Int. J. Biosci.
2015 International Journal of Biosciences | IJB | ISSN: 2220-6655 (Print), 2222-5234 (Online) http://www.innspub.net Vol. 6, No. 10, p. 142-148, 2015
RESEARCH PAPER
OPEN ACCESS
Genetic characterization of local chicken from Taif region in Saudi Arabia using RAPD markers Amena M. Ibrahim1,2, Ayman M. Sabry3,4, Mohamed M. Hassan3,5, Ehab I. ElHallous3,6, Alaa A. Mohamed3,7 1
Medical Laboratory Department, Faculty of Applied Medical Sciences, Turabah Branch, Taif
University, KSA 2
Chlamydia Unit, Animal Health Research Institute, Agriculture Research Center
3
Scientific Research Deanship, Taif University, KSA
4
Cell Biology Department, National Research Centre, Dokki, Giza, Egypt
5
Department of Genetics, Faculty of Agriculture, Minufiya University, Egypt
6
Zoology Department, Faculty of Science, Al-Arish, Suez Canal University, Egypt
7
Department of Animal Reproduction and AI, Veterinary Research Division, National Research
Center, Dokki, Giza, Egypt Key words: RAPD markers, Taif region, genetic distance andlocal chickens.
http://dx.doi.org/10.12692/ijb/6.10.142-1348
Article published on May 08, 2015
Abstract The Taif domestic chicken (Gallus gallus, 2n = 78) is believed to have descended from the wild Indian and Southeast Asian red jungle fowl. The molecular characterization of local chickens in different districts of Taif region in Saudi Arabia was done using RAPD markers of the variable region. The twelve random primers were used to generate fingerprint patterns for these chickens. Twenty five DNA of blood samples from individual chicken were tested to confirm the fingerprinting and genetic distance among these chickens. Specific results for fingerprinting were obtained by the twelve primers retained for RAPD analysis produced different fragment patterns with varied number of bands. The primers yielded a total of 187 distinct bands 34.7% were considered as polymorphic bands and 65.3% were considered as monomorphic bands. The OPA-06 primer has showed the highest polymorphism 83.3% and total of 18 bands ranged from 250 bp-1700 bp. While, the OPA-03 primer has showed the lowest polymorphism 7.8% and total of 14 bands ranged from 200 bp-1650bp.The Dendrogram based on RAPD results grouped the twenty five individual local chicken samples into two different clusters with about 80% genetic similarity. Moreover, the genetic distance among native chicken was relatively low. The smallest genetic distance (0.09) was estimated between sample No. 23 and sample No. 19.This work aims to estimate the genetic resources in the local Saudi chickens reared using RAPD markers in Taif region as preliminary work to established the basis for genetic conservation program for local chicken in Saudi Arabia. * Corresponding
Author:
142 Ibrahim et al.
[email protected]
Int. J. Biosci.
2015
Introduction
exposed to hot arid environment which alter their
Chickens are domesticated fowl belonging to the
capacities
subspecies Gallus gallus domesticus and is raised all
immune response compared to other chicken lines
over the world for its delicious meats and eggs. Native
(Ahmed, 2011;Ahmed and Alamer, 2011;Ahmed et al.,
breeds are considered as a national asset and a key
2014), so body weight and immune response of the
factor in creating sustainable agriculture inSaudi
current lines could be good indicator for physiological
Arabia. Therefore, precise assessment of such native
parameters differences next to DNA polymorphism.
genetic resources is of great importance and could be
The objectives of the present study were to evaluate
utilized for the purpose of their conservation,
the genetic variation within and between local
management,
exploitation
chicken in Taif region, KSAand the molecular
(Shahbaziet al., 2007, Ahmed and Alabbad, 2014).
characterization for the local chicken using RAPD
Native chicken are known to be good foragers and
markers.
reproduction
and
regarding
growth,
physiological
and
efficient mothers and minimal care is required for their growth (Alabbad, 2014). Just few studies
Materials and methods
showed comparisons between local Saudi Arabia
Sample collection
chicken lines and commercial layers or broiler lines in
The sample collection and laboratory work of this
terms of productive traits, physiological performance
study was conducted from December 2014 to March
and some genetic parameters (Ahmed and Alamer,
2015.The chicken samples of this experiment was
2011;Alamer and Ahmed, 2012). The previous
conducted to study the efficiency of RAPD marker for
diversity studies of the local chickens reported in
generating
Saudi Arabia were based mostly on morphological
populations (Mollahet al., 2005), which collected
characterizations, including adult body weight, egg
from the several indigenous chicken populations from
weight, reproduction performance and immune
different districts in Taif governorate, from different
responses to various diseases (Ghanemet al., 2012;
personal farms distributed allover Taif region. The
Ahmed and Alabbad, 2014).Manynew markers have
laboratory work was performed in the Biotechnology
been developed over the past two decades and used to
and Genetic Engineering Unit and Scientific Research
detect and estimate thegeneticdiversity within and
Deanship, Taif University.
polymorphism
in
different
chicken
between chicken populations. Few studies have been recently
performed
to
assess
the
genetic
Blood sampling and DNA extraction
polymorphismin chicken in Saudi Arabia using DNA
Blood was collected and prepared for DNA isolation
fingerprinting technique. The Randomly Amplified
by using the procedure suggested by Hoelzel (1992).
Polymorphic DNA (RAPD) assays have been used for
Genomic DNA was extracted from the whole blood by
estimating genetic diversity among different breeds
using a DNA extraction Kit (QIAGEN). DNA quality
and verities in poultry (Salem et al., 2005; Rabie and
was checked by electrophoresis in a minigel and
Abdou, 2010; Nikkhooet al., 2011;Ghanemet al.,
quantified using a spectrophotometer (Spectronic
2012;Ahmed and Rezk, 2015). It is a quick and
Genesys, Thermo Electron Corporation).
effective method that can be applied to generate genotype specific banding patterns (El-Gendyet al.,
RAPD analysis
2005; Ahmed and Alabbad, 2014). It was used for the
For RAPD analysis, twelve 10-mer random primers
analysis of genetic diversity in Saudi Arabia chicken
were used (supplied by Amersham Pharmacia
which had a significant impact on the breeding and
Biotech. NJ. USA.). Names and sequences of the
conservation of native chicken genetic resources in
primers are illustrated in Table 1.Following the
Saudi Arabia (Ahmed and Alamer, 2011;Alamer and
experiments
Ahmed, 2012),In Saudi Arabia, accompany the
concentrations, PCR amplification of random primers
suspected DNA polymorphism of local chicken
were carried out according to Williams et al. (1990)
143 Ibrahim et al.
for
optimization
of
component
Int. J. Biosci.
2015
and Alyet al., (2010) in 25 μl volume containing 1μl
Results and discussion
(20 ng) of genomic DNA, 12.5μl of Go Taq® Green
RAPD analysis of local chicken from Taif region,
Master Mix, Promega, USA. 1μl of primer (20 p.mol),
KSA
deionized distilled water (up to a total volume of 25
Molecular markers are efficient tools for cultivar
μl). For DNA amplification, the C1000TM Thermo
identification and estimation of relatedness through
Cycler Bio-Rad, Germany, was programmed under
DNA fingerprinting. RAPD markers where developed
the conditions involving denaturation at 94°C for 5
by Williams et al. (1990). RAPD technique using
min; 40 cycles of denaturation at 94°C for 30 Sec,
single arbitrary 10-mer oligonucleotides primers to
primer annealing at 35°C for 1.5 min and primer
amplify discrete fragments of DNA using Polymerase
extension at 72°C for 2.5 min; final extension step at
Chain Reaction (PCR). This technique has been used
72°C for 7 min. Amplified DNA products were
extensively in many different applications and in
analyzed by electrophoresis in 1.5% agarose gel run in
different plant species because of its simplicity
TBE. The gels were stained with ethidium bromide (5
(Hassan et al., 2014).Genomic diversity of chicken
μg ml-1). 100 pb. DNA Ladder RTU, (Gene Direx®)
samples was investigated by RAPD analysis. The
was used as a standard. DNA was visualized by UV
RAPD results illustrated in Table (2) and Figures (1, 2
illumination and then photographed by a Bio-Rad Gel
and 3) showed polymorphic numbers of the genetic
Doc 2000 device.
bands, which were the electrophoretic products of PCR for chicken samples. RAPD-PCR reactions were
Data analysis
performed with twenty five individual local chicken
The amplification products of RAPD-PCR were scored
samples collected from Taif region, KSA and twelve
for the presence “1” or absence “0” and missing data
different 10-mer primers, which were pre-selected for
as “9”. The genetic associations between isolates were
their performance with chicken DNA. Out of the
evaluated by calculating the Jaccard's similarity
twenty primers twelve retained for RAPD analysis
coefficient for pair wise comparisons based on the
produced different fragment patterns with varied
proportion of shared bands produced by the primers.
number of bands. The primers yielded a total of 187
The similarity matrix was subjected to cluster analysis
distinct bands (RAPD markers), (34.7%) of which
by un-weighted pair group method for arithmetic
were considered as polymorphic and 65.3% of which
mean (UPGMA) and a dendrogram was generated.
were considered as monomorphic. Table (1 and 2)
The computations were performed using the program
record the number of amplified fragments scored for
NTSYS-PC version 2.01 (Rohlf, 2000). The Jaccard׳s
each individual local chicken samples. The amplified
similarity
products were highly polymorphic among the twenty
matrix
was
component analysis.
subjected
to
principal
five different individual local chicken samples.
Table 1. Names and sequences of the random primers used in this study. Primer OP-A1 OP-A2 OP-A3 OP-A6 OP-A8 OP-A10 OP-B6 OP-B7 OP-B8 OP-C2 OP-C4 OP-C6
144 Ibrahim et al.
53 sequence CAGGCCCTTC TGCCGAGCTG AGTCAGCCAC GGTCCCTGAC GTGACGTAGG GTGATCGCAG TGCTCTGCCC GGTGACGCAG GTCCACACGG GTGAGGCGTC CCGCATCTAC GAACGGACTC
Int. J. Biosci.
2015
Table 2. Polymorphic bands of each genetic primers and percentage of polymorphism in twenty five individual local chicken samples. Primers OPA-01 OPA-02 OPA-03 OPA-06 OPA-08 OPA-10 OPB-06 OPB- 07 OPB- 08 OPC- 02 OPC- 04 OPC- 06 Total
Total Bands 17 16 14 18 19 11 15 20 10 15 18 14 187
No. of Monomorphic Bands 5 4 13 3 5 4 7 7 4 5 4 4 65
No. Polymorphic Bands 12 12 1 15 14 7 8 13 6 10 12 10 122
%Monomorphic bands 29.4 25.0 92.2 16.7 26.3 36.4 46.7 35.0 40.0 33.3 22.2 28.6
%Polymorphic bands 70.6 75.0 07.8 83.3 73.6 63.6 53.3 65.0 60.0 66.7 77.8 71.4
A total of 187 fragments from all analysis were
PCR results using primer (OPA-06) has showed the
enough for the identification and the evaluation of
highest polymorphism, a total of 18 bands in these
genetic similarities and designing the phylogenetic
twenty five different individual local chicken samples
tree for these twenty five different individual local
ranged from 250 bp-1700 bp. Three common bands
chicken samples.The total number of bands as shown
were observed in all isolates which exhibited about
in Table (2) varied from 20 bands with primer OPB-
16.7% monomorphism, while the other 15fragments
07 (Figure 2) to 10 bands with primer OPB-08
have showed 83.3% polymorphism (Table 2). In case
(Figure 3). The total of monomorphic amplicons was
of
65 and the total of polymorphic amplicons was 122. It
polymorphism a total of fourteen fragments have
can be concluded from our study that RAPD markers
showed 7.8% polymorphism among the twenty five
are effective in detecting similarity between chicken
different individual local chicken samples. The
strains and they provide a potential tool for studying
molecular size of the amplicon products ranged from
the
260 bp-1750 bp. Also, this primer has not recognized
inter-strain
genetic
similarity
and
the
establishment of genetic relationships. The RAPD-
OPA-03
primer
has
different unique fragments.
Fig. 1. RAPD profile of 25 individual local chicken samples generated by primer OPA-10.
Fig. 2. RAPD profile of 25 individual local chicken samples generated by primer OPB-7.
145 Ibrahim et al.
showed
the
lowest
Int. J. Biosci.
2015
Cluster analysis of local chicken from Taif region,
Our future research based on integrating RAPD and
KSA
microsatellite marker will provide more details about
According to genetic similarity and intra-species
this matter. Finally, we can say that RAPD markers
differentiation, the twenty five individual local
found sufficient nuclear DNA level variations among
chicken samples were grouped into two different
different chicken populations in Taif region, Saudi
clusters with about 80% genetic similarity. Eleven
Arabia. The RAPD data presented here might be a
individual local chicken samples were grouped in the
good source of information about the diversity of
first cluster and fourteen individual local chicken
native chicken in Saudi Arabia.
samples were grouped in the second cluster (figure 4).
Fig. 3. RAPD profile of 25 individual local chicken samples generated by primer OPB-8.
Fig. 4. Dendrogram analysis among the twenty five individual local chicken samples collected from Taif region in Saudi Arabia based on the twelve RAPD primers. Conclusions
determine the genes detected by RAPD experiments.
The effectiveness of RAPD in detecting polymorphism
Further studies with other molecular methodologies
between different samples from individual local
are
chicken
relationships among local chickens in Saudi Arabia
in
Taif
applicability
in
region,
Saudi
population
Arabia,
studies
their
and
the
essential
to
clarify
and
confirm
genetic
depicted using Microsatellite Markers.
establishment of genetic relationships demonstrated with this study. It is important to mention the fact
Acknowledgment
that data results from RAPD assays can be extended
The authors would like to express their thanks and
to further dissect traits in a more refined way to
appreciation to Taif University, KSA, for financial
exactly knowledge on specific genes and genetic
support to carry out this work. This work was
pathways using other molecular methodologies.
supported by Taif University, KSA under project No.
There is also the opportunity and need to study
1-435- 3519.
sequences
of
specific
146 Ibrahim et al.
polymorphic
bands,
to
Int. J. Biosci.
2015
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