Novel single nucleotide polymorphism identificationin interleukin-6 gene of Pakistani sheep

Safdar Ali • Masroor Ellahi Babar •

A. Hossain Farid • Pervez Akhtar • Ali Raza Awan

Received: 8 March 2010 / Accepted: 4 September 2010 / Published online: 29 September 2010

� Springer Science+Business Media B.V. 2010

Abstract The present study aimed to identify single-

nucleotide polymorphism (SNP) in coding and non-coding

regions of interleukin-6 (IL-6) gene of Pakistani sheep. The

IL-6 gene of 205 animals from nine sheep breeds were

sequenced for screening of SNP. Characterizing the IL-6

gene revealed thirteen SNP sites within the intronic region

of IL-6 gene. The novel SNPs found in the present study

can serve as genetic marker for association studies with

susceptibility/resistance to parasite infection in sheep. This

is first report of SNP polymorphism of IL-6 gene of

Pakistani sheep.

Keywords Sheep � Polymorphism � SNP � Pakistan �Interleukin � Ovine

Introduction

Pakistan has one of the biggest and diversified genetic pool

of sheep with 27 million animals is classified into more

than 20 indigenous breeds (Economic Survey of Pakistan;

http://www.finance.gov.pk/finance_survery_chapter.aspx?

id=21; 08-03-2010). The sustainable control of diseases

caused by internal parasites in livestock requires not only

management practices but also dependent on the

enhancement of host resistance. Host resistance (or

immunity) is the ability of an animal to cure a parasitic

infection and to prevent reinfection by utilizing both innate

and acquired immune responses. Sheep internal parasites

are included Dictyocaulus (Lung worm), Haemonchus

(abomasum), Nematodirus, Cooperia (small intestine),

Oesophagostomum (large intestine), Taenia (tapeworms),

Teladorsagia (Ostertagia) and Trichostrongylus (aboma-

sum and small intestine). Host genotype influences

resistance to worms, with significant genetic variation for

resistance to a number of nematode species reported both

between and within sheep breeds [1, 2]. Cytokines are the

immune regulators, which have a significant role in para-

site resistance in sheep [3]. IL-6 is a proproliferative, anti-

apoptotic cytokine [4]. The several complete-genome

projects have led to the emergence of single-nucleotide

polymorphisms (SNPs) as most modern genetic markers.

SNPs occur frequently in the mammalian genome [5, 6]

and are useful for rapid, large-scale, and cost-effective

genotyping [7–10] for ecological and conservation studies

[10–12] and for population and evolutionary studies

[13–15]. The gene encoding for sheep IL-6 gene is present

on sheep chromosome 4 (www.ncbi.nlm.nih.gov; 01-06-

2010). Polymorphisms within IL genes have been reported

to be associated with susceptibility/resistance to infectious

agents especially internal parasites [16]. However, infor-

mation regarding the polymorphisms within the sheep IL-6

gene is lacking. In the present study, we describe through

DNA sequencing, the locus specific polymorphisms within

different regions of sheep IL-6 gene in different breeds of

Pakistani sheep.

S. Ali � P. Akhtar

Department of Animal Breeding and Genetics, University of

Agriculture Faisalabad, Faisalabad, Pakistan

M. E. Babar � A. R. Awan (&)

Institute of Biochemistry & Biotechnology, University of

Veterinary and Animal Sciences, Outfall Road, Civil Lines,

Lahore, Pakistan

e-mail: arawan77@uvas.edu.pk

A. Hossain Farid

Department of Agriculture and Animal Sciences,

Nova Scotia Agricultural College, Truro, NS, Canada

123

Mol Biol Rep (2011) 38:2151–2154

DOI 10.1007/s11033-010-0342-5

Materials and methods

To explore the variations within sheep IL-6 gene, unrelated

animals (different families having no blood relation) of

sheep breeds with typical phenotypic features known for a

given breed were selected from several respective breeding

tracts and from the government livestock farms. Blood

samples were collected from a total of 205 animals

belonging to nine well-recognized breeds of Pakistani

sheep, which included Buchi (20), Sipli (23), Thalli (22),

Pak karakul (28), Kachi (24), Lohi (24), Awaissi (19),

Hissardale (22) and Kajli (23). All these different sheep

breeds exist in different geographic and agro-climatic

conditions in Pakistan.

Blood was collected from jugular vein into EDTA con-

taining vacutainer tubes, and DNA extraction was performed

from whole blood following standard phenol–chloroform

extraction method [17]. Primers designed using sheep

sequences (where available) or the consensus sequences of

the closest species in GenBank were used for Polymerase

Chain Reaction (PCR) amplification of the corresponding

genomic fragment. Purified PCR products were sequenced

with both forward and reverse primers using BigDye ter-

minator cycle sequencing kit (Applied Biosystems, USA) on

ABI 3100 Genetic Analyzer. Sequence data were edited

manually using Chromas Ver. 1.45, http://www.tech

nelysium.com.au/chromas.html). Nucleotide sequence of

sheep IL-6 gene was submitted to GenBank (NCBI) under

the accession no. FJ409227. Multiple sequence alignments

were performed with ClustalW freeware. The coding DNA

sequences of different regions were conceptually translated

to amino acid sequences using Bioedit software. Allelic

Frequencies and Hardy–Weinberg Equilibrium was calcu-

lated by using POPGENE3.2 freeware.

Results and discussion

Results of analysis of sequencing of IL-6 gene of nine sheep

breed revealed distinct pattern of variance (Table 1). At

nucleotide position 1372 two variant VS1A and VS1B was

observed. Variant VS1A was monomorphic with 1.00

(100%) frequency in all breeds except Sipli and Thalli,

which have both monomorphic and heteromorphic variants.

Among the nine sheep breeds analyzed variant VSB1 was

completely absent in breed Buchi, Kajli, Pak-Karakul,

Kachi, Lohi, Awassi and Hissaredale while it was predom-

inant in Sipli and Thalli. Three different variants VS2A,

VS2B and VS2C were observed at nucleotide position 1829

analyzing sheep breeds. VS2C was least frequent with over

frequency of 0.166. VS2A and VS2B were almost equally

predominant in all sheep breeds with over all frequency of

0.585 and 0.249, respectively. At the locus 3036 the

monomorphic genotype (VS4A) was predominant with

average frequency of 0.937. VS4A had maximum frequency

i.e. 1.0 in all breeds except Sipli (0.479) and Thalli (0.955).

Locus 4171 was all sheep breeds were highly heteromorphic

with three variants VS5A, VS5B and VS5C with overall

frequency of 0.537, 0.346 and 0.117 but in Kajili there are

only two variants (VS5A and VS5B).

A multiple sequence alignment revealed that for locus

1372 variant VS1A was conserved only in Ovis aries and

Table 1 Genotypic and allelic frequencies of IL-6 variants

Genotypes Locus Breeds (sample size)

Name Alleles B (20) Kaj (23) S (23) T (22) P (28) Kac (24) L (24) A (19) H (22) All breeds

(205)

VS1A CC 1372 1.0 1.0 0.479 0.955 1.0 1.0 1.0 1.0 1.0 0.937

VS1B CT 0.0 0.0 0.521 0.045 0.0 0.0 0.0 0.0 0.0 0.063

VS2A AA 1829 0.5 0.652 0.609 0.773 0.536 0.458 0.667 0.368 0.682 0.585

VS2B AG 0.35 0.217 0.304 0.091 0.250 0.334 0.125 0.369 0.227 0.249

VS2C GG 0.15 0.131 0.087 0.136 0.214 0.208 0.208 0.263 0.091 0.166

VS3A ??a 2450 0.9 0.391 0.217 0.636 0.536 0.792 0.542 0.474 0.455 0.546

VS3B ?- 0.1 0.609 0.783 0.273 0.464 0.208 0.333 0.526 0.545 0.430

VS3C -? 0.0 0.0 0.0 0.091 0.0 0.0 0.125 0.0 0.0 0.024

VS4A CC 3036 1.0 1.0 0.479 0.955 1.0 1.0 1.0 1.0 1.0 0.937

VS4B CT 0.0 0.0 0.521 0.045 0.0 0.0 0.0 0.0 0.0 0.063

VS5A TT 4171 0.4 0.913 0.348 0.727 0.321 0.458 0.542 0.684 0.5 0.537

VS5B TG 0.4 0.087 0.565 0.227 0.572 0.375 0.292 0.211 0.318 0.346

VS5C GG 0.2 0.0 0.087 0.046 0.107 0.167 0.166 0.105 0.182 0.117

a Refers to insertion (?)/deletion (-) of CCT

B Buchi, Kaj Kajli, S Sipli, T Thalli, P Pak-Karakul, Kac Kachi, L Lohi, A Awassi, H Hissardale

2152 Mol Biol Rep (2011) 38:2151–2154

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Sus scrofa (Fig. 1). At locus 1829 and 4171 the variant

VS2A and VS5A were present in all species while at locus

3036 none of the variants were found in aligned species.

All variants except above mentioned were not found in

other species. Although these SNPs were found in interonic

region of sheep IL-6 gene but the importance of these

mutations cannot be overlooked as it had been found that

the mutation in interonic regions can alter the gene

expression by effecting regulation and splicing pattern

[18, 19]. The present study, revealed a total of 13 novel

SNPs within IL-6 gene of sheep. These SNPs can also be

used as genetic markers for breed characterization and it

will also help the future association studies for suscept-

ibility/resistance to parasite infections. This is first report of

gene characterization and SNP polymorphism of IL-6 gene

of Pakistani sheep breeds.

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Fig. 1 Multiple alignment of IL

6 gene the conservativeness of

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