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Molecular Characterization of Mitochondrial D Loop of Indian Red Jungle Fowl

Shahaj Uddin Ahmed Sanjeev Kumar Shukla Hemen Das
Vol 7(6), 144-152

Mitochondrial DNA has the characteristic of quick evolution, matrilineal inheritance, and simple molecular structure, and it serves as the most used marker for molecular study. As an important role of genomics, studying it can help understand the origins, history, and adaptation of domestication. A 1325 bp fragment of the mtDNA D-loop region was amplified; sequenced and phylogenetic study was done. Out of 400 sites about 12 % were polymorphic between the Gallus gallus (G.g.) subspecies and most of them (60%) were transitions. Indian RJF showed very low genetic distances (0.008-0.013) with G.g. gallus birds from Thailand as compared to those from Japan and Indonesia. The G.g. murghi showed comparatively high genetic distance i.e. 031-0.034 with G.g. spadiceus and from 0.061-0.066 with G. gallus bankiva. The phylogenetic tree showed that G.g. banikva is well separated from the other three subspecies as it made a separate cluster. G.g. gallus makes two separate clusters i.e. one those from Thailand and other those from Japan and Indonesia. Within each chicken breed, there is an excess of homozygosity, but there is no significant reduction in the nucleotide diversity. Phenotypic modifications of chicken breeds as a result of artificial selection appear to stem from ancestral polymorphisms at a limited number of genetic loci.

Keywords : Mitochondrial D Loop Indian Red Jungle Fowl Subspecies


It is postulated that chickens (Gallus gallus domesticus) became domesticated from wild jungle fowls in Southeast Asia nearly 10,000 years ago. In the recent past, molecular genetics have provided several powerful tools such as DNA based genetic markers that can exploit the great wealth of polymorphism at DNA level. DNA-based markers can be grouped into clone/sequence based (CSB) markers and fingerprint markers (West and Zhou 1989). The CSB markers include microsatellite (Zeuner, 1963), RFLP, Sequence Tagged Site (STS) and Expressed Sequence Tags (EST). On the other hand, the Fingerprint (FP) markers include Variable Number of Tandem Repeats (VNTR). These VNTR are often called minisatellites for large loci (West and Zhou, 1989) and microsatellites for smaller VNTR loci (Kawabe et al., 2014). The primary advantage of mtDNA is that it is present in higher copy numbers within the cells and therefore more likely to be recovered even from highly degraded specimen (Fumihito et al., 1996). Ulfah (2016) described mtDNA as a potent tool for understanding human evolution, owing to its high copy number, apparent lack of recombination, high substitution rate and maternal mode of inheritance. The SNPs that occur in the mitochondrial genome are very important tool in the species identification, forensic studies as well as anthropological and evolutionary research (Yacoub et al., 2013). Jia et al. (2016) observed the single nucleotide polymorphism in the D-loop region in chicken mitochondrial DNA and reported 11 SNPs. For six of the SNP sites, polymerase chain reaction (PCR) primers that had each base (A, C, G and T) as the penultimate base at the 3’ end were produced to type the polymorphisms. Twenty-one out of 96 primers succeeded in distinguishing the SNPs by the presence or absence of PCR product. This method provides an easy way to discriminate SNP in chicken mitochondrial DNA.

Some studies were made to establish phylogenetic relationship between domestic fowl, different red jungle fowl subspecies and other jungle fowls. Fumihito et al. (1994) suggested a strong possibility of a single domestication event being the G.g. gallus, a major or sole contributor. This theory was further substantiated by Fumihito et al. (1996) who suggested that G.g. gallus is the real matriarchic origin of all the domestic poultry. They also found no discernible differences among G.g. spadicus and G.g. gallus, while G.g. bankiva showed differences with both (Ulfah et al., 2016) the genetic and phylogenetic relationships among these species. It provides a framework for genetic studies in wild jungle fowls and native and domestic chicken breeds. These studies have excluded the two other subspecies of red jungle fowl i.e. G.g. murghi and G.g. jabouillei. In view of strong evidences of domestication of chicken in Indus valley, it may be interesting to study the genetic relatedness between Indian red jungle fowl and other red jungle fowl subspecies including G.g. domesticus and other jungle fowls (Kawabe et al., 2014) studied the genetic diversity of native fowls in Laos by analyzing a mitochondrial DNA (mtDNA) sequence polymorphism, multiple maternal lineages were involved in the origin of domestic chicken in Laos. Moreover, there appear to be at least two maternal lineages, one from China and the other from the Southeast Asian continent.

Hence in present study an attempt has been made to study the nucleotide sequence variation in most variable region of mitochondrial D loop region to establish phylogenetic relationship among them.

Material and Methods

Colony PCR

Four individual white colonies were picked up and subjected to colony PCR. The primers were designed to amplify the D loop region. The forward primer was taken from tRNA-Glu (5’-AGG ACT ACG GCT TGA AAA GC-3’), while the reverse primer was taken from tRNA-Phe (5’-CAT CTT GGC ATC TTC AGT GCC A-3’). As evident in amplification of a single product of 1325 bp was seen (Fig. 1).


Fig. 1: Amplification of 1325 bp fragment in RJF from the colony PCR (Lane 1-2. M 100 bp ladder plus

PCR reactions were set up in 25 µl reaction volume containing 2.5 µl of 10 X Assay buffer (100 mM Tris- HC1, pH 9.0, 15 mM MgCl2, 500mM KC1 and 0.1% gelatin), 200 µM of dNTP mix, 10 pm of forward and reverse primer, 1U Taq DNA polymerase (Thermo Fisher Scientific), 50 ng of genomic DNA and autoclaved milliQ water to make up the volume. The amplification was carried out in an i-cycler (Bio-rad). Protocol for PCR reaction consisted of an initial denaturation at 94°C for 5 min. followed by 35 cycles of PCR, each cycle consisting of 1 min at 94°C, 1 min at 55 °C and 2 min at 72°C, and followed by a final extension step of 10 min at 72 °C. The PCR products were resolved on 1.6 % agarose gel in 1x TBE. Electrophoresis was done at 90 volts for 10 minutes, then at 50 volts for 2 hour. Gel was viewed under a UV light. The amplified product was purified, cloned in pTZ57R/T vector, MBI Ferments and sequenced using automated sequencer using Sanger’s dideoxy chain termination method. The related sequences were obtained from Gene bank ( The sequences were edited by using GENETOOL software to get comparable sequences. Subsequently, the sequences were aligned using CLUSTALW (Thompson et al., 1994), website ( Jukes-Cantor genetic distances were estimated using the computer program Molecular Evolutionary Genetic Analysis (MEGA Version 4.0). Jukes-Cantor estimates were used because all mitochondrial sequences were not much divergent (< 6 % divergence from raw counts) and no strong transition bias was evident. The Molecular Evolutionary Genetic Analysis (MEGA Version 4.0) software was used to estimate nucleotide as well as amino acid variability. The genetic distances were estimated as Kimura 2-parameter distances using MEGA software. Phylogenetic trees were constructed with neighbor joining (NJ) procedure using MEGA Version 4.0. Support of the clusters was evaluated by bootstrap, as percentage recurrence of clusters based on 100 bootstrapped replications with MEGA Version 4.0.


The complete D loop in Indian red jungle fowl was 1235 bp and among them, the Domain I of the D loop region was found to be most polymorphic (Fig. 1). Hence the nucleotide sequence variability in this region was used to establish the phylogenic relationship of Indian red jungle fowl (G. gallus murghi) with the other three subspecies of Red Jungle Fowl. The sizes of the different domain of d loop region in different gallus species. Table 1, including those from Indian Red Jungle Fowl (G.g. murghi) was used to study the nucleotide variability in this region. Out of 400 sites about 12 % were polymorphic between the Gallus gallus subspecies. Among the 45 nucleotide substitutions, 60 % were transitions (22 T/C and 5 G/A), while 18 were transversions (4 T/G, 10 Y/A, 1 C/G and 3 C/A).

Table 1: The sizes of the different domain of d loop region in different gallus species

Species Accession number Domain I Domain II Domain III Total
G. gallus murghi 386 462 384 1232
G. gallus domesticus AP003317 386 462 383 1231
G. gallus domesticus AP003318 386 458 383 1227
G. gallus domesticus AP003319 386 462 383 1231
G. gallus spadicus AP003321 386 462 384 1232
G. gallus gallus AP003322 386 462 384 1232
G. gallus bankiva AP003323 386 462 384 1232
G. sonneratii AP006741 448 457 383 1288
G. varius AP003324 385 462 381 1228
G. lafayettei AP003325 447 462 383 1292

Restriction Enzyme Digestion

Consequent upon the fact that the cloning vector pTZ57R/T has one restriction site for EcoR I(GAA TIC) and one site for Pst I (CTGCA↓G) on either side of the Multiple cloning site (MCS). Accordingly, EcoR I and Pst I double-digestion of plasmid DNA was done in 10 l reaction mixture volume. Presence of two bands i.e. 1401 bp and 2840 bp on resolution of restriction digestion mixture on 0.8 % agarose gel confirmed the release of an insert of expected size of 1325 bp in positive clones (Fig. 2).


Fig 2: Resolution of 4241 bp fragment representing whole plasmid plus insert (lane 1) and EcoR Iand Pst I digest 1401 bp fragment (insert) along with 2840 bp fragment (remaining plasmid DNA) (lane 2) and M : molecular weight marker

Among red jungle fowl subspecies, showed comparatively higher genetic distances (0.008 to 0.036) with each other in G.g. gallus birds as compared to the G.g. spadiceus birds (0.003-0.008) and G.g. bankiva birds (0.013-0.015). It suggested comparatively more within-population variability in G.g. gallus as compared to other two subspecies. While Indian RJF (G.g. murghi) showed very low genetic distances (0.008-0.013) with some G.g. gallus birds (GG_gallus8, GG_gallus10 and GG_gallus3322), with other G.g. gallus samples i.e. GG_gallus11, GG_gallus39 and GG-gallus58, the genetic distances were comparatively higher (0.018-0.034). With other two RJF subspecies, G.g. murghi showed comparatively high genetic distance i.e. 031-0.034 with G.g. spadiceus and from 0.061-0.066 with G.g. bankiva. A plot of number of variable sites in non-overlapping 50 bp segments was used to examine the distribution of variation across the control region (Fig. 3).

Phylogenetic Relatedness among Gallus Species

The phylogenetic tree construced using NJ method based on nucleotide sequence variability of complete D loop region has been shown in Fig. 3. Among the different jungle fowls, three jungle fowls i.e. G. sonneratiiG. lafayattei and G. varius seemed to be well separated out from the G. gallus as all these three made a separate cluster.

Fig. 3: Plot of Gallus species control region variability in 50 base windows. Substitutions are indicated with black bars and indels with grey bars

Among the G. gallus subspecies, G. g. spadiceus falls as separate branch, while G. g. murghi and G. g. domesticus falls in one sub-cluster, while the other two sub species i.e. G. g. bankiva and G. g. gallus made another subcluster (Fig. 4).

Fig. 4: Phylogenetic tree constructed by NJ method based on the nucleotide sequences of D loop region in Indian RJF, other subspecies of RJF other jungle fowls a domesticated fowl

Fumihito et al. (1996) based on nucleotide divergence of 480 bp of D loop region, showed that domestic fowls including Indonesian races belong to the same cluster as a continental population of G. g. gallus and G. g. spadiceus sampled from Thailand and its adjacent areas. On the other hand, three specimens of G. g. gallus from South Sumatra form a separate cluster. Presence of Indian RJF in the first cluster showed its closeness to the other two Gallus gallus subspecies i.e. G.G. gallus and G.g. spadiceus, which are supposed to have contribution towards evolution of domestic fowl (Jia et al., 2016; Yacoub et al., 2013). We found that the genetic divergence between these types of chickens was very low and the phylogenetic tree revealed that each strain of native chicken belonged to each other with the same cluster. In addition, each strain has its own cluster in some individuals.


Since most of the sequences of mt D loop region from three species of RJF were taken from Fumihito et al. (1996), hence the trend for phylogenetic relationship among them were similar as described by them. But we were more interested to know the phylogenetic relationship of Indian RJF with these RJF subspecies. Our results placed the Indian RJF very close to the G.g. gallus from Thailand (continental population), which were more nearer to G.g. spadiceus. Very few reports are available on phylogenetic relationship of Indian RJF with other RJF subspecies. Gupta et al. (2009) studied the genetic divergence between Indian Red Jungle Fowl (G.g. murghi), Gallus gallus subspecies (G.g. spadicusG.g. gallus and G.g. bankiva) including G.g. domesticus(domestic fowl) and three other Gallus species (G. variusG. lafayeteiG. sonneratii) in two ribosomal genes (12S rRNA and 16S rRNA) and found that G.g. murghi was more close to Gallus gallus subspecies in comparison to other jungle fowls, however between Indian RJF and other RJF subspecies, the divergence was very low. Very recently, Kanginakudru et al. (2008) suggested the evidence for domestication of Indian birds from G.g. spadiceus and G.g. gallus as well as from G.g. murghi, corroborating multiple domestication of Indian and other domestic chicken (Maw et al., 2015; Mekchay et al., 2014). The proposed SNP panel can effectively be used to characterize the four Thai indigenous chickens; these indigenous chicken breeds were more closely related to red jungle fowls than those of the commercial breeds (Maw et al., 2015). Neighbor joining tree using genetic distance revealed that the native chickens from two countries were genetically close to each other and remote from Red and Green jungle fowls of Java Island (Osman et al., 2014; Silva et al., 2009). The phylogenetic analysis showed the close genetic relationship within and between the populations of each country and molecular information on genetic diversity revealed may be useful in developing genetic improvement and conservation strategies to better utilize of precious genetic reserve. The genetic information from this study is the initial investigation using these populations in Thailand, Indonesia, Japan and India which may be useful in developing future strategies for conservation and improvement of valuable genetic resource.


Authors are highly thankful to the Director and technical staff, Central Avian Research Institute (CARI), Izatnagar, Bareilly, Uttar Pradesh, India for providing necessary facilities to carry out this work at CARI. I am highly thankful to late Dr. Deepak Sharma (Principal Scientist) for valuable advice and suggestion in my doctoral research work.

Conflict of Interest

None of the authors of this paper have a financial or personal relationship with other people or organization that could inappropriately influence or bias the content of the paper.


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