Genetic relationships of the anabas (anabas sp) using mitochondrial cytochrome oxidase subunit I marker gene

Southeast-Asian J. of Sciences: Vol. 7, No. 1(2019) pp. 80-88 GENETIC RELATIONSHIPS OF THE ANABAS (ANABAS SP.) USING MITOCHONDRIAL CYTOCHROME OXIDASE SUBUNIT I MARKER GENE Truong The Quang Institute of Applied Science and Technology Van Lang University, Vietnam e-mail: quangtruongthe@gmail.com Abstract Total DNA of the anabas specimens (A1, A2, A3, A4) in Vietnam were extracted using PHUSA-IHHNV kit and according to the process of Phu Sa Biochemical Company. The mitochondrial cytochrome oxidase subunit I (COI) gene were amplified using PCR reaction. The obtained PCR products were checked using 2 % agarose gel electrophoresis and were sequenced with both the forward and reverse primers using an auto- mated sequencer ABI 3730XL of Applied Biosystems by Sangers method. The genetic distances (Dij) between the anabas species in Vietnam (A1, A2, A3, A4) are very small from 0.0046 to 0.0156 and they are all in group 4 with Anabas testudineus in Malaysia so they are the same An- abas testudineus. The researched species of Anabas genus in Vietnam (A1, A2, A3, A4) and some countries in South and Southeast Asia were divided into four groups based on COI marker gene. The groups with far genetic distance include group 2 and group 4 (D24 = 0.1055), group 1 and group 4 (D14 = 0.1041), group 1 and group 3 (D13 = 0.0910). The groups with medium genetic distance include group 2 and group 3 (D23 = 0.0849), group 1 and group 2 (D12 = 0.0772). The groups with near genetic distance include group 3 and group 4 (D34 = 0.0285). The species Anabas testudineus in India and Anabas testudineus in Myanmar should be re-identified Anabas cobojius in India and Anabas cobojius in Myanmar belong to group 1. Key words: Anabas, Cytochrome oxidase I gene, Genetic relationships, Phylogeny tree. 80 Truong The Quang 81 Background The Anabas genus includes two species of the climbing perch (Anabas tes- tudineus Bloch, 1792) [16], see Figure 1 and the gangetic koi (Anabas cobojius Hamilton, 1822), see Figure 2. They are called the anabas. Living areas of the anabas are South and Southeast Asia including India, Sri Lanka, Bangladesh, Myanmar, Malaysia, Thailand, Vietnam and Philippines. They can survive in brackish and freshwater environments [22]. Figure 1. Anabas testudineus Figure 2. Anabas cobojius The anabas is an aquaculture species with many prospects because they are likely to like high suspicion due to the respiratory organs on the gills. The aquaculture of anabas is developing strongly in the Mekong Delta. Therefore it is necessary to classify several species of Anabas genus in Vietnam and identify the names of these species. The complete mitochondrial genome of Anabas testudineus was sequenced with length as 16,603 bp including 13 protein coding genes, 2 ribosomal RNA coding genes, 22 transfer RNA coding genes and a region control [23]. The research of species identification using molecular biological methods in the world has applied in genetic checking and genetic modification [7]. For many species, some genes of mitochondrial genome (mitochondrial DNA), such as COI (cytochrome oxidase subunit I), NADI (nicotinamide dehydrogenase subunit I), COB (cytochrome b) and gene of nuclear genome (nuclear DNA) ITS-2 (internal transcribed spacer 2) which has been considered important molecular markers for species identification and classification [3-5, 7, 8]. How- ever, mitochondrial genes evolve faster nuclear genes. Therefore, mitochondrial genes are often used to analyze the type-generation relationship for low-level taxa such as familia, subfamilia, genus, species, subspecies. In which the COI gene evolved rapidly and was used to analyze the relationship between species of genus or subspecies [19]. In this research, the genetic relationships of the anabas species in Vietnam and neighboring countries were researched using 82 Genetic relationships of the anabas... molecular biology method based on COI marker gene to supplement reliable information in the identification and classification of the species of this genus. 1. Materials and methods 1.1. Sample collection and DNA extraction The anabas specimens were collected in Vietnam including Tri An reservoir, Dong Nai province (specimens A1); Can Gio district, Ho Chi Minh city (spec- imens A2); Vam Co Dong river, Long An province (specimens A3) and Dau Tieng lake, Tay Ninh province (specimens A4) by gill net. These specimens were processed and stored at -200C for total DNA extraction. Total DNA was extracted from fish tissue by PHUSA-IHHNV kit according to the procedure of Phu Sa Biochemical Company. Concentration and purity of total DNA were checked using UV-VIS molecular absorption spectrometer of Bio-Rad measur- ing two wavelengths 260 nm and 280 nm. 1.2. Amplify, purify and sequencing Forward primer (5’-AACCCAAAAGACATTGGCACC-3’) and reverse primer (5’-GGCCAAAGAATCAAAACAAGT-3’) used to amplify mitochondrial cy- tochrome oxidase subunit I(COI) gene were designed using Primer-BLAST tool of the National Center for Biotechnology Information (NCBI) and were synthesized at Oligo laboratory of Phu Sa Biochemical Company. The thermo- cycler conditions were slightly modified as follows: 1 initial cycle of 3 minute at 960C followed by 35 cycles of 960C for 15 seconds and 520C for 15 seconds, 600C for 4 seconds. This is followed by a final step of 40C for 5 minutes [10]. The PCR products were checked using 2 % agarose gel electrophoresis and were sequenced with both the forward and reverse primers using an automated sequencer ABI 3730XL of Applied Biosystems by Sangers method [18]. The bands of PCR product must be clear, the width of the bands is large and size about 650 bp as successful amplification reaction. Then, the sequences of COI gene were edited using GENtle version 1.9.4 software and Nucleotide BLAST tool of NCBI. 1.3. Phylogenetic analysis Phylogenetic analysis was conducted based on the multiple sequence alignment of COI gene of the anabas in Vietnam (A1, A2, A3, A4) and the anabas in India, Bangladesh, Myanmar, Malaysia, Thailand and Philippines collected from GenBank database (Table 2) using ClustalW algorithm [2, 9]. The genetic Truong The Quang 83 distances were computed using the Maximum Composite Likelihood method [20]. Phylogenetic tree was built using Neighbor - Joining Tree algorithm [17]. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches [1]. Evolutionary analyses were conducted in MEGA version 10.0.4 software [6]. 1.4. Genetic relationships analysis The groups were classified based on phylogenetic tree and genetic distance between groups (Dij). From there, the groups were identified with far, medium and near genetic distances. Single nucleotide polymorphism (SNP) sites were analyzed based on pairwise sequence alignment of COI gene of the anabas in same group or other group using DnaSP version 6.12.01 software. SNP ratio Kij (%) of the anabas i and j is computed by equation (1) [10]. Kij = 100Sij Nij Where Sij is the number of SNP sites; Nij is the total number of sites in pairwise sequence alignment of COI gene of the anabas species i and j. 2. Results and discussion 2.1 Total DNA extraction The checking results of total DNA samples are high concentrations from 81.0 to 191.3 g/ml. Absorption rates at wavelengths of 260 nm and 280 nm (OD260 / OD280) are within the limits of 1.8 to 2.0, indicating that good pure DNA samples are not contaminated with proteins and organic substances (Table 1). Table 1. Results of checking the concentration and purity of total DNA 2.2. Sequencing of COI genes The COI genes were successfully amplified by PCR. The PCR products were checked using 2 % agarose gel electrophoresis obtained the clear and wide DNA 84 Genetic relationships of the anabas... bands of about 650 bp (Figure 3). The COI genes of A1, A2, A3 and A4 speci- mens were sequenced and were submitted in GenBank of NCBI having respec- tively accession number MK351908, MK368519, MK368520 and MK368521 for world-wide publish [11, 12, 13, 14]. Figure 3. Results of PCR products electrophoresis 2.3. Genetic relationships analysis The genetic distance matrix (Figure 4) and the phylogenetic tree (Figure 5) were established using multiple sequence alignment of COI gene of the anabas (Table 2). Table 2. List of the anabas for multiple sequence alignment of COI gene In terms of genetic distance between groups Dij < 0.020, the researched anabas are classified into four groups: Group 1: Anabas testudineus India (JX260824), Anabas testudineus Myan- mar (LC190180), Anabas cobojius Bangladesh (KY124377), Anabas cobojius India hap.AnoH1 (KC774635), Anabas cobojius India hap.AnoH2 (KC774636); Group 2: Anabas testudineus Indonesia (KU692243), Anabas testudineus Philip- pines (HQ682664); Group 3: Anabas testudineus Thailand (JQ661369); Group 4: Anabas testudineus Vietnam (A1, A2, A3, A4), Anabas tes- tudineus Malaysia (JF781185). Truong The Quang 85 Figure 4. Distance and standard error matrix of the anabas based on COI marker gene The genetic distances between the anabas in Vietnam (A1, A2, A3, A4) are very small from 0.0046 to 0.0156 (Figure 4) and they are all in group 4 with Anabas testudineus Malaysia (Figure 5) so they are the same Anabas testudineus in accordance with the research results of Trinh et al., 2013 and Rainboth, 1996. Figure 5. Phylogenetic tree of the anabas based on COI gene The genetic distance and corresponding standard errors between the groups based on COI gene are shown in figure 6. Based on the genetic distance matrix of the groups (Figure 6), the groups with far genetic distance include group 2 and group 4 (D24 = 0.1055), group 1 and group 4 (D14 = 0.1041), group 1 and group 3 (D13 = 0.0910). The groups with medium genetic distance include group 2 and group 3 (D23 = 0.0849), 86 Genetic relationships of the anabas... Figure 6. Distance and standard error matrix of the groups based on COI gene group 1 and group 2 (D12 = 0.0772). The groups with near genetic distance include group 3 and group 4 (D34 = 0.0285). Table 3. SNP ratio Kij (%) using pairwise sequence alignment of COI gene Result of genetic diversity analysis using pairwise sequence alignment of COI gene of the anabas was shown in table 3. The SNP ratio of Anabas testudineus India (group 1) and Anabas cobojius India hap. AnoH1 (group 1) is K11 = 3/664 = 0.45 %; The SNP ratio of Anabas testudineus Myanmar (group 1) and Anabas cobojius India hap. AnoH1 (group 1) is K21 = 2/651 = 0.31 %; The SNP ratio of Anabas testudineus India (group 1) and Anabas testudineus Indonesia (group 2) is K12 = 54/652 = 8.28 %; The SNP ratio of Anabas testudineus Myanmar (group 1) and Anabas testudineus Indonesia (group 2)K22 = 45/655 = 6.87 %. The SNP ratioK12 = 8.28 % andK22 = 6.87 % is much larger than K11 = 0.45 % and K21 = 0.31 % so Anabas testudineus India, Anabas testudineus Myanmar should be re-identified Anabas cobojius India, Anabas cobojius Myanmar respectively belong to group 1 (Gangetic koi group). Conclusions The genetic distances between the anabas species in Vietnam (A1, A2, A3, A4) are very small from 0.0046 to 0.0156 and they are all in group 4 with Anabas testudineus in Malaysia so they are the same Anabas testudineus. The researched anabas in Vietnam and some countries in South and Southeast Asia were divided into four groups using COI marker gene. The groups with far genetic distance include group 2 and group 4 (D24 = 0.1055), group 1 and group 4 (D14 = 0.1041), group 1 and group 3 (D13 = 0.0910). The groups with Truong The Quang 87 medium genetic distance include group 2 and group 3 (D23 = 0.0849), group 1 and group 2 (D12 = 0.0772). 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