Composition of Mitochondrial DNA 16 S Nucleotide of Dwarf Snakehead ( Channa gachua Hamilton , 1822 ) from Keji River , Magelang , Central Java

Indonesia has a high marine and freshwater biodiversity including freshwater fish biodiversity. One of freshwater fish which is commonly consumed by Indonesian people is dwarf snakehead (Channa gachua Hamilton, 1822). However, research on genetic characterization, especially the composition of mtDNA 16S nucleotide of dwarf snakehead has poorly understood. Therefore, the aim of this study was to determine the composition of mtDNA 16S nucleotide of dwarf snakehead as a part of genetic characterization of the fish species taken from Keji River, Magelang, Central Java which has not been previously examined. This study analyzed 16S mtDNA of two samples of dwarf snakehead from Keji River (KTS-01 and KTS-02). In addition, two sequences of Channa gachua with accession number KU986900, KU238074, and HM117234HM117238 taken from GenBank were used as a comparison. A method used in this research was a PCR method and primers used in this research were 16Sar and 16Sbr. The results revealed that the average of nucleotide composition T, C, A and G of the fish species was 23.04%, 25.13%, 29.06% and 22.77% respectively whereas the average rate of nucleotide composition A+T and G+C was 52.10% and 47.90% respectively. The two dwarf snakehead had similar T and C composition but different in A and G composition. In addition, the G+C content in KTS-01 and KTS-02 had the highest frequency compared to other dwarf snakehead taken from GenBank. From this finding it could be assumed that there is genetic variation between the two dwarf snakehead from Keji River which is important genetic data for breeding program of the fish species in the future. Article history: Received 18/05/2018 Received in revised form 05/07/2018 Accepted 07/07/2018


Introduction
Fish has important nutritional values especially protein, vitamin and minerals.Dwarf snakehead (Channa gachua) is one of freshwater fish which is commonly consumed by Indonesian people.The dwarf snakehead belonging to the family Channidae and considered as the important freshwater food fish in tropical Asia due to possess high economic value (Benziger et al., 2011;Kottelat, 2013).In addition, the fish are commonly used as ornamental fish in aquarium due to the beautiful colorization (Milton et al., 2011).
The fish is native to Asia and has widely distribution in Middle East and South Asia, including Indonesia (Berra, 2007;Kottelat, 2013).This species mainly inhabit most any type of wetland including streams, creeks and rivers with pH 6.0-7.0 and temperature 10-28 o C. The species can reach 28 cm in length and feeds on small fish, aquatic insects and crustaceans (Chaundhry, 2010).
In order to increase the production of the fish, research attention has to be focused on the fish genetic characterization using molecular approaches.Mitochondrial genes are widely used as efficient molecular tools not only for identification unambiguous species but also for examination genetic variation and biodiversity with high levels of accuracy (Pereira et al., 2008;Yang et al., 2014;Hammer et al., 2014;Satoh et al., 2016).One of the mitochondrial genes that can be used for molecular marker is 16S mtDNA.The 16S mt-DNA is often used for studies genetic characterization of the DOI: 10.22146/jtbb.35613© 2018 JTBB species and genetic variation at inter-specific levels, such as Coilia mystus (Cheng et al., 2008), Epinephelus lanceolatus (Cheng et al., 2015), Monopterus albus (Arisuryanti, 2016), and Labeo spp.(Jahan et al., 2017).For the analysis, the 16S mtDNA marker is first amplified by PCR using primers (universal or specific primers) and the amplicons are sequenced.Sequencing data are then aligned and compared using appropriate bioinformatic tools (Arif and Khan, 2009;Arif et al., 2009).
Despite the importance of this species as a food consumption resource, little is known about genetic characterization and genetic diversity of dwarf snakehead in the Indonesian waters.The information of 16S mtDNA varieties in C. gachua collected from Keji River (Magelang, Central Java) has never been reported.Failure to detect population units of the fish species coupled with local overfishing will ultimately lead to decrease in populations.In order to implement conservation and management strategies for a declining species, it is important to investigate composition of mitochondrial DNA 16S nucleotide of dwarf snakehead as a part of the fish genetic characterization throughout its natural habitat.Therefore, the objective of this research was to find the basic nucleotide data of the 16S mtDNA of dwarf snakehead from Keji River (Magelang, Central Java) to complete the genetic information of Indonesian dwarf snakeheads.It is expected that this finding of this study is able to give genetic information which is beneficial for improving the genetic quality of dwarf snakeheads in Indonesia through breeding program.

Sample collection and storage
The dwarf snakehead samples were collected from Keji River, Magelang, Central Java (7 o 35'34.85"S110 o 16'19.48"E)(Figure 1).The fish were caught by net and then documented (Figure 2).Approximately 100 mg of muscle tissue from two individuals (KTS-01 and KTS-02) was preserved in 99% ethanol and each was placed into 1.5 ml tube.Fish samples were then brought to Laboratory of Genetics and Breeding, Faculty of Biology, Universitas Gadjah Mada and stored at 4 o C until processed.Genetika Science (Jakarta) for purification and sequencing.

Data analysis
Chromatograms of the two dwarf snakeheads were checked and assembled using SeqMan, and edited using EditSeq Pro Program Lasergene DNASTAR software package (DNASTAR Inc., Madison, USA).Consensus sequences of the 16S mtDNA were checked from forward and reverse.The sequence of each sample was verified using BLAST.The composition of mtDNA 16S nucleotide of each fish sequence obtained in this study were then calculated using DNA Statistics from EditSeq menu.The composition of C+G was validated using DnaSP v.5.10.01 (Librado and Rozas, 2009).
The comparison nucleotide composition of dwarf snakehead fish used was taken from GenBank (accession number KU986900, KU238074, and HM117234-HM117238).

Results and Discussion
The amplification product for the 16S mitochondrial gene of the two dwarf snakeheads investigated in this study (KTS-01 and KTS-02) generated 573 bp in fragment length (Figure 3).The analysis using BLAST showed that the two dwarf snakeheads investigated in this study have 98% similarity with C. gachua deposited at GenBank.    from India) and it can be shown in

Figure 1 .
Figure 1.Map of sampling collection site for C. gachua samples in Keji River, Magelang, Central Java

Figure 3 .
Figure 3.The DNA band profiles of 16S mitochondrial DNA of C. gachua (1) KTS-01 and (2) KTS-02.M = 1 kb DNA ladder (Bioline).Based on the range of the analysis 573 bp nucleotide, the result of nucleotide parallelization of the two dwarf snakehead taken from Keji River (Magelang, Central Java) is shown on theTable 1.From the Table 1, it can be seen that respectively.Both dwarf snakeheads had similar nucleotide T and C but different A and G.The nucleotide divergence between the two C. gachua taken from Keji River indicated that there is intra-population genetic variation This is due to the 16S mitochondrial gene is considered as a highly conserved and usually used for interspecies or intergeneric level of identification and diversity(Arif and Khan, 2009).Therefore, the finding of 16S mtDNA nucleotide divergence between the two samples indicate that there is intrapopulation genetic variation which is important genetic data for breeding program of the fish species in the future.Next, the 16S mtDNA sequence data of C. gachua collected from Keji River (Magelang, Central Java) were compared to C. gachua taken from GenBank (KU986900 from Malaysia, KU238074 from China and HM117234-HM117238

Table 1
. From the Table1, it can be seen that the average of nucleotide T, C, A and G was 23.04%, 25.13%, 29.06% and 22.77% respectively whereas the average rate of nucleotide composition A+T and G+C was 52.10% and 47.90%

Table 2 .
From the Table 2, it can be seen that the composition of nucleotide C from C.
gachua examined in this study has the highest frequency compared to the two other C. gachua from Malaysia and China.Similarly, the composition of C+G of C. gachua from Keji River is also higher than that of C+G from Malaysia and