Isolation and characteristics of polymorphic microsatellite

markers in Parabramis pekinensis

Abstract
The bleeker (Parabramis pekinensis) is an economically important freshwater fish
which are widely distributed in China. In order to obtain a set of genetic markers for
evaluating the genetic diversity of bleeker, we developed thirty-four microsatellites
from a (AC) 13-enriched genomic library in 30 individuals collected from Liangzi
Lake. Seventeen of those loci were polymorphic with allele numbers ranged from 4 to
7. The observed, expected heterozygosities and polymorphism information content
range from 0.5000 to 1.0000, 0.6328 to 0.8211 and 0.571 to 0.797, respectively. The
polymorphic microsatellite loci developed in this study will be of potential use in
understanding population genetic structure and conservation genetics of P. pekinensis.
Keywords Parabramis pekinensis, microsatellite, polymorphism
Introduction
The bleeker (Parabramis pekinensis) is the most common and widespread species in the genus of
Parabramis in China. It is characterised by an increased body height that is 34.48% to 40.00% of
standard length, a protracted diamond body and skinny body breadth (Wu XW et al., 1964).
Several species are considered endemic to the Heilong River and the Liaohe River

including P. pekinensis P. strenosomus and P. liaohonensis. However, it has a great

commercial importance as food resource since its a crucial aquaculture income for
the coastal communities. Various factors such as water pollution, overfishing,
construction of dams for power generation and so on affected the population
drastically which led to a decline in population of P. pekinensis. Despite decades of
study, there is not much information pertaining to genetics of this species (Li et al.
2008; Miao et al. 2013). Microsatellite markers are commonly used to assess genetic
population structure because of their high level of polymorphism and co-dominant
inheritance (Goldstein and Schltterer 1999). So far, Polymorphic microsatellite loci
have been isolated from various fish species such as Amblema neislerii (DazFerguson et al. 2011), Siniperca chuatsi (Liu et al. 2011) and Pelteobagrus fulvidraco
(Hu et al. 2009). But no Microsatellite markers have been developed for P. pekinensis.
Therefore, in this study we focused isolate novel polymorphic microsatellite loci of P.

pekinensis using FIASCO (Fast Isolation by AFLP of Sequences Containing Repeats)

for future population genetic studies.
Methodology
Genomic DNA of P. pekinensis was extracted from caudal fin by using proteinase-K
digestion and Ammonium acetate chromatography with RNase treatment, and then an
enriched partial genomic library for the repeat motif (AC) 13 was constructed using the
FIASCO protocol (Zane et al. 2002) with some modifications (Zhu et al. 2005).
Enriched fragments were ligated into pMD19-T vector (TaKaRa) and then
transformed into Escherichia coli DH5 (TaKaRa) cells. Grown overnight in a SOC
solid medium with ampicillin, clones were confirmed by polymerase chain reaction
(PCR) amplifications using M13 forward and reverse primers. After PCR
confirmation, 57 positive clones were sequenced by Sangon (Shanghai). Sequences
were analyzed for the repeat region using software SSRHunter 1.3. The software
Primer 5.0 was used to design 57 pairs of primers flanking the repeat regions of
interest.
Thirty individuals of adult P. pekinensis were sampled from Liangzi Lake in Hubei
province of China. Genomic DNA of P. pekinensis was extracted using ammonium
acetate high salt chromatography, and these DNA samples were used for the test and
characterization of polymorphism of the isolated microsatellite loci.
The conditions for polymerase chain reaction were optimized for each pair of
primers, and the tests of polymorphism were performed in 30 individuals of P.
pekinensis population. The PCR amplifications were carried out in 10 L volume on a
Veriti 96 well Thermal Cycler (ABI), the mixture containing 1 PCR buffer (10mM
Tris-Cl, 1.5mM MgCl2, 50mM KCl), 10ng genomic DNA, 0.1M for each primer,
100mol/L dNTPs and 0.2 U rTaq DNA polymerase (TaKaRa). The amplification
system was set as a initial denaturation at 95 for 5 min; 33 cycles including
denaturation for 45s, annealing at annealed temperature (Table 1) for 45s and
elongation at 72 for 30s; and a final extension at 72 for 10 min. Amplified
fragments were size-fractionated on 10% non-denaturing polyacrylamide gels running
at 135V for 200 min and visualized by silver nitrate staining, photographed, and
analyzed using the Ultraviolet Gel Document System (Biotech). The analyses of
polymorphism, including allele diversity, observed (H0), polymorphism information
content (PIC), expected heterozygosities (HE), and the exact test for Hardy-Weinberg

equilibrium

(HWE)

was

performed

using

GENEPOP

online

version

(http://genepop.curtin.edu.au/) (Zeng, 2011).

Results and Discussion
Out of the

57 pairs of primers designed, 23 failed to amplify PCR products. In sum,

17 of the 34 successfully amplified microsatellite loci were polymorphic, with the

number of alleles per locus ranging from 4 to 7. For those 17 polymorphic loci, H0,
HE and PIC ranged from 0.5000 to 1.0000, from 0.6328 to 0.8211 and from 0.571 to
0.797, respectively (Table 1). The remaining 18 loci were monomorphic. The tests for
HWE revealed that the majority (3 out of 17) of these polymorphic microsatellites
were in HWE, and Ppe5, Ppe10, Ppe12 of the 17 loci showed significant departure
from HWE (P<0.005), which may suggest population subdivision or occurrence of
null allele may be detected as homozygotes. These 17 polymorphic microsatellite loci
of P. pekinensis will facilitate future studies on conservation genetics in P. pekinensis
populations.

Table 1 Characterization of 17 polymorphic microsatellites of P. pekinensis in Liangzi Lake

GenBank
Accession No.

Repeat motif

Ppe1

KF057769

(AC)20(CA)5(CA)5
(CA)15(CA)5(CA)5
(CA)12(CA)36

Ppe2

KF057770

(CA)6(AC)5

Ppe3

KF057771

(ATTC)6(CA)20(AC)10
(CA)5(AC)20

Ppe4

KF057772

(CA)42

Ppe5

KF057773

(GT)43

Locus

Ppe6

(TG)34(TG)7
KF057774

Ppe7

(GT)16

Ppe8

KF057775

(TG)14

Ppe9

KF057776

(GT)21(GT)5

Ppe10

KF057777

(TG)22(TG)13(TG)7(TG)10

Ppe11

KF057778

(TG)28

Primer sequence (5'-3')

F: ACTTTTTCATCTTGTCAT
R: GCATTTAGTCTTCTGGAG
F: TTACAGTGACCCTCATCCG

R: GAACTATCCCCTCCCCAG
F: AAGATTCTGAAAGCGTAC

R: GCTCTGTGATAGTGTGGT
F: TCTTATTACAGTTACCCTCC

R: TGGCGTCTACACTCACAT
F: GTAACTACTTCCTCCGTCC
R: GGGTTTTAGGTGTTTTGTG
F: TCCCTACATCAACGACGA
R: TCTTTCACCCATTCTCCC
F: CTCACACAGCGGGAGAAT
R: GCGGGACCAGAGACTAATC
F: TGTGGGAAGGAAGAGGTC
R: TGCTTTGTTTTGAATGGC
F: ACAGTCTGTGCTGAATGA
R: TACACGGGAAAACAATAC
F: TGTGAGTAGATTGGAAGG
R: GAAGCAGTGTTTTAGGAG
F: ATGAATATCTGCTGAGGTAC
R: AGAGACATCTGAGACGAAG

T
()

size
range
(bp)

Na

HO

HE

PIC

50

384-396

1.0000

0.8056

0.777

58

409-451

0.9333

0.8133

0.788

53

386-396

0.8000

0.7444

0.705

54

229-251

0.7667

0.7056

0.668

55

271-303

0.9000

0.7694

0.731*

55

409-457

0.9667

0.7672

0.729

58

195-225

0.5000

0.6550

0.604

54

214-250

0.7000

0.7172

0.666

51

176-186

0.6333

0.6844

0.629

52.7

203-217

0.4333

0.6811

0.627*

54

308-338

0.6333

0.6989

0.655

Ppe12

KF057779

(CA)24

Ppe13

KF057780

(CA)71(AC)12(CA)14(CT)5

Ppe14

KF057781

(AC)8(CA)7(AC)5(CA)10
(CA)10(AC)13

Ppe15

KF057782

(CA)20(AC)17

Ppe16

KF057783

(CA)27

Ppe17

KF057784

(TG)22(TC)6(TG)27

F: CTGGTGTTTGTGTGATTGTG
R: GACGGCTTCCATTCCTCT
F: GAGTAAGGATGGGTGTAT
R: TGGTAACTGAGCAAGGT
F: TAGGAGAGGTGTGGAGTG
R: CCCGAGATAGGTTTGTTT
F: ACACCAGGCAACAAAGAC
R: TCAGATTCGCCCTCATAC

56

293-335

0.6333

0.8211

0.797*

52.5

419-451

0.5000

0.6328

0.571

55

336-360

0.7000

0.7189

0.669

55

215-251

0.8000

0.7717

0.736

56

202-224

0.6333

0.7683

0.731

53
240-280 5 0.5333 0.7700
R: GTTCTTTATTGGTTGCTTGC
T: annealing temperature (); Na: number of alleles; HO: observed heterozygosity; HE: expected heterozygosity; PIC: polymorphism

0.730

F: CCTAACAACCAACCCGAC

R: AGGCTTCTTATTCCTGACG
F: ATCCTTTGTGTTTGCCCT

information content; * indicated deviation from Hardy-Weinberg equilibrium (P<0.05) after Bonferroni correction

Acknowledgements
This study was supported by grants from the Modern Agroindustry Technology
Research System, Staple Freshwater Fishery Industry Technology System (No.
CARS-46-05), Fundamental Research Funds for the Central Universities (No.
2011PY0232011PY0432011SC27, 2011ZC011 and 2012YB08), and Guangdong
Haid Group Co., Ltd.
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