Thermo, USA).
In order to study the polymorphism in exonic region of Leptin gene, two sets of primers (F 5’CCC GGG AAG GAA AAT GCG CTG TGG ACC3’ and R 5’CTC ATC TTC CAG TCC TTC CCT ACC GTG3’) were used. This primer pair was designed on the basis of DNA sequence of caprine LEP (GenBank Acc. No. JQ739232) to amplify 180bp of LEP exon 1/2. While, 275bp exon -3 of LEP was amplified by using primer set F: 5′-GCT CCA CCC TCT CCT GAG TTT GTC C3′ R: 5′-TGT CCT GTA GAG ACC CCT GTA GCC G3’ as described by Tahmoorespur et al. (2009).
PCR was carried out in a total volume of 25 µl using Mastercycler Nexus gradient thermocylcer (Eppendorf, Germany). The reaction mixture consisted of 10X ‘Dreamtaq’ green PCR Buffer, 20 mM MgCl2, 0.2 mM of each dNTPs, 5 pmol of each primer, 1 U Taq polymerase (Thermofisher scientific) and ≈50-100 ng genomic DNA. For amplification of LEP exon 1/2, the PCR cycle was accomplished by initial denaturation for 5 min at 950C, 35 cycles of denaturation at 940C for 30 sec, annealing at 590C for 30 sec., extension at 720C for 1 min. and final extension at 720C for 10 min. While, for amplification of exon -3 region, PCR cycle was consisted of initial denaturation for 10 min at 950C, 35 cycles of denaturation at 940C for 30 sec, annealing at 66.50C for 30 sec., extension at 720C for 30 sec. and final extension at 720C for 10 min. The PCR products were visualized on 2.5% agarose gel stained with 1% EtBr.
Single Strand Confirmation Polymorphism (SSCP)
3.2 μl of PCR products were mixed with 7.7 μl denaturing solution (95% formamide deionized, 25 mM EDTA, 0.025% xylene- cyanole and 0.025% bromophenol blue), incubated at 95°C for five minutes and was snap chilled on ice. This denatured DNA was subjected to 12% native PAGE (polyacrylamide gel electrophoresis) in 0.5 per cent TBE buffer at a constant voltage of 200V for 3-4 hrs. These polyacrylamide gels were stained with 0.23 % silver nitrate for observing SSCP band pattern.
The present investigation was undertaken to identify Leptin gene polymorphism in Madgyal Sheep using PCR-SSCP method. The extracted genomic DNA from the blood samples (n = 100) had the O.D. ratio in the range of 1.8 to 2.0 (OD 260: 280) and its concentration ranged between 50-285 ng/μl. The required amount of DNA was diluted with ultra-pure water to get approximately 50-100 ng/μl as a final DNA concentration for further use. For optimization of PCR suitable annealing temperature was tested from a range of 55-68°C in the gradient thermal cycler, however primers of exon 1/2 and exon 3 of LEP annealed at 590C and 66.50C, respectively. The 180 bp fragment comprising exon 1/2 (Plate 1) and 275 bp fragment comprising third exonic region (Plate 2) of LEP was successfully amplified. The locus specific clear dark bands were visualized in all samples.
Plate 1: EtBr Stained 2.5% agarose gel showing 180bp LEP exon 1-2 PCR amplicons generated from Madgyal Sheep (lanes 1-21). Lane: L, 50 bp DNA ladder
Plate 2: EtBr Stained 2.5% agarose gel showing 275bp LEP exon 3 PCR amplicons generated from Madgyal Sheep (lanes 1-22). Lane: L, 50 bp DNA ladder
For detection of allelic variation in the amplified exonic regions of LEP, the PCR products were further processed for SSCP. The non-denaturing gel electrophoresis enabled the visualization of ssDNA and was analyzed for SSCP band patterns. The present study revealed, uniform PCR-SSCP band pattern for LEP exon 1/2 and exon 3 (Plate 3 and 4 respectively) in all DNA samples.
Plate 3: PCR-SSCP band pattern in the LEP exon 1-2 fragment of Madgyal leptin gene
Plate 4: PCR-SSCP band pattern in the LEP exon 3 fragment of Madgyal leptin gene
The conformation pattern LEP exon 1/2 was characterized with two uniform bands while four alternate light and dark bands were visualized for LEP exon 3 on PAGE. Both LEP exon 1/2 and exon 3 were designated as homozygote. The present investigation could not detect any mutation in the LEP exon 1/2 and exon -3 region of ovine LEP. The absence of polymorphisms indicated the probable lack of mutation/s suggesting high degree of conservation of Leptin gene in Madgyal breed of sheep.
Several Single Nucleotide Polymorphism (SNPs) have been reported across different livestock species. Three SNPs were detected in the Exon 3 of LEP in Poll Dorsets, Suffolk, Texels and Tan sheep all of which resulted in amino acid changes (Li et al., 2008). Zhou et al. (2009) reported four SNPs in exon 3 of the ovine LEP out of which three SNPs were non-synonymous and resulted in amino acid changes at codon positions 105, 120 and 144. Singh et al., (2009) reported five major haplotypes in exon 2 region and six major haplotypes in intron 2 region of LEP gene in Barbari and Jamunapari goat breeds of India. The studies about LEP exon 3 genotypes were carried out in Baluchi and Kermani sheep breeds (Tahmoorespur et al., 2010; Tahmoorespur and Ahmadi, 2012 and Shojaei et al., 2010) of Iran. Tahmoorespur et al., 2010 observed significant association of three genotypes of LEP with additive EBVs for weaning weight at 90 days in Baluchi and Kermani sheep and it was concluded that LEP polymorphism might be the one of the important genetic factor influencing growth traits. Some workers have reported five genotypes for Exon 3 of LEP gene in Makoei sheep of Iran (Hashemi et al., 2011; Hajihosseinlo et al., 2012). Genotyping of Assaf, Awassi and Dorper breeds of sheep in the Exon 3 of LEP gene, resulted in three synonymous and three nonsynonymous mutations (Reicher et al., 2010; Reicher et al., 2011). Lara et al. (2012) reported one SNP305 of exon 2 and four genotypes in exon 3 of LEP gene of native, commercial and crossbred sheep. The sequencing of LEP exon 3 of Nilagiri revealed two SNPs as 16973 G>A (SNP-L1) and 17476 C>T (SNP-L2). Ozge and Oztabak (2016) observed the highest haplotype number with nucleotide and haplotype diversity both in exon 2 and intron 2 in the Anatolian Black goat breed while only one haplotype was found in both exon 2 and intron 2 in Angora goat breed.
Our observations of monomorphic exon 1/2 and 3 of ovine LEP 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 LEP. Qureshi et al. (2015) reported monomorphic pattern at position A2262T, C2256G, -2730C in intron 2 and A3201G at exon 2 sequence of LEP in three sheep breeds of Pakistan using PCR RFLP technique. However, they observed polymorphism at position C1467T and A3050- in intron 2 of Ovine LEP. The monomorphic nature of Leptin gene in the present study indicates the lack of mutation in LEP gene, therefore the association studies of nucleotide variation with growth rate could not be established.
The present study revealed that Leptin gene was monomorphic at exon 1/2 and exon 3 locus in the screened samples of Madgyal Sheep. This might be due to the relatively small population of Madgyal sheep in which the Leptin gene might have more protected genetic structure. These findings are in contrast with the earlier reports of polymorphic nature of ovine Leptin gene. The lack of nucleotide variation in ovine LEP in the present study could not establish association with growth rate. Furhter research on other candidate growth related genes can be targetted for development of a suitable marker in sheep.