Phylogenetic Analysis of Native Chicken from Bangladesh and

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doi:10.2141/ jpsa.0120007
Copyright Ⓒ 2012, Japan Poultry Science Association.
Phylogenetic Analysis of Native Chicken from Bangladesh and
Neighboring Asian Countries Based on Complete Sequence
of Mitochondrial DNA D-loop Region
Mohammed A. Islam1, 2 and Masahide Nishibori2
1
Department of Animal Science, Bangabandhu Sheikh Mujibur Rahman Agricultural Uniuversity, Gazipur-1706, Bangladesh
2
Department of Bioresource Science, Graduate School of Biosphere Science,
Hiroshima University, Higashi-Hiroshima 739-8528, Japan
The complete mitochondrial D-loop region was sequenced for a total of 18 individuals of Bangladeshi native
chickens (BNC); full feathered (nana) (n=7), naked neck (Nana) (n=8) and Red junglefowl (RJF) (n=3). The
alignment of mitochondrial D-loop sequence of these chicken populations with 39 reference sequences from DNA
databank; White Leghorn, G. g. murghi, G. g. bankiva, G. g. spadiceus, G. g. gallus and other Asiatic chickens was
done to identify the phylogenetic position of BNC for the conservation and improvement of chicken genetic resource.
The nucleotide variation of sequence among haplotypes for within and between populations of BNC supported the
phenotypic variation of individual of the populations. Phylogenetic analysis showed that 57 individuals were grouped
into 6 clades. Of the BNC populations, 6 nana, 7 Nana and 2 RJF individuals (83.3%) were closely related with each
other and only 1 nana and 1 Nana (11%), and 1 RJF (5.5%) individuals were divergent from them. Therefore, the
phylogenetic tree showed low genetic distance and close relationship within and between the chicken populations of
Bangladesh, which were closely related with G. g. murghi of Indian origin, and also related with G. g. bankiva, G. g.
gallus implying the origin of gene flow to Bangladesh. The genetic information from this study may serve as an initial
step to make future plans to assess more molecular information on genetic diversity for the characterization,
conservation and improvement of valuable chicken genetic resource of Bangladesh.
Key words: Asian country, Bangladesh, mtDNA, native chicken, phylogenetic position
J. Poult. Sci., 49: 237-244, 2012
Introduction
It is widely believed that currently recognized domesticated chickens are descended from a single ancestor, the Red
junglefowls (Gallus gallus) (Hashiguchi et al., 1981; Okada
et al., 1984; Yamashita et al., 1994; Fumihito et al., 1996)
which originated from Southeast Asia (Fumihito et al., 1994;
1996). The Red junglefowls were further divided into five
subspecies; Gallus gallus gallus, G. g. jaboullei, G. g.
spadiceus, G. g. bankiva and G. g. murghi based on morphological features and geographical distribution (Johnsgard,
1999, Sibley and Monroe, 2000). The habitat of G. g.
spadiceus is mainly in Myanmar (Burmese bird) whose
earlobes are red in color (Jonsgard, 1999). Fumihito et al.
(1996) determined the phylogenetic position and geographic
Received: January 12, 2012, Accepted: April 28, 2012
Released Online Advance Publication: June 25, 2012
Correspondence: Dr. Md. A. Islam, Department of Animal Science,
Bangabandhu Sheikh Mujibur Rahman Agricultural University, Gazipur1706, Bangladesh. (E-mail: aminul_dgvc@yahoo.com)
distribution of three subspecies of Red junglefowls namely;
G. g. gallus (China, Cambodia, Laos, Thailand and Vietnam), G. g. spadiceus (Thailand, Myanmar, northern Laos,
Malaya, and northern Sumatra) and G. g. bankiva (Sumatra,
Java, Bali of Indonesia) based on the nucleotide sequences of
a part of the mitochondrial D-loop region (Johnsgrad, 1999).
Nishibori et al. (2002) determined the complete sequence of
mitochondrial D-loop of Red junglefowls in Laos, which
placed Laos native chickens in a clade. Nowadays, Red
junglefowls may be hybridized with native chickens in Asian
countries (Nishibori et al., 2005).
In Bangladesh, various types of non-descriptive indigenous chickens; i.e., naked neck, fully feathered and Red
junglefowls are found, and also in India, Myanmar, Bhutan,
Thailand, Laos, Cambodia and Vietnam. Therefore, chicken
germ plasm from neighbor countries may have migrated to
Bangladesh. Islam (2000) found that Bangladeshi indigenous chickens are well adapted to tropical climate, and naked
neck chicken is especially a promising and probable genetic
resource in terms of productive adaptability.
238
Journal of Poultry Science, 49 (4)
In terms of growth rate, egg production, egg quality and
meat yield traits, indigenous naked neck chicken is superior
to indigenous full feathered and exotic egg type or exotic
naked neck counterparts. Indigenous naked neck can
produce double the standard number of eggs under improved
nutrition and management conditions (Islam and Nishibori,
2009). Crossbred of indigenous naked neck with exotic
chicken can perform even better than exotic chicken in terms
of productive and reproductive traits (Islam and Nishibori,
2009; Islam and Nishibori, 2010). Therefore, indigenous
naked neck as well as other types of indigenous chicken may
be a valuable genetic resource for breed development in the
future.
A work has been done on genetic diversity and genetic
relationship within native chicken of Bangladesh (Yamamoto
et al., 2011). Recently some authors investigated the origin,
and genetic diversity, genetic relationship and variation
within the indigenous chickens of Iran, Sri Lanka, Indonesia,
China, Nigeria, Japan, Thailand, and especially India using
microsatellite markers, partial and complete mitochondrial
D-loop sequences (Yamamoto et al., 2000; Nishibori et al.,
2004; Qu et al., 2006; Guan et al., 2007; Oka et al., 2007;
Shahbazi et al., 2007; Sulandari et al., 2008; Adebambo AO
and the Chicken Diversity Consortium, 2009; Silva et al.,
2009; Dorji et al., 2012). Recently a study on characterization of birds showed the differences between Indian native
Aseel and Kadakanath chicken breeds for growth, production, egg and semen quality, and behavioral traits, but not on
welfare traits, although female Aseel birds exhibited greater
fearness (Haunshi et al., 2011).
An investigation showed high market demand and price of
Benin indigenous chickens makes their products profitable
which suggests making policies for the conservation of Benin
indigenous chicken genomes (Rodriguez et al., 2011).
Therefore, it is desirable to investigate the origin, phylogeny,
genetic variation and relationship within and between the
indigenous chicken of Bangladesh and neighboring Asian
countries.
Nucleotide sequence of the mitochondrial Displacementloop (D-loop) region is a useful and powerful tool to
investigate the origin and phylogeny of fowls (Nishibori et
al., 2004). The D-loop region has nucleotide variation
which is quite high, more polymorphic compared to other
regions of mtDNA (Quinn and Wilson, 1993; Ishida et al.,
1994) and is often used for phylogenetic analysis within the
species (Brown et al., 1982; Fumihito et al., 1994). The Dloop has a central domain, is conserved, and has hyper variable segments flanking it on either side. This control region
serves as a point of initiation for the replication of mitochondrial DNA.
A few works have been done to evaluate the genetic variability and interrelationship among red jungle fowls, Myanmar indigenous and commercial chickens based on the complete sequence of mtDNA genome (Nishibori et al., 2002;
2003; 2004; 2005; Oka et al., 2007). Considering the above
points, the complete sequence of the mitochondrial D-loop
was used to investigate the phylogenic relationship within,
and between the native chickens, Red junglefowls from
Bangladesh, and neighboring Asian countries to identify
unique population for characterization, conservation and
further improvement of native chicken genetic resource.
Materials and Methods
Geographically Bangladesh is located in the northern part
of the South-Asia between 20°
34′and 26°
38′north latitude
and 88°
1′and 92°
41′longitude. Bangladesh is a tropical
country where the temperature (℃), humidity (%) and rainfall (mm/month) in winter (November-February) are 10.729.7, 75.4, 3.9, and in summer (March-October) 15.8-32.0,
82.5, 261.0, respectively (Islam, 2000). As neighbor country, Indian provinces; Tripura, Asam and Megalayan are
adjacent to the east, northeast and northern, and Myanmar is
located in the northern verge of Bangladesh.
Blood Sample Collection
A total of 52 samples belonging to 3 types of Bangladeshi
native chickens and Red junglefowls (17 naked neck, 32
fully feathered and 3 Red junglefowls) from 5 locations in 3
districts (Trishal, Nandail and Haluagut of Mymensingh
district; Gazipur Bazar of Gazipur district, and Nikhongchari
of Rangamati district) in Dhaka and Chittagong divisions,
Bangladesh were collected with blotting paper and used as
DNA materials in this study (Table 1).
Extraction and Amplification of Mitochondrial DNA, and
D-loop DNA Sequence
DNA was extracted from all the blood samples using the
recommended protocol described by Sambrook and Russell
(2000) and Nishibori et al. (2002, 2003). The mtDNA fragments were specifically amplified from mitochondrial genome in long PCR using the KOD-FX polymerase
List of samples collected from Bangladeshi
native chickens and Red junglefowls
Table 1.
Name of
Sample
Name of population
Sex
Samples from the districts of Dhaka and
Chittagong division)
BNGM8
BNGM7
BFGF9
BFTM10
BFTF1
BFTF4
BFTF5
BNNM19
BNNM22
BNNM27
BFNM25
BNHF28
BNHF29
BNHF38
BFHM33
BRJF1
BRJF 2
BRJF 3
Naked neck
Naked neck
Full feather
Full feather
Full feather
Full feather
Full feather
Naked neck
Naked neck
Naked neck
Full feathered
Naked neck
Naked neck
Naked neck
Full feathered
Red junglefowl-RJF
Red junglefowl-RJF
Red junglefowl-RJF
♂
♂
♀
♂
♀
♀
♀
♂
♂
♀
♂
♀
♀
♀
♂
♂
♂
♀
Gazipur
Gazipur
Gazipur
Trishal
Trishal
Trishal
Trishal
Nandail
Nandail
Nandail
Nandail
Haluagut
Haluagut
Haluagut
Haluagut
Naikhongchari
Naikhongchari
Naikhongchari
Islam and Nishibori: Phylogenetic Analysis of Bangladeshi Chicken
(TOYOBO, Osaka Japan) with specimen DNA as a template
and the LA-PCR primer sets, following the procedure recommended by the manufacturer. The LA-PCR primer set
was prepared as described by Nishibori et al. (2001). The
LA-PCR was conducted at 94℃ for 2 mins, followed by 30
cycles consisting of 10 sec denaturizing at 98℃, and then
annealing and extension at 68℃ for 5 min using a GeneAmp
PCR system 9700 (Applied Biosystems, Forester, CA). Amplified fragments with about 5 kb were then isolated through
agarose gel electrophoresis as described by Nishibori et al.
(2001), and then used for amplification of complete D-loop
region with a primer pair of GalF (5′
-AGG ACT ACG GCT
TGA AAA GC-3′and GalR (5′
-TGC TTA AGG TTA ATT
ACT GCT G-3′
) (Nishibori et al., 2001). The PCR products
were electrophoresed on a 1.0% agarose gel (Nippon Gene,
Osaka, Japan), and visualized by staining with ethidium
bromide via ultraviolet transilluminator.
The DNA fragments were obtained through the amplification which was sequenced using ExoSAP-IT (Amershan
Biosciences, Buckinghamshire, England) and a Big Dye terminator v3.1 Cycle Sequencing kit (Applied Bio-Foster City,
CA). Two primers for sequencing were added to the primers
for D-loop those were the GalF and GalR. Labeled D-loop
DNA fragments were analysed on a Model 3130xl genetic
analyzer (Applied Biosystems Inc.).
Nucleotide sequences were analyzed using the computer
program AutoAssembler (Genetic Assembler, AB: 3130xl),
Data collection software v.3.0) (Applied Bio-systems).
Data Analysis
The mtDNA D-loop fragment on the first 1,232 base pairs
length was used for analysis in the present study. Sequence
data were obtained after being edited from complete D-loop
sequence. Sequence data were analyzed using various kinds
of computer software: GPROF software, GENETYX (Tokyo,
Japan). GPROF software was used for viewing and editing
the sequence results. GENETYX package program was used
for sequence data analysis. Nucleotide sequences were
performed using multiple alignments through ClustalX (ver.
1.83) program (Thompson et al., 1997). Molecular phylogenetic analysis was performed for complete D-loop sequences of Bangladeshi native chickens and Red junglefowls
together with those registered in the nucleotide DNA
databank (Accession numbers presented in Table 3). The
Neighbor-joining (NJ) program (Saitou and Nei, 1987) was
employed for complete sequences of mitochondrial D-loop
using Kimura’s 2 parameter model (Kimura, 1980). Bootstrap values were estimated with 1000 repetitions.
Results
Bangladeshi population to determine complete mitochondrial D-loop variability, 18 mitochondrial D-loop sequences
from the populations of Bangladeshi native chickens and Red
junglefowl were produced in this study. The chicken populations are of native full feathered (n=7), naked neck (n=8),
and Red junglefowls (n=3) (Table 1).
Alignment of mitochondrial D-loop sequences was done
with reference sequences from DNA databank as White
239
Leghorn (AP003317), G. g. murghi (GU261709), G. g.
bankiva (AP003323), G. g. spadiceus (AP003321), G. g.
gallus (AB007725) and other Asiatic chickens. Sixteen
haplotypes of 18 complete mitochondrial D-loop sequences
(1232 bp) were identified, of which 7, 6 and 3 from
Bangladeshi full feathered, naked neck, and Red junglefowl
populations were transitions, respectively. The distribution
of sequence variations among the mitochondrial D-loop
region which was up to 1225 bp nucleotides is shown in
Table 2. In addition, the sequence variations for mitochondrial D-loop region of G. g. bankiva, G. g. spadiceus, Red
junglefowls from Laos and the Philippines, and Indonesian
and Myanmar native chickens as per findings of Nishibori et
al. (2004) were added to compare with the present findings
(Table 2). The sequence variation of nucleotide between
haplotypes of within and between Bangladeshi populations
was found by comparing with the sequence of standard
White Leghorn chickens, and Myanmar native chickens, and
Red junglefowls of the Philippines, (G. g. gallus), Indonesia
(G. g. bankiva) and Laos (G. g. jabouillei).
Phylogenetic Analysis of Bangladeshi Chickens and Reference Chickens from GeneBank
Phylogenetic analysis of Bangladeshi native chickens and
Red junglefowls was done along with those from neighboring countries and other reference sequences and shown to
represent six clades (Table 3). The reference sequences in
this study were used to identify the phylogenetic position of
Bangladeshi native chickens and Red junglefowls. As
mentioned in the materials and methods, the 18 individuals of
3 populations from Bangladesh and 39 reference sequences
from DNA databank (neighboring countries, Silkie and
commercial breeds of chickens) were used to construct the
phylogenetic tree for a total of 57 individuals to determine
the phylogenetic relationships and genetic distance within
and between the populations of Bangladeshi, and those from
neighboring countries (Fig. 1).
Fig. 1 shows that almost all individuals (83.3%) of Bangladeshi populations are in clade 4 except BRJF2 (5.5%) and
BFNM25 and BNGM7 (11%) which are in clade 3 and clade
5, respectively. However, they are in the neighbor of clade
4. This figure clearly shows that Bangladeshi individuals
(native chickens and Red junglefowls) of 3 populations are
closely related with each other and are also closely related
with G. g. murghi (Indian origin), fowls of China, Cambodia,
Laos, Vietnam, Thailand (G. g. gallus), and Indonesian fowls
(G. g. bankiva). Fig. 1 shows the Myanmar (G. g.spadiceus)
and most of the reference fowls and chickens are far away
from Bangladeshi populations. Few chickens from Bangladesh like BFNM25 (full feathered) and BNGM7 (naked
neck) are closely related with commercial White Leghorn
(AP003317) and other reference chickens (Japanese native
and Silkie chickens) shown in clade 5 (Table3). Notwithstanding small genetic distance as per phylogenetic tree
among Bangladeshi populations in clade 4, this may represent the phenotypic variability within and between
populations. The phylogenetic tree revealed that Bangladeshi Red junglefowls are related with Indonesian fowls (G. .
240
Journal of Poultry Science, 49 (4)
Table 2.
Sequence variations observed among mitochondrial D-loop sequences of Bangladeshi and neighboring Asian countries chickens.
g. bankiva), which has a close relationship with India,
Cambodia, Laos, Thailand, Vietnam fowls. But the
genetic distance of BRJF is found far away from
Myanmar and other reference chickens or fowls of
clades 1, 2, 6 (Table 3).
Discussion
Bangladeshi population to determine complete
mitochondrial DNA D-loop variability.
Sequence variation of Bangladeshi chickens and
Red junglefowls was performed by comparing with
the standard sequence of White Leghorn (WLH)
(AP003317), and also with Myanmar native chickens,
and Red junglefowls of Indonesia, Philippines and
Laos as references of Nishibori et al. (2004). The
sequence variation of nucleotides was found in comparison with the standard WLH chickens and the
commercial breed of WLH and native chicken or Red
junglefowl of Bangladesh. This variation was corroborated with the findings of Nishibori et al. (2004)
except the transverse position of nucleotides at 986 bp
in case of Myanmar native chicken from Mawlamynie as found by Nishibori et al. (2004), whereas
Bangladeshi chickens and fowls had no such transverse position. On the basis of nucleotide sequence
variation, there were 7 haplotypes in full feathered
(nana), 6 haplotypes in naked neck (Nana) chicken
and 3 haplotypes in Red junglefowls (BRJF). Therefore, the total haplotypes of 16 was found within
Bangladeshi chickens and Red jungle fowls, where
sequence variation between haplotypes of within and
between populations was present representing phenotypic variation among the individuals which partially
consistent with the findings of Adebambo AO and the
Chicken Diversity Consortium (2009). They found
16 haplotypes in Nigerian indigenous and Anak titan
chickens which are grouped under one clade 4. They
showed 97.32% sequence variation between haplotypes within populations, and 2.68% variation between
populations.
Phylogenetic Analysis of Bangladeshi Chickens and
Reference Chickens from GeneBank
All individuals in the phylogenetic tree were
grouped into 6 clades. Of these experimental individuals of Bangladeshi populations were observed in
clade 3, clade 4 and clade 5. Most of them were
distributed under clade 4 while BFNM25 and
BNGM7 were found in clade 5, and BRJF2 in clade
3. Experimental individuals of 3 populations (Bangladeshi full feathered: nana, Bangladeshi naked
neck: Nana and Red junglefowls) were closely related
within, and between the Bangladeshi populations and
also with neighboring Asiatic countries populations
like India, China, Cambodia, Laos, Thailand, Vietnam and Indonesia, which supported the findings of
Oka et al. (2007); Silva et al. (2009); Dana et al.
(2011). Oka et al. (2007) reported the multiple
Islam and Nishibori: Phylogenetic Analysis of Bangladeshi Chicken
Table 3.
Group of
individual
241
Distribution of individuals under clade of the phylogenetic tree
Name of individuals
Experimental individuals
Reference individuals (GeneBank)
No.
individuals
in clade
Clade 1
E01 (AB268539), E02 (AB268540),
G. g. spadeceus (P003321)
3
Clade 2
B07 (AB294232), RxP_GDG1
(AB263973), SilkieD-loop (AB
086102), B04 (AB268519), B01
(AB268516), B06 (AB268521)
6
2
Clade 3
Sam2comp (BRJF2)
G01 (AB268545)
Clade 4
BFGF9, SamR1comp (BRJF1),
BNHF28, BFTF4, Sam22comp
(BNNM22), BNNM27, BFHM33,
BNHF29, SamR3comp (BRJF3),
Sam10 (BFTM10), Sam38comp
(BNHF38), Sam19comp (BNNM19),
BNGN8, BFTF5, BFTF1
Gggallus (AB007725), G. g. murghi
(GU261709), Ggbankiva
(AP003323), C06 (AE268527), C07
(AB268528), G. g. gallus
(AP003322), C03 (AB268524),
C05 (AB268526), C03 (AB268529)
24
Clade 5
Sam25comp (BFNM25), BNGM7
SAW1_SLKAW1, F02 (AB268544),
F01 (AB268543), SMb2_SLKBM4
(AB263966), A09 (AB268514), A04
(AB268509), A07 (AB268512),
WLH (AP003317), A06
(AB268511), A05 (AB268510),
SAb2_SLKBW2 (AB263953),
NHR2_AY235571, SAb1_SLKBW1
(AB263952), SAb3_SLKBW3
(AB263954)
16
SMw2_SLKWM6 (AB263962), D02
(AB268531), D06 (AB268535), D04
(AB268533), D05 (AB268534), D09
(AB268538)
6
Clade 6
Total
origins of Japanese native chicken populations which were
originated from South East Asia: China, Korea and Indonesia. Non-game birds also have relationship with red jungle
fowl and game bird developed in each area of South East
Asia. Dana et al. (2011) showed the origin of Dutch fancy
and commercial chicken from the Indian sub-continent.
Silva et al. (2009) found the genetic relationship of SriLankan chickens with Red junglefowls which originated
from China, Cambodia, Laos, Vietnam and Thailand of
South East Asia.
Although the individuals of BFNM25, BNGM7 and
BRJF2 were scattered in clade 5 and clade 3, they are related
with Japanese native and Silkie chickens (reference sequence) and have relationship with South East Asian
populations from China, Cambodia, Laos, Vietnam, Thailand
and Indonesia (Oka et al., 2007; Sulandari et al., 2008; Dana
et al., 2011). Sulandari et al. (2008) reported five distinct
clades within Indonesian indigenous chickens. They found
67.85% total sequence variation within population and 32.15
% between populations. Moreover, these populations shared
the multiple maternal origins under the same maternal an-
57
cestor and supported by the present findings.
Out of 3 populations of Bangladeshi chickens two populations are native full feathered, and naked neck had the small
genetic distance observed in clade 4 and clade 5. Clade 4
shared more than 83.3% individuals of nana and Nana, and
clade 5 only 11% individuals of nana and Nana chickens, and
5.5% Red junglefowls in Clade 3 of the total Bangladeshi
individuals measured as per phylogenetic tree. This partially
coincided with the results obtained by Shahbazi et al. (2007)
who found small genetic distance among the four Iranian
populations except one population-Isfahans Yazd (Central
Iran). They found the largest genetic distance in Isfahan
from 4 other populations of Iranian chickens. According to
clade 4 in the phylogenetic tree, a small genetic distance
within populations of Bangladesh samples was observed and
supported the phenotypic variation of these populations as
depicted in accordance with Lujiang et al. (2006); Yamamoto et al. (2011). Lujiang et al. (2006) found high genetic
diversity in Chinese indigenous chicken in agreement with
great phenotypic variation of these breeds. Yamamoto et al.
(2011) found the lower degree of genetic differentiation
Journal of Poultry Science, 49 (4)
242
Phylogenetic tree was constructed based on 57 individuals
(18 individuals from Bangladesh, and 39 from reference DNA databank) representing 6 clades. The numbers at the nodes represent the
percentage bootstrap values for interior branches after 1000 replications
Fig. 1.
among Bangladeshi native chicken populations.
Therefore, it can be assumed that Bangladeshi populations
may have migrated from India, and other Asiatic countries
like Indonesia, Cambodia, Laos, Thailand and Vietnam (Fig.
1). Moreover, this tree indicates that the chickens and fowls
of the said countries may be genetically related with each
other.
Conclusion
The results of the present study reveal that mtDNA especially its complete D-loop region is an important and
powerful molecular tool for genetic information showing
phylogenetic relationship, genetic distance and variability
within and between populations. Haplotype variation within
and between the Bangladeshi population was present, which
represents the phenotypic variation within and between the
populations. The phylogenetic analysis showed the close
genetic relationship within and between the populations of
Bangladeshi native full feathered, naked neck, and Red
junglefowls. Bangladeshi chickens also have the close relationship with G. g. murghi (Indian origin) and also with references of Asian countries populations that imply the origin
of gene flow to Bangladesh. The genetic information from
this study was the initial investigation using these populations in Bangladesh that may be helpful in developing future
strategies for the characterization, conservation and improvement of valuable native genetic resource. However, this
study suggests doing more research using these genetic resources to answer different questions relevant towards the
development of the local poultry industry.
Acknowledgments
We are grateful to the Japan Society for the Promotion of
Science (JSPS), for providing a fellowship to carry out the
researches in Japan and prepare the scientific articles.
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