Novel polymorphic microsatellites useful for population analysis in ...

Page 1 of 10
Molecular Ecology Resources
1
2
Novel polymorphic microsatellites useful for population
analysis in Meretrix meretrix (Bivalva: Veneroidae)
3
4
Shu-Yin Chen*†, Lei. Wang*, Wen. Sun*, Hong-Jiu Ji†, Xiao-Feng Xu* 1
5
6
7
8
*Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences,
Nanjing Normal University, Nanjing 210097, China; †Jiangsu Marine Fisheries
Research Institute, Nantong 226007, China
9
10
11
12
13
14
15
16
17
18
19
Abstract
The asiatic hard clam Meretrix meretrix is one of the largest species in size among
the Venus-shells (Veneridae). It is of great commercial value throughout China,
however little is known of the genetic diversity or structure of its wild populations. In
order to evaluate its genetic diversity, we have isolated eleven polymorphic
microsatellite DNA loci from M. meretrix in this study. The numbers of alleles
observed for a single locus ranged from four to 16 per locus, with polymorphic
information content from 0.420 to 0.808. These genetic markers will be valuable
tools for genetic diversity studies of both cultivated and wild populations of M.
meretrix.
20
21
Keywords: Meretrix meretrix, microsatellite locus, polymorphism, bivalve
Correspondence: X.F. XU, E-mail: xuxiaofeng@njnu.edu.cn, Fax: +86-25-85891513.
1

Molecular Ecology Resources
Page 2 of 10
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
The asiatic hard clam Meretrix meretrix (Linnaeus), found in sandy-mud habitats from
the intertidal zone to 6 m depth, is one of the largest species in size among the
Venus-shells (Veneridae) and an endemic along the coasts of China (Wang et al.1998).
This species is of great value and commercial importance in Asia, and has been widely
cultured in China. Moreover, this clam, proclaimed the ‘number one delicacy on earth’
by Qianlong (emperor of the Qing Dynasty), is a very popular seafood throughout
China. Being such an encouraged farming species, a number of previous research
studies of asiatic hard clam were mostly focused on reproductive biology and culture
technology (Zhang et al. 2005; Wang et al. 2006). However, the clam farming is
dependant on natural stocks and clam famers often purchase a large number of wild
juvenile clams and transport them from one place to another for commercial cultivation.
This method of immigration might result in new genetic variants to the established gene
pool. Genetic diversity study could provide useful information for conservation of
natural stocks and selective breeding of M. meretrix. For example, the identification of
genetically distinct populations of M. meretrix (e.g., Chen et al. 2004; Du et al. 2004;
Cheng et al. 2007) and the analysis of artificially cultured and wild populations of this
clam (He et al. 2008) are of high value to the evaluation of genetic diversity in this
species.
Microsatellites are evenly dispersed throughout genomes. High polymorphism and
relative ease of scoring represent two major features that make microsatellites useful for
2

Page 3 of 10
Molecular Ecology Resources
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
many genetic studies (Zane et al. 2002). Microsatellites have become very useful
markers for the research of genetic diversity, pedigree tracing and quantitative trait loci
(QTL). No microsatellite markers have so far been reported for M. meretrix. To assist
genetic studies in this species, here we report the characterization of eleven
polymorphic microsatellite loci of M. meretrix, and evaluate the use of these loci to
quantify the genetic diversity of a wild population.
Genomic DNA was extracted from the adductor muscle of one individual using the
DNeasy Tissue Kit (Qiagen) according to the manufacturer’s protocol. The DNA was
then subjected to restriction digestion with Mse I (NEB), and simultaneously ligated to
adaptors: oligo 1: 5′-TAC TCA GGA CTC AT-3′ and Oligo 2: 5′-GAC GAT GAG TCC
TGA G-3′ at 37 °C for 4 h. The ligated fragments were amplified by PCR using the
Mse-N (5′-GAT GAG TCC TGA GTA AN-3′) as a primer, and the amplification
condition was performed by 23 cycles of 40 s at 94 °C, 50 s at the annealing 53 °C and
30 s at 72 °C, with a final extension time of 5 min at 72 °C. PCR products were selected
ranging from 300-800 bp according to a DNA standard marker and hybridized to two 5′
58
biotinylated probe (CA) 15 and (GA) 15
at room temperature for 30 min. The hybridized
59
60
61
62
63
fragments were captured by streptavidin-coated magnetic beads (Promega),
unhybridized DNA was washed away and the remaining DNA was eluted from the
beads. The eluted fragments were amplified again with the same PCR reaction
conditions as mentioned above, and the products were finally purified. The purified
DNA were cloned into pGEM-T Easy vectors (Promega) at 4 °C overnight, and then
3

Molecular Ecology Resources
Page 4 of 10
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
transformed into JM109 competent cells. Transformed cells grew at 37 °C for 16 h on
an LB agar plate containing ampicillin (100 mg/mL), X-gal and IPTG for blue/white
selection.
A total of 67 clones were sequenced and 56 contained microsatellites with over five
repeats. Sixteen primer sets were designed using PRIMER 3 (Rozen & Skaletsky 2000)
and synthesized (Invitrogen). The forward primer of each set primers was synthesized
with a modified 19 bp M13 tail (f-29: 5′-CACGACGTTGTAAAACGAC-3′) ( LI-COR)
added to the 5′-end. The PCR amplifications were optimized using a PTC-200
Thermocycler (MJ Research) and performed in a 20 µL reation mixture, including 10
µL Ex Taq DNA polymerase premix buffer (Takara) , and about 50 ng of DNA template,
0.8 µM of each primer, and 0.5 pmol of fluorescent labeled primer (IRDye700 or
IRDye800) (LI-COR). The conditions for amplification were 8 min at 94 °C followed
by 34 cycles of 94 °C for 40 s, 50 s at the annealing temperature (Table 1) and 72 °C for
30 s, and finally followed by 8 min at 72 °C. PCR products were electrophoresed on
6.5% polyacrylamide gels using a LI-COR 4300 DNA analyzer. Allele sizes were
determined by comparison to a standard size ladder (LI-COR) and then analyzed using
LI-COR SAGA GT software.
Allelic variation at each microsatellite locus was assessed by genotyping 28
specimens, which were collected from the coastal area of Qidong (QD, E121°36′,
N32°10′), Jiangsu province, China. Genomic DNA was extracted with a genomic DNA
kit (Axygen). Eleven of the 16 microsatellite loci were polymorphic. The alleles number
4

Page 5 of 10
Molecular Ecology Resources
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
(N A ) at each polymorphic locus, polymorphic information content (PIC) and observed
and expected heterozygosities at each polymorphic locus were calculated using
CERVUS 2.0 software (Marshall et al. 1998). The observed heterozygosities (H O ) were
between 0.154 and 0.773 (Table 1). Nine of the eleven loci were highly polymorphic
(PIC > 0.5). GENEPOP 3.4 software (Raymond & Rousset 2004) was used to test both
Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium (LD). Four loci were
found to depart from HWE and LD was not identified between any pairs of loci (P
>0.05). We used MICROCHECKER software (van Oosterhout et al. 2004) to check
microsatellite data for null alleles and scoring errors. Analysis of the microsatellite
dataset indicated significant deficits of heterozygotes present at the four loci that were
out of HWE, and the most probable cause of these were null alleles. From the results of
previous studies on bivalve, e.g., zebra mussel Dreissena polymorpha (Astanei et al.
2005), pacific oyster Crassostrea gigas (Hedgecock et al. 2004), eastern oyster C.
virginica (Reece et al. 2004; Carlsson et al. 2006), and bay scallop Argopecten
irraidians (Zhan et al. 2007), null alleles also seem to be a explanation for heterozygote
deficiencies. In addition, multiple pairwise comparisons were determined using the
sequential Bonferroni method (Rice, 1989). After the correction, departures from HWE
was observed only in WG604 (P < 0.05/11).
The result obtained in this study revealed that most of the microsatellites developed
here are highly polymorphic, and the genetic diversity of wild population was at high
levels according to the PIC. This information will be essential to acquaculture
5

Molecular Ecology Resources
Page 6 of 10
106
107
108
109
development and management of genetic resources of M. meretrix. The microsatellites
should be useful tools for a better understanding of genetic population structure in the
wild, the conservation and maintenance of wild populations as well as aiding in
management of genetic breeding programs.
6

Page 7 of 10
Molecular Ecology Resources
110
111
112
Table 1 Characteristics of the eleven polymorphic microsatellite loci isolated from Meretrix meretrix.
Locus Accession no. Repeat motif Primer sequence (5′−3′) Size(bp) Ta(°C) N A H O H E PIC
WG307 FJ232978 (CA) 17 F: AGCTACGCTATACTCCACACTC 182-220 58 8 0.741 0.843 0.808
R: GGACCAGACACAATGTATAAACT
WG312 FJ232979 (TG) 8 F: GTCACAGGGACTCAAAGAT 239-281 58 7 0.571* 0.790 0.747
R: ATCAAAATAGAATAACTAGATGT
WG323 FJ232980 (GT) 27 F: GCCATAACAGTCCTACAAACGG 214-262 60 7 0.621* 0.842 0.805
R: CGATCAATGATAGGCGTTCAAAG
WG335 FJ232981 (CA) 17 F: ATAACTCTCCCACTCTAAAACTC 214-240 52 10 0.679 0.829 0.790
R: TAGAATGGGAGCATGTGATAG
WG337 FJ232982 (GT) 35 F: TCATGGCATTGTTTTAGAAGG 134-212 52 12 0.517* 0.764 0.730
R: TTTATCGTTGTGATTGACAGTTC
WG347 FJ232983 (GCGT) 8 (GT) 25 F: GTGATTCCAATATAGCTTCCCC 206-216 58 4 0.321 0.486 0.420
R: GCACATAGGCAAACAATAGACAG
WG604 FJ232984 (TC) 8 G(TC) 22 F: TTATAGGTCACCGCCATTTACTG 309-329 58 6 0.154* 0.837 0.783
R: AATCCACATCATAGACAAACGCT
WG607 FJ232985 (GA) 14 F: ATGAATCTTGAAAACTCTGGGTG 260-274 60 5 0.538 0.691 0.618
R: AATCAAATGTTTCCTGGCGG
WG609 FJ232986 (TACC) 7 F:TAAAGTTTGGGCAGACACTCGGT 185-247 59 8 0.621 0.569 0.531
R: CGTTTCCAAGCAACAGGGCATAC
WG610 FJ232987 (CT) 23 F: TCATTGCTTAGTATTGGTTGGTC 224-262 62 9 0.448 0.563 0.493
R: TCGATTGCGAAACAAGTTCTAAC
WG647 FJ232989 (CT) 10 F: ATTGACAATGATAATTCGGTGTG 116-336 51 16 0.773 0.835 0.805
R: ACCTTGGTATGATAGTTGCGG
N A is number of alleles, Ta is annealing temperature, H O and H E are observed and expected heterozygosities, and PIC is polymorphic
information content, asterisks denote significant deviation form Hardy-Weinberg Equilibrium (P

Molecular Ecology Resources
Page 8 of 10
113
114
115
116
117
Acknowledgements
We are grateful to Dr. Lang Chen, Dr. Zi-wei Zhang and Mr. Hui-rong Wan for
technical guidance and assistance in the laboratory. We are very grateful to the
Subject Editor for her efforts in improving the manuscript.
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
References
Astanei I., Gosling E., Wilson J. & Powell E. (2005) Genetic variability and
phylogeography of the invasive zebra mussel, Dreissena polymorpha (Pallas).
Molecular Ecology, 14 (6), 1655-1666.
Carlsson, Morrison & Reece (2006) Wild and aquaculture populations of the eastern
oyster compared using microsatellites. Journal of Heredity, 97(6), 595-598.
Chen D.P., Shen H.S., Ding Y.P.,Yang J.X., Shen S.D. & Xu P. (2004) Using
inter-simple sequence repeats(ISSR)technique in two populations of Meretrix
meretrix. Journal of Nanjing Normal University (Natural Science), 27(3), 74-77.
Cheng H.L., Xia D.Q., Wu T.T., Meng X.P., Ji H.J., Dong Z.G. & Chen S.Y. (2007)
Sequence analysis of mitochondrial COI gene fragment of six Veneridae clams
(Mollusca : Bivalvia) and four populations of Meretrix meretrix. Acta
Oceanologyca Sinica, 29(5), 109-116.
Du X.D., Deng Y.W., Ye F.L. & Wang H. (2004) Genetic diversity of seven wild
populations of Meretrix meretrix. Journal of Fishery Sciences of China, 11(1),
41-47.
8

Page 9 of 10
Molecular Ecology Resources
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
He C.B., Cong L.L., Ge L.L., Liu W.D., Zhou Z.C. & Gao X.G. (2008) AFLP
analysis of cultured and wild hard clam (Meretrix meretrix) populations. Journal of
Fishery Sciences of China, 15 (2), 215-221.
Hedgecock D., Li G., Hubert S., Bucklin K. & Ribes V. (2004) Widespread null alleles
and poor cross-species amplification of microsatellite DNA loci cloned from the
Pacific oyster, Crassostrea gigas. Journal of Shellfish Research, 23, 379-385.
Marshall T.C., Slate J., Kruuk L.E.B. & Pemberton J.M. (1998) Statistical confidence
for likelihood-based paternity inference in natural populations. Molecular Ecology,
7, 639-655.
Raymond M. & Rousset F. (2004) Genepop (version 3.4): population genetics
software for exact tests and ecumenicism. Available at: http://wbiomed.curtin.edu.
au/genepop/. html.
Reece K.S., Ribeiro W.L., Gaffney P.M., Carnegie R.B. & Allen S.K. Jr (2004)
Microsatellite marker development and analysis in the eastern oyster, Crossaster
virginica: confirmation of null alleles and non-Mendelian segregating ratios.
Journal of Heredity, 95, 255-361.
Rice W.R. (1989) Analyzing tables of statistical tests. Evolution, 43, 223-225.
Rozen S. & Skaletsky H.J. (2000) Primer3 code available at http://frodo.wi.mit.edu/
primer3 /primer3_code.html. In: Bioinformatics Methods and Protocols: Methods
in Molecular Biology (ed. by S. Krawetz & S. Misener), pp. 365-386. Humana
Press, Totowa, NJ, USA.
van Oosterhout C., Hutchinson W.F., Wills D.P.M. & Shipley P.F. (2004) The
9

Molecular Ecology Resources
Page 10 of 10
157
158
159
160
161
162
163
164
165
166
167
168
169
University of Hull Micro-Checker User Guide. Available at: http://www.hull.ac.uk/
evolution/Software/ microchecker.html.
Wang M.Z., Chen H.C. & Chen X.L. (2006) Comparison of Several Meretrix
meretrix Culture Models in Ponds. Fisheries Science, 25(8), 417-419.
Wang R.C., Wang Z.P. & Zhang J.Z. ( 1998) Marine mussel culture, pp. 318-329.
Ocean University of Qingdao Press, Qingdao, Shandong, China.
Zane L., Bargelloni L. & Patarnello T. (2002) Strategies for microsatellite isolation: a
review. Molecular Ecology, 11, 1-16.
Zhan A.B., Bao Z.M., Hui M.,Wang M.L., Zhao H.B., Lu W., Hu X.L. & Hu J.J.
(2007) Inheritance pattern of EST-SSRs in self-fertilized larvae of the bay scallop
Argopecten irradians. Annales Zoologici Fennici, 44, 259-268.
Zhang A.G., Li T.W. , Su X.R. & Liu B.Z. (2005) Current Status and Prospect of
Meretrix meretrix Culture. Fisheries Science, 24(2), 31-33.
10