Molecular characterization of Tapah fish (Wallago leerii, Bleeker 1851) from Riau Province based on Cytochrome b gen

DOI: 10.13170/depik.12.2.33161 Tapah fish (Wallago leerii) is a fish with high economic value in Riau Province. Molecular research on W. leerii is very important to do. This study aims to determine the molecular characteristics of W. leerii from Riau Province based on the Cytochrome b gene. The universal primer Cytochrome b is used for the Polymerase Chain Reaction (PCR) process. The PCR result is a partial Cytochrome b gene with a length of 373 bp. Multiple alignments were performed on nucleotide sequences of the Cytochrome b gene of W. leerii from river of Tapung and Indragiri Riau with the cytochrome b gene of other fish from Genbank and analyzed using the MEGA program version 6.0. Phylogenetic construction using the Cytochrome b gene can distinguish W. leerii from river of Tapung and Indragiri Riau, with other species.


Introduction
Riau Province is a geographical area that has four major rivers.These rivers include the Kampar, Siak, Indragiri and Rokan Rivers.The river in Riau Province has the characteristics of a flood plain river (Elvyra, 2009).
The flood plain river ecosystem is a complex system consisting of riverbeds, tributaries, and flooded lakes (Rollet et al., 2014).This complex system has certain functions to fulfill the liberation of fish life in that habitat.The bottom of the river is used as a shelter for fish from predators; tributaries, especially on the banks of the river, can be used as shelter and foraging; flooded lakes are used by fish as a place of refuge, foraging, spawning and laying eggs until they hatch because there is riparian vegetation which is submerged when the rainy season enters (Hartoto et al., 1998).
The Tapah fish (Wallago leerii Bleeker, 1851) is a unique fish that inhabits flood plain river areas, including the Kampar, Tapung and Indragiri Rivers in Riau Province.According to Kottelat et al. (1993), W. leerii are included in the catfish group or fish that have antennae.In the classification structrure, the W. leerii belongs to the order Siluriformes, family Siluridae, genus Wallago and species W. leerii.
W. leerii is a fish that has high economic value and have a maximum weight of up to 35 kg and total length 1.5 m (Kottelat et al., 1993).Catches that are continuously carried out by fishermen and no conservation efforts are carried out, it is feared that extinction will occur in the future.
The development of molecular techniques is currently very helpful in obtaining scientific information genetically.Genetic information obtained can be used to differentiate species and see kinship and diversity between species (Muchlisin et al., 2017;Yulianto et al., 2020;Batubara et al., 2021).Polymerase Chain Reaction (PCR) is a method that can be used to reproduce the DNA of an organism (Garibyan and Avashia, 2013).PCR-based methods are often used in the identification of organisms, either through DNA finger printing or through DNA barcoding (Hebert et al., 2004).Genetic identification  Elvyra et al. (2023) methods using the PCR method have been developed a lot, and have been carried out on various marine organisms, including various corals hard (Shearer and Coroth, 2008).

Location and time of research
This research was conducted in December 2017 -May 2018 at the Genetics Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, University of Riau.Sampling was obtained from fishermen as many as 30 fish samples (10 individuals per species) from three different rivers, namely the Kampar (0 o 11'38" N, 101 o 28'59" E), Tapung (0 o 40'07" N, 101 o 17'16" E) and Indragiri (0 o 24'16" S, 102 o 36'45" E) Rivers of Riau Province.

mtDNA Extraction and Isolation
In the mtDNA extraction and isolation process, three fish samples were taken from 30 individuals in each river.The sample was taken in the form of fish muscle tissue near the tip of the tail which was cut with a size of 1 cm.The tissue is stored in absolute alcohol.The extraction and isolation processes refer to the Dneasy Blood and Tissue Kit (Qiagen) DNA isolation and purification kit protocol.
Fish muscle tissue was dried and finely chopped, added with TE Buffer, vortexed for 15 seconds, then centrifuged at 3000 rpm for 2 minutes.Discard the TE Buffer liquid and dry the sample and then put into a 1.5 ml tube.The sample was added with 180 μl of ATL buffer to lyse the tissue.After being lysed, the samples were vortexed with 20 µl Proteinase K for 10 seconds and incubated for 1-2 hours in a water bath at 56 o C until all samples dissolved.
Samples that had been incubated were removed and vortexed 200 μl AL buffer for 15 seconds, then 200 μl absolute ethanol was added and vortexed for 2 minutes.After that, the sample was transferred to a 2 ml spincolumn, centrifuged at 8000 rpm for 1 minute.Next, discard the column tube which contains the supernatant.
In the washing process, add 500 µl of Buffer AW 1, centrifuge at 8000 rpm for 1 minute.Then added 500 µl of Buffer AW 2 and centrifuged at 13000 rpm for 3 minutes.After that, 200 µl of AE Buffer was added to obtain total DNA stock, incubated at room temperature for 1 minute and centrifuged at 8000 rpm.The DNA stock that has been obtained is stored at 4 o C.

Electrophoresis DNA extraction product
Visualization of DNA bands can be done by electrophoresis method.The medium used was made using 1% agarose gel solution, 1X TBE buffer (Trisborate-EDTA pH 8.0) and 1 µg/ml ethidium bromide heated until dissolved and poured into the mold.The electrophoresis process was carried out at a voltage of 50 volts for 30 minutes.Electrophoresis results can be observed using a UV transilluminator and photographed with a camera.

Cytochrome b gene amplification from mitochondrial DNA
The primers used for amplification (PCR) are the universal primer pair Cytochrome b consisting of: L14841 5'AAAGCTTCCATCCAACATCTCAGCATGAT GAAA-3' (Forward) and H15149.5'AAACTGCAGCCCCTCCAGAATGATATTTGTCCTCA-3' (Reverse) (Kocher et al., 1989).The DNA used as a template at the amplification stage was W. leerii DNA which had been previously isolated.
Predenaturation at 94 o C for 3 minutes, denaturation at 94 o C for 30 seconds, annealing or primary attachment stage at 57.4 o C for 1 minute, elongation stage at 72 o C for 1 minute, final extinction at 72 o C for 10 minutes.The PCR process was carried out for 35 cycles.

PCR product electrophoresis and sequencing
Visualization of the DNA bands of PCR products was carried out using the electrophoresis method as before.The electrophoresis results of a properly amplified PCR product will show one band without impurities.The PCR products were then sequenced.Sequencing was carried out by sending 50 µl of PCR results to PT. Genetics Science Indonesia, Jakarta.

Results
The total DNA molecule of W. leerii that had been extracted and isolated was then visualized using the electrophoretic method, 9 samples were selected from 30 samples (three samples per river) (Figure 1).DNA that has good quality will show DNA bands like lines.
The results of DNA products that have good quality are then amplified by the PCR method.The PCR amplification product uses the universal Cytochrome b gene with a different band thickness in each DNA (Figure 2).leerii DNA obtained fragment lengths of around 300-350 bp is then translated into amino acids.The amino acid sites obtained show the differentiation for the genus at the 10 th site which is at the 28 th , 29 th and 30 th nukeotides (Table 2).

Discussion
Cytochrome b gene amplification in the PCR process using mitochondrial DNA fragments from W. leerii obtained a sequence of 338 bp.Based on the length of the fragments obtained, the results were not much different referring to the research that had been carried out on Kryptopterus limpok and K. apogon from the Kampar and Indragiri Rivers using the universal Cytochrome b gene L14841 (F) and h15149 (R) obtained sequences of the same size 373 bp (Elvyra et al., 2009).Other studies have revealed that Cytochrome b gene fragments reach 300-400 bp in fish (Kiril'Chik and Slobodyanyuk, 1997;Akasaki et al., 2006;Hsieh et al., 2010).
Based on the analysis of the genetic distance between W. leerii originating from the Kampar, Tapung and Indragiri rivers, the value is relatively small, ranging from 0.003-0.015(Table 1).The genetic distance between W. leerii from the Kampar, Indagiri, Tapung rivers, and W. leerii from GenBank had a genetic distance of 0.015-0.018,0.018-0.021,and 0.018-0.024.The genetic distance found in W. leerii intraspecies revealed that there were variations in the DNA structure that might have occurred due to random crossing over of genetic information in the fertilization process (Féral, 2002).The genetic distance that has the greatest value is between O. bimaculatus and S. microdorsalis 0.169.A genetic distance value > 0.03 indicates that one fish species is different from another, so intraspecies occurs if the genetic distance value is <0.03 (Muchlisin et al., 2022;Nur et al., 2022).
Based on the construction of the phylogenetic tree W. leerii from the Kampar, Tapung and Indragiri rivers and W. leerii comparator from GenBank formed one cluster and separated from O. pabo, O. bimaculatus, and S. microdorsalis which became an outgroup (Figure 3).The kinship in the first cluster, namely W. leerii from the Kampar, Tapung and Indragiri rivers with W. leerii from GenBank, has a bootstrap value of up to 100% (identical).In a separate outgroup cluster due to a different genus.
The 338 bp sequence was then translated using the MEGA version 6.0 program to produce 112 amino acid sites.Based on the site obtained, there is one genus coding site, namely at the 10 th site position,  Elvyra et al. (2023) with the nucleotide composition there are sites 28 th , 29 th and 30 th (Table 2).The occurrence of transition substitution in the nucleotides of the Cytochrome b gene in W. leerii is presented in Table 3.Based on the values, it can be analyzed that transitional substitutions between W. leerii from the Kampar, Tapung and Indragiri rivers have relatively differences, ranging from 0-4 nucleotides.In W. leerii from the Kampar river and W. leerii from GenBank, there was a transitional substitution of 2-3 nucleotides, between W. leerii from the Indragiri, Tapung rivers, and W. leerii from GenBank, ranging from 3-4 nucleotides.The transitional substitution value that has the greatest value is between O. pabo and S. microdorsalis reach of 35 nucleotides.
The transversion substitution events in the nucleotides of the W. leerii Cytochrome b gene are presented in Table 4.The smallest transversion substitution value was 0, which means that no nucleotide transversion substitution occurred in some W. leerii DNA samples from Riau Province.Meanwhile, the biggest one is between O. bimaculatus and S. microdorsalis reaching 24 nucleotides.
W. leerii nucleotide transversions and substitutions cause variations in DNA structure.This allows fish to have variations in morphology, for example differences in color, number of fin rays, size and behavior (Hrbek and Larson, 1999;Jang et al., 2019).However, this variation does not cause the fish to become a separate species.It is evident from the phylogenetic tree which shows the monophylletic relationship between W. leerii collected from different locations.

Conclusion
The alignment of the nucleotides L14841 (F) and h15149 (R) in the 338 bp DNA of W. leerii was then translated into 112 amino acids.The amino acid sites obtained show a differentiator for the genus at the 10t h site which is at the 28 th , 29 th , and 30 th nukeotides.The construction of the phylogenetic tree of W. leerii f from Riau Province and W. leerii from GenBank formed one cluster.

Figure 3 .
Figure 3. Phylogenetic tree construction using the neighbor-joining method and bootstrap analysis (1000 times

Table 1 .
Genetic p-distance based on the Cytochrome b gene of W. leeri.

Table 2 .
Sites encoding the amino acid gene for Cytochrome b of W. leerii.