Genetic diversity based on SSR analysis of

the cultured snakehead fish, Channa argus,

(Channidae) in China
S.-R. Zhu1,2, J.-L. Li1, N. Xie3, L.-M. Zhu3, Q. Wang2 and G.-H. Yue4

1
Key Laboratory of Freshwater Aquatic Genetic Resources Certified by
Ministry of Agriculture, Shanghai Ocean University, Shanghai, China
2
Department of Biology, East China Normal University, Shanghai, China
3
Institute of Fisheries, Hangzhou Academy of Agriculture Science, Hangzhou,
China
4
Molecular Population Genetics Group, Temasek Life Sciences Laboratory,
National University of Singapore, Singapore, Singapore

Corresponding author: J.-L. Li

E-mail: [email protected]

Genet. Mol. Res. 13 (3): 8046-8054 (2014)

Received December 11, 2012
Accepted May 13, 2013
Published February 13, 2014
DOI http://dx.doi.org/10.4238/2014.February.13.6

ABSTRACT. The snakehead fish Channa argus is an important food

fish in China. We identified six microsatellite loci for C. argus. These
six microsatellite loci and four other microsatellite markers were used to
analyze genetic diversity in four cultured populations of C. argus (SD, JX,
HN, and ZJ) and determine their relationships. A total of 154 alleles were
detected at the 10 microsatellite loci. The average expected and observed
heterozygosities varied from 0.70-0.84 and 0.69-0.83, respectively, and
polymorphism information content ranged between 0.66 and 0.82 in
the four populations, indicating high genetic diversity. Population JX
deviated from mutation-drift equilibrium and may have experienced a
recent bottleneck. Analysis of pairwise genetic differentiation revealed
FST values that ranged from 0.028 to 0.100, which indicates a moderate
level of genetic differentiation. The largest distances were observed

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 Genetic variations of cultured Channa argus 8047

between populations HN and SD, whereas the smallest distances were

obtained between populations HN and JX. Genetic clustering analysis
demonstrated that the ZJ and HN populations probably share the same
origin. This information about the genetic diversity within each of the
four populations, and their genetic relationships will be useful for future
genetic improvement of C. argus through selective breeding.

Key words: Channa argus; Population genetic differentiation;

Microsatellite;

INTRODUCTION

Channa argus, commonly called the snakehead fish, belongs to the family Channidae;
it is native to eastern Russia and China, as well as parts of North Korea (Courtenay and Wil-
liams, 2004). Former Soviet Republics of Uzbekistan, Kazakhstan and Turkmenistan have
experienced relatively recent introductions of snakehead fish (Amanov, 1974; Dukravets and
Machulin, 1978). In many areas of the world, the snakehead fish is a cultured freshwater fish
species, which grows very fast and has a high nutritious and economic value, especially in
China (Guo et al., 2004). However, the wild snakehead fish has declined sharply because of
overfishing and improper management (Li and Yang, 1998). In this regard, the snakehead fish
breeding industry has become very important.
Identifying population structure is one of the cornerstones of aquaculture population as-
sessment (Begg and Waldman, 1999). Microsatellites (simple sequence repeats, SSRs) are a form
of repetitive DNA discovered in the 1980s (Tautz and Renz, 1984). Their repeat numbers are vari-
able, which makes microsatellites polymorphic. Characterized by codominant inheritance, high
polymorphism and good reproducibility, microsatellites have become instrumental as genetic
markers in areas such as population genetics, parentage assignment, marker-assisted breeding and
phylogenetic evolution (Liu and Cordes, 2004; Yang et al., 2008; Liu et al., 2009). In addition,
SSRs could help to assess the genetic and selective breeding of snakehead fish population.
In the present study, eight wild northern snakehead populations were analyzed using
five microsatellite loci (Zhuo et al., 2012). Here, we report the isolation and characterization
of five polymorphic microsatellite loci from the C. argus genome and one microsatellite locus
from the C. maculate genome, and investigation of spatial genetic structure, interpopulation di-
versity and population genetic differentiation of four cultured C. argus populations, which will
provide additional insight and help to facilitate the selective breeding of C. argus populations.

MATERIAL AND METHODS

Sample collection and DNA extraction

In China, Shandong, Jiangxi, Hunan, and Zhejiang are the major breeding provinces
for the snakehead fish (Li and Yang, 1998). Places in these provinces, namely Luqiao in Shan-
dong, Wujia in Jiangxi, Ruanjiang in Hunan, and Yuhang in Zhejiang, have a long history of
breeding snakehead on a large scale (Li and Yang, 1998; Zhu et al., 2011). Sixty cultured C.
argus specimens were respectively collected from these four areas (Table 1). Pectoral fins

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 S.-R. Zhu et al. 8048

were collected from each fish and preserved in 95% alcohol. Genomic DNA was extracted
from one individual by the standard phenol-chloroform extraction method (Sambrook and
Russell, 2001).

Table 1. Sampling sites and sample sizes of Channa argus used in the experiment.

Stock Sampled site Location Sample No.

SD Luqiao, Shandong 34°89ꞌN117°08ꞌE 60
JX Wujia, Jiangxi 29°64ꞌN115°91ꞌE 60
HN Ruanjiang, Hunan 28°61ꞌN112°85ꞌE 60
ZJ Yuhang, Zhejiang 30°39ꞌN120°18ꞌE 60

Microsatellite isolation

About 600 ng genomic DNA was digested with MseI restriction enzyme (NEB) at
37°C for 2 h. These fragments were ligated to MseI adapters and then amplified by PCR us-
ing MseI-N primers (5ꞌ-GATGAGTCCTGAGTAAN-3ꞌ) following the program of 94°C for 4
min, 30 cycles of 94°C for 30 s, 53°C for 1 min and 72°C for 1 min, and 72°C for 5 min. The
PCR products were then hybridized with biotinylated probe (CA)15 in a 300-μL hybridization
solution (4X SSC, 0.1% sodium dodecyl sulfate, 0.5 μM probe) at 55°C for 30 min. Subse-
quent probe-bound DNA fragments were enriched for CA repeats using streptavidin-coated
magnetic beads (Promega, Shanghai, China) at room temperature for 30 min, followed by
two washing steps. Recovered DNA fragments were amplified with MseI-N primers as de-
scribed above. The PCR products, after being purified with Gel Extraction kit (Tiangen),
were ligated to pGEM-T vector (Promega) and transformed into Escherichia coli DH5α com-
petent cells (Tiangen). Fifty-five positive clones were picked out and tested by PCR using
MseI-N primers.
Positive clones were sequenced on an ABI 3700 automated DNA Sequencer (Applied
Biosystems Incorporation), of which 46 screened clones containing microsatellite repeats.
Primers for these loci were designed with the PRIMER3 online software. A total of five primer
pairs produced polymorphic DNA products from C. argus genome and one primer produced
a polymorphic DNA product from C. maculata, including CHA7, CHA13, CHA25, CHA31,
CHA41, and CHM9 (Table 2). In addition, we designed four primers according to the NCBI se-
quence from the C. argus genome, and they showed high polymorphism in C. argus (Table 2).

Microsatellite genotyping

All ten microsatellite loci that amplified specific products were selected for further
investigation using fluorescently labeled probe (either 6-FAM or HEX) on 240 samples from
four populations (SD, JX, HN and ZJ). The PCR amplifications were performed in a 25-μL
volume, including approximately 20 ng genomic DNA, 0.2 mM dNTPs, 1.5 mM MgCl2, 0.5
μM of each primer, and 1 U Taq DNA polymerase (Tiangen). The amplification program in-
cluded an initial denaturation at 94°C for 3 min and 35 cycles of 30 s at 94°C, 30 s at 50-64°C
depending on the primer pair (Table 2) and 1 min at 72°C, followed by a final extension step
for 10 min at 72°C. Fluorescent dye-labeled fragments were separated and analyzed using the
ABI3730xl sequencer GeneMapper software (Lo and Yue, 2008).

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 Genetic variations of cultured Channa argus 8049

Table 2. Microsatellite marker primers and their repeat sequences used in the experiment.

GenBank No. Primer sequence (5ꞌ-3ꞌ) Size Repeat motif Annealing temp.
HM015826 F: CACTGGTCACTGTTGAAATCT 253-305 (TACA)7 58
TLL01 R: CAGCGAGTACAAAATTACAGC
HM015833 F: TGATTGGTCCCAAATGTCTG 257-327 (TAGA)32 55
TLL02 R: GGACGCACCACCTAGAGAAG
HM015836 F: ATGACAGTGTTCCTGCTCTATG 273-329 (TAGA)12 56
TLL03 R: GGTGCAACTTCCTCAGTGA
HM015842 F: CACACCCAAAGGTTACACC 326-418 (TAGA)35 55
TLL04 R: AATGTGGGCACTGATCTTC
GQ131294 F: ATTGGGTGCTGCCATCATAC 164-185 (AC)22 60
CHA7 R: GGAGGAAACGGTTTGACTGA
GQ131300 F: GTTTCAAAGGTCGGGAGAGG 221-258 (AC)15 64
CHA13 R: AGCAAAAGCATGATCCTTGG
HQ404179 F: CCTGGCATCTTCGGTAAAAG 241-280 (TG)19 58
CHA25 R: CTCCAAGGGCGAAAACTCTT
HQ404185 F: CCTCTGATGCCCTTAGAGACA 151-198 (AC)31 60
CHA31 R: TCAGACTTCGTCTGCACCAG
HQ404195 F: TGCCAGTTTATTGGAAAGCA 135-180 (TG)5TC(TG)26 56
CHA41 R: GTAGGCACCTCAGCCATGAT
HQ404206 F: CGCAGCAAGTATCAGCAT 142-165 (GT)6GC(GT)9 58
CHM9 R: GGTCCCAACTAGGAAGAAC

Data analysis

The numbers of alleles (NA) and effective alleles (NE), observed heterozygosity (HO),
expected heterozygosity (HE), and pairwise linkage or gametic phase disequilibrium were ana-
lyzed using GDA (Lewis and Zaykin, 2000). Polymorphic information content (PIC) was cal-
culated using a formula (Botstein et al., 1980). Pairwise genetic differentiation F-statistics and
analysis of molecular variance (AMOVA) were determined using ARLEQUIN 3.1 (Excoffier
et al., 2005). Phylogenetic trees were constructed using FST genetic distance based on the un-
weighted pair group method with arithmetic averages (UPGMA). The genetic structure of dif-
ferent C. argus populations was analyzed using Structure 2.3 (Pritchard et al., 2000; Evanno
et al., 2005). In the assumption populations menu, we set the assumed K value from 1 to 4.

RESULTS

Genetic diversity

There was significant variation in the 10 microsatellite loci in this study, with 154 dif-
ferent alleles in 240 individuals from four populations. The mean number of alleles observed
over all markers in four populations was 15.40. PIC showed that all 10 loci were highly infor-
mative (mean = 0.81 ± 0.10). HE and HO varied 0.64-0.94 and 0.56-0.98, respectively, and PIC
between 0.57 and 0.93 in 240 individuals. Among them, NA, NE, HE, and PIC of locus TLL04
were the highest. Allelic diversity (NA and NE) and gene diversity (HE and HO) of each locus in
every population are shown in Table 3. Due to the important parameters for measuring allele
polymorphism, we used HE and PIC to compare different population, and found that the mean
HE and PIC were highly informative. In the four populations, the ZJ population showed the
highest HO (0.83), followed by HN (0.79), JX (0.74), and SD (0.69) (Table 3). The highest es-
timates of HE were obtained from ZJ (0.84), followed by HN (0.83), JX (0.80), and SD (0.70),

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 S.-R. Zhu et al. 8050

while PIC was the highest in ZJ (0.82), followed by HN (0.81), JX (0.77), and SD (0.66). The
allele equilibrium status of the four stocks was tested for Hardy-Weinberg equilibrium, and
some microsatellites deviated significantly from Hardy-Weinberg equilibrium (P < 0.05), such
as CHM9 in the SD population, TLL01, CHA7, CHA13, CHA41, and CHM9 in the JX popula-
tion, and TLL01 and CHA25 in the HN population.

Table 3. Genetic diversity and P values of the four Channa argus populations.
Population Index TLL01 TLL02 TLL03 TLL04 CHA7 CHA13 CHA25 CHA31 CHA41 CHM9 Mean
SD NA 3 15 11 19 4 5 8 11 8 7 9.1
NE 1.9 7.4 4.2 3.9 2.2 2.0 4.1 4.9 5.2 3.1 3.9
HO 0.45 1.00 0.77 0.49 0.65 0.43 0.77 0.86 0.75 0.72 0.69
HE 0.48 0.87 0.77 0.75 0.55 0.50 0.76 0.80 0.82 0.69 0.70
PIC 0.38 0.85 0.73 0.74 0.50 0.46 0.72 0.77 0.78 0.65 0.66
P 0.39 0.21 0.75 0.57 0.31 0.05 0.98 0.21 0.13 0.00*
JX NA 4 15 12 26 8 5 12 12 8 8 11.0
NE 2.5 10.1 5.5 16.7 3.4 4.5 5.2 4.7 5.1 6.4 6.41
HO 0.47 0.97 0.80 0.67 0.68 0.67 0.75 0.90 0.68 0.83 0.74
HE 0.60 0.91 0.83 0.95 0.71 0.79 0.81 0.80 0.81 0.85 0.80
PIC 0.51 0.89 0.80 0.94 0.67 0.75 0.78 0.76 0.78 0.83 0.77
P 0.01* 0.35 0.29 1.00 0.04* 0.02* 0.89 0.36 0.01* 0.00*
HN NA 5 21 10 27 11 8 13 15 12 11 13.3
NE 3.0 11.6 6.0 16.0 7.6 4.7 6.6 7.7 4.3 5.5 7.3
HO 0.67 0.97 0.85 0.87 0.83 0.72 0.62 0.84 0.65 0.92 0.79
HE 0.67 0.92 0.84 0.95 0.88 0.80 0.86 0.88 0.78 0.82 0.83
PIC 0.62 0.91 0.81 0.93 0.85 0.76 0.83 0.86 0.75 0.79 0.81
P 0.04* 1.00 0.23 1.00 0.06 0.14 0.04* 0.93 0.30 0.08
ZJ NA 5 17 11 23 13 6 14 13 10 11 12.3
NE 3.1 7.5 5.4 14.2 9.0 4.6 6.1 6.5 6.0 9.6 7.2
HO 0.67 0.97 0.75 0.88 0.87 0.83 0.77 0.92 0.88 0.82 0.83
HE 0.68 0.87 0.82 0.94 0.90 0.79 0.84 0.85 0.84 0.90 0.84
PIC 0.61 0.85 0.79 0.93 0.88 0.75 0.82 0.83 0.81 0.89 0.82
P 0.58 0.85 0.69 1.00 0.29 0.22 0.98 0.99 0.27 0.16
NA = number of alleles; NE = number of effective alleles; HE = expected heterozygosity; HO = observed heterozygosity;
PIC = polymorphism information content. *Significant difference (P < 0.05).

Bottleneck analysis was conducted using the Bottleneck software under the two-
phrased model of mutation (TPM) of microsatellites. P > 0.05 indicates that the population has
not experienced a recent bottleneck. According to the sign test, population JX deviated from
mutation-drift equilibrium and experienced a recent bottleneck (Table 4).

Table 4. Analysis of the possibility of a recent bottleneck using sign tests in the four Channa argus populations.
Population TPM
HE/HD P
SD 5/5 0.40
JX 6/4 0.61
HN 9/1 0.04
ZJ 8/2 0.16
TPM = two-phrased model of mutation. HE/HD = ratio of number of individuals with heterozygosity excesses to the
number with heterozygosity deficiency. P > 0.05 indicates that the population has not experienced a recent bottleneck.

Population genetic differentiation

Pairwise genetic differentiations based on allele frequency were determined for the

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 Genetic variations of cultured Channa argus 8051

four cultured stocks of C. argus (Table 5). All pairwise FST was statistically significant (P <
0.05). The highest differentiation occurred between populations HN and SD (0.028), while the
lowest differentiation observed was between populations JX and HN (0.100). Similarly, the
greatest distances were observed between populations HN and SD, whereas the smallest dis-
tances were obtained between populations HN and JX. The phylogenetic tree was constructed
by UPGMA using the MEGA5 software (Figure 1). AMOVA of microsatellites revealed that
the variation within individuals, between individuals within populations, and between popula-
tions was 90.85, 3.57 and 5.58%, respectively (Table 6).
Genetic clustering analysis using the Structure program indicated that the number of
genetic clusters was three (K = 3; Figure 2). The four stocks were divided into three potential
populations. Populations ZJ and HN were assigned to the same genetic background, suggest-
ing that they may share the same origin (represented in green in Figure 2).

Table 5. Pairwise genetic differentiation (FST) in the four Channa argus populations.
Population SD JX HN ZJ
SD 0.000
JX 0.045 0.000
HN 0.100 0.028 0.000
ZJ 0.097 0.037 0.029 0.000
All pairwise FST is statistically significant (P < 0.05).

Figure 1. UPGMA molecular trees based on genetic distance of four Channa argus populations.

Table 6. AMOVA of microsatellite in four populations of Channa argus.

Source of variation Sum of squares Variance components Percentage of variance

Among populations 96.67 0.23 5.58
Among individual within populations 970.45 0.15 3.57
Within individuals 915.45 3.81 90.85
Total variation 1982.12 4.41 100.00

Figure 2. Structure version 2.3.1 analysis of Channa argus populations using microsatellite genotype data from 10
microsatellite loci (the inferred clusters, K = 3).

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 S.-R. Zhu et al. 8052

DISCUSSION

Genetic variation is the basis of a selective breeding program. In past few years, many
popular genetic makers were used for assessing genetic variation in selective breeding pro-
grams (Liu and Cordes, 2004), such as mitochondrial DNA, and SSR. Due to many advantages,
microsatellites as genetic markers have been extensively used in genetic diversity studies in
fish species (Shimoda et al., 1999; Aguilar and Garza, 2006; Narum et al., 2006; Sekino and
Hara, 2007). In this study, we constructed a partial genomic DNA enriched for AC- and TG-
microsatellites with six primers designed here, and four primers designed by the sequence of
NCBI for studying genetic variation and relationships of four cultured populations of C. argus.
Among them, the primer CHM9 was designed by the sequence of Channa maculate and showed
high polymorphism in the four C. argus populations. All 10 microsatellites were found highly
polymorphic. Highly polymorphic loci provided better estimates of genetic distances than less
polymorphic loci (Kalinowski, 2002). In comparison to the published microsatellites of C. ar-
gus (Wen and Sun, 2010), these 10 microsatellite loci seemed to be much more polymorphic
in terms of allele number. Several factors may account for this difference, such as the repeat
length of microsatellites may be different. Normally, a longer repeat microsatellite shows high-
er polymorphism (Goldstein and Schlotterer, 1999). Thus, allele number is usually positively
associated with the sample size (Goldstein and Schlotterer, 1999). Compared with the wild
snakehead populations (Zhuo et al., 2012), the average HE in these four cultured populations
varied from 0.70 to 0.84 while the average HE in those eight wild populations ranged from 0.70
to 0.85. Compared with other freshwater species (DeWoody and Avise, 2000), it indicated that
C. argus has high allelic diversity. Actually, breeding methods inevitably influence the genetic
variability of any farmed population (Romana-Eguia et al., 2004). Normally, cultured popula-
tions contain lower genetic diversity than wild populations as indicated by previous research
(Norris et al., 1999). However, due to no regulation system in breeding program in China, fish
farmers have been using wild snakehead as parents to produce offspring. The high diversity in
cultured snakehead fish populations may be good news for fish breeders.
It is important to determine whether the population had experienced a recent bottle-
neck, because bottlenecks can increase demographic stochasticity, loss of genetic variation,
rates of inbreeding and fixation of mildly deleterious alleles, thereby reducing evolutionary
potential and increasing the probability of population extinction (Frankel and Soule, 1981;
Lande, 1988; Ralls et al., 1988; Hedrick and Miller, 1992; Jimenez et al., 1994; Mills and
Smouse, 1994). In the present study, the JX population was shown to have experienced a small
bottleneck, which indicates that the genetic variability of the population is expected to decline
rapidly. One explanation could be that the JX samples were collected from a cohort based on
a small number of parent fish. However, as soon as population size becomes larger, it starts to
increase owing to new mutations (Nei et al., 1975). When population size is restored, the aver-
age number of alleles per locus increases faster than the average heterozygosity. In other words,
it is necessary to increase effective population sizes in the JX population for better breeding.
The FST value and AMOVA are useful measurements of genetic differentiation among
populations. The analysis of pairwise genetic differentiation revealed that the FST values
ranged from 0.028 to 0.100, which was in line with a moderate level of genetic differentia-
tion (Hartl and Clark, 1997). Based on microsatellite genotypes, the UPGMA-phylogenetic
tree showed that JX and HN were the nearest clusters while HN and SD showed the farthest

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 Genetic variations of cultured Channa argus 8053

genetic distance. This clustering result is related to their geographic distribution distance. The
relativity between geographic isolation and genetic distance shows that the genetic differentia-
tions between populations are mainly because of the geographic proximity (Li et al., 2007).
AMOVA revealed that genetic variation within individuals was 90.85%, and the genetic varia-
tion between populations was 5.58%, which suggested a moderate level of genetic differen-
tiation. These different degrees of differentiation between populations and the variations in
populations were mainly attributed to genetic drift and gene flow or the diffuseness between
populations (Wright, 1978). Future studies are aimed at understanding whether the crosses
between fishes from different populations will generate hybrid vigor.
The present study focused only on four cultured snakehead fish populations subjected
to a supportive breeding program. High genetic diversity was detected in these four snake-
head fish populations, which could provide the basis for future breeding program. This study
demonstrates a strong indication of reduced effective numbers of breeders in the JX popula-
tion. Thus, attention should be paid to routine genetic monitoring of breeding activities and to
identifying whether there is a decrease in the effective population sizes of the snakehead fish.

ACKNOWLEDGMENTS

Research supported by the Shanghai University Knowledge Service Platform (#ZF1206).

We acknowledge the help of Narayan P. Pandit for improving the English of the manuscript.

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