INTRODUCTION
The diversity of local chicken species in Indonesia is a wealth of Livestock Genetic Resources which has the potential to be used and developed as a source of germplasm for both conservation and utilization. This has an important and strategic meaning in the effort to encourage food security and prevent a decline in the potential of local chickens. Local chickens are known as chickens that come from the domestication of the jungle fowl (Gallus gallus) and are grouped into several types, including types of broiler, laying, dual-purpose and ornamental (fancy) types. According to Nataamijaya [1], there are 31 types of local Indonesian chickens that have been identified, one of which is the Cemani chicken.
Cemani chickens (Cemani in Javanese means “black”) are commonly found in the Temanggung area, Central Java, and are kept as ornamental chickens or raised to produce eggs which will be hatched. The Cemani chicken, also known as the black Kedu chicken, has specific characteristics marked by the entire color of its feather which is black. Every part of its body, from the skin and flesh to the bones, beak, cloaca, comb, face and legs, is black. The black body of the Cemani chicken is the result of a genetic mutation involving duplication in an area of the genome with 5 genes, resulting in fibromelanosis or hyperpigmentation [2,3]. One of the genes within the duplication area is endothelin 3 (EDN3) which plays a role in the formation of melanocyte-producing proteins. This causes the overexpression of melanocyte-forming proteins while the Cemani chicken is still an embryo [4]. This chicken commands a relatively high price, because apart from its exotic physical appearance, it is often used by the local rural community as a tribute at ancient religious ceremonies, especially the Cemani rooster with its black tongue.
So far, several researchers have carried out genetic characterization of local chickens, including the Cemani chicken, both qualitatively, quantitatively and molecularly. Particularly for genetic characterization using molecular techniques, it has been limited to mitochondrial DNA segments such as Dloop [5,6], or microsatellite [7]. Complete genetic information data regarding the mtDNA of Cemani chicken is not yet available. Moreover, research on genetic characterization of complete mtDNA (whole genome mtDNA) needs to be carried out to complement the results of previous studies and to enrich information regarding the existence of the Cemani chicken as one of Indonesia’s local chickens. Therefore, this study aimed to identify, discover and explore the characteristics of the mtDNA genomes of Cemani chicken (Gallus gallus).
MATERIALS AND METHODS
Ethical approval
The experimental procedures were approved by the Animal Research Ethics Committee of the Faculty of Animal and Agricultural Sciences, Universitas Diponegoro (No. 59–01/A-01/KEP-FPP).
Tissue collection and DNA extraction
This study used gDNA of Cemani chicken (Figure 1) isolated from liver tissue as it is known that cells requiring large amounts of energy tend to have a higher abundance of mtDNA. A Cemani chicken was slaughtered and dissected to collect the liver tissue. Tissue samples (10 g) were stored in Falcon tubes containing ethanol. These tissue samples were used to obtain gDNA, which was extracted according to the manufacturer’s standard protocol using the gSYNC DNA Extraction Kit (Geneaid, New Taipei, Taiwan). The collected gDNA was then selected based on quality and quantity, and genomic mtDNA enrichment was conducted using the REPLI-g Mitochondrial DNA Kit (Qiagen, Hilden, Germany). The library preparation process used the enhanced mtDNA.
mtDNA sequencing and bioinformatic analysis
mtDNA sequencing was performed using WGS mtDNA analysis with nanopore technology [8,9] by Oxford Nanopore Technologies GridION facilitated by Genetika Science (Tangerang, Indonesia). Bioinformatics analysis was then performed. The workflow procedure for WGS, mtDNA, and bioinformatic analysis is shown in Figure 2. The MinKNOW (v21.11.17) program was used to run the sequencing output from the Oxford Nanopore Technologies GridION sequencing. Guppy (v5.1.13) performed base calling in high-accuracy mode [10]. NanoPlot (v1.40.0) was used to visualize read quality [11]. Using minimap2 (v2.24), all readings were mapped to the mitochondrial reference sequence from GenBank [12]. Flye (v2.8.3) was used to perform the assembly using the filtered mapped reads [13]. A Nanoplot (v1.40.0) was used to assess the quality of mapped and filtered reads. Racon (v1.5.0) was used to polish the constructed sequence four times and Medaka (v1.5.0) was used three times [14] (https://github.com/nanoporetech/medaka). MitoZ (v2.4) was used to annotate and visualize the final sequences [15]. Quast (v5.0.2) assessed the quality of constructed sequences [16].
Data analysis
Data analysis was conducted using MEGA11 software [17]. Complete mtDNA genome sequences of Cemani chicken were aligned to identify the genetic characteristics, including gene sequence, position, size, amino acid length, amino acid alteration, and nucleotide composition. mtDNA genome sequences of Cemani chicken were also aligned with another 35 Gallus gallus complete mtDNA genome sequences obtained from GenBank as reference (Table 1) to identify mutation, diversity and visualize the genetic relationship through a phylogenetic tree constructed based on the maximum likelihood method [18].
RESULTS AND DISCUSSION
The results of the analysis showed that the length of the mtDNA genome of Cemani chicken was 16,789 bp, which consisted of 2 ribosomal RNA (l-rRNA, s-rRNA); 22 transfer RNA genes (trnF, trnV, trnL, trnl, trnQ, trnM, trnW, trnA, trnN, trnC, trnY, trnS, trnD, trnK, trnG, trnR, trnH, trnS, trnL, trnT, trnP, trnE); 13 protein coding genes (ND1, ND2, COX1, COX2, ATP8, ATP6, COX3, ND3, ND4L, ND4, ND5, ND6, CYTB) and control region (Dloop) (Figure 3). The results of reading the mtDNA sequences of Cemani chicken are shown in Table 2. As many as 12 of the 13 protein-coding genes of Cemani chicken begin with the ATG start codon, while the rest start with the GTG (COX1) start codon. The stop codons in the Cemani chicken protein coding gene were dominated by TAA (ND1, COX2, ATP8, ATP6, ND3, ND4L, ND5, CYTB, ND6), while the other stop codons were TAG (ND2); AGG (COX1); CTT (COX3); and TAT (ND4).
The positions of the trnQ, trnM, trnA, trnN, trnC, trnY, trnS, trnP, trnE, and ND6 genes are in the light strand, while the rest are in the heavy strand. The I-rRNA gene is known to be the longest gene in mtDNA, which has a size of 1,620 bp. On the other hand, the shortest gene in mtDNA is the trnC gene, which is 66 bp long. The longest amino acid translation is owned by the COX1 gene, which is 517 amino acids length, and the shortest amino acid translation is owned by the ATP8 gene, which is 64 amino acids length. Overall, the DNA strand in Cemani chicken mtDNA is dominated by adenine (A) and thymine (T) bases. The most abundant composition of Adenine (A) was found in the trnD gene at 37.7%, and the least was found in the ND6 gene at 10.3%. The most abundant Thymine (T) base composition was found in the ND6 gene by 41.4%, and the least was found in the trnF gene. The ATP8 gene was identified as the gene that had the least amount of Guanine (G) base composition, namely 4.8%, whereas the ND6 gene was identified as the gene that had the most Guanine (G) base composition, namely 41.4%. Meanwhile, the most abundant composition of Cytosine (C) was found in the ATP6 gene, namely 38.6%, and the least found in the ND6 gene, namely 9.6%.
The overall nucleotide composition of Cemani chicken mtDNA was 23.7% for T; 32.5% for C; 30.3% for A; and 13.5% for G, which was quite similar with references as well as their mtDNA length (Table 3). The result of alignment between Cemani chicken in this study and references mtDNA nucleotide sequence from GenBank showed polymorphism in most of the genes (Table 4). Dloop gene had the highest polymorphic sites (3.65%), followed by ND6 gene (1.91%); ND4 gene (1.52%); ND3 gene (1.42%); CYTB gene (1.31%); ATP6 gene (1.17%); COX1 gene (1.09%); ND1 gene (0.92%); COX3 gene (0.89%); COX2 gene (0.88%); ND2 gene (0.67%); ND5 gene (0.44%), while ND4L genes had the lowest polymorphic sites (0.33%). Otherwise, ATP8 gene showed monomorphism that indicated 100% conservation. According to the parsimony form, ND4L gene had the highest site (100%), due to only one mutation point. On the other hand, ND2 genes had 71.4% parsimony form sites, while ND4 gene had 28.57% parsimony form sites. Conversely, there was a negative correlation as in the singleton form. It was shown that ND4 gene had the highest sites (71.42%), while ND2 gene had the lowest sites (28.6%). This study also found indel mutation that located in the Dloop gene, specifically in site (859indel/C) in parsimony form and in site (860indel/C) in singleton form. Those two indel mutations were thought to have occurred due to the presence of repeat sequence of C as much as 7 to 9 times [30]. Among all polymorphic sites that found based on alignment with all references, site (988T>indel) in Dloop genes and site (328A>G) in ND3 gene were specific markers that were only found in Cemani chicken and had never been reported by previous researchers. Moreover, site (988T>indel) in Dloop gene showed there was insertion in “T” form only in Cemani chicken, while site (328A>G) in ND3 gene showed only Cemani chicken that had “A” in that site, while references showed “G”.
The result of alignment between Cemani chicken in this study and references mtDNA in amino acid sequence from GenBank showed there were amino acid alterations in most of the protein coding genes (PCGs) (Table 5). ND6 gene experienced many alterations in amino acid (4.62%), followed by ND3 gene (4.27%); ND4 gene (1.96%); COX1 gene (1.35%); CYTB gene (0.79%); COX3 gene (0.76%); ND5 gene (0.67%); ND1 gene (0.61%); COX2 and ATP6 genes (0.44%) as the lowest. Most of the amino acid alterations in this study were due to mutations in the 1st and 2nd bases in the triplet codon unit, as stated by Nei and Kumar [41]. At the same time, amino acid alteration was not found in ND4L and ATP8 genes. Those facts were due to mutation in ND4L gene being in the 3rd bases in the triplet codon unit leading to a synonymous form of amino acid, so that it wasn’t causing amino acid alteration, while in ATP8 gene it was due to its gene being monomorphic. Furthermore, the notable amino acid alteration was found in ND3 gene, specifically in codon (110AGG> GGG) which showed glycine in all references but which showed as a stop codon only in Cemani chicken.
Based on phylogenetic analysis, Cemani chicken had a very close relationship with Kedu chicken from Indonesia and Manticao chicken from the Philippines, which were still in the Southeast Asia region (Figure 4). Moreover, Daweishan Mini chicken from India and Hawaiian chicken were also known to have a slightly close relationship with Cemani chicken, because they were still in the same branch of phylogenetic tree based on Tamura and Nei [18] methods. In addition, Figure 5 showed the phylogenetic tree based on the D-loop sequence, which indicated three main clusters differently. It showed that Cemani chicken was in the same cluster as Daweishan Mini chicken, while Kedu chicken and Manticao chicken formed another sub-cluster.
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