Molecular Ecology Resources (2009)

doi: 10.1111/j.1755-0998.2008.02410.x

PERMANENT GENETIC RESOURCES
Blackwell Publishing LtdIsolation of eight microsatellite markers from Moina
macrocopa for assessing cryptic genetic structure in the wild
H A R U K I TAT S U TA ,* I Z U M I YA O † and Y O S H I N A R I TA N A K A **Research Center for Environmental Risk, National Institute for Environmental Studies, Onogawa, Tsukuba, Ibaraki 305-8506, Japan,†21st COE Program (Neoscience of Natural History), Graduate School of Science, Hokkaido University, Kita-ku, Sapporo, Hokkaido
060-0810, JapanAbstract
We isolated eight polymorphic microsatellite loci from the zooplankton Moina macrocopa(Straus), which is sensitive to pollutants such as insecticides and heavy metals. The isolatedloci were polymorphic, with three to seven alleles among 23 individuals. Expected heterozygosities ranged from 0.167 to 0.787. These loci can be used to examine cryptic genetic
structure and to infer the connectivity among metapopulations.
Keywords: genetic structure, littoral, magnetic particles, Moina macrocopa, Moinidae
Received 28 July 2008; revision accepted 7 September 2008

Moina macrocopa (Straus) is a littoral zooplankton that is
distributed in ponds and rice fields in Asia (Chuah et al.
2007). Research on the species has mainly centred on ecotoxicological studies for hazard assessment of agrochemicals
or heavy metals. Because such hazardous chemicals do not
generally show homogeneous distribution in water bodies,some variation in resistance against these chemicals can beexpected among sites because of different levels of selection
pressure for the resistance.
Widely distributed zooplankton such as M. macrocopa
are suitable for assessing relationships between the levelsof resistance to chemicals and different water environment.
For such assessments, however, it is necessary to know theextent to which gene flow among metapopulations constrains
the evolution of resistance. Particularly in large ponds andlakes, it is difficult to infer the connectivity of metapopulations
and to find genetic discontinuities without a direct approach
using genetic markers. Accordingly, we identified polymerase
chain reaction (PCR) primers for the amplification of eight
loci, which can be used for acquiring the necessary population
genetic parameters. These primers may also be useful inother closely related Moina species.
We constructed an enriched library based on the modified
method of Fischer & Bachmann (1998). Samples of M.
Correspondence: Haruki Tatsuta, Laboratory of Subtropical Zoology,
Graduate School of Agriculture, University of the Ryukyus,
Nishihara, Okinawa 903-0213, Japan. Fax: +81 (0)98 895 8734,
E-mail: htatsuta@agr.u-ryukyu.ac.jp© 2009 The Authors
Journal compilation © 2009 Blackwell Publishing Ltd

macrocopa were collected from Lake Kasumigaura and its
vicinity, Ibaraki, central Japan. Immediately after thecollection, we established isofemale lines from each adultindividual and reared them in plastic containers until needed.
Ten individuals were randomly chosen from each containerand genomic DNA was extracted from whole bodies using
the QIAamp DNA Mini Kit (QIAGEN). Five microgramsof genomic DNA were digested with 50 U of Sau3AI(TaKaRa), and fragments were ligated to Sau3AI cassettes(TaKaRa). PCR amplification was carried out with CassettePrimer C1 (TaKaRa) in a GeneAmp 2720 Thermal Cycler(Applied Biosystems). The PCR products were resolved inbinding buffer (10 mm Tris-HCl, 1 mm EDTA, 100 mm NaCl,pH 7.5) and hybridized to 5′ biotin-labelled oligonucleotide
probes (CA)10 after denaturation. The hybrids were subsequently isolated by binding them to Streptavidin
Magnetic Particles (Roche). After rinsing the particles inwashing buffer (10 mm Tris-HCl, 1 mm EDTA, 1 m NaCl,
pH 7.5), target DNAs were recovered by resuspending theparticles in elution buffer (6 m guanidine-HCl). The obtainedfragments were then amplified by PCR and digested with
Sau3AI to remove the cassettes. The enriched fragments
ranging in size from 300 to 1000 bp were ligated into BamHIcut pUC118 (TaKaRa) and transformed into competentEscherichia coli cells (Competent High-DH5α, Toyobo).
A total of 960 recombinant colonies were chosen andsuspended independently in 20 μL of TE (10 mm Tris-HCl,1 mm EDTA, pH 8). Inserts were amplified by PCR using
an M13 primer pair for the multicloning site of pUC18.

2 PERMANENT GENETIC RESOURCES
Table 1 Characteristics of the eight microsatellites: repeat motif, number of alleles (Na), size range in PCR product, primer sequences, locusspecific annealing temperature (Ta), observed (HO) and expected (HE) heterozygosities and results of the test for Hardy–Weinberg (HW)
proportions.

Locus

Repeats

NaSize range
(bp)

MM-01

(GT)4 ... (GGA)3

4200–220

MM-02(GCT)6

7

230–248

MM-03(GT)4

4

158–180

MM-04(GTC)53

195–201

MM-05(CA)57220–238

MM-06

3

164–170

MM-07(GCA)3 ... (TA)3(CA)3A(CA)3 ... (GT)6(TTG)5

7220–240

MM-08(CT)55155–169

Primer sequences (5′–3′)Ta (°C)HOHE

HW
P values

GenBank
Accession no.

GAATTTCACAGTCCGCCAATGGCTCCCCTATTTCCATCTC
AGACGAGAGAGCCCGAAAAGCATGCGTCACTCGTACACTTCAGTGTCAATGGCCTGAAATTGCTTTGAGGTTCGTTCAATATACGTCGAGCAGCCAGTG
CGTGTTACCATCTCCCGTTACGCCGGTAACTAGGTCAGAAGTTGGCAATTTTTCGTCGATAGCGGCGAAGAAGAGTAACACGTTGGCCTAAACACTTTGC
ACCTCTCGTCTCGTCTCGTC
CCTTTACTCGAGGGCCGTATCAACGGGCAAGAAAGAACATAACGAACGAAACAGATGTTGG

590.1740.167

1.0000AB449261

600.3480.636

0.0002

AB449262

60

0.261

0.310

0.0370AB449263

59

0.565

0.530

1.0000AB449264

60

0.652

0.777

0.0064

AB449265

60

0.087

0.414

< 0.0001

AB449266

60

0.087

0.787

< 0.0001

AB449267

60

0.783

0.673

0.1852

AB449268

After PCR, 1 μL of each amplified product was dropped
separately on a positively charged nylon membrane (Roche).
After drying at room temperature, DNAs on the membrane
were blotted by normal alkaline transfer. PCR products
containing microsatellite regions were detected using adigoxigenin (DIG) nucleic acid detection kit (Roche) and5′ DIG-labelled oligonucleotide probes mentioned above.
Sixty-seven of 960 clones were positive. For positive clones,
1 μL of the remaining PCR product was checked for its
length on 10% polyacrylamide gels in TBE. There were 17
unique inserts (length 300–1000 bp). Inserts of the positive
recombinants were amplified independently using the M13
primers and sequenced using BigDye version 3.1 cycle
sequencing kit (Applied Biosystems) and an automatedsequencer (ABI 3730, Applied Biosystems). There were ninedifferent loci in these positive clones and the nine primer
pairs were designed using the online primer design software
Primer 3.0 (Rozen & Skaletsky 1998).
To test the effectiveness of the obtained microsatellite loci,we checked amplification and polymorphism of the primer
pairs in 23 individuals of M. macrocopa collected from a
locality of Lake Kasumigaura in Ibaraki Prefecture, central
Japan. The PCR amplifications were performed on a GeneAmp 2720 Thermal Cycler (Applied Biosystems) in 10-μL
reaction volumes containing 1 μL (50 ng) of template DNA,0.5 pmol of each microsatellite primer, 1 μL of 10× reaction
buffer [20 mm/L Tris-HCl (pH 8.0), 100 mm/L KCl, 0.1 mm/
L EDTA, 0.5% Tween20, 0.5% Triton X-100, 1 mm/L DTT,50% glycerol], 1.5 mm/L MgCl2, 200 mm/L dNTP, 0.05 Uof Gene Taq NT (Nippon Gene), and 6.15 μL of double-

distilled water. Temperature cycles were as follows: 2 min
at 94 °C; 30 cycles of 30 s at 94 °C, 30 s at 50–55 °C, 90 s
at 65 °C. After optimization of PCR conditions, forwardprimers were labelled with TaqMan Dye fluorescence(Applied Biosystems). To determine allele sizes, fragment
analysis was performed on an automated sequencer (ABI3730, Applied Biosystems). A LIZ size standard (AppliedBiosystems) was run with samples to calculate fragment
size. Eight primers successfully amplified the target regions
across the 23 individuals. Of these eight loci, all showedpolymorphism, with three to seven alleles per locus (Table 1).
We tested for genotype frequency deviations from
Hardy–Weinberg equilibrium for each locus among thepolymorphic loci using GenePop on the Web (Raymond &Rousset 1996). A significant deviation was observed at fiveof eight loci (P < 0.05, Hardy-Weinberg exact tests withBonferroni correction for multiple comparisons). This species
is parthenogenetic that may explain the deviation from
Hardy—Weinberg equilibrium. Null allele frequencies
were also estimated by the method of Brookfield (1996).
Their significance was tested by probability test using MicroChecker software (version 2.2.3; van Oosterhout et al. 2004).
Significant evidence of null alleles (P < 0.05, probabilitytest) was found for three of eight loci (MM-02, MM-06 andMM-07). There was no evidence of linkage disequilibrium
for all loci (P > 0.05, Fisher’s exact probability test withBonferroni correction for multiple comparisons). We conclude that the markers are useful for searching cryptic
population structure and for inferring the levels of geneflow between metapopulations of M. macrocopa.
© 2009 The Authors
Journal compilation © 2009 Blackwell Publishing Ltd

PERMANENT GENETIC RESOURCES 3

Acknowledgements
We thank Takehiko Hayashi, Hiroyuki Mano, and Masaki Sakamoto
for assistance of collecting materials. Thanks are also due to Tomomi
Sawabe, Fumika Shimokoube, Midori Sugimoto, Mie Shibuya,and Keiko Ikeda for rearing specimens. This study was supportedin part by a Grant-in-Aid for Scientific Research (No. 16770069 forH.T.) and by a 21st Century Center of Excellence Program on‘Neo-Science of Natural History’ at Hokkaido University (Leader, H.
Okada), financed by the Japan Society for the Promotion of Science.

References
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© 2009 The Authors
Journal compilation © 2009 Blackwell Publishing Ltd

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