Sixteen Polymorphic Simple Sequence Repeat Markers from Expressed Sequence Tags of the Chinese Mitten Crab Eriocheir sinensis

Abstract

The Chinese mitten crab (Eriocheir sinensis) is an economically important aquaculture species in China. In this study, we developed and evaluated simple sequence repeat markers from expressed sequence tags of E. sinensis. Among the 40 wild E. sinensis individuals tested, 16 loci were polymorphic. The number of alleles per locus ranged from two to ten. The observed heterozygosity ranged from 0.0667 to 0.9667, whereas the expected heterozygosity ranged from 0.0661 to 0.9051. These markers have the potential for use in genetic studies of population structure and intraspecific variation in E. sinensis.

Full text

Int. J. Mol. Sci. 2010, 11, 3035-3038; doi:10.3390/ijms11083035
International Journal of
Molecular Sciences
ISSN 1422-0067
www.mdpi.com/journal/ijms
Communication
Sixteen Polymorphic Simple Sequence Repeat Markers from
Expressed Sequence Tags of the Chinese Mitten Crab Eriocheir
sinensis
Xiang-Gang Gao 1, Hong-Jun Li 1, Yun-Feng Li 1, Li-Jun Sui 2, Bao Zhu 2, Yu Liang 3,
Wei-Dong Liu 1 and Chong-Bo He 1,*
1 Liaoning Key Laboratory of Marine Fishery Molecular Biology, Liaoning Ocean and Fishery
Science Institute, 50 Heishijiao St, Dalian, Liaoning 116023, China;
E-Mail: xiangganggao@hotmail.com (X.-G.G.); oceanlhj@163.com (H.-J.L.); yfli2008@yahoo.cn
(Y.-F.L.); cnliu51@tom.com (W.-D.L.)
2 College of Life Science, Liaoning Normal University, 100 Huanghe Rd. Dalian, Liaoning 116029,
China; E-Mail: suilijun2006@126.com (L.-J.S.); swx04250zb@126.com (B.Z.)
3 College of Life Sciences and Biotechnology, Dalian Ocean University, 52 Heishijiao St, Dalian,
Liaoning 116023, China; E-Mail: xiaoyu325000@hotmail.com
* Author to whom correspondence should be addressed; E-Mail: hechongbo@hotmail.com;
Tel.: +86-411-846-970-03; Fax: +86-411-846-710-27.
Received: 23 June 2010; in revised form: 17 August 2010 / Accepted: 18 August 2010 /
Published: 24 August 2010
Abstract: The Chinese mitten crab (Eriocheir sinensis) is an economically important
aquaculture species in China. In this study, we developed and evaluated simple sequence
repeat markers from expressed sequence tags of E. sinensis. Among the 40 wild E. sinensis
individuals tested, 16 loci were polymorphic. The number of alleles per locus ranged from
two to ten. The observed heterozygosity ranged from 0.0667 to 0.9667, whereas the
expected heterozygosity ranged from 0.0661 to 0.9051. These markers have the potential
for use in genetic studies of population structure and intraspecific variation in E. sinensis.
Keywords: Chinese mitten crab; Eriocheir sinensis; expressed sequence tags; simple
sequence repeats
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Int. J. Mol. Sci. 2010, 11
3036
1. Introduction
The Chinese mitten crab (Eriocheir sinensis) is a native of East Asia that lives predominantly in
freshwater but migrates seawards to breed. Economically, E. sinensis is an important cultured decapod
crustacean in China because of its good taste. In order to protect genetic diversity and prevent
population degradation, understanding the genetic diversity of E. sinensis is important. However, few
studies have focused on the population structure of E. sinensis at the DNA level [1–3].
Microsatellites, also called simple sequence repeats (SSRs), are short tandem repeated sequences
(1–6 bp) that are widely dispersed in eukaryotic genomes [4]. Because of high polymorphism,
codominant inheritance, and even distribution throughout the genome, SSRs have been used
extensively for studying genetic diversity and population structure in many species [5–7]. The
expressed sequence tag (EST) represents part of the transcribed sequence and is an important resource
for microsatellite and SNP screening [8,9]. In this study, we identified microsatellites from Chinese
mitten crab ESTs and analyzed the polymorphisms present in the EST-SSRs.
2. Results and Discussion
A total of 16,961 Chinese mitten crab ESTs from the GenBank database
(http://www.ncbi.nlm.nih.gov/Genbank/) were screened for microsatellites using the software MISA
(http://pgrc.ipk-gatersleben.de/misa/) with the following parameters: at least eight repeats for di-, six
repeats for tri-, five repeats for tetra-nucleotides, four repeats for penta-nucleotides, and three repeats
for hexa-nucleotides. A total of 1,768 (10%) SSRs were derived from the 16,961 EST sequences.
Analysis of these SSRs revealed that the dinucleotides (1189), hexanucleotides (616), and
trinucleotides (615) were major motifs that accounted for 47%, 24%, and 24% of the total,
respectively. CA/TG was the most frequent motif and accounted for 32%, followed by GA/TC
(268, 11%). Forty SSR-containing ESTs that contained sufficient flanking sequences of good quality
were chosen for polymerase chain reaction (PCR) analysis in E. sinensis. Primers were designed using
the software Primer3 (http://biotools.umassmed.edu/bioapps/primer3_www.cgi). The results indicated
that 20 (50%) of primer pairs could successfully amplify scorable products. The remaining primer
pairs failed to amplify any PCR product, perhaps because the primer sequences spanned introns and/or
contained mutations and/or indels (insertion or deletion). Among the 20 functional primer pairs, 16
loci showed polymorphism in the 40 E. sinensis individuals (Table 1), with the allele number ranging
from 2 to 10. The observed heterozygosity ranged from 0.0667 to 0.9667, whereas the expected
heterozygosity ranged from 0.0661 to 0.9051. Eight of the 16 loci (ESMS03, ESMS13, ESMS15,
ESMS19, ESMS20, ESMS21, ESMS25 and ESMS35) departed from HWE (P < 0.05). This might be
due to the limited sample size, and/or presence of overdominant selection, and/or a high degree of
outbreeding. Further studies would be necessary for clarification. These polymorphic microsatellites
derived from E. sinensis would be useful for population genetic structure analysis and genetic diversity
assessment in crab populations and will facilitate breeding programs.

Int. J. Mol. Sci. 2010, 11
3037
Table 1. Characterization of EST-SSRs from the Chinese mitten crab (Eriocheir sinensis).
T, annealing temperature; NA, number of alleles detected; HO, observed heterozygosity; HE,
expected heterozygosity; PHW < 0.05 indicates significant departure from Hardy-Weinberg
equilibrium calculated for data from 40 E. sinensis individuals.
3. Experimental Section
Forty E. sinensis individuals were randomly collected from the Liaohe River in northeastern China.
The leg muscles were removed from live individuals and stored in 80% ethanol until use. DNA was
extracted following the traditional phenol/chloroform extraction method [9]. Each reaction was
conducted in a 25 µL volume containing 50 ng of genomic DNA, 1 × PCR buffer, 1.5 mM MgCl2,
Locus
(Acc. No.)
Repeat
motif
Primer pair sequence
(5′-3′)
Expected
Size (bp)
T
(°C)
NA
HO
HE
PHW
ESMS03
(FG359457)
(AC)18
F:CTGACGGCTACCTCCACTTC
R:TTTCCTTCCATCCTGAGTCC
223
53
7
0.966
7
0.848
6
0.000
0
ESMS04
(FG359097)
(AGG)6
F:GCCTGCCTCAAGAATGGGTT
R:GGTTGGTCTCCAGGAAGTGAAT
133
62
3
0.066
7
0.066
1
0.998
3
ESMS05
(FG359055)
(TCA)7...
(CCT)6
F:ACGATACCCAAAGCAGAGGAC
R:ATGATGACGGAGACGACGAA
211
62
3
0.333
3
0.287
6
0.612
1
ESMS07
(FL574574)
(ACT)12
F:GTCACCACTGCTGCTTCTGC
R:ACATTTGACGGTGGGACTGC
168
60
3
0.333
3
0.552
0
0.065
7
ESMS11
(FG983239)
(AC)10
F:TAGAGGTGGAAGATACTAGATGG
R:TTGGAGGGTGGTAGGTTGAT
246
57
3
0.466
7
0.424
9
0.504
3
ESMS13
(FG981455)
(AC)15
F:CGCACGGGAAATGGAACAGA
R:GAGGCATTTGAAAAGATGAAGCAC
249
53
6
0.966
7
0.814
7
0.000
5
ESMS15
(FG982058)
(CCA)6
F:GTGAAAGGACGGACGTATTGA
R:GGAGGAAGAGGAGTGCGAGT
217
62
3
0.133
3
0.243
5
0.012
8
ESMS16
(FG982584)
(CCA)9
(ACA)8
F:ACTGATGCCTGACGAAGACTACCA
R:CCTTTATGCCTTTATTGACCGAGAC
184
62
6
0.866
7
0.774
0
0.486
7
ESMS17
(FG983201)
(CA)13
F:GTATCCACAAGAGCATAAAGCAA
R:AGCCAAACCTGAGAACCACT
183
57
3
0.200
0
0.187
6
0.906
2
ESMS19
(FG360290)
(TG)13
F:CTGAAGGTTTGCCTCGTGTT
R:GGTGAAATGGACCAAATGAC
205
60
7
0.900
0
0.842
9
0.005
0
ESMS20
(FG359986)
(TC)35
F:TTGCGGTATCTTGCGTCTCG
R:ATGTACCACAGCAACGCCTC
220
62
10
0.966
7
0.905
1
0.000
0
ESMS21
(FG359967)
(AC)37
F:GCAAACGAACTGATAAGCAC
R:CTTTATGTTCCCAGGTGATG
192
56
9
0.933
3
0.852
0
0.039
6
ESMS24
(FG358074)
(CAC)6
F:CTTATCTCAGCGATGATTTGC
R:AGCAGTGCCTGGTTTGTATT
239
62
2
0.100
0
0.096
6
0.745
5
ESMS25
(FG360197)
(TG)17
F:AACAGTTTGTAAGGTTCAGCAC
R:TAGGGTGTAAATCCTCTGGC
203
56
7
0.966
7
0.833
9
0.020
5
ESMS26
(GE339913)
(AC)17...
(CGCA)5
F:ACGCACAAAGGCAACAAACTG
R:AGGAAACGGCTGGCGAGACAA
153
62
2
0.333
3
0.333
3
0.999
7
ESMS35
(GE340314)
(GAG)8
F:TTGCCGAGAAGATCGCTTTGG
R:GCCCGTCGCAGATACTGGTTT
184
62
5
0.266
7
0.587
0
0.005
8

Int. J. Mol. Sci. 2010, 11
3038
0.2 mM dNTPs, 200 nM of each primer, and 1U of Taq polymerase (Takara). The PCR program was
as follows: initial denaturation for 5 min at 94 °C followed by 35 cycles of 30 s at 94 °C, 30 s at
annealing temperature (see Table 1), and 30 s at 72 °C, with a final extension at 72 °C for 10 min. The
amplified PCR products were separated on a 10% non-denaturing polyacrylamide gel at 280 V for
1–2 h, stained with ethidium bromide, and visualized under ultraviolet light. The genetic diversity
indices, including observed and expected heterozygosities and tests for departures from Hardy-
Weinberg equilibrium (HWE), were performed using POPGENE32 version 1.32 [10].
Acknowledgements
This work was supported by National Natural Science Foundation of China (No.30972246) and
Liaoning Scientific Research Program of Ocean and Fisheries Department (No.200801).
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