Aquaculture and Fisheries xxx (xxxx) xxx

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Aquaculture and Fisheries

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Assessment of genetic diversity, detection of strain-specific single

nucleotide polymorphisms and identification of the Bangladesh and
Vietnam strain of Channa striata by PCR-RFLP analysis of the mitochondrial
COI gene fragment
Md Samsul Alam *, Foyjunnesa Projna, Mst. Sadia Zafrin, Rituparna Das,
Mohd Golam Quader Khan
Department of Fisheries Biology and Genetics, Bangladesh Agricultural University, Mymensingh, 2202, Bangladesh

A R T I C L E I N F O A B S T R A C T

Keywords: Striped snakehead (Channa striata) has enormous fisheries and aquaculture importance due to its high market
COI demand and food value. Recently, Vietnam strain of the species has been introduced to Bangladesh for aqua­
Channa striata culture. Therefore, it is essential to develop a reliable molecular technique that can distinguish the Bangladesh
snakehead
strain from the Vietnam ones as biological invasion by feral exotic fish to the wild may cause enduring damage to
Genetic diversity
SNP
ecosystem and species diversity. Fin clips from snakehead samples were collected from six locations and total
PCR-RFLP DNA was extracted. The cytochrome oxidase c 1 (COI) gene fragment was amplified by PCR using the Fish F1/R1
primer set and sequenced for analysis of intra- and inter-strain genetic diversity and detection of single nucle­
otide polymorphism to distinguish the Vietnam strain from Bangladesh ones. A total of 15 haplotypes were
identified with an overall haplotype diversity of 0.888. The median-joining network created based on COI gene
sequences clearly differentiated the Bangladesh strain from the strain of Vietnamese origin by a total of 18 single
nucleotide polymorphisms. An In silico restriction analysis revealed AluI was the most suitable restriction enzyme
that produced diagnostic restriction profiles of the two strains of C. striata. PCR-RFLP analysis by the restriction
enzyme AluI created distinguishing restriction profile similar to that in the In silico analysis. This work illustrates
a simple, reliable and inexpensive molecular tool for the identification of the Bangladesh and Vietnam strains of
C. striata by one-step PCR and restriction of the COI gene fragment.

1. Introduction snakeheads was 73,358 MT in 2017-2018, 97% of which came from the
open water sources (DoF, 2018). Snakeheads in Bangladesh include four
The striped snakehead Channa striata (Bloch, 1793) belongs to the major species- C. striata, C. marulius, C. gachua and C. punctata. However,
Family Channidae of the Order Perciformes. It is native to Bangladesh, C. striata is the most common and predominant species contributing
Cambodia, China, India, Indonesia, Malaysia, Myanmar, Nepal, maximum to the total production of snakeheads since C. marulius is
Pakistan, Sri Lanka, Thailand and Vietnam (Chaudhry, 2010). The fish endangered and C. punctata is a small sized fish (IUCN, 2015). The
lives in swamps, ponds and streams; attains a known maximum length of snakeheads in Bangladesh suffered severely during the late 1980s and
up to 75 cm with a common size of 30–40 cm (Talwar & Jhingran, early 1990s from epizootic ulcerative syndrome (EUS) resulting in a
1991). heavy decline in the availability of the species across the country (Barua,
C. striata is a popular and high priced fish and commercially cultured 1989, 1994; Lilley et al., 2002). The current production statistics may
in Thailand and Vietnam. In 2006, the production of capture fisheries have been the results of population expansion after the bottleneck.
and aquaculture of this species was 70, 802 MT and 21, 721 MT, Examining the spatial distribution of genetic diversity, within and
respectively (FAO, 2018). In Bangladesh, the total production of among populations, is important in the management and conservation of

* Corresponding author.
E-mail addresses: samsul.alam@bau.edu.bd (M.S. Alam), foyjunnesa.bau@gmail.com (F. Projna), sadiazafrinbsmrau@gmail.com (Mst.S. Zafrin),
rituparna50546@bau.edu.bd (R. Das), khanmgq@bau.edu.bd (M.G.Q. Khan).

https://doi.org/10.1016/j.aaf.2020.12.006
Received 4 July 2020; Received in revised form 3 December 2020; Accepted 12 December 2020
2468-550X/© 2021 Shanghai Ocean University. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article as: Md Samsul Alam, Aquaculture and Fisheries, https://doi.org/10.1016/j.aaf.2020.12.006
M.S. Alam et al. Aquaculture and Fisheries xxx (xxxx) xxx

a taxon (Avise et al., 1989; Lacy et al., 1987). Fragmentation and

isolation of habitat may cause significant decrease in effective popula­
tion size with consequences on the genetic structure of a species through
increasing the levels of inbreeding and genetic drift. This, in turn, results
in reduction in the amount of genetic diversity within the gene pool
(Cornuet et al., 1996; Frankham et al., 1996; Pavlova et al., 2017).
Reduced genetic diversity can have damaging effects on the ability of a
species to recover from demographic, environmental and genetic sto­
chasticity, and can contribute to reduced viability of endangered species
(Lande, 1998). Past evolutionary history and contemporary de­
mographic processes might be responsible for the patterns of population
genetic variation that we observe today. Understanding the underlying
mechanisms that produced the patterns is central in designing pragmatic
plans for conservation of vulnerable species. Information on genetic
diversity in the population can be obtained by sequence and haplotype
information from random samples collected from different populations.
C. striata has been introduced to Bangladesh from Vietnam in 2011
by a few private farms for aquaculture and its popularity as an aqua­
culture species is growing day by day (Alam and Pandit, per. com.).
Efforts for domestication and captive breeding of Bangladesh strain of
C. striata has also been going on since 2015 and progress has already
been achieved. Since Vietnam and Bangladesh strain of C. striata belong
to the same species (Chaudhry, 2010), we apprehend inadvertent or
intentional crossbreeding between the two strains in the near future
when the Bangladesh strain will also be included in commercial aqua­
culture once the captive breeding and large-scale fingerling production
technique is optimized. Moreover, biological invasion by feral farmed
fish to the wild may cause a serious damage to the ecosystems. The
exotic strain, when established in the introduced environment, can Fig. 1. Bangladesh map showing the sampling locations. NG: Naogaon; MG:
negatively affect the native populations through competition and hy­ Muktagacha; SK: Sutiakhali; MH: Mohangonj; BH: Bhairab.
bridization (Kitanishi et al., 2018). Therefore, it would be useful if
reliable phenotypic and molecular techniques are developed that can
easily identify the two strains. Morphometric and meristic analysis are
not always sufficient to differentiate strains of the same species. In the located in Bailor, Trishal, Mymensingh, Bangladesh. Another batch of
past, morphometric analysis did not succeed to differentiate Bangladesh twelve specimens of Vietnam origin was collected from Asian Scientific
and Vietnam strains of C. striata (Projna, 2018). Therefore, a suitable Fish Hatchery and Nursery, located in Dhola, Trishal, Mymensingh,
molecular technique would be useful in differentiating the two strains. Bangladesh to supplement and validate the PCR-RFLP analysis. Both the
The mtDNA cytochrome c oxidase 1 (COI) gene fragment is used as a farms imported one thousand fingerlings each directly from Hanoi,
species identifying marker. Nonetheless, we hypothesize that since there Vietnam in 2011 (Alam and Pandit, pers. com.) and conducted natural
is a long geographic distance between Bangladesh and Vietnam and and artificial spawning in the hatchery. The collected specimens (12 +
there exists hardly any chance of gene flow between the two strains, 12) were progeny of the imported parent fish.
variations might have occurred in the same gene sequence of the two
strains that can assist identify them. Polymerase Chain Reaction- 2.2. Isolation of DNA and PCR amplification of COI gene fragment
Restriction Fragment length Polymorphism (PCR-RFLP) analysis of the
mitochondrial cytochrome b gene and COI gene fragment has long been Fin clips were collected from all the specimens and stored in 95%
found to be an effective technique for rapid identification of fish species ethanol in 1.5 ml microfuge tube following the guidelines approved by the
(Akasaki et al., 2006; Pappalardo et al., 2018; Xu et al., 2016). In this animal welfare and ethics committee (AWEC) of Bangladesh Agricultural
article, we present genetic diversity assessed by surveying variation in University (Approval No. BAURES/ESRC/Ag/06). Total DNA was isolated
an approximately 650 bp region of the mitochondrial COI gene in from the fin clips following SDS-proteinase-K digestion, phenol:chloroform:
samples collected from six populations including one originating from isoamyl alcohol extraction, and alcohol precipitation method as described
Vietnam. We also present here the distinguishing single nucleotide by Islam and Alam (2004). Approximately 650bp DNA barcoding region of
substitutions and a PCR-RFLP technique that distinguish the two strains the mitochondrial COI gene was amplified using the fish barcoding uni­
of C. striata. versal primer set: Fish F1: 5′ -TCAACCAACCACAAAGACATTGGCAC-3′ ,
Fish R1: 5′ -TAGACTTCTGGGTGGCCAAAG-AATCA-3’ (Ward et al., 2005).
2. Materials and methods For sequencing purpose, PCR was conducted in a reaction volume of 50 μl
containing 5 μl of 10× buffer containing 1.5 mM MgCl2, 4 μl of dNTPs (2.5
2.1. Collection of experimental fish mM each), 5 μl (10 μM) of each primer, 1.5 unit of Taq DNA polymerase
(DreamTaq, Thermo Fisher Scientific) and 100 ng of DNA template. Ther­
For genetic diversity analysis, a total of seventy two specimens of mal cycler parameters were set to 94 ◦ C for 3 min initial denaturation step,
C. striata were collected of which sixty were from five populations followed by 35 cycles each consisting of 30 s at 94 ◦ C, 30 s at 47 ◦ C and 1
(12×5 = 60) in Bangladesh (Fig. 1) and twelve specimens originating min at 72 ◦ C followed by a single extension step of 10 min at 72 ◦ C. PCR
from Vietnam were collected from Agro3 Fish Hatchery Complex, products were purified using commercial kit (FAVO Green spun column,
Thermo Fisher Scientific). DNA sequencing was performed on an ABI 3500

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M.S. Alam et al. Aquaculture and Fisheries xxx (xxxx) xxx

Genetic Analyzer using Big Dye Terminator Cycle Sequencing method reaction mixture were incubated at 37 ◦ C for 3 h for complete restric­
(outsourced from Macrogen Inc., South Korea). tion. For generating the restriction profile, 2 μl restricted product of each
specimen was electrophoresed on 8% polyacrylamide gel (19:1
2.3. Statistical interpretation of the sequence data Polyacrylamide:bis-acrylamide). Electrophoresis was conducted at 50V
for 2:30 h and the gel was stained with ethidium bromide and visualized
The chromatograms of individual sequences of each specimen were by a UV-Transilluminator placed in a Gel documentation system.
inspected visually for reading errors (if any) using the software Bioedit
5.0.9 (Hall, 1999) and a consensus sequence was obtained for each DNA 3. Results
fragment of each specimen. Nucleotide sequences from all the specimens
were compared and aligned among themselves using the ClustalW 3.1. MtDNA diversity in C. striata populations
software implemented in MEGA 7.0 (Kumar et al., 2016). We used
Kimura-2-Parameter model (Kimura, 1980) for calculating the sequence A total of 62 sequences of the mitochondrial COI gene fragment from
divergence among the haplotypes. The standard genetic diversity C. striata specimens, 53 from five locations in Bangladesh and 9 from
indices, such as mean nucleotide composite, the number of transitions Vietnam, were aligned of which 53 sequences of sizes 647bp or larger
and transversions, polymorphic sites (Ps), number of segregating sites were used for further analysis. The 53 sequences were grouped into 15
(S), the number of haplotypes (H), haplotype diversity (hd), and haplotypes with an overall haplotype diversity of 0.8880 ± 0.0190.
nucleotide diversity (π: Pi) and the mean number of pairwise differences Among the 15 haplotypes, 11 haplotypes were found only in a single
(k) for each population were calculated using DnaSP 5.10 (Librado & population (private haplotypes). The numbers of segregating poly­
Rozas, 2009). We used an exact test (Raymond & Rousset, 1995) of a morphic sites, parsimony informative sites and singleton variable sites in
contingency table based on haplotype frequencies and pairwise com­ the data set were 39, 26 and 13, respectively. For all the sequences of
parisons of the FST using analysis of molecular variance (Excoffier et al., Bangladesh and Vietnam specimens, the average frequencies (%) of the
1992) implemented in Arlequin 3.5 (Excoffier & Lischer, 2010). A hi­ nucleotide thymine (T), cytosine (C), adenine (A), and guanine (G) were
erarchical molecular variance analysis (AMOVA) was performed in 29.44, 29.00, 24.11 and 17.45%, respectively, with the A+T content
Arlequin 3.5 to test the significance in overall population genetic dif­ higher than the G+C content (Table 1).
ferentiation and partition the variance within and among populations. A The haplotype diversity (Hd = 0.889 ± 0.084) of Muktagacha pop­
median-joining network of the haplotypes was drawn using NETWORK ulation was found to be the highest, followed by the Bhairab, Sutiakhali
Ver. 10.0.0.0 (Bandelt et al., 1999). and Mohangonj population, respectively, while, the nucleotide diversity
(Pi = 0.0053 ± 0.0017) of Sutiakhali population was found to be the
highest followed by the Muktagacha, Bhairab and Mohangonj popula­
2.4. In silico RFLP and PCR-RFLP analysis
tion. The lowest haplotype diversity (among those obtained) was found
in the Vietnam population (0.378 ± 0.181). However, no haplotype
An In silico analysis involving 115 restriction enzymes was performed
diversity (0.000 ± 0.000) and nucleotide diversity (0.000 ± 0.000) was
for the evaluation of a suitable restriction enzyme to differentiate the
observed in the Naogaon population. The number of polymorphic sites
Bangladesh and Vietnam strain of C. striata. The 15 COI haplotype se­
in the Sutiakhali population was the highest (S = 13) followed by the
quences of 647bp each, two from C. striata of Vietnam origin and 13
Bhairab (S = 7), Muktagacha (S = 6) and Mohangonj (S = 2) population,
from Bangladesh origin, were subjected to In silico restriction digestion
respectively. The number of polymorphic sites in Vietnam population
analysis using a web-based restriction site analysis tool (Restriction
was 1 (Table 1).
Comparator: molbiotools.comrestriction comparator.html) to predict
the product sizes in each of the haplotypes. For validation of the In silco
RFLP data to differentiate the Bangladesh and Vietnam strains of 3.2. Haplotype distribution
C. striata, we conducted a PCR-RFLP analysis involving the specimens of
Bangladesh origin and Vietnam origin. For PCR-RFLP analysis, PCR was The GenBank Accession numbers of the 15 haplotypes detected in the
conducted in a reaction volume of 25 μl following the same conditions as present study and their distribution among the six populations analyzed
used for generating sequencing template (described above). The crude are presented in Table 2. Out of the 15 haplotypes, two haplotypes (H1
PCR product was digested with AluI in a restriction digestion reaction and H2) were found to be specific for the Vietnam population, four (H3,
volume of 50 μl containing 5 μl 10× CutSmart buffer (New England H5, H6 and H8) were specific for the Bhairab population, three (H9, H10
Biolab), 23 μl of PCR product, 1 μl of AluI restriction enzyme (New and H11) were specific for the Sutiakhali, and two (H13 and H15) were
England Biolab) and 26 μl of deionized water. The tubes containing the specific for the Muktagacha population (Table 2). The highest six

Table 1
Summary statistics of molecular diversity among five Bangladesh and one Vietnam population of C. striata (N- number of sequences; Ps-polymorphic sites; #h-number
of haplotypes; Hd-haplotype diversity; pi-nucleotide diversity; k-average number of nucleotide differences, ts-number of transitions; tv-number of transversions).
Parameters Naogaon Mohangonj Bhairab Muktagacha Sutiakhali Vietnam1

N 8 8 9 9 10 9
#h 1 3 6 6 6 2
Hd 0 0.6790± 0.8330± 0.8890± 0.7780± 0.3780±
0.1220 0.1270 0.0840 0.1370 0.1810
Ps 0 2 7 6 13 1
Pi (π) 0 0.0013± 0.0032± 0.0036± 0.0053± 0.0003±
0.8214 0.0008 0.0006 0.0017 0.0006
K 0 0.8214 2.0560 2.3330 3.4444 0.2220
Ts 0 2 5 4 9 0
Tv 0 0 2 2 4 3
G+C 0.4606 0.4627 0.4626 0.4629 0.4640 0.4660
A+T 0.5394 0.5373 0.5373 0.5371 0.5360 0.5340

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M.S. Alam et al. Aquaculture and Fisheries xxx (xxxx) xxx

Table 2
mtDNA COI haplotype distribution in the samples of five Bangladesh and one Vietnam populations of C. striata.
Haplotype Accession No. Naogaon Mohangonj Bhairab Muktagacha Sutiakhali Vietnam1 Total

H1 MT269924 8 8
H2 MT269925 1 1
H3 MT269926 1 1
H4 MT269927 3 4 2 1 10
H5 MT269928 1 1
H6 MT269929 1 1
H7 MT269930 4 1 1 1 7
H8 MT269931 1 1
H9 MT269932 5 5
H10 MT269933 1 1
H11 MT269934 1 1
H12 MT269935 3 1 4
H13 MT269936 1 1
H14 MT269937 8 1 1 10
H15 MT269938 1 1
Total 15 8 8 9 9 10 9 53

haplotypes were found in the Bhairab population and the lowest - only 3.5. In silico RFLP and PCR-RFLP analysis
one haplotype (H14) was found in the Naogaon population. The single
haplotype of the Naogaon population was, however, not specific, rather The In silico digestion of COI sequences allowed the comparison of
it was also shared by the Mohangonj and Muktagacha populations. the relative restriction profiles between the Bangladesh and Vietnam
Haplotypes H4 and H7 were shared by four of the five Bangladesh strain of C. striata. We tested a total of 115 restriction enzymes including
populations except the Naogaon population. No haplotypes were found eight-base cutters, six-base cutters, five-base cutters and four-base cut­
to be shared among the Vietnam and Bangladeshi populations of ters. Many of the enzymes did not have restriction sites in any of the COI
C. striata. sequences from C. striata, some enzymes cut the Vietnam sequence but
The median joining network constructed involving the 15 haplotypes not the Bangladesh sequences and vice versa (data not shown). We
of 647bp COI sequences of 44 Bangladesh and nine Vietnam individuals found a few enzymes, which cut sequences of both origin and resulted in
of C. striata is shown in Fig. 2. The network comprised of two major sub- the same or different restriction profiles. The AluI (recognition site, AG/
networks corresponding to Bangladesh and Vietnam specimens of CT), was found to show the most distinguishing RFLP profiles to identify
C. striata, respectively. Haplotypes H1 and H2 belong to the Vietnam the Bangladesh and Vietnam strains. We therefore used AluI for PCR-
population, whereas haplotypes H3-H15 belong to the Bangladesh RFLP analysis of C. striata.
populations. The Vietnam haplotypes are separated from the Bangladesh There were two cuts at positions 231bp and 536bp in the COI bar­
haplotypes by 18 mutations which were specific for the country speci­ code sequence of the Bangladeshi strain of C. striata, resulting in three
mens. Haplotypes H9, H10 and H11 belonging to Sutiakhali population bands of sizes 305bp, 231bp and 111bp. On the other hand, there were
formed a subcluster among the Bangladeshi populations (Fig. 2). three cuts at positions 231, 386 and 536bp, resulting in four fragments of
sizes 231, 155, 150 and 111bp in the COI barcode sequence of the
3.3. Population structuring Vietnam strain. It is to be noted that all of the eight GenBank sequences
of C. striata of Vietnam origin (for accession Nos. please refer to the
The population genetic differentiation index (FST) values among the previous section) showed the same in silico restriction profile. The
population pairs were found to be high. Out of the 15 pairs of compar­ 305bp fragment is unique for the Bangladesh strain which is absent in
ison, 14 FST values were found to be significant. The FST values between the specimens of Vietnam origin. On the other hand, the 155bp and
the single Vietnam and five Bangladesh populations were very high, 150bp fragments are unique for the Vietnam specimens which are absent
ranging from 0.9040 to 0.9941 while the FST value between the in the specimens of Bangladesh origin (Fig. 4a, 4b, 4c). Thus, we found a
Mohangonj and Bhairab population was found to be the lowest and not one-step PCR with restriction endonuclease AluI digestion allowing the
significant (p > 0.05) (Table 3). The results of AMOVA attributed differentiation of the Bangladesh and Vietnam strains of C. striata.
82.57% of the variance to among-populations with an overall FST value
of 0.8257 (P = 0.0000) (Table 4). Among-population variance was, 4. Discussion
however, reduced to 31.17% (FST = 0.3339; p = 0.0000) when the
Vietnam specimens were excluded from the AMOVA (Table 4). Population genetic structure of a species may provide critical infor­
mation for developing conservation and management strategies for
natural fish populations. Though C. striata in Bangladesh is categorized
3.4. Diagnostic single nucleotide substitutions between Bangladesh and
as a taxon of least concern (IUCN, 2015), the species had suffered
Vietnam C. striata
severely in an outbreak of EUS between 1988 and 1994 (Barua, 1994;
Lilley et al., 2002). In the present study, we examined the genetic di­
We identified 18 diagnostic single nucleotide polymorphisms that
versity using mitochondrial COI gene sequence in an attempt to distin­
could differentiate the Bangladesh and Vietnam strains of C. striata. The
guish the indigenous (Bangladesh) and exotic (Vietnam) strains of
SNPs were consistent in all the 44 sequences of Bangladesh origin and
C. striata as well as to assess the genetic diversity of selected C. striata
nine sequences of Vietnam origin (Fig. 3). In order to further check the
populations. This is the first report on the genetic diversity analysis of
reproducibility of the results, we collected eight COI sequences of
C. striata in Bangladesh.
C. striata originated from different regions of Vietnam submitted by
different authors to the GenBank (Accession Nos. MK368522.1,
MK368523.1, MK368524.1, MK368525.1, KT001934.1, KT001935.1, 4.1. Mitochondrial DNA sequence variation and genetic diversity
KT001938.1, MH721193.1) and found that 14 out of the 18 SNPs were
still diagnostic between the Bangladesh and Vietnam strains (Results not In this study, we analyzed 647 bp fragment of the COI gene in five
shown). indigenous and one exotic (originated from Vietnam) populations of

4
M.S. Alam et al. Aquaculture and Fisheries xxx (xxxx) xxx

observed in 368bp sequences of Malaysian populations (Jamaluddin

et al., 2011). The G+C content obtained in the present study is similar to
that reported by Jamaluddin et al. (2011).
We observed higher levels of genetic variation (in terms of Hd and Pi
(Table 1) in the Muktagacha and Bhairab population. Among the six
populations analyzed, no genetic variation was observed in the Naogaon
population of C. striata where only one haplotype was detected.
The level of genetic diversity maintained in a species is attributable
to a number of factors, such as the population size, the mating pattern
(random or non-random), physical distribution of the species, habitat
fragmentation and isolation mechanism, mutation and migration. The
absence of genetic variation in the Naogaon population might be due to
reduction in population size through habitat fragmentation and over-
exploitation as it is a small natural depression not connected to any
other larger waterbodies. This type of population shows rapid increases
in the proportion of identical haplotypes (Weber, 2000) because some
haplotypes are lost through genetic drift and the subsequent generations
are descended from a few surviving female ancestors (Dadi et al., 2012),
as mtDNA is inherited maternally. Progressive overexploitation of such
population and increased fragmentation of the population would
accelerate reductions in genetic diversity by either loss or fixation of
alleles (Pavlova et al., 2017) through genetic drift. The Tapti river
population (Central India) of C. striata also showed zero haplotype and
nucleotide diversity in mitochondrial ATPase 8/6 sequences (Baisvar
et al., 2015) that conformed to genetic drift through bottleneck (Spiel­
man et al., 2004). In contrast, the higher genetic variation in the Muk­
tagacha and Bhairab population may be accounted for by a relatively
broader distribution range and connectivity with transboundary rivers
(i.e., the Brahmaputra River). Another concern behind the increased or
decreased genetic variability of the population of this species lies with
the nature of the parental care along with the sampling method and size.
C. striata displays a very strong parental care (Paray et al., 2013).
Sampling from a school of C. striata at a particular confined location
would mean a higher possibility of obtaining the siblings resulting in low
(or none) genetic variation, a phenomenon that might also explain the
absence of nucleotide and haplotype diversity in the Naogaon popula­
tion. It is to be noted that the samples of Naogaon population were
fingerlings whereas the samples of other populations of Bangladesh
origin were either juvenile or adult. Furthermore, the sample size is an
important criterion of analyzing the population genetics structure. The
Fig. 2. Median-joining networks of mtDNA COI haplotypes of C. striata of appropriate sizes of sample, however, depends on the nature and num­
Bangladesh and Vietnam origin. The network involved 15 haplotypes calculated ber of markers used for the analysis. For mitochondrial DNA genetic
from 647 bp sequences of 44 Bangladesh and nine Vietnam individuals. Each
variation analysis Mandal et al. (2012) used 4 to 9 specimens per pop­
circle represents one unique haplotype and bars across branches indicate
ulation of Chitala chitala while Garg and Mishra (2018) used only five
number of substitutions separating two haplotypes. The Median-joining
haplotype network has formed two sub-networks corresponding to the
specimens per population of Catla catla. Zhou et al. (2019) studied the
Bangladesh and Vietnam specimens. Haplotypes H1 and H2 belong to nine genetic diversity of the Northern snakehead Channa argus based on COI
Vietnam specimens and the rest 13 (H3-H15) belong to 44 Bangladesh speci­ gene sequences of nine populations and six of them had either 3 or 2
mens. The Vietnam haplotypes are separated from the Bangladesh haplotypes specimens each. Boonkusol and Tongbai (2016) analyzed genetic vari­
by a total of 18 single nucleotide polymorphisms illustrated by 18 bars. ation in the Mekong river basin populations of Channa striata using COI
gene sequences involving samples sizes of 8–10 per population. While,
C. striata. The number of segregating sites (S = 39) obtained in this study Baisvar et al. (2015) analyzed the genetic diversity in nine populations
is relatively lower compared to those observed in 396 bp COI gene of India using sample sizes ranging from 3 to 10. We started with 12
sequence (S = 66) of C. striata reported by Boonkusol and Tongbai specimens from each population but due to low sequencing quality, we
(2016) in Thailand, but higher than the segregating sites (S = 10) had to reject some of sequences and used sequences of 8–10 specimens
from each of the six populations.

Table 3
Pair-wise FST values (below diagonal) and their respective p values (above diagonal) of six C. striata populations.
Sample Vietnam1 Bhairab Muktagacha Mohangonj Naogaon Sutiakhali

Vietnam1 - 0.0000 0.0000 0.0000 0.0000 0.0000

Bhairab 0.9490 - 0.0180 0.5856NS 0.0000 0.0000
Muktagacha 0.9413 0.2436 - 0.0180 0.0360 0.0000
Mohangonj 0.9766 0.0313 0.2574 - 0.0000 0.0000
Naogaon 0.9941 0.5182 0.2795 0.7013 - 0.0000
Sutiakhali 0.9040 0.3719 0.2723 0.3882 0.3248 -

NS=Not significant (p > 0.05).

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M.S. Alam et al. Aquaculture and Fisheries xxx (xxxx) xxx

Table 4
Results of hierarchical analysis of molecular variance (AMOVA) within and among five Bangladesh and one Vietnam population of C. striata.
Sources of variation d.f. Sum of squares Variance components Percentage of variation Fixation Index (FST)

Among Population 5 167.5960 3.7104 Va 82.57 0.8257 (p = 0.0000)

Within populations 47 36.8190 0.7834 Vb 17.43
Total 52 204.4150 4.4938
When the Vietnam sample was excluded from the analysis
Among Population 4 19.9100 0.4618 Va 31.17 0.3339 (p = 0.0000)
Within populations 39 35.9310 0.9213 Vb 66.61
Total 43 55.8410 1.3831

Fig. 3. Polymorphic sites among 15 haplotypes calculated from COI sequences

of 44 Bangladesh and nine Vietnam individuals. Eighteen unique substitutions
b
(* marked) which are present in all the 44 Bangladesh and nine Vietnam se­
quences, thus are considered as diagnostic SNPs.

However, future research using samples from different schools of

such species with increased number from a wider range of locations and
distributions are recommended. Based on the present study, we presume
that establishing a sanctuary may prevent further loss of genetic varia­
tion of the population of Noroil Beel, Naogaon. Establishing a sanctuary
may prevent further loss of genetic variation of such a population. More
studies, however, are required to ascertain whether this is an “endan­ c
gered” habitat to other organisms as well.

4.2. Population differentiation

The population differentiation analysis revealed a strong population

structuring among the C. striata populations of Bangladesh. Fragmen­
tation and physical separation of the populations allows a species to
acquire different genetic structures due to random mutation and genetic
drift resulting in high genetic differentiation, especially in freshwater
species (Habib et al., 2010; Ward et al., 1994). The population pairwise
FST values obtained in the present study indicates a high genetic struc­ Fig. 4. PCR-RFLP profiles of the COI gene fragment of C. striata analyzed by
turing characteristic to non-migratory freshwater fishes (Domi­ polyacrylamide gel electrophoresis. DNA amplification product was digested by
nguez-Dominguez et al., 2008; Kamarudin & Esa, 2009; Michel et al., restriction enzyme AluI. Lane M, 100bp DNA ladder. (a) Lanes B1-B6 are
specimens of Bangladesh strain of C. striata; VN1 and VN2: The specimens of
2008). Baisvar et al. (2015) analyzing the mitochondrial ATPase8/6
Vietnam strain of C. striata of the first batch (Agro3 Fish Hatchery Complex). (b)
gene sequence obtained a very high overall FST value of 0.9387 (range:
Lanes B7-B16, specimens of Bangladesh strain; V1,V2: Specimens of Vietnam
0.0198–0.9805) among nine C. striata populations while Baisvar et al. strain of the second batch (Asian Scientific Fish Hatchery and Nursery). (c)
(2019) analyzing the control region of mtDNA obtained an overall fix­ Lanes V3-V12: Specimens of Vietnam strain of the second batch; VN3: Specimen
ation index of 0.56 (range: 0.273–0.813) among seven C. striata pop­ of Vietnam strain of the first batch; B17, B18: Specimens of Bangladesh strain
ulations in India. When migration occurs, adjoining populations may of C. striata.
effectively become panmictic, sharing or exchanging alleles as observed
in this study between the geographically closer Bhairab and Mohangonj structured. Therefore, in one AMOVA, we excluded the Vietnam popu­
populations showing relatively higher level of haplotype sharing and lation which indicated that a rather high (31.17%) proportion of the
small non-significant FST value (tbl2Tables 2 and 3tbl3). However, the total variance was attributed to among-population differences with a
sharing of haplotype H14 between the two isolated highly significant p-value (p= 0.000) for the fixation index which con­
populations-Naogaon (the Northern) and Mohangonj (East Central) with firms the population structure in this species in Bangladesh. Except the
a highly significant FST value (p = 0.0000) cannot be explained. Mohangonj-Bhairab population pair, the FST values were found to be
We wanted to know how the Bangladesh populations of C. striata are

6
M.S. Alam et al. Aquaculture and Fisheries xxx (xxxx) xxx

significant. The among-population variance obtained in the present the PCR product amplified by the universal FishF1/FishR1 primer set
study is very much consistent with the findings of Vrijenhoek (1998) without sequencing. As mentioned in the results section, we have tested
who reported among-population variance for migratory and GenBank sequences of eight C. striata originating from different regions
non-migratory fishes as 15% and 32.4%, respectively, as C. striata is a of Vietnam and found the same In Silico restriction profile.
non-migratory or local migrant species.
4.6. Validation of in silico analysis by PCR-RFLP analysis for strain
4.3. Network analysis differentiation

We constructed a median joining network to reveal the phylogenetic The ability of In Silico RFLP of the COI gene to identify the
relationships among the haplotypes of the studied six C. striata pop­ Bangladesh and Vietnam strains of C. striata was validated by real PCR-
ulations (Fig. 2). The clustering resulted in two distinct lineages from RFLP experiments. Using the enzyme AluI on crude PCR product, we
haplotype network, which suggests that the two lineages could have found the same restriction profile as was obtained through the In silico
evolved from genetically different ancestors. The haplotypes belonging analysis (Fig. 4).
to the Vietnam population formed a separate cluster completely There are two cuts at positions 231bp and 536bp in the COI barcode
different from the Bangladesh populations (Fig. 3). sequence of the Bangladeshi strain of C. striata resulting in three bands of
sizes 305bp, 231bp and 111bp. of which the 305bp fragment is internal.
4.4. Diagnostic single nucleotide polymorphisms (SNPs) Depending on the total size of the PCR fragment generated by the Fish
F1/R1 primer set the two terminal fragments may, however, change
The mtDNA genes have a very high sequence evolution rate, 10–20 their positions. On the other hand, there are three cuts at positions 231,
times that of comparable nuclear genes (Wallace et al., 1987). The rate 386 and 536 bp resulting in four fragments of sizes 231, 155, 150 and
of mutation accumulation, and thus genetic divergence among the 111bp in the COI barcode sequence of the Vietnam strain. Among them,
population, is expected to increase more rapidly when the effective fragments 155bp and 150bp are internal which are absent in the
population size is reduced and genetic drift is increased (Lanfear et al., Bangladesh strain. Since 155 and 150bp fragments are very close in size,
2014). This rapid divergence leads to the accumulation of mutations only one band may be visible at this position but the band would be
exclusively in one population, which may serve as diagnostic single relatively thicker compared to the other two bands (≥231bp and
nucleotide polymorphisms (SNPs) (Rianti et al., 2015). SNP represents ≥111bp) which indicates that there are two bands at this position
the most widespread type of sequence variation in the genome, which (Fig. 4). Therefore, we have detected diagnostic restriction fragments
has emerged as valuable genetic markers for revealing the evolutionary that can differentiate the two strains of C. striata. The 305bp band is
history of populations (Brumfield et al., 2003). It is clear that the diagnostic for the Bangladesh strain and the 155/150 band is diagnostics
occurrence of SNPs within a gene creates different variants or alleles of for the Vietnam strain. In order to reconfirm the diagnostic properties of
the gene, which tend to be inherited unchanged across generations. the AluI restriction pattern, we collected another batch of 12 fish of
One of the objectives of the present study was to detect nucleotide Vietnam origin from a different farm and found the same results. It is
mutations in the mtDNA COI gene that are diagnostics and could be used worth mentioning that based on the COI gene sequence, we have ob­
to differentiate the Vietnam strain of C. striata from the Bangladesh tained a single AluI restriction profile for all the specimens of Bangladesh
strain. Alignments of 647bp COI nucleotides revealed 18 polymorphic origin though we have observed significant differentiation among the
sites distinguishing the Vietnam strain from the Bangladesh strain of five Bangladeshi population. This finding indicates that the same AluI
C. striata. Due to a relatively high interspecific and low intraspecific restriction pattern may be obtained if fish from other sources in
variation, the COI gene sequence is efficiently used as a species identi­ Bangladesh are analyzed suggesting the PCR-RFLP is a reliable tech­
fication marker for a wide range of animals. The present study, however, nique for identification of the Bangladesh strain of C. striata. Baffoni
established that the intraspecific variation in the COI gene sequence can et al. (2013) developed a PCR-RFLP technique for efficient discrimina­
also be used as a strain identifying marker. Developing diagnostic SNP tion of the species and subspecies in order to differentiate the most
markers to distinguish the native and exotic strains of a species has widely distributed Bifidobacterium species and subspecies of the genus by
important practical implications. For example, Kitanishi et al. (2018) PCR-RFLP analysis of the hsp60 gene fragment.
developed a simple SNP genotyping method to assess invasions of In conclusion, the present study has revealed significant differenti­
non-native haplotypes in the pale chub, Opsariichthys platypus in Japan ation among the analyzed populations. Considering the fact that many
while Verspoor et al. (2012) suggested that mtDNA SNPs could be exotic species/strains introduced for aquaculture may escape into the
detected by restriction enzyme digestion and used as potential simpler wild and hybridize with the native counterparts there is an growing need
and more cost-effective markers for assignment of European salmon, for the development of rapid and reliable methods for detecting non-
Salmo salar to region and river of natal origin. native conspecifics. We have detected 18 diagnostic SNPs that has
created an opportunity to develop genotyping methods that could
4.5. Strain differentiation by In Silico and PCR-RFLP mapping accurately discriminate between native and non-native haplotypes of
C. striata. We have successfully distinguished C. striata of Vietnam origin
As a sequence-free molecular approach, web-based restriction map­ introduced to Bangladesh. We validated our claim by checking eight COI
ping tools have made it possible to rapidly identify species or strains sequences of C. striata of Vietnam origin available in GenBank which
(Ferrito et al., 2019; Ovalle et al., 2014; Wong et al., 2014). The species contained the same three AluI restriction sites at the same position (231,
or strains are discriminated through specific restriction profiles obtained 386, 536 bp) suggesting that the technique may be applicable for most
with appropriate restriction enzymes. As we have identified 18 single C. striata in Vietnam. We, therefore, conclude that we have developed a
nucleotide substitutions in a 647bp fragment of the COI gene between PCR-RFLP technique that can reliably identify the Bangladesh and
the Bangladesh and Vietnam strains of C. striata, we hypothesized that Vietnam strains of C. striata.
there would be a good number of strain-specific restriction sites in the
two sets of sequences. From among 115 restriction enzymes tested, we CRediT authorship contribution statement
have chosen AluI, which created clearly distinguishing restriction pro­
files of Bangladesh and Vietnam strain of C. striata. A restriction frag­ Md Samsul Alam: Conceptualization, Writing - review & editing,
ment of 305bp, which is present in all the 13 haplotypes of Bangladesh Supervision. Foyjunnesa Projna: Formal analysis, Sample collection,
origin is absent in the haplotypes of Vietnam origin. Hence, AluI can be Laboratory analysis, Data curation. Mst. Sadia Zafrin: Formal analysis,
used to distinguish the two strains of C. striata by restriction digestion of Sample collection, Laboratory analysis, Visualization. Rituparna Das:

7
M.S. Alam et al. Aquaculture and Fisheries xxx (xxxx) xxx

Formal analysis, Sample collection, Laboratory analysis, Visualization. Garg, R. K., & Mishra, V. (2018). Molecular insights into the genetic and haplotype
diversity among four populations of Catla catla from Madhya Pradesh revealed
Mohd Golam Quader Khan: Supervision, Validation, Writing - review
through mtDNA cyto b gene sequences. Journal of Genetic Engineering and
& editing. Biotechnology, 16, 169–174.
Habib, M., Lakra, W. S., Mohindra, V., Khare, P., Barman, A. S., Singh, A., Lal, K. K.,
Punia, P., & Khan, A. A. (2010). Evaluation of cytochrome b mtDNA sequences in
Declaration of competing interest genetic diversity studies of Channa marulius (Channidae: Perciformes). Molecular
Biology Reports, 38(2), 841–846.
None. Hall, T. (1999). BioEdit: A user-friendly biological sequence alignment editor and
analysis program for windows 95/98/NT. Nucleic Acids Symposium Series, 41, 95–98.
Islam, M. S., & Alam, M. S. (2004). Randomly Amplified Polymorphic DNA analysis of
Acknowledgements four different populations of the Indian major carp Labeo rohita (Hamilton). Journal
of Applied Ichthyology, 20, 407–412.
IUCN Bangladesh. (2015). Red list of Bangladesh volume 5: Freshwater fishes. Dhaka,
This work is supported by a grant of Bangladesh Agricultural Uni­ Bangladesh: IUCN, International Union for Conservation of Nature, Bangladesh
versity Research System (Grant No. 2017/53/BAU). We are very much Country Office. xvi+360p.
thankful to Agro3 Fish Hatchery Complex and Asian Scientific Fish Jamaluddin, J. A. F., Pau, T. M., & Siti-Azizah, M. N. (2011). Genetic structure of the
snakehead murrel, Channa striata (Channidae) based on the cytochrome c oxidase
Hatchery and Nursery for providing the Vietnam specimens. subunit I gene: Influence of historical and geomorphological factors, 2011 Genetics
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