1151

Development and Isolation of 17 polymorphic Microsatellite 
Loci in Coptoterms formosanus (Isoptera: Rhinotermitidae)

by

Bing-rong Liu1,3, Jun-hong Zhong2*, Ming-fang Guo2, Zhi-qiang Li2                            
& Wen-hui Zeng2

ABSTRACT

  Seventeen polymorphic microsatellite DNA loci for Copototermes formo-
sanus were isolated and characterized. Polymorphism of these loci was assessed 
in a sample of 32 unrelated C. formosanus individuals. An average of 4.6 alleles 
per locus (3-8 alleles) was detected. Observed and expected heterozygosities 
ranged from 0.2500 to 1.0000 and from 0.5591 to 0.8562, respectively. Six 
loci were found to have deviated from Hardy-Weinberg equilibrium in the 
sampled population after Bonferroni correction. No significant linkage dis-
equilibrium was detected. These markers will be useful in population genetics, 
phylogenies and other relevant studies of C. formosanus.

Key words: Coptotermes Formosanus, Microsatellite loci, Polymorphism 

INTRODUCTION

The Formosan subterranean termite (FST) Coptotermes formosanus Shiraki 
(Isoptera: Rhinotermitidae) is a major economic pest worldwide. It is thought 
to have originated in China, but has spread to many areas around the world 
where it is a highly destructive pest of wood structures (Su & Tamashiro 
1987). Recently, mitochondrial DNA sequencing ( Jenkins et al. 2002; Austin 
et al. 2006; Fang et al. 2008) has been used to study the genetic diversity of 
C. formosanus, mainly in China, America and Japan, but this information is 
insufficient for investigating the genetic diversity and detailed spatial structure 
of these populations. Microsatellite DNA loci as polymorphic genetic markers 
are necessary for elucidating the details of colony organization, population 
structure, and relationships among introduced and native populations (Ross 

1South China Botanical Garden, The Chinese Academy of Sciences, Guangzhou 510650, China.
2Guangdong Entomological Institute, Guangzhou 510260, China. *Corresponding author, e-mail: 
zhong@gdei.gd.cn.
3Graduate School of the Chinese Academy of Sciences, Beijing 100049, China.



1152  Sociobiolog y Vol. 59,  No. 4, 2012

2001, Vargo et al. 2003). Although Vargo & Henderson (2000) identified 
12 microsatellite loci in C. formosanus, few subsequent studies have been 
performed. Therefore we isolated and characterized 17 polymorphic micro-
satellite loci for C. formosanus following the protocol modified from Yu et al. 
(2010), so as for further study of population genetics, phylogenies and other 
relevant study of C. formosanus. 

MATERIALS AND METHODS

Total genomic DNA was extracted from the heads of twenty C. formosanus 
individuals, using the regular phenol-chloroform method as described by 
Zhang and Hewitt (1998). Approximately 4 ug high quality genomic DNA 
was digested with restriction enzyme Sau 3AI (Promega), and the size-selected 
DNA (400 - 900 bp) was isolated from an agarose gel using DNA purification 
kit (TaKaRa). Fragments were then ligated to a blunt-end adapter (SAULA: 
GCGGTACCCGGGAAGCTTGG, SAULB: GATCCCAAGCTTC-
CCGGGTACCGC) with T4 DNA ligase (Takara) at 16°C for 14h. The 
ligation products were amplified by polymerase chain reaction using the adapter 
SAULA as primers. After denaturation at 95°C for 10 min, microsatellite-
bearing amplified fragments were selected with the biotin-labeled (AC)

12 
and 

(AGG)
8
 (Sangon) oligonucleotide probes in sodium phosphate buffer (0.5 

M sodium phosphate, 0.5% SDS, pH 7.4) at 50°C for 16h. The target frag-
ments were amplified by PCR and the size-selected DNA (400 - 900 bp) were 
excised from agarose gel and recovered. These recovered DNA product were 
ligated with pMD19-T vector (TaKaRa) and transformed to E. coli DH5a 
competent cells (TaKaRa). Eighty-two positive clones were identified from 
196 recombinant colonies via PCR with the adapter SAULA as primers, and 
were sequenced with M13 primers in one direction.

Forty-eight sequences contained microsatellites, of which thirty-five pos-
sessed sufficient flanking sequences appropriate for primer design. The others 
had either insufficient or inappropriate flanking regions on one or both sides 
of their microsatellites. These thirty-five pairs of primers were designed by 
the Premier 5.0 program (PREMIER Biosoft International, Silicon Valley, 
USA), and synthesized by Sangon Biotech (Shanghai) Co., Ltd. 

A range of annealing temperatures (50 °C - 68 °C) were tested and the 



1153 Liu, B-R. et al. —Microsatellite Loci in Coptotermes formosanus

temperature producing the cleanest and strongest PCR product when ob-
served on an 1.5% agarose gel stained with Goldview was selected for PCR. 
After these optimization procedures, seventeen of the thirty-five primer sets 
designed amplified successfully and were used to assess polymorphism based 
on 32 unrelated C. formosanus individuals.

Following Genomic DNA extraction, PCR was performed in a volume of 
20 ul containing 50 - 100 ng of total DNA, 0.25 - 0.5 units of Taq polymerase 
(Tiangen, China), 19 PCR buffer, 1.0 - 2.0 mM MgCl

2
, 0.2 mM dNTPs, and 

0.2 - 1 uM of each primer. The PCR profile was as follows: initial denaturation 
at 95 °C for 3 min, followed by 30 cycles of 94 °C for 30 s, a primer-specific 
annealing temperature for 30 s and 72 °C for 40 s, with a final extension at 
72 °C for 10 min. The PCR products were checked by electrophoresis on 
8% non-denaturing polyacrylamide gel, and visualized with silver staining. 
Allele size was determined against a 50-bp DNA ladder (Tiangen, China) 
with software Gel-Pro Analyzer 4.5. Number of alleles, heterozygosity, test 
of Hardy–Weinberg equilibrium (HWE) and linkage disequilibrium (LD) 
were analyzed using GENEPOP4.0.6 (Raymond and Rousset 1995). 

RESULTS

Conditions and characteristics of the 17 loci are shown in Table 1. The 
number of observed alleles per locus ranged from 3 to 8. The expected and 
observed heterozygosity values ranged from 0.2500 to 1.0000 and 0.5591 
to 0.8562, respectively. Six loci (Copf02, Copf04, Copf09, Copf10, Copf13 
and Copf17) significantly deviated from Hardy-Weinberg equilibrium after 
Bonferroni correction (P<0.0029). No significant linkage disequilibrium was 
detected. We conclude that the markers obtained will be useful in population 
genetics, phylogenies and other relevant study of C. formosanus.

ACKNOWLEDGMENTS

We acknowledge the support of the National Natural Science Foundation 
of China (31172163) and the Guangdong Academy of Sciences Foundation 
for Excellent Young Scientists and Technicians (200901). And we are grateful 
to Dr. Yuchun Wu for revising the draft of the manuscript.



1154  Sociobiolog y Vol. 59,  No. 4, 2012
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1155 Liu, B-R. et al. —Microsatellite Loci in Coptotermes formosanus

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