International Journal of Animal and Veterinary Advances 3(1): 6-9, 2011
ISSN: 2041-2908
© Maxwell Scientific Organization, 2011
Received: August 07, 2010
Accepted: September 01, 2010
Published: February 05, 2011
Estimation of Genetic Diversity in Sheep (Ovis aries) using
Randomly Amplified Polymorphic DNA
1
M. Qasim , 1H. A hmad, 1S. Ghafoor, 1S.G. Afridi, 1I. Muhammad and 1,2 A.K. Imtiaz
1
Department of Genetics, Hazara University, Mansehra, Pakistan
2
Department of Genetics, University of Karachi, Karachi, Pakistan
Abstract: Domestic sheep (Ovis aries) belo ngs to o rder Artiod actyla. Despite its great economic importance,
not much work has been done in Pakistan on breeding and genetics of sheep. Present research wa s initiated to
analyze DNA based genetic diversity in Pakistani breeds of sheep. Total genomic DNA was isolated from
blood of Rambo uillet, Kaghani and R amghani breeds. Po lymerase Chain Reactions (PCR) were carried out
using six Ran dom ly Amp lified Polymo rphic DNA (RAPD) primers. Amplified fragments ranged in size from
500 bp to 2000 bp. On average, 3 alleles per genotype were amplified. Average genetic distance estim ate
ranged from 30-1 00% . Eleven com pariso ns showed maxim um ge netic diversity (G D = 100 %) while 6
comparisons sowed minimum genetic diversity (GD = 30%) for the loci detected using 6 RAP D prim ers.
Phylo genetic elaboration among the 14 sheep accessions were carried out using dendrogram analysis. The 14
accessions o f sheep were group ed into 5 clusters com prising 3 , 3, 3, 3 and 2 accessions, respectively.
Key w ords: Dendro gram, genetic diversity, Ovis aries, phylogeny, RAPD , sheep
INTRODUCTION
Do mestic sheep (Ovis aries) are quadra p edal,
ruminant mam mals typically kep t as livestock.
Archaeological evidence shows that the sheep was
dom esticated approxim ately 90 00 year ago in the border
region between Syria and Iraq (Hecker, 1983; Simm ons
and Ekarius, 2001). Sheep are distributed allover the
world in wild. It is a general consensus that some breeds
of sheep retain som e of the c harac teristics of the ir wild
relative, such as short tails. In Pakistan most of the short
tailed sheep are reared in northern areas. The fat-tailed
sheep are common in western part of the country
including FATA and B alochistan. Depending upon the
breed and selective pressure, sheep show range of height
and weight. Typically sheep weight between 45-100 Kg
and with the larg er ram s between 4 5-16 0 K g.
Pakistan is home to seven species of sheep with 11
sub-species of sub family Ca prinae. Two sp ecies with
very limited ranges in Pakistan are Blue Sheep and
Marcopolo sheep, both restricted to northern Pakistan.
Urial on the other hand is the mo st widespread breed in
Pakistan. Pakistan has 28 indigenous breeds of sheep
(Ha snain and Shah, 1985). Punjab and Khyber
Puktoonkhawa have seven each; Balochistan and Azad
Jammu and Kashmir (AJK) have four each while Sindh
and Northern Are as have three breeds each. In P akistan
sheeps are kept for wool and mutton. Milk is important
only in Damani breed. Most of the sheep breeds produce
coarse wo ol. Co mmercially important breeds of sheep in
Pakistan include Rambouillet, Awassi, Kaghani,
Ram ghani, Dam ani, Kachhi and L ohi (Ishaq, 1982 ).
During recent years, DNA technology has been used
to study variation in sheep genetic material. Much of
these studies have concentrated on the use of more
difficult, time consuming Mitochondrial DNA. For
example, Zhao et al. (200 4) used PCR and R FLP to
study paternal inheritance of mitochondrial DNA.
Tapio et al. (200 6) Studied variation in sheep
mitoc hond rial DNA and concluded that some
mitochondrial lineage arrived in Europe from the near
east. Despite high economic value of sheep, not much
work has been done in sheep breeding and genetics in
Pakistan. The present study was aimed to (i) establish
molecular procedures suitable for sheep using locally
available cond itions, and (ii) estimatio n of genetic
variation existing in local sheep bre eds using easy,
cheaper and user’s friendly technique of Randomly
Amplified D NA .
MATERIALS AND METHODS
Fourteen healthy male sheep (Ovis aries) were used
during prese nt study. A ccess to the stock was kindly
provided by D r. Muqarrab A li Khan, Director Livestock
Research Station Jaba, Mansehra. These accessions
belonged to Rambouillet, Kaghani and Ramghani breeds
(Table 1). Experimen tal work was conducted at the
Department of Gene tics, Hazara U niversity, Mansehra
during 2009.
The blood samples were collected from the ulnar
superficial vein using 27 Gua ge hypode rmic needle and
5ml disposible syringe. Each blood sample was
transferred immediately to a 3 mL E DT A (Ethylene
Diamine Tetra Acetate) tube. EDTA tubes were kept in
Corresponding Author: A.K. Imtiaz, Department of Genetics, University of Karachi, Karachi, Pakistan
6
Int. J. Anim. Veter. Adv., 3(1): 6-9, 2011
Table 1: Some basic information regarding experimental sheep stock
S. N o.
Access ion #
Stra in
1
E 3 -4
Ram bouillet
2
C 8-86
Ram bouillet
3
6R-36
Ram bouillet
4
7K -6
Kaghani
5
X2 -16
Ramghani
6
C3-82
Ram bouillet
7
C3-84
Ram bouillet
8
X3-24
Ramghani
9
C3 -52
Ram bouillet
10
D3-76
Ram bouillet
11
R2-26
Ram bouillet
12
E3 -4
Ram bouillet
13
C3 -8
Ram bouillet
14
R2-26
Ram bouillet
Table 2: List of RAPD primers used during present study
O lig o n am e
Seque nce(5`-3`)
GLA-19
CAAACGTCCGG
GLB-20
GCACCCTTAC
GLC-11
AAAGCTGCGG
GLC-14
TGCGTGCTTG
GLC-15
GACGGATCAG
GLC-20
ACTTCGCCAC
Ye ar of b irth
2004
2003
2006
2007
2002
2003
2003
2003
2003
2003
2002
2004
2003
2002
Mol wt
3,037
2,988
3077
3050
3077
2947
ice box until stored at -20ºC in refrigerator. T he small
scale DNA isolation procedure developed by W eining and
Langridge (1991) was used with m odifica tion to iso late
total genomic DNA from the blood. The EDT A tubes
containing blood samples were thawed and 300 :L blood
was taken in an Eppendorf tube. Three hundred :L DNA
extraction buffer (20% SDS, 100 mM Tris-Cl, 400 mM
NaCl, 10 mM EDT A, pH = 8 .5) was added to the
Eppend orf tube and mixed well. Then 300 :L of P heno l:
Chloroform : isoamyalcohol (in ratio of 25:24:1) was
added and the Eppendorf tubes were placed in ice on a
shaker for approximately 1 h. Shaking was continued
until homogenous mixture was obtained. The tubes were
centrifuged at 12000 rpm for 5 min. The aqueous phase
was transferred to fresh tubes. Then 30 :L 3M Sodium
Acetate (pH = 4.8) and 300 :L isopropanol was added
and mixed gently until the DNA precipitate was visible.
The Eppendorf tubes were then centrifuged at 12000 rpm
for 10 min to make the DNA pellet. Supernatant was
discarded and DNA pellet was wa shed four time s with
70% ethano l, partia lly dried at room temperature and
dissolved in 40 :L TE buffer. Q uality and quantity of the
DNA was checked on 1% agaro se/TBE gel. Six
Random ly Amplified P olymo rphic DNA (RAPD) primer
viz; GLA-19, GLB-20, GLC-, 11, GLC-14, GLC-15 and
GLC-20 (obtained from Gene Link, Inc, 10532, NY,
USA) were used to amplify genomic DNA isolated from
sheep (Table 2). PCR were formulated following Devos
and Gale (19 92). Denaturation, annealing and extension
were carried out at 94, 34 and 72ºC, respectively. The
cycling was continued for 40 cycles. PCR products were
separated on 2% agarose/T BE gels.
For statistical analyses, every DNA band was
considered as a single allele / locus. The loci were scored
as present (1) or absent (0). The bivariat (1-0) d ata ma trix
W eight (Kg)
55 to 60
65 to 70
65 to 70
42 to 45
60 to 65
65 to 70
65 to 70
60 to 65
65 to 70
65 to 70
65 to 70
55 to 60
65 to 70
65 to 70
G C (% )
60
60
60
60
60
60
was generated and genetic distances (GD) among the
genotypes were estimated using “Unweighted Pair Group
of Arithm etic Means” p rocedure as described by Nei and
Li (1979). The Bivariate data was also used to construct
a dandrogram using computer program ”Popgene ver 3.2”
(Yeh et al., 1999).
RESULTS AND DISCUSSION
An example of PCR amplification profile of DNA
isolated from sheep accessions using Randomly
Amplified Polymorphic DN A Primers is presented in
Fig. 1. Various kinds of amplification profiles were
observed. Size of the DNA fragments was estimated using
1 kbp DNA ladder (Gene Link, Inc., USA). The DNA
fragments amplified ranged in size from 250 bp to
app roxim ately 200 0 bp . Average ge netic distance
estimates among the 14 sheep accessions b ased upon six
RAPD primers ranged from 30 to 100%. Six comparisons
showed minimum ge netic diversity (G .D = 30 %) while 11
comparisons showe d maximum genetic diversity
(G.D = 100%) using RAPD primers GLA-19, GLB-20,
GLC-11, GLC-14 , GLC -15, G LC-20 (Table 3).
Khan et al. (2007) reported high amo unt of genetic
diversity in Pakistani sheep breeds based upon
morphological
charac ters.
Stahlberger-Saitbekova
et al. (200 1) using Simple Sequence Rep eat (SSR)
primers repo rted m edium to high amou nt of genetic
diversity in Swiss sheep breed.
Biva riate 1-0 data matrix was also used for the
construction of dendrogram (Fig. 2). Fourteen accessions
were grouped in five clusters (A-E) com prising 3 , 3, 3, 3
and 2 accessions, respectively. It was observed that
accessions 1 (#E3-4, Rambouillet breed) and Accessions
7
Int. J. Anim. Veter. Adv., 3(1): 6-9, 2011
Table 3: Average gen etic distance
1
2
2
0.7
3
0.8
0.5
4
0.8
1.0
5
0.5
0.4
6
0.4
0.5
7
0.8
0.5
8
0.7
0.5
9
0.9
0.6
10
0.7
0.4
11
1.0
0.7
12
0.7
0.4
13
1.0
0.6
14
0.7
0.4
estimates am ong 14 acces sions of sheep u sing 6 RA PD primers
3
4
5
6
7
8
0.8
0.8
1.0
0.6
0.8
0.8
0.8
1.0
0.8
1.0
0.8
0.5
0.7
0.8
0.8
0.9
0.8
1.0
0.8
1.0
0.8
0.4
0.5
0.6
0.7
0.6
0.8
0.6
0.8
0.6
0.8
0.7
0.9
0.7
1.0
0.7
1.0
0.7
0.6
0.6
0.7
0.6
0.7
0.5
0.5
0.3
0.4
0.6
0.9
0.7
1.0
9
10
11
12
13
0.6
0.6
0.5
0.6
0.5
0.7
0.3
0.6
0.3
0.6
0.3
0.6
0.3
0.3
0.6
Fig. 1: PCR amplification profile of seven accessions sheep
using Randomly Amplified Polymorphic DNA (RAPD)
primer GLC-14 1 = accession # X3-24, 2 = accession
# C3-52, 3 = accession# D3-76, 4 = accession # R2-26,
5 = accession# E3.-4, 6 = accession# C3-8, 7 =
accession# R2-26. M = Molecular size marker 1Kb
DNA ladder (Gene Link). Size of DNA fragment (in
bp) is presented on right 1 = Accession # E 3-4, 2 =
Accession# C3-86, 3 = Accession 6R-86, 4 = Accession
# 7K-6, 5 = Accession # X2-16, 6 = Accession# C3-82,
7 = Accession# C3-84, 8 = Accession # X3-24, 9 =
Accession # C3-52, 10 = Accession# D3-76, 11 =
Accession # R2-26, 12 = Accession# E3-4, 13 =
Accession# C3-8, 14 = Accession# R2-26
7 (C3-84, Ramb ouillet breed) we re most distantly related
to each other.
Estimation of genetic diversity is a prerequisite for
improving of any species or genetic material. Various
procedures (M orp holo gical, cyto logica l and/o r
biochemical markers) have been utilized in the past for
the estimation of genetic diversity in various plants and
animal species of comm ercial importance. The se markers
were not co nsidered suitable for large scale utilization
mainly because of their limited numb er and /or difficult,
expensive and time consuming assay procedures (Islam
and Shepherd, 1991; Paterson et al., 1991). With the
recent introduction of DNA technology, Marker Assisted
Fig. 2: Dendrogram constructed for 14 sheep accessions using
data obtained from PCR involving six Randomly
Amplified Polymorphic DNA primers. 1 = Accession
# E 3-4, 2 = Accession# C3-86, 3 = Accession 6R-86,
4 = Accession # 7K-6, 5 = Accession # X2-16, 6 =
Accession# C3-82, 7 = Accession# C3-84, 8 =
Accession # X3-24, 9 = Accession # C3-52, 10 =
Accession# D3-76, 11 = Accession # R2-26, 12 =
Accession# E3-4, 13 = Accession# C3-8, 14 =
Accession# R2-26
Selection (MAS) of suitable genotypes has been utilized
extensively. These DN A based markers include
8
Int. J. Anim. Veter. Adv., 3(1): 6-9, 2011
Polyme rase Chain Reaction (PCR ), Restriction Fragment
Length Polymorphism (RFLP), Amp lified Fragment
Length Po lymorphism (AFLP), etc., Among PCR based
assays, various primer systems viz; Allele Specific
Am p l if ic a t io n (ASA) , Cleav age A mplific atio n
Polymorphic Sequences (CAPS), Sequence Tag Site
(STS), etc., have been used (Dubcovsky, 2004).
Random ly Amplified Polymorphic DNA (RAPD) has an
extra advantage that it does not require any sequence
information on the target genome. The P resent results are
among the initial documented reports of utilization of
DNA techno logy for the characterization and ultimate
improvement of sheep breeds in Pakistan.
Ishaq, S.M., 1982. Research and deve lopm ent.
Proceeding International Seminar on Sheep and
W ool. Pakistan Agricultural Research C ounc il,
Islama bad , pp: 14-16 .
Islam, A.K.M.R. and K.W. Shepherd, 1991. Alien
Genetic Variation in Wheat Improvement. In:
Gupta, P.K. and T . Tsuchiya (Ed.), Chromoso me
Engineering in Plants: Genetics, Breeding and
Evolution. Vol. A, E lsevier Scienc P ublishers,
Amsterdam, pp : 291 - 312.
Khan, M.S., M .A. Khan, S. Ahmad an d S. M ahmood,
2007. Genetic Resources and Diversity in Pakistani
Sheep. Int. J. Agr. Bio., 9: 941-944.
Ne i, N. and W . Li, 1979. M athem atical mode l for
studying genetic variation in terms of restriction
endonucleases. Proc. Natl. Acad . Sci., 76:
5269-5273.
Paterson, A.H., S.D. Tanksley and M.E. Sorrells, 1991.
DNA markers in plant imp rovement. Adv. Agron.,
46: 39-90.
Simmo ns, P. and C. Ekarius, 2001. S torey's Guide to
Raising Sheep. North Adams, Storey Publishing
LLC, MA. ISBN: 978-1-58017-262-2.
Stahlberger-Saitbekova, N., J. Schlapfer, G. Dolf and
C. Gaillard, 2001. Genetic relationships in Swiss
sheep breeds based on microsatellite analysis. J.
Anim. Breed. Genet., 118: 379-387.
Tapio, M., N. Marzanov, O. Mikhail, C. Mirjana,
G. Galina, T. Kiselyova, M. Maciej, V. Haldja and
K. Juha, 2006. Sheep mitochondrial DNA variation
in European, Cauca sian, and Central Asian Areas.
Mol. Biol. Evol., 23(9): 1776-1783.
W eining, S. and P. Langridge, 1991. Identification and
mapping of polymorphism in cereals base on
polymera se chain re action. Theor. A ppl. G enet., 82:
209-216.
Yeh, F.C., R.C. Yang, T.B.J. Boyle, Z.H. Ye and
J.X. Mao, 1999. POPGENE 3.2, the User-Friendly
Shareware for Population Genetic Ana lysis.
Molecular Biology and Biotechnology Centre,
University of Alb erta, Edmo nton, A lberta, Canada.
Retrieved from: http://www.ualberta.ca./~fyeh/fyeh.
Zhao , X., N. Li, W. Guo, X. Hu, Z. Liu, G. Gong,
A. Wang, J. Feng and C. Wu, 2004. Further evidence
for parental inheritance o f mitochondrial DN A in
the sheep (Ovis aries). Heridity, 93: 399-403.
CONCLUSION
It is concluded that high amounts of genetic diversity
(GD max = 100%) is present in the local breeds of sheep
which can be utilized in Marker Assisted Selection for the
better genotypes. It is recommended that similar kinds of
research should be conducted on larger scale so that better
picture regarding existing genetic diversity in Pakistani
sheep breeds can be estimated which will help in
formulating better strategy for the improvement of sheep
industry in Pakistan.
ACKNOWLEDGMENT
Technical help and access to experimental material
provided by Director Livestock Research Station Jaba,
Mansehra, is highly ackno wledged.
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