A note on a DNA polymorphism study of leptin gene in Sahiwal and crossbred cattle using PCR-RFLP technique

A Dandapat, D Kumar, A K Ghosh and V Umapathi*

Department of Genetics and Animal Breeding, College of Veterinary and Animal Sciences,
G B Pant University of Agriculture and Technology, Pantnagar, Uttarakhand – 263145, India
dr_dandapat@yahoo.co.in
* Department of Veterinary Biochemistry

Abstract

Leptin performs important roles in the control of body weight, feed intake, immune function, and reproduction. The present study was carried out to indicate polymorphisms at the bovine leptin gene locus in Indian purebred Bos indicus (Sahiwal) and crossbred cattle Bos taurus x Bos indicus (Jersey X HF X Sahiwal).

The 330bp leptin gene fragment is polymorphic in Bos taurus x Bos indicus crossbred cattle and monomorphic in purebred Bos indicus (Sahiwal) cattle with respect to HphI PCR-RFLP. In crossbred cattle the genotype and gene frequency was found to be 0.57, 0.36 and 0.07 for AA, AV and VV genotypes and 0.75 and 0.25 for A and V alleles, respectively. Since no mutation was found in Sahiwal cattle and only A allele was present throughout the population studied, the frequency of A allele was one.

Keywords: Crossbred cattle, DNA polymorphism, leptin gene, Sahiwal

Introduction

Leptin is a hormone which is mainly produced in white adipose tissue and secreted into the bloodstream as a 16 kDa protein. It performs important roles in the control of body weight, feed intake, energy expenditure, immune function and reproduction and regulates through a neuroendocrine pathway. It was initially reported that the mutant gene ob of mouse is closely associated with obesity (Zhang et al 1994). It has been shown that leptin gene influences milk performance in cattle (Liefers et al 2002; Madeja et al 2004). It seems that leptin has a large effect in coordinating whole body energy metabolism and may be classified as a “metabolism modifier” (Houseknecht et al 1998). In cattle, leptin gene (LEP) is located on 4^th chromosome (Pomp et al 1997) and consists of three exons. Several polymorphic studies on the bovine leptin gene have been reported (Pomp et al 1997; Lien et al 1997; Wilkins and Davey 1997; Haegeman et al 2000; Buchanan et al 2002). Pomp et al (1997) verified that the frequency of a restriction fragment length polymorphism (LEP Sau3AI) in bovine leptin gene was different between Bos taurus and B. indicus cattle breeds. Madeja et al (2004) showed that the HphI polymorphism had an effect on the breeding values of yield traits. The recognition site of HphI restriction enzyme is 5′...GGTGA (N) _8↓…3′ and 3′...CCACT (N) _7↓…5′. The aim of this study was to indicate polymorphisms at the bovine leptin gene locus in Indian purebred Bos indicus (Sahiwal) and crossbred cattle Bos taurus x Bos indicus (Jersey X HF X Sahiwal).

Materials and methods  

The present investigation was carried out by randomly selecting 70 crossbred cattle and 30 Sahiwal cattle from a cattle herd of 760 crossbreds and 87 Sahiwal cattle maintained at Instructional Dairy Farm (IDF), G.B. Pant University of Agriculture and Technology, Pantnagar, Uttarakhand. A total of 70 blood samples from 70 crossbred cattle and 30 blood samples from 30 pure Sahiwal cattle were collected.

About 5-10 ml of the blood was collected from each animal from the jugular vein in sterile polypropylene vials containing 0.5 M EDTA (pH 8.0) solution as an anti-coagulant under sterile conditions. After collection of blood, the vials were tightly capped and shaken gently to facilitate proper mixing of blood with the anti-coagulant. The vials were then kept immediately in icebox containing ice and gel cool packs and transported to the laboratory immediately. In the laboratory, samples were kept in deep freeze at -20ºC till the isolation of DNA.

The genomic DNA was extracted from blood samples by using phenol-chloroform extraction protocol (Sambrook and Russell 2001) with suitable modification. Quality and quantity of the extracted DNA were analyzed using gel electrophoresis and UV Spectrophotometry, respectively, and only good quality samples were used for further study. 

A 330bp region of the leptin gene exon 2 was amplified using a pair of primers (Forward primer-5′ GGGAAGGGCAGAAAGATAG 3′ and Reverse primer-5′ TGGCAGACTGTTGAGGATC 3′) described by Haegeman et al (2000). PCR was performed in a final volume of 50 µl containing 100 µm dNTPs mix, 20 pmol of each primer, 5 µl of 10X PCR assay buffer, 1.5 mM MgCl₂, 1.2 U of Taq DNA polymerase, 150 ng of the bovine genomic DNA and 34.3 µl autoclaved triple distilled water (pH 7.0). The PCR conditions were 5 min denaturation followed by 35 cycles of 30 sec at 94°C, 30 sec at 55°C and 45 sec at 72°C with an additional 5 min extension in the last cycle. The amplification of the 330bp region was carried out using a thermal cycler (PTC-200, M J Research). The PCR products were separated by 2% (w/v) agarose gel electrophoresis at 60V for one hour (Figure1).

The amplified fragments were digested overnight with 5 U of HphI (Genetix Biotech Asia Private Limited, India) at 37^°C in water bath. The reaction was stopped by adding a drop of 0.5M EDTA (pH 8.0). The digested products were then kept at 4°C till further study.  The restriction fragment of different lengths was separated in 2.5% (w/v) agarose gel run at 60V for 1 h. After completion of gel electrophoresis the digested products were visualized by keeping the gel over UV transilluminator and documented by photography with the help of gel documentation system to detect the genotype of each sample (Figure 2).

Statistical analysis for estimation of gene and genotypic frequency was performed using the standard methods as suggested by Falconer and Mackay (1998).

Results and discussion   

Digestion of the PCR product with HphI restriction enzyme revealed three patterns, one with 330bp i.e no HphI site (AA genotype); second with 330bp, 310bp and 20bp (AV genotype) and third with 310bp and 20bp (VV genotype) in crossbred cattle (Figure 2). The smaller fragment of 20bp could not be visualized on gel. Only one undigested fragment of 330bp was found in Sahiwal cattle (AA genotype). Similar results were also observed in different breeds of cattle (Haegeman et al 2000; Liefers et al 2002; Choudhary 2004). In the present study, the result shows that the mutation has produced recognition site for HphI restriction enzyme in crossbred cattle.

In crossbred cattle the genotype and gene frequency was found to be 0.57, 0.36 and 0.07 for AA, AV and VV genotypes and 0.75 and 0.25 for A and V alleles, respectively (Table 1).

Since no mutation was found in Sahiwal cattle and only A allele was present throughout the population studied, the frequency of A allele was one (Table 1). In contrast to the present findings, lower frequency of the mutant allele (V) was reported in Belgium Blue crossbred (0.125) (Haegeman et al 2000). The present results in crossbred are in agreement with the findings of Choudhary (2004) who reported frequency of AA, AV and VV genotypes in crossbred (HF X Hariana) as 0.53, 0.42 and 0.05, respectively. Liefers et al (2002) reported the frequency of AA, AV and VV genotypes as 0.581, 0.329 and 0.090, respectively in HF breed. The present results show that the low frequency of VV genotype in crossbred cattle may be due to absence of V allele in Sahiwal cattle. HF and Jersey both might be the only source of V allele in crossbred population.
 

Conclusions  

• In the present study, the 330bp leptin gene fragment is polymorphic in Bos taurus x Bos indicus crossbred cattle and monomorphic in purebred Bos indicus (Sahiwal) cattle with respect to HphI PCR-RFLP. In crossbred cattle the genotype and gene frequency was found to be 0.57, 0.36 and 0.07 for AA, AV and VV genotypes and 0.75 and 0.25 for A and V alleles, respectively. Since no mutation was found in Sahiwal cattle and only A allele was present throughout the population studied and the frequency of A allele was one. This genetic information of bovine leptin gene could be useful for further genetic studies of this gene.

References 

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Received 8 September 2009; Accepted 28 October 2009; Published 1 January 2010

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