http:// www.jstage.jst.go.jp / browse / jpsa 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. References Adebambo AO and the Chicken Diversity Consortium. Mitochondrial DNA D-loop analysis of Southwestern Nigerian chicken. Archivos de Zootecnia, 58: 637-643. 2009. 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