Indonesian Aquaculture Journal, 13 (2), 2018, 51-56

Available online at: http://ejournal-balitbang.kkp.go.id/index.php/iaj

CORRELATION OF MICROSATELLITE DNA MARKERS WITH GROWTH TRAITS
IN STRIPED CATFISH (Pangasianodon hypophthalmus)
Huria Marnis#, Evi Tahapari, and Jadmiko Darmawan
Research Institute for Fish Breeding
Jl. Raya Pantura No. 2, Patokbeusi, Subang, Sukamandi 41263, West Java
(Received 22 March 2018; Final revised 3 May 2018; Accepted 4 May 2018)
ABSTRACT
Marker-assisted selection in genetic improvement of striped catfish is useful in the breeding program of
the fish. Five microsatellite markers were characterized in the largest (4.03 kg ± 1.67 kg) and smallest (1.41
kg ± 0.22 kg) individuals. Five polymorphic loci were then used to genotype 160 individuals and the
associations between their genotypes and growth traits were examined. The result showed that twentyseven alleles were detected in striped catfish. The number of alleles per locus (NA) ranged from 4 to 7, with
an average of 5.4 alleles per locus. The effective number of alleles per locus (NE) ranged from 3.940 to
6.939, with an average of 5.32 alleles per locus. HO and HE ranged from 0.125 to 0.944 (mean value of
0.472) and from 0.564 to 0.775 (mean value of 0.697), respectively. PIC ranged from 0.573 to 0.799 (mean
value of 0.706), showing that they were highly polymorphic loci. Only one microsatellites loci (Pg13) that
showed significant differences (P<0.01) in the associations between their genotypes and growth traits,
while Pg3 and Pg14 were significantly associated with the standard length (P<0.01) and body weight
(P<0.05). However, the Pg1 and Pg2 were not significantly associated with the body weight and standard
length. Four genotypes of three loci were positively correlated with the growth traits (body weight and
standard length) i.e. genotypes 194/194 for Pg3, 227/227 and 229/229 for Pg13, 279/279 for Pg14. These
four genotypes can be used to identify growth traits in the molecular marker-based selection of a breeding
program.
KEYWORDS:

microsatellite; growth traits; allele; fast-growing; genetic diversity; Pangasianodon
hypophthalmus

INTRODUCTION
Nowadays, the development of molecular genetic
markers has stimulated a renewed interest in the
study of growth trait improvement of fish. In aquatic
animals, molecular markers are not only used in genetic monitoring of selective breeding lines (Hao et
al., 2010; Yu et al., 2011) but also in the association
analysis of target traits (Kang et al., 2002). Many researchers have reported that certain genotypes of
microsatellite loci are positively correlated to growth
traits (Fan et al., 2009; Liu et al., 2012).
Microsatellite markers have been used as functional markers to identify genes that play important
roles in productive traits. The characterization of
#

Correspondence: Research Institute for Fish Breeding.
Jl. Raya Pantura No. 2, Patokbeusi, Subang, Sukamandi 41263,
West Java, Indonesia
Phone: + 62 260 520500
E-mail: marnis.huria@gmail.com

gene-associated microsatellites in some species including Oreochromis sp. (Yue & Orban, 2002), Cyprinus
carpio (Yue et al., 2003), Salmo salar (Vasemägi et al.,
2005), Gadus morhua (Stenvik et al., 2006), Epinephelus
bruneus (Kessuwan et al., 2016), Larimichthys crocea
(Han et al., 2017), and Colossoma macropomum (Ariede
et al., 2018) had already demonstrated the usefulness
of these markers in the detection of economically
important quantitative traits and its application in
breeding studies. Nonetheless, this characterization
has not been observed in striped catfish.
This study was conducted to study the growth
performance of striped catfish and identify molecular markers that are associated with the growth traits
in striped catfish using microsatellite. The markers
could provide a valuable theoretical basis for markerassisted selection (MAS) to breed faster growing strain
and to preserve fish germplasm.

Copyright @ 2018, Indonesian Aquaculture Journal, p-ISSN 0215-0883; e-ISSN 2502-6577

51

Correlation of microsatellite DNA markers with growth traits ..... (Huria Marnis)

MATERIALS AND METHODS
Fish and DNA Samples
The research spanned in the period of six years
from January 2012 until December 2017. The identification of broodstock bearing fast growth markers
was performed at the Genetic and Physiology Laborator y, Research Institute for Fish Breeding
Sukamandi, Ministry of Marine Affairs and Fisheries
of the Republic of Indonesia.
Three generations of striped catfish have been
produced from the fish breeding selection program
i.e. G-0, G-1, and G-2 populations. These fish were
tagged using microchips. The average weight of the
largest individuals was 4.03 kg ± 1.67 kg and the
smallest was 1.41 kg ± 0.22 kg from a total of 160
fish of an isolated population at the Research Institute for Fish Breeding. Approximately 1 cm2 of the
caudal fin of each fish was cut using a sterile section
scissor. Each sample was placed in a microtube containing 1 mL of 70% ethanol and stored at room temperature until the DNA extraction process was performed.
DNA Extraction
The genomic DNA of each sample was extracted
using DNA extraction kit (GeneJet Genomic DNA Purification Thermo Scientific, Lithuania) following the
protocols recommended by the manufacturer (Thermo
Scientific, Lithuania). The genome sample was analyzed in a mini horizontal gel electrophoresis. The
sample was loaded into 1.5% (w/v) agarose gel, powered with 65-volt electricity and run for 60 min. The
gel was then stained with peqGREEN (Vwr, UK) 1 µg/
mL and viewed using ultraviolet transilluminator gel
documentation system.
Microsatellite Primers
Five microsatellite loci (Pg1, Pg2, Pg3, Pg12, and
Pg14) were used in this study (Na-Nakorn et al., 2006;
Na-Nakorn & Moeikum, 2009). It showed polymorphisms in Pangasianodon hypophthalmus. The primers characteristics used to amplify the five
microsatellite loci are presented in Table 1.
Amplification of Microsatellite Locations and
Scoring
A total of 2 L ( 200 ng) of the extracted genomic DNA was amplified using type-it microsatellite
PCR (Qiagen, Germany). The PCR process was carried out using the following settings: 95°C for 5 minutes; (95°C for 30 seconds, 6°C-62°C for 90 seconds

52

according to the degree of attaching temperature (Tm)
per primer (Table 1); 72°C for 30 seconds) of 28 cycles;
and 72°C for 30 minutes. The PCR amplification results were loaded on 2% (w/v) agarose gel by an electrophoresis method. The electrophoresis results
observed under UV transilluminator and photographed
using a Canon EOS 1100D digital camera.
Microsatellite locus polymorphisms were screened
using the QIAxcel (Qiagen, Germany) fragment analyzer and using QIAxcel DNA High resolution (Qiagen)
kit, and the size of the alleles was determined based
on the PCR product size relative to the size of the
DNA fragment on QX size marker 50-800 bp (Qiagen,
Germany) and alignment marker 50-1,000 bp (Qiagen,
Germany). The patterns of DNA band and
electrophoregram data were analyzed using QIAxcel
screen Gel software 1.5 (Qiagen, Germany) to scan
alleles that emerged. The allele score data were then
used for the analysis of the relevant genetic parameters.
Statistical Analysis
Allelic frequencies, genotype frequencies, HardyWeinberg equilibrium, and observed (HO) and expected (HE) heterozygosities were statistically analyzed using POPGENE software version 1.32, Fstat’s
statistical genetic software version 2.9.3. (Goudet,
2001) and Arlequin (Excoffier et al., 2006). The associations between genotypes and growth traits were
considered significant if two-way P values were less
than 0.05. The statistical analyses were carried out
using the SPSS software (Version 19.0; SPSS Inc.,
Chicago, IL, USA).
RESULTS AND DISCUSSION
A total of five simple sequence repeat (SSR) markers were screened from the 160 samples of the isolated fish population. Three of these loci were polymorphic in striped catfish and significantly associated with the body weight and standard length (Table
2).
A random population was amplified using these
five microsatellite loci (Table 2), and 27 alleles were
detected in striped catfish. The number of alleles per
locus (NA) ranged from 4 to 7, with an average of 5.4
alleles per locus. The effective number of alleles per
locus (NE) ranged from 3.940 to 6.939, with an average of 5.32 alleles per locus. HO and HE ranged from
0.125 to 0.944 (average of 0.472) and from 0.564 to
0.775 (average of 0.697), respectively. Heterozygosity is one of the indicators of the degree of genetic
variation (Allendorf & Luikart, 2007). PIC ranged from

Copyright @ 2018, Indonesian Aquaculture Journal, p-ISSN 0215-0883; e-ISSN 2502-6577

Indonesian Aquaculture Journal, 13 (2), 2018, 51-56

Table 1. Primer sequences and characteristics of five polymorphic microsatellites loci of
striped catfish populations
Loci

Repeat motif

Pg-1

(CA)18

F : GGCCTGTCACAATGTGTATTGC
R : GTCTGAGGTAGGCCTGTGAGGAG

231-239

64

Pg-2

(GA)14N116 (GT)11

F : TGTGTCTAATCTTGTCCGTGCTG
R : TACTGTTGGACCAGACGTTCCTC

258-276

60

Pg-3

(GT)16

F : CCAGCCCACATTAGGTAGCATC
R : ACTAAAAGGCCTGACCCTTAGC

204-220

60

Pg-13

(CA)25

F : GTTTTCCATCCAGGTTGTTTTCC
R : TAAGTCCATGTGGGTTTCCTCTG

232-262

58

(GT)5 AT(GT)11

F : ACCGTGCATGTGCATTATCATAG

AT(GT)13

R : AGAATGTGACCTGGAAATGAGCA

289-293

60

Pg-14

Primer sequence (5’-3’)

0.573 to 0.799 (average of 0.706), indicating that they
were highly polymorphic loci.
These novel markers will facilitate further studies
on genetic diversity evaluation, conservation genetics, construction of high-density linkage map, and
molecular marker-assisted breeding of striped catfish and its related species. PIC is the change of
function of allele frequency and the allele’s number,
and it is a good indicator of genetic information capacity of a genetic marker (Zhu et al., 2008). Moreover, PIC is also an indicator of the degree of genetic
variation (Li et al., 2017). The five loci of this study
showed a high polymorphism (PIC>0.5).
In this study, one microsatellites loci (Pg13)
showed a significant difference (P<0.01) in the associations between their genotypes and growth traits,
while Pg3 and Pg14 were significantly associated with
the standard length (P<0.01) and body weight
(P<0.05). However, Pg1 and Pg2 were not significantly associated with the body weight and standard
length (P>0.05). Four genotypes of three loci were
positively correlated with growth traits (body weight

Range size (bp) Temperature (°C)

and standard length), i.e. genotypes 194/194 for Pg3,
227/227 and 229/229 for Pg13, 279/279 for Pg14 (Table
4). These significantly correlated loci carry an important function in the evolution because they control
the viability of individuals bearing different genotypes
of the locus (Xu, 2008). Therefore, these kinds of
genotypes could indirectly be used to select preferred
growth traits in striped catfish. The study about correlated marker to growth traits also has been reported by Sun et al. (2015) for Mandarin fish. The
result of the study showed that the eight genotypes
of six loci were positively correlated with growth
traits (body weight, length, and height) in the mandarin fish population.
CONCLUSION
This study had confirmed that the identified
microsatellite DNA markers are closely associated with
growth traits in striped catfish. There are four genotypes of three loci positively correlated with growth
traits (body weight and standard length) in striped
catfish. It could be useful for marker-assisted selection in breeding programs of striped catfish. It also

Table 2. Genetic diversity in Pangasianodon hypophthalmus population
Locus
Pg-1
Pg-2
Pg-3
Pg-13
Pg-14

NA

NE

Ho

HE

PIC

7
5
4
5
6

6.939
5.000
3.940
4.985
5.785

0.944
0.563
0.213
0.125
0.513

0.775
0.742
0.564
0.696
0.709

0.799
0.755
0.573
0.702
0.703

Copyright @ 2018, Indonesian Aquaculture Journal, p-ISSN 0215-0883; e-ISSN 2502-6577

53

Correlation of microsatellite DNA markers with growth traits ..... (Huria Marnis)

Table 3. Association between five microsatellite loci to body weight and standard length of Pangasianodon hypophthalmus
Locus

Body weight (g)

Standard length (cm)

0.49
0.5
0.012*
0.003**
0.0422*

0.5
0.14
0.001**
0.0006**
0.001**

Pg-1
Pg-2
Pg-3
Pg-13
Pg-14
Note:

* significantly correlated with markers at P<0.05
** significantly correlated with markers at P<0.01

Table 4. Association between three microsatellite loci with body weight and standard
length of Pangasianodon hypophthalmus
Locus Genotype N

Pg-3

Standard length
(cm)

Smallest fish

Largest fish

1,283a

42a

198/206

1

192/198

4

2,775.10 ± 1,939.40

194/196 10

a

2,534.9 ± 2,099.41

a

194/194 14 3,023.727 ± 1,507.33

b

192/192

a

192/206
198/198

8 4,144.667 ± 1,730.96
1
1

231/231 12

6,800

a

7,400

a

3,364 ± 1,536.21a

1

0

49.85 ± 12.07

a

2

2

48.22 ± 10.37

a

5

5

55.33 ± 8.76

b

2

12

59.59 ± 7.34

a

4

4

69.5

a

1

1

70.5

a

0

1

55.94 ± 9.15 a

2

10

229/229 31 3,147.323 ± 1,890.74 b

53.01 ± 10.22

b

5

26

3,774.455 ± 945.04 a

58.79 ± 4.79 a

5

6

49.51 ± 8.16

b

2

15

59.00 ± 4.58

a

0

3

69.25 ± 0.35

a

0

2

0

1

0

1

0

2

1

0

1

0

6

6

233/233 11

227/227 17 2,492.471 ± 1,624.29
Pg-13 229/233
227/231
229/233
231/233

3

3,872 ± 1,197.14

2

a

1
1

7,200 ± 565.69
7,400

a

7,600

a

b

a

a

69.33

a

70.01

a

227/233

2

3,671± 1,030.96

285/291

1

2,028a

47.5 a

1

a

a

289/289

279/291 12
279/285
Pg-14

Number of individual

Body weight
(g)

1

285/285 10

4,600

5,364 ± 746.12
6,000

60.25 ± 7.42

64
a

45.90 ± 6.58

a

6,337 ± 1,175.92

279/279 11 3,182.70 ± 2,162.71
279/291
279/289
281/281
281/291

2

25,438 ± 1,859.3

a

0

1

54.75 ± 8.13

5

5

59.95 ± 7.81

b

0

11

a

53.65 ± 10.36

a

1

1

a

57.32 ± 9.88

a

2

2

55.50 ± 8.18

a

3

2

a

2

2

4

30,949 ± 2,132.96

5

a

4

b

a

a

67.5
a

a

58,221 ± 1512.24

75,321 ± 1,940.78

a

55.90 ± 10.31

Data labeled with b superscript letters in the same column by individual locus mean significant difference. In
contrast, data labeled with a superscript letters in the same column by individual locus mean insignificant
difference.

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Copyright @ 2018, Indonesian Aquaculture Journal, p-ISSN 0215-0883; e-ISSN 2502-6577

Indonesian Aquaculture Journal, 13 (2), 2018, 51-56

provides new information on striped catfish growth
traits to guide future breeding programs of this species. The prospective microsatellites highlighted in
this study could be used in the validation of MAS in
fish species belong to the striped catfish family.
ACKNOWLEDGEMENTS
The research was supported by the funding from
APBN 2016 and 2017 of the Research Institute for
Fish Breeding, Sukamandi. We thank Ika Nurlela,
Kamlawi, Arsyad Tirta Subangkit, Ahmad Suryana, and
other researchers for their help in this research.
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