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Experiments on the D-Loop of Mitochondrial Dna on 6 Chickens

Updated August 8, 2022
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Improved tropically adapted chicken breeds (iTABs) are characterized as dual purpose, low-input high-output birds suitable for smallholder poultry (SHP). A 550bp sequences of mitochondrial DNA (mtDNA) D-loop region of 77 iTABs from six populations were used to access the genetic differentiation of six iTABs introduced to smallholder poultry farmers in Imo State by the African Chicken Genetic Gains (ACGG) project.

The specific objectives were to determine the level of polymorphism among and within the breeds, origin, phylogenetic relationship and evolutionary distance between and among these breeds. The haplotype diversity and nucleotide diversity of the iTABs were 0.80±0.025 and 0.391±0.013, respectively. 14 haplotypes were identified from 62 polymorphic sites. These haplotypes clustered into three clades with 99.7 % of the total maternal genetic variations occurring within population. Sasso and ShikaBrown had the least (0.385) genetic distance. The results indicate the existence of three distinct maternal lineages from Southeast Asia, Indian subcontinent and Southwest China evenly distributed among the iTABs. The high genetic diversity observed within population can be utilized for the long term genetic improvement and stabilization of the breeds.


The improved tropically adapted chicken breeds (iTABs) are low-input high-output chickens suitable for smallholder poultry (SHP). The establishment of an effective long-term genetic improvement, multiplication and delivery system is essential for the adoption and continuous supply of the iTABs to SHP farmers. Understanding the genetic diversity and phylogenetic architecture of the iTABs using mitochondrial DNA (mtDNA) will contribute to the development of the long-term genetic improvement program. MtDNA is an extremely variable genome which has unique characteristics that make it an ideal genetic marker (Avise, 2004).Therefore this study was conducted to determine the level of mitochondrial DNA polymorphism, and establish phylogenetic relationships among the iTABs introduced to SHP farmers in Imo State, Nigeria.

Materials and methods

A total of 77 blood samples were collected from six populations of iTABs (Noiler (No), FUNAAB Alpha (Fu), ShikaBrown (Sb), Kuroiler (Ku), Sasso (Sa) and Fulani (Fi)) from three African Chicken Genetic Gains (ACGG) project sites in Imo State, Nigeria. Genomic DNA was extracted using standard phenol-chloroform method.

Figure 1: PCR Gel electrophoresis result of mtDNA D-loop region showing a 592-bp amplicon size for the iTABS: Noiler (No), FUNAAB Alpha (Fu), ShikaBrown (Sb), Kuroiler (Ku), Sasso (Sa), and Fulani (Fi), M= 200-bp molecular –weight size marker.

PCR reactions were conducted at 96℃ for 15 mins, followed by 35 cycles consisting of 30 sec denaturation at 95℃, 30 sec annealing at 56℃ and 30 mins extension at 70℃, with final extension at 70℃ for 5mins using a Gene Amp PCR System 9700 (USA). The PCR products were electrophoresed (120 V, 20 min) on 1.5% agarose gels, and purified before sequencing. A 592-bp of the mtDNA D-loop region was amplified, and sequenced (3730XL DNA Analyzer sequencer, Applied Biosystems) using primers from Mobegi et al. (2005): L16750 (5′-AGGACTACGGCTTGAAAAGC-3′, accession No.: NC_001323, Desjardins and Morais, 1990) as the forward primers and H547 (5′-ATGTGCCTGACCGAGGAACCAG-3′, Accession No.: AB098668, Komiyama et al., 2003) as the reverse primer.

Genetic analysis: A 315-bp sequence fragment was subsequently analysed. Fintch TV version 1.4.0 (Patterson et al., 2004) was used to assemble, view and edit the sequences. The MEGA 7.0 (Tamura et al., 2015) was used to align theD-loop sequence to the Gallus gallus RefSeq (Accession No:KX987152.1), following 1000 bootstrap replicates, a maximum Likelihood (ML) tree was generated. 3 D-loop sequences of Galliformes; Meleagrisgallopavo, Cortunixjaponica and Anasplatyrhynchos (Accession No: EF153719, AP003195 and MF069250, respectively), were the out-groups. Eight RefSeq of Gallus sp. and two sub-species from the most common haplotypes of different clades found in Asian domestic chickens were included in the analysis. Genetic distances and diversities were analysed using Mega 7.0 and DnaSP 6.11.01 (Librado et al., 2017).

Results and Discussions

The 77 sequences from 6 iTABs populations generated 14 haplotypes from 62 polymorphic sites (Figure 2). All variable sites were due to substitution mutations, and 94.6% of these mutations were transitions.

Figure 2: Nucleotide polymorphism and their frequencies (N) based on the haplotypes. Vertically oriented numbers indicate the RefSeq position, dots (.) indicate identity with the RefSeq, Hap 1-14are haplotype numbers.

In this study, Hd were higher than the values previously reported for African chicken populations by Adebambo et al. (2010) (Nigeria: 0.421), and Nwacharo et al. (2011) (Ethiopia: 0.374, Sudan: 0.413, Uganda: 0.322). Among the populations under study, Sasso showed the highest sequence conservation (29.2%) whereas FUNAAB-Alpha and Kuroiler both had the lowest sequence conservation (23.1%). Diversity indices Table 1 further revealed high mtDNA polymorphism within populations and low among populations. Table 2 shows that the highest genetic distance (0.457) was between Sasso and FUNAAB Alpha, and lowest (0.385) between Sasso and ShikaBrown. It was observed that all the breeds shared a common ancestry, with Sasso and ShikaBrown being more closely related. Furthermore, Analysis of Molecular variance shows that 99.67% of the maternal genetic variation occurred within populations while the remaining variation (0.38%) was found among populations.

Below diagonal represents distance estimate (d); above diagonal represents Standard Error of Mean (SEM). Analysis was computed in Bootstrap model. (No=Noiler, Fu=FUNAAB Alpha, Sb=ShikaBrown, Ku=Kuroiler, Sa=Sasso and Fi=Fulani).

The phylogenetic analysis (Figure 3) grouped the iTABs into three main clades (clade II, III and IV) out of the seven clades identified in Asian domestic chicken (Bjornstad et al., 2003). Mobegi et al. (2006) observed that the majority of haplotypes in West African village chicken populations cluster in clade IV,

Figure 3.Phylogenetic relationship of the iTABS constructed using the haplotypes.

Muchadeyi et al. (2008) observed two distinct clades amongst both Zimbabwe and Malagasy chickens, while Adebambo et al. (2010) observed a single clade among Nigerian chicken populations, indicating a closer history of domestic chicken between West African village chicken and iTABs, but different between Nigerian chicken and iTAB which suggests the absence of admixture. The phylogram revealed a shared ancient lineage between iTABs and the individuals of Gallus species. A recent common ancestor was observed between G. varius and iTABs haplotypes found in clade IV. Individuals in hap 11 (Sasso and FUNAAB Alpha) were found to have an intermixed lineage with G. bankiva and G. lafayetii. This distinct distribution patterns suggests that the divergent clades originated from divergent regions of Japan, Serbia and the Indian subcontinent, which supports the theory of multiple origins in South and Southeast Asia (Liu et al.,2006; Kanginakudru et al.,2008).

Conclusion and Recommendation

The high genetic diversity within the population can be utilized for further genetic improvement of the breeds. This result can also guide in the conservation of the local germplasm, and evaluate the rate of admixture with the iTABs.


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