DNA BARCODES OF THE PLECO ( LORICARIIDAE , PTERYGOPLICHTHYS ) IN THE CILIWUNG RIVER

Rosnaeni 1 , Dewi Elfidasari 1 and MeltaRini Fahmi 2 . 1. Study Program of Biology, Faculty of Science and Technology, University of Al Azhar Indonesia.Kompleks Masjid Agung Al-Azhar, Jl. Singamangaraja, KebayoranBaru, Jakarta 12110, Indonesia. 2. Center for Research and Development of Ornamental Fish Aquaculture, Fisheries Research and Development Center, Ministry of Maritime Affairs and Fisheries. Jl. Perikanan No. 13 Pancoran Mas, Depok, West Java City 16436. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


ISSN: 2320-5407
Int. J. Adv. Res. 5(2), 33-45 34 The abundance of PlecoinCiliwung river for Plecohave a high level of adaptation in a polluted environment and the body of Plecocovered with plates of hard scales (Rachmatika&Wahyudewantoro 2006). Therefore, Plecodidn'thave predators and be a competitor in obtaining food on the river ecosystem (Haiti 2007), so the species is considered to have contributed to decline in fish endemic ecosystem species Ciliwung river ecosystem (Kusumah 2011).
Results of Hadiaty research (2011) shows the data rate of loss the species endemicfromCiliwung river in2009 reached 92.5% of the initial number of about 187 species and decreased to 20 species, including five species of which is the introduction of fish species. This type of that fish is lost in the waters of the Ciliwungriver is betutu fish and Balidafish (Wowor 2010) Morphological diversity and genetic information of an organism is very useful for the characterization of the type, the development, distribution by thetime and space. Characterization, development and distribution of populations are needed to determine the step of conservation, management and sustainable utilization. The level of diversity among the population, especially genetic diversity can be used as a step to estimate the level of risk of extinction and the abundance of an organism (Singkam et al., 2011). One of themethod to analyze the genetic diversity is molecular detection.
Molecular detection method was developed to analyze the genetic diversity of a species. Polymerase chain reaction (PCR) is a molecular detection method of in vitro without the use of living organisms to amplify a specific territory of a strand DNA. The reaction is limited by the primer pair (short oligonucleotide) using DNA polymerase enzyme and dNTPs as a number (Widowati 2013). The result of PCR is used to stage a reading strand bases in analyzing phylogenetic kinship.
One of the methods in tracing the phylogenetic relationships of a species commonly did is see the resemblance of mitochondrial DNA (mtDNA). Many studies related phylogenetic studies both invertebrate and vertebrate animals used mtDNA CO1 as the gene sign or genetic barcode (Maramis&Warouw 2014). DNA barcoding is a molecular technique to identify species using differences in nucleotide sequence from standardized gene regions (Hubert et al. 2003). DNA barcoding is based on fragments of mtDNA gene Cytochrome Oxidase I (COI), which serves as a 'barcode' for identifying species (Ward et al., 2005). CO1 genes of the mitochondrial genome are a gene that is often used as standard sign genes in animal identification. The superiority of CO1 gene is to have a universal primer solid, so as to identify the 5 'end from the most groups of animals. CO1 gene also has the highest molecular evolutionary compared with other genes in the mitochondria, thus having a low intraspecific variation and interspecific in height between adjacent taxa (Hajibabaei et al. 2006) Until now there has been no research results related molecular identification of the originate from of PlecoCiliwung river. Therefore it is necessary for the molecular analysis of DNA PlecofromCiliwungriveroriginate using DNA barcoding techniques.

Objectives and Benefits Research:-
This study was conducted to identify Pleco(Pterygoplichthys sp.) As a molecular marker gene CO1, knowing the genetic diversity, and the identification of changes in the nucleotide sequence Plecoin theCiliwung river Section of South Jakarta. The results are expected to be a source of information and databases for the genetic diversity of Pleco(Pterygoplichthys sp.) in the Ciliwungriver. The next stage is the binding of DNA by adding 200 µL of absolute alcohol in the supernatant and homogenized. Samples were then transferred on columntube2 mL and the collecting tube which then centrifuged at 15 000 g for 1 minute. The liquidsolutioncolumn tubedisposedand then collecting tube was transferred to a new tube.
Washing stage by adding 400 µL of Wash Bufferin1 coloumn tube and centrifuged at 15,000 g for 30 seconds. The solution liquid to the collecting tube was removed and reassembled. For the drying process is done centrifugation at 15,000 g for 3 min. Coloumn phase of displacement by moving the tube into a new micro tube and added 100 µLNuclease Free Water (NFW) that has been heated at 60 ° C after that silenced for the last 3 minutes and centrifuged at 15,000 g for 30 seconds to get the DNA.  The PCR process is carried out as many as 35 cycles. Components PCR 50 µL consists of nuclease free water (NFW) as much as 11 µL, forward primer F1 and revers R1 respectively of 2 µL, master mix 25 µL (containing dNTP, buffer and taq polymerase), and the DNA samples of 10 µL , Visualization:-Visualization of DNA using agarose gel (1.5%) with peq green dyes DNA and RNA dye. The result of extraction with the addition of loading dye was electrophoreses in 1 µL and a DNA sample as much 5 µL. Electrophoresis was performed on the current strength of 100 volts for 30 minutes and then photographed using a gel doc with UV rays of a wavelength of 302 nm. Measurement of DNA concentration using a Gene-Quant has a principle wavelength spectrophotometer with 260λ. A beam of UV light passed through a sample of a specific wavelength to see the purity and concentration of DNA. This is done to measure the purity, DNA concentration, protein concentration, and absorbance. Absorbance value is the value measured using a wavelength of 260, so that the absorbance value is the reference number of the DNA concentration (Tiara et al. 2014).

Readings strand bases|:-
Extraction of DNA can be obtained from muscle tissue and blood. Muscles on the fins are a system of organs that have a central role in the movement of fish. Striated muscle groups are named according to the place of attachment, such as enforcement muscle dorsal fin and pectoral fins towing muscle (Rahardjo et al. 2010). The use of fin muscle tissue as a source of the DNA showed that the muscle tissue of fish fins Pleco fish can be a good source of DNA for molecular analysis.

37
Based on one way ANOVA statistical test showed that the source of the DNA of the dorsal fin, pectoral and pelvic fins have DNA purity values were not significantly different (Figure 2). Significance probability value of 0541 which means> 0.05 showed no significant difference between types of fins are used as a source of DNA (Appendix 2). According to Newson (2013) if the significance value of <0.05 means that there are significant differences among the treatments. Visualization the Results of DNA ExtractionPlecooriginate fromCiliwungriver:-Visualization of the extracted DNA Pleco fish on a 1.5% agarose gel shows that DNA can be clearly seen ( Figure 3). This is consistent with the concentration and purity of DNA was good so the quality of the electrophoresis results is clearly visible on a 1.5% agarose gel. Electrophoresis using a marker size of 100 bp.
38  6,7,9,12,13,15,20,21,22,24,25,26 = No. Sample fish) Electrophoresis is the movement of electrically charged substances due to the influence of an electric field (Wardani& Sari 2015). DNA molecules including negatively charged compounds so that the process of electrophoresis, DNA migrate toward the positive pole. DNA molecule migration velocity depends on the concentration of the gel used, the size of the molecules being analyzed, as well as the power supply voltage is supplied. Agarose gel used to separate, identify, and purify DNA fragments. The movement of DNA fragments in agarose gels is strongly influenced by the composition and solubility of the ion electrophoresis buffer. If the concentration of ions is very little the electrical conductivity is very small and DNA migration becomes slow. Excessive ion concentration will result in the gel melts and denatured DNA. DNA electrophoresis technique also requires loading buffer. This buffer works to increase the sample density that is at the bottom well and gives color to the DNA fragment to facilitate observation of the process of electrophoresis (Sambrook&Russel 2001). CO1 gene amplification Fish Pleco fishoriginate fromCiliwungRiver:-CO1 gene amplification product Pleco fishoriginate fromCiliwungRiver is clearly visible on a 1.5% agarose gel. This indicates that the Primer F1 and R1 successfully amplify the COI gene Pleco fishfishoriginate fromCiliwungRiver in fragment length of 650 bp (Figure 4). The nucleotide sequences of 13 samples of Pleco fishoriginate fromCiliwungRiver then compared with the nucleotide sequences in GenBank NCBI (National Center for Biotechnology Information). It aims to determine the similarity of the reading strand fragments to the data base GenBank using BLAST (Basic Local Alignment Search Tool) on megablast-high similarity (Table 3). CIL 006-C 100% Pterygoplichthys pardalis JF769357.1 Results blast on NCBI genbank indicates that nucleotide sequence Pleco fishoriginate fromCiliwungriver showed an average value of 100% identification accuracy with Pterygoplichthys pardalis species (Table 3). This suggests that the originate from of the Pleco fish from Ciliwung river is one species.  2015) shows the similarity of 100% and 0% genetic distance between species P. pardalis, P. disjunctivus and P.ambrosetti. These results suggest that differences in the pattern of the abdomen is not the main character to identify fish species Pleco fish. TA A T   110  G T CA T C G T TA   120  C T GCACA TG C   130  C T TCG T AA TA   140  A T T T TC T T TA   150  TAG T AA TAC C   160  A A T T A TA A T T   170  G G AG GC T TCG   180  G AA AT T G AC T   190  A G T CCCAC TA   200  A T G A T T G GAG   210  CACC CG   A CAG A   580  CCG A A A T T TA   590  A A T AC T A CC T   600  T C T T TG A T CC   610  T GCAGG AGG T   620  GG AG AC CCA A   630  T TC T A T AC CA   640  A CAC T T A T T T   650  TG A T TC T T TG   660  The analysis shows that there are different variations of the arrangement of nucleotides in Pleco fishoriginate fromCiliwungriver. Differences in the nucleotide arrangement causes the Pleco fishoriginate fromCiliwung river splits into two on the construction of phylogenetic clade ( Figure 7). Therefore, the fourth point of nucleotides serves as the main characteristics of each clade and distinguishing nucleotide sequences between individuals. Ubaidilah and Sutrisno (2009) states that if the DNA sequence emerges from a common ancestor sequence, the sequence will gradually separate offspring through nucleotide differences due to mutations or point mutations. Changes in the nucleotide variation four positions do not change the amino acid sequence translated. According to Lynch and Jaryl (1993) amino acid sequence changes occur more slowly in CO1 gene so that accuracy in phylogenetic. The substitution of base pairs (base-pair substitution) is the turn of one nucleotide and partner with 41 another pair of nucleotides. Some substitution called silent mutations (silent mutation) from an excess of genetic code. Thats not influental the amino acid sequence translated. In other words, nucleotide mutations do not change the amino acid translation of the same. Some codons can translate the same amino acids if there is a difference in the third base of the triplet codon (Campbell et al., 2008), as in the TTT and TTC codon that translates the same amino acid that is Phenilalanine (F).

Construction Phylogenetic Fish Sweep broom:-
The results of phylogenetic construction Pleco fishoriginate fromCiliwungriver and out group genus Synodontis (Synodontisdecoratus, S.euterus, and S.leopard) showed genetic distance apart. Plecofishoriginate fromCiliwung river is divided into two clade this is due to changes in the four nucleotide variations (Figure 7).

Figure 7.
Construction of phylogenetic Pleco fish originate fromCiliwungriver with NJ-boostrap 1000x (649 bp) According Mahardika and Parede (2008) the method most commonly used method is the Neighbor-Joining (NJ). The branching pattern of phylogenetic tree based on the distance matrix is formed between the pair populations. Long branches of the phylogenetic tree describes the number of nucleotide substitutions in the form of DNA polymorphism. Skala is located under a phylogenetic tree showing the size of the distance between sequences. Numbers located on the branches of the phylogenetic tree shows the value boostrap (Mahardika&Parede 2008). Bootstrap value in fish samples Pleco fish showed a value of 100%. Bootstrap analysis was conducted to test the validity of the construction of phylogenetic trees. Phylogenetic trees giving information about the classification of the population based on evolutionary relationships. In the reconstruction of phylogenetic trees, the molecular data more widely used because it is considered more stable in the process of evolution compared with the morphological data (Dharmayanti 2011).
42 Figure 8. Construction of fish genetic distance Pleco fishoriginate fromCiliwungriver Fish genetic distance Pleco fishoriginate fromCiliwung river is 0.0-0.03 (Figure 8). This suggests that the genetic distance were lower in Pleco fishoriginate fromCiliwungriver, so the Pleco fishoriginate fromCiliwung river is the same species. According to Hebert et al. (2004) and Ward et al. (2009) said that the genetic distance of more than 0:03 can show different types. This is evident with the genetic distance Pleco fishoriginate fromCiliwung river with out group genus Synodontis has a genetic distance of 0:18 to 0:20 ( Figure 10).

Conclusion:-
Pleco fishoriginate fromCiliwung river had been identified using DNA barcodes CO1 on a fragment length of 650 bp. The nucleotide sequences Pleco fish aligned on NCBI genbank showed an average value of 100% identification accuracy with Pterygoplichthyspardalis species. There transversion nucleotide substitution at nucleotide position to 306 (CT), 339 (GA), 387 (CT), and 471 (TC), but this should not affect the amino acid sequence changes in Pleco-baby fish originate fromCiliwung river.