Identification of a Novel Single Nucleotide Polymorphism in Porcine Beta-Defensin-1 Gene.

D R Pruthviraj,A P Usha,R T Venkatachalapathy

doi:10.5713/ajas.15.0638

Abstract

Porcine beta-defensin-1 (PBD-1) gene plays an important role in the innate immunity of pigs. The peptide encoded by this gene is an antimicrobial peptide that has direct activity against a wide range of microbes. This peptide is involved in the co-creation of an antimicrobial barrier in the oral cavity of pigs. The objective of the present study was to detect polymorphisms, if any, in exon-1 and exon-2 regions of PBD-1 gene in Large White Yorkshire (LWY) and native Ankamali pigs of Kerala, India. Blood samples were collected from 100 pigs and genomic DNA was isolated using phenol chloroform method. The quantity of DNA was assessed in a spectrophotometer and quality by gel electrophoresis. Exon-1 and exon-2 regions of PBD-1 gene were amplified by polymerase chain reaction (PCR) and the products were subjected to single strand conformation polymorphism (SSCP) analysis. Subsequent silver staining of the polyacrylamide gels revealed three unique SSCP banding patterns in each of the two exons. The presence of single nucleotide polymorphisms (SNPs) was confirmed by nucleotide sequencing of the PCR products. A novel SNP was found in the 5′-UTR region of exon-1 and a SNP was detected in the mature peptide coding region of exon-2. In exon-1, the pooled population frequencies of GG, GT, and TT genotypes were 0.67, 0.30, and 0.03, respectively. GG genotype was predominant in both the breeds whereas TT genotype was not detected in LWY breed. Similarly, in exon-2, the pooled population frequencies of AA, AG, and GG genotypes were 0.50, 0.27, and 0.23, respectively. AA genotype was predominant in LWY pigs whereas GG genotype was predominant in native pigs. These results suggest that there exists a considerable genetic variation at PBD-1 locus and further association studies may help in development of a PCR based genotyping test to select pigs with better immunity.

Highlights

Disease occurrence is a major threat to any livestock farming and pig production is no exception
Defensins are a subclass of cationic Antimicrobial peptides (AMPs) with broad spectrum antimicrobial activity against various bacteria, fungi and Pruthviraj et al (2016) Asian Australas
Blood samples were collected from 50 Large White Yorkshire (LWY) and 50 Ankamali pigs reared at Centre for Pig Production and Research, Mannuthy, Thrissur, Kerala, India

Summary

Introduction

Disease occurrence is a major threat to any livestock farming and pig production is no exception. Selection for disease resistance employing molecular markers would help in evolving a population with greater immunity. Modern techniques like polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) are helpful in identifying such molecular markers. AMPs are polypeptides made up of less than 100 amino acid residues (Ganz, 2003). Based on the net charge present, AMPs are broadly classified into anionic and cationic peptides (Hancock, 1997). Defensins are a subclass of cationic AMPs with broad spectrum antimicrobial activity against various bacteria, fungi and Pruthviraj et al (2016) Asian Australas. They possess six to eight highly conserved cysteine residues that form intramolecular disulfide bonds based on which, three families of defensins are defined viz. They possess six to eight highly conserved cysteine residues that form intramolecular disulfide bonds based on which, three families of defensins are defined viz. α-, β- and θ-defensins (Lai and Gallo, 2009)

Objectives

Findings

Methods