* Corresponding: [email protected]
Received: 09–8–2021; Reviewed: 19–10–2021; Accepted: 25–10–2021
GENETIC DIVERSITY OF GIANT MOTTLED EEL (Anguilla marmorata Quoy & Gaimard, 1824) BY RAPD
IN THUA THIEN HUE PROVINCE, VIETNAM
Kieu Thi Huyen1*, Ha Thi Hue1, Nguyen Quang Linh1, 2
1 Faculty of Fisheries, University of Agriculture and Forestry, Hue University, 102 Phung Hung street, Thua Thien Hue, Vietnam
2 Department of Nutritional Diseases and Systems for Livestock and Aquaculture, Institute of Biotechnology, Hue University, Phu Thuong, Phu Vang, Thua Thien Hue, Vietnam
Abstract. Anguilla marmorata is a high economic value species with and increasingly interested by organizations and scientists. So far, many of the eel's biological characteristics remain mysterious, and they are often classified according to morphological features such as pigmentation patches, number of vertebrae, ... It is even difficult to distinguish one individual from another in some species, especially in the larval stage. In this study, the Random amplification of polymorphic DNA (RAPD) molecular marker was used to evaluate the genetic diversity of 48 eel samples collected in Thua Thien Hue province. Results showed that the genetic diversity of individuals in the eel population studied is quite high. With 8 random primers via PCR, 77 DNA tapes with 76 polymorphic tapes were obtained, the tape size ranged from 170-2,500 bp, in which primer S10 showed the highest diversity with an average Ho value of 0.563, followed by primer S8 (Ho = 0.558). The lowest diversity was in the OPD5 primer (Ho = 0.300). The OPG17 primer is the primer that produces the most polymorphic tapes (13/13 tapes) and the S3 primer for the least amplified tapes polymorphism (9/10 DNA tapes). The diversity coefficient in each random primer ranged from about 0.300 to 0.563, with an average of 0.433. The genetic variation in the Eel population is random. Genetic variation can be attributed mainly to different eel breeding conditions and origins. Genetic similarity coefficients among the Eels varied from 0.660 to 0.910 and were divided into two main groups in genetic similarity coefficient 0.660.
Keywords: Anguilla marmorata, RAPD, genetic diversity, Thua Thien Hue.
1 Introduction
The eel of the genus Anguilla Schrank (1798) was identified, including 16 species and 3 subspecies by Ege, (1939) [4], Watanabe et al., (2003; 2005) [26, 27] based on morphological and molecular indicators. Ambient factors, such as salinity, temperature, altitude, river size, tolerance, and ecological competitiveness, such as ecological competition between countries affect diversity, distribution, and habitats among the Anguilla eels [2]. The combination of the hydrological regime disturbances, climate change, the effects of environmental degradation, and overfishing in
continental waters have negatively affected the size of the Anguilla eels population [15].
Examining the change and identification of the Anguilla eel's genetic and biodiversity characteristics concerning geographic and climatic factors [1] essential to formulating strategies for resource recovery and development.
In the Anguilla genus, A. marmorata is the second largest species [4] with the widest geographic distribution on two different oceans (tropical and subtropical central Pacific and Indian oceans) [19]. In Vietnam, A. marmorata is distributed in central Vietnam [18] and has high commercial value [9]. However, the eel resources here are being threatened by fishing pressure for commercial demand [7] and seed in farms [14]; impacts of environmental pollution [12] and the decline of natural populations due to migration [3]. Although there have been preliminary studies on eels in Vietnam such as: Mai Dinh Yen et al., (1994) [28], Nguyen Huu Phung (2001) [20], Vo Van Phu et al., (2008) [21, 22]; Kieu Thi Huyen et al., (2012, 2014, 2015) [6 – 8], Nguyen Quang Linh et al., (2010) [14], Nguyen et al., (2018) [18], ... but in-depth studies on the biological characteristics and genetic diversity of the Marbled eel based on molecular markers is still limited.
Therefore, to provide more biological data on population genetic diversity and species adaptation for conservation, protection, and development of eel resources in Thua Thien Hue province. We used RADP indicators to evaluate eel populations' genetic diversity in different areas in Thua Thien Hue province.
2 Materials and methods
2.1 Materials
Eel (48 samples) collected from different water bodies in Thua Thien Hue province were placed in a sterile polymer sample bag and stored in an icebox then transferred to the laboratory before carrying out further studies (Table 1).
Table 1. Samples of Eel used in the study
No. Sample locations
No. of
sample Code of sample No. of PCR
wells W (g) TL (mm)
1 Thao Long
dam (TL) 10 TL01, TL02, TL03, TL04, TL05,
TL10, TL13, TL21, TL25, TL28 1 - 10
52.3 - 3200.0 683.8 ± 937.3
303.0 - 1080.0 556.5 ± 221.7
2 Truoi dam
(TR) 05 TR01, TR02, TR03, TR04, TR05 11 - 15
24.5 - 196.5 71.7 ± 72.6
215.0 - 435.0 295.2 ± 93.7
3 Nam Dong
distrisc (ND) 09 ND1, ND2, ND3, ND4, ND5,
ND8, ND14, ND15, ND16 16 - 24
13.7 - 511.1 97.6 ± 145.0
183.0 - 620.0 345.9 ± 117.3
No. Sample locations
No. of
sample Code of sample No. of PCR
wells W (g) TL (mm)
4 Phong Dien distrisc (PD) 14
PD2, PD3, PD4, PD5, PD6, PD7, PD8, PD9, PD10, PD12, PD13, PD15, PD19, PD20
25 - 38
15.7 - 493.9 212.1 ± 150.6
210.0 - 610.0 435.0 ± 111.7
5 Phu Loc
distrisc (PL) 03 PL1, PL2, PL7 39 - 41
37.5 – 89.0 59.3 ± 26.6
291.0 – 362.0 320,7 ± 36.9
6 Bu Lu river
(BL) 05 BL8, BL15, BL18, BL20, BL22 42 - 46
7.0 – 46.0 20.2 ± 15.1
169.0 – 307.0 225.6 ± 36.9
7 Lang Co
lagoon (LC) 02 LC1, LC2 47 - 48
11.5 – 18.5 15.0 ± 4.9
189.0 – 214.0 201.5 ± 17.7
Total/Average 48 48 48
7.0 – 3200.0 245.2 ± 484.6
169.0 – 1080.0 396.1 ± 168,47
2.2 Methodology Total DNA extraction
Total DNA of Anguilla marmorata eel genome was extracted and purified according to Sambrook's (1989) [24] description and modified for eel [10]. Eel meat (0.5g) was chopped into pieces and put into a tube (2 ml) of the type that was autoclaved. The sample was then finely ground with a pestle and re-suspended in 1.5 ml of extraction buffer (10 mM Tris.HCL, 10 mM EDTA, 10 mM NaCl, 1.0 % SDS, pH 8.0) and 1 ml of protein K (10 mg / ml). The suspension is then incubated at 65 ° C for 3 h and centrifuged 5000 g at 4 ° C for 5 minutes. The supernatant was transferred to a new tube and purified with an equivalent phenol volume (repeated 3 times). The supernatant is then further purified with an equivalent volume of the phenol mixture: chloroform: isoamyl alcohol (25 : 24 : 1). The resulting solution will be DNA precipitate with 0.5 NH4Ac and 2 ethanol in - 20 ° C bodies for 60 minutes, then centrifuge 6,000 rpm for 10 minutes to obtain a precipitate, then left to dry at room temperature for 20 minutes, the DNA particles were re-dissolved in TE buffer (10 mM Tris and 1 mM EDTA, pH 8.0). Total DNA concentration and purity were determined by spectroscopy on a NanoDrop ND-1000 (Thermo, USA) and 1% agarose gel electrophoresis. The DNA solution was stored at - 20 ° C for use in PCR - RAPD reactions [10], [24].
RAPD technical
Amplified randomization segment polymorphism (RAPD) was used to study the eel's genetic diversity [23]. Eel samples were selected based on their non-overlapping origins; the total samples
analyzed were 48 samples presented in Table 1. The number of primers used in the study varied was 8 (Table 2).
Table 2. Sequence of primers used in PCR-RAPD
No. Primer Sequence 5'-3' No. Primer Sequence 5'-3'
1 S1 GTTTCGCTCC 5 S9 TGGGGGACTC
2 S3 CATCCCCCTG 6 S10 CTGCTGGGA
3 S5 TGCGCCCTTC 7 OPD5 TGAGCGGACA
4 S8 GTCCAACCGG 8 OPG17 ACGACCGACA
PCR-RAPD reaction was performed according to the method described by Qiu and Li (1999) [23] with random primers (Operon Technologies, CA) (Table 2) on thermocycler heaters (SimpliAmp, ThermoFisher Scientific, USA). The reaction mixture consisted of 0.4 µM primers, 2.55 mM MgCl2, 2.0 μl 5 buffers (50 mM Tris.HCl, 250 mM KCl, 0.005% gelatin), dNTPs (0.1 mM each); 2 U Taq DNA polymerase (Bioline), 30 ng of template DNA with a total reaction volume of 10 µl with a thermal cycle including: denatured 94 ° C / 5 minutes; 40 cycles: 94 ° C / 2 minutes, 35 ° C / 2 minutes, 72 ° C / 2 minutes; and finally 72 ° C / 10 minutes. The PCR-RAPD product was electrophoresis on 2% agarose gel and stained with ethidium bromide. Electrophoresis images were captured by the Gel Documentation system and analyzed by Quantity One program (Bio- rad, USA) [23].
Data analysis
The PCR - RAPD product electrophoresis spectra of samples with primers were analyzed according to the principle of presence or absence of tapes, number "1" if tape appears and the number "0" if no tape appears. Genetic diversity coefficients are calculated using the following equations [25]. Genetic diversity in each study area (Shannon's index): Ho = -∑pilog2pi (in which pi is the frequency of occurrence of the PCR - RAPD product in the population) using PopGen 3.2 software. Constructing a pedigree diagram according to the UPGMA algorithm of 48 studied Eel samples was done by NTSYS 2.1 program (Exeter Software, USA) based on Jaccard genetic similarity coefficient (1908) [5].
3 Results and discussion
3.1 Results of total DNA extraction
The muscle of 48 Eel samples collected from different locations was used to extract and purify the genome's total DNA. The electrophoresis test results on 1% agarose gel shown in figure 1
show that the total DNA obtained is representative of the research samples for a single, clean, unbroken, clear tape. Total DNA quality is guaranteed to be the raw material for further experiments.
Fig. 1. Mitochondrial cpDNA electrophoresis on 1% agarose gel
M1, M2: the weight of standard DNA scale (Lambda DNA / HindIII Markers (564 - 23130 bp), Promega);
wells from 1 to 48 are images of cpDNA 48 samples.
Genetic diversity analysis based on PCR - RAPD directive.
We used 20 randomized primers (RAPD) to screen 3 Eel samples randomly selected in 48 study samples to look for RAPD primers with high polymorphic tape rate. As a result, we selected 8 randomized RAPD primers to analyze the genetic relationships of 48 eel samples (Table 1).
Analysis of genetic relationships of 48 Eel samples collected in different places in Thua Thien Hue province with 8 RAPD indicators showed that all primers showed polymorphism. A total of 1,671 DNA tapes were recorded, averaging 34,813 tapes per sample studied. In which, the Eel with the symbol BL18 was the one with the most amplified number of tapes with 49 DNA tapes (accounting for 2,930% of the total number of tape forming), followed by individual BL22 (45 DNA tapes) and TL03 (44 DNA tapes). Meanwhile, individuals with the least amplification tapes were TL25 (22 DNA tapes), LC2 (25 DNA tapes), and LC1 (24 DNA tapes). Thus, the number of amplified tapes in different Eels for 8 study primers is different (the lowest is 22 DNA tapes, accounting for 1.317%, and the most are 49 tapes, accounting for 2,930%. The average number of DNA tapes/sample in the primers ranged from 2.583% to 6.979% (Table 3).
Table 3. Number of DNA amplification tape of eels in each primer
No. Sample
RAPD primers
Total
S1 S3 S5 S8 S9 S10 OPD5 POG17
1 TL01 4 8 2 7 6 7 3 5 42
2 TL02 1 8 1 4 5 7 3 7 36
3 TL03 3 7 4 6 5 6 3 10 44
4 TL04 4 8 3 4 5 4 3 5 36
5 TL05 3 8 3 0 5 5 4 6 34
6 TL10 5 9 2 5 4 4 5 8 42
7 TL13 3 6 4 5 5 6 4 7 40
8 TL21 1 7 3 5 5 6 4 6 37
9 TL25 2 6 2 1 4 3 2 2 22
10 TL28 2 5 2 1 4 1 6 4 25
11 TR01 0 2 2 5 6 3 3 9 30
12 TR02 4 4 4 2 0 2 3 9 28
13 TR03 3 5 4 2 5 4 4 7 34
14 TR04 5 4 4 2 4 4 3 10 36
15 TR05 2 6 4 3 5 4 4 6 34
16 ND1 3 5 4 2 2 4 3 8 31
17 ND2 6 4 3 2 4 4 3 9 35
18 ND3 3 4 3 2 4 6 3 8 33
19 ND4 3 3 3 7 6 6 4 9 41
20 ND5 1 6 4 3 5 5 4 9 37
21 ND8 2 4 2 7 1 4 4 4 28
22 ND14 3 5 1 7 3 4 4 6 33
23 ND15 2 2 1 8 4 4 3 6 30
24 ND16 3 5 2 8 3 4 5 5 35
25 PD2 2 7 2 8 4 5 4 8 40
26 PD3 2 7 4 6 4 5 4 6 38
27 PD4 2 7 3 1 3 7 5 9 37
28 PD5 2 6 3 1 3 6 4 10 35
No. Sample
RAPD primers
Total
S1 S3 S5 S8 S9 S10 OPD5 POG17
29 PD6 4 5 2 7 3 2 4 5 32
30 PD7 3 5 3 5 3 3 4 8 34
31 PD8 2 5 3 8 2 6 4 9 39
32 PD9 1 5 4 8 3 5 3 10 39
33 PD10 1 7 3 8 3 5 2 9 38
34 PD12 0 5 2 8 1 0 3 5 24
35 PD13 3 7 4 8 4 4 4 7 41
36 PD15 1 5 2 8 3 0 4 5 28
37 PD19 0 5 2 8 3 1 5 5 29
38 PD20 1 8 0 7 3 2 4 6 31
39 PL1 8 7 2 3 3 6 4 6 39
40 PL2 5 8 2 8 3 4 3 7 40
41 PL7 6 8 2 6 4 4 4 9 43
42 BL8 4 5 1 6 4 3 3 8 34
43 BL15 6 4 1 8 4 0 3 8 34
44 BL18 8 8 2 9 4 7 5 6 49
45 BL20 1 6 2 6 4 4 3 6 32
46 BL22 6 5 4 7 5 5 5 8 45
47 LC1 0 6 2 3 3 4 0 6 24
48 LC2 0 7 2 1 4 1 4 4 23
Total DNA tapes
obtained 136 279 124 246 180 196 175 335 1,671.0
Number of DNA
tapes/sample 2.833 5.813 2.583 5.125 3.750 4.083 3.646 6.979 34.813 The results of the study in Table 4 showed that there were 77 DNA tapes amplified from 8 random primers, of which there are 76 polymorphic DNA tapes (the number of polymorphic tapes per primer is an average of 9.5), the tape size ranges from 170 - 2,500 bp. In which, the primers S3, S8, OPG17 had the most amplified number of individuals (100%) with 10, 9, 13 DNA tapes, respectively formed (Figure 3), followed by S5, S9, and OPD5 (97.92%) with the number of DNA tapes creating 8, 8, and 9 DNA tapes, respectively (Figure 2). The OPG17 primer is the primer that produces the most polymorphic tapes (13/13 tapes), and the S3 primer for the least
amplified tapes polymorphism (9/10 DNA tapes), the ratio of polymorphic tape per primer in our study is quite high, ranging from 90 to 100% (Table 4).
Table 4. Number of individuals and number of amplification tapes of each primer No. Primers % individual
amplification
Total the most amplified tape/primer
Number of polymorphic
tapes
DNA tape size range
(bp)
Polymorphism rate (%)
1 S1 89.58 11 11 170-1700 100
2 S3 100 10 9 250-1700 90
3 S5 97.92 8 8 250-2200 100
4 S8 100 9 9 380-1600 100
5 S9 97.92 9 9 300-1500 100
6 S10 93.75 8 8 250-1250 100
7 OPD5 97.92 9 9 300-2000 100
8 OPG17 100 13 13 200-2500 100
Total 97.14 77 76 98.75
According to Nei et al. (1978), the more the number of DNA tapes is amplified, the greater the ability to distinguish patterns on the genealogical tree, in which the minimum number of polymorphic tapes is 50 for the correct genealogy tree to be built [16]. With 8 primers used, we have obtained 77 polymorphic DNA tapes from 48 different Eel samples to research genetic diversity and build a family tree. Therefore, the data obtained after analyzing 8 RAPD primers is sufficient for the study.
Fig. 2. Picture of PCR - RAPD electrophoresis with S5 primer
M: Weight of standard DNA ladder (Marker DNA 100 bp ladder, Bioline); 1 - 48: PCR - RAPD products of individual samples eels from 1 - 48.
Fig. 3. Picture of PCR - RAPD electrophoresis with OPG17 primer
M: Weight of standard DNA ladder (Marker DNA 100 bp ladder, Bioline); 1 - 48: PCR - RAPD products of individual samples eels from 1 - 48.
Table 5. Genetic diversity of Eel populations in Thua Thien Hue province
Primers na* ne* h* Ho*
S1 2.000 1.302 0.203 0.330
S3 1.900 1.592 0.339 0.502
S5 2.000 1.329 0.213 0.346
S8 2.000 1.674 0.381 0.558
S9 2.000 1.378 0.233 0.363
S10 2.000 1.683 0.384 0.563
OPD5 2.000 1.257 0.178 0.300
OPG17 2.000 1.544 0.325 0.495
Average 1.987 1.470 0.282 0.433
± SE 0.114 0.339 0.166 0.217
* na = Number of alleles is observed [16]; * ne = Number of effective alleles [11]; * h = Nei's genetic diversity (1973) [17]; * Ho = Shannon Genetic Diversity Index [13].
Analyzing the diversity of individuals in the population (Ho) shows that there is great diversity in the study sample (Table 5). Of the 8 randomized primers used in the study, the S10 showed the highest diversity with the average Ho value of 0.563, followed by the S8 primer (Ho = 0,558). The lowest diversity was in the OPD5 primer (Ho = 0,300). Diversity coefficients in each random primer ranged from about 0.300 to 0.563, with an average of 0.433 (Table 5). Genetic similarity coefficients between individual eel ranged from 0.66 - 0.91. Based on genetic similarity coefficients studied eel population is divided into 2 main groups (figure 4).
Group I includes 43 individuals, in this group with the genetic similarity coefficient of 0.685 individuals divided into two subgroups: Group Ia (including 12 individuals) and Ib (including 31 individuals). The remaining individuals in this group have similarity coefficients ranging from 0.700 to 0.910. In which, the eels had symbols ND8, ND15, PD12, PD15, PD19, PD20 and individuals with symbols TR02, TR04, ND1, ND3, ND4 ; although they are far from each other in the genetically generated plants, they all share the same genetic similarity coefficient when divided into two groups (0.807). Similarly, the eels with symbols BL8, BL20, ND8, ND15, PD2, PD3, PD6, ND2, ND3, TR03, TR05 had the same genetic similarity coefficient (0.843) and individual eel with the symbols TL10, BL7, TR04, ND1 (0.853); ND14, ND16, PD2, PD3 (0.867); PD4, PD5, PD8, PD9 (0.895). The individual with the highest similarity coefficients are PD12 and PD19 (0.910), and the individual with the lowest similarity coefficient is BL22 (0.697). Group II consists of 5 individuals. In this group,
the eels were also divided into two subgroups IIa (including 1 individual) and subgroup IIb (including 4 individuals) at the genetic similarity coefficient of 0.697.
The analysis of the DNA genealogy diagram shows that there is a massive diversity between individuals in the same population. This phenomenon can be due to different reproductive conditions and origins of eels, leading to differences in genetic characteristics between individuals in the same species.
Fig. 4. UPGMA algorithm tree of eel population in Thua Thien Hue, Vietnam
4 Conclusion
Eight randomized amplification primers were used in this diverse study for 48 Eel individuals that obtained 77 DNA tapes. The total number of polymorphic tapes is 76 (the number of polymorphic tapes on the average primer is 9.5), the tape size ranges from 170 - 2,500 bp.
Analysis of the diversity of individuals in the (Ho) population shows a large diversity of the sample. Of the 8 randomized primers used in the study, the S10 showed the highest diversity with the average Ho value of 0.563, followed by the S8 primer (Ho = 0,558). The lowest diversity was found in OPD5 primers (Ho = 0,300). The diversity coefficient in each random primer ranged from about 0.300 to 0.563, with an average of 0.433. The OPG17 primer is the primer that produces
the most polymorphic tapes (13/13 tapes) and the S3 primer for the least amplified tapes polymorphism (9/10 DNA tapes). The diversity coefficient in each random primer ranged from about 0.300 to 0.563, with an average of 0.433. The genetic variation in the Eel population is random. Genetic variation can be attributed mainly to different eel breeding conditions and origin. Genetic similarity coefficients between individual Eels ranged from 0.66-0.910 and divided into two main groups in genetic similarity coefficient 0.660.
Acknowledgments
To complete this study, the authors would like to sincerely thank the students at the Faculty of Fishery, Hue university of Agriculture and Forestry and fishermen who assisted with the collection of specimens; MSc Dang Thanh Long and Biotechnology Institute - Hue University assisted in sample analysis. Sincere thanks to the Hue University Project (Code: DHH 2019-113) for funding part of the sample funding for research.
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