TECHNICAL NOTE
Isolation and characterization of 13 microsatellite loci
in Rhinolophus pusillus (least horseshoe bat)
with cross-amplification in five related species
Panyu Hua Æ Tingting Guo Æ Wenchao Liu Æ
Shuyi Zhang Æ Stephen J. Rossiter
Received: 5 April 2008 / Accepted: 12 April 2008 / Published online: 29 April 2008

Springer Science+Business Media B.V. 2008
Abstract We used the enriched genomic library method
to isolate and characterize dinucleotide microsatellite loci
in the least horseshoe bat, Rhinolophus pusillus. Seventeen
loci were obtained and tested on 31 individuals sampled
from Guangxi Province in southern China. Thirteen of
these markers were polymorphic with expected heterozy-
gosity ranging from 0.821 to 0.909. A total of 164 alleles
were detected and the number of alleles per locus ranged
from 9 to 16 (mean 12.6). These polymorphic markers will
be used to assess population structure in R. pusillus. In
addition, successful cross-amplification in five congeneric
bat species suggests most of these markers will also be
useful for studying related species.
Keywords Microsatellite loci
Rhinolophus pusillus

Genetic structure
Introduction
The least horseshoe bat, Rhinolophus pusillus, occurs
across India, Nepal, Thailand, Myanmar, Laos, South
China, Peninsular Malaysia, and Indonesia (Simmons
2005) and might also be conspecific with taxa in Taiwan
and Japan (Li et al. 2006). It roosts in moist and dark caves
and, like many cave roosting bats, has suffered from
declines in suitable roosting sites and foraging habitats.
Here, we describe the development of a set of microsat-
ellite markers in this species, which will be used to assess
population structure.
We collected tail membrane tissue using a dermatological
biopsy punch (Stiefel Laboratories Limited, Buckingham-
shire, UK) from several individuals of R. pusillus from
Guilin in Guangxi Province (South China). Tissue was stored
in 95% ethanol and genomic DNA was extracted with
DNeasy Tissue Kits (Qiagen). We constructed a partial
genomic DNA library enriched for AC dinucleotide repeat
units based on Kandpal et al. (1994) and Karp et al. (1998)
with some additional modifications (Hua et al. 2007).
Briefly, DNA from five individuals was pooled and digested
with the restriction enzyme MboI (TaKaRa). Digested
fragments were run out on an agarose gel and those between
400 and 800 bp were cut out and recovered with a DNA
Purification Kit (Tiangen). Purified fragments were ligated
to a blunt-ended linker (adapter A: GCGGTACCCGGG
AAGCTTGG; adapter B: GATCCCAAGCTTCCCGGGTA
CCGC) with T4 DNA ligase (Takara) at 16C for 16 h, after
which, the ligation products were amplified by polymerase
chain reaction (PCR) with an annealing temperature of 67C
and using the adapter A sequences as primers. Purified PCR
products were denatured and hybridized with a biotin-
labeled dinucleotide repeat (CA)15 probe (Sangon) in
sodium phosphate buffer (0.5 M sodium phosphate, 0.5%
SDS, pH 7.4) at 50C for 15 h. The hybridization mixture
was incubated with VECTREX Avidin D (Vector Labora-
tories) and washed with TBS buffer (100 mM Tris, 150 mM
NaCl, pH 7.5) four times at different temperatures to remove
unbound fragments (37C for 30 min, 55C for 30 min, 65C
for 30 min, and 65C for 30 min in 0.19 TBS buffer). The
bound fragments were eluted with dH2O, amplified by PCR
and ligated into a PMD-19-T vector (TaKaRa), which was
P. Hua
T. Guo
W. Liu
S. Zhang
School of Life Science, East China Normal University,
Shanghai 200062, China
S. J. Rossiter (&)
School of Biological and Chemical Sciences, Queen Mary,
University of London, London E1 4NS, UK
e-mail: s.j.rossiter@qmul.ac.uk
123
Conserv Genet (2009) 10:597–600
DOI 10.1007/s10592-008-9586-1

T
ab
le
1
D
et
ai
ls
of
13
m
ic
ro
sa
te
ll
it
e
lo
ci
fo
r
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.
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si
ll
us
L
oc
us
Pr
im
er
se
qu
en
ce
(5
0 –
30
)
C
or
e
se
qu
en
ce
s
T
a
(
C
)
N
um
be
r
of
al
le
le
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Si
ze
ra
ng
e
of
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le
le
s
(b
p)
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E
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O
G
en
B
an
k
ac
ce
ss
io
n
nu
m
be
r
A
4
F:
G
A
T
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A
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A
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C
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T
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(T
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)2
1
56
16
14
8–
19
8
0.
88
6
0.
90
3
E
F4
23
56
0
R
:
T
C
A
G
T
A
A
G
C
C
C
T
G
T
T
T
A
T
T
C
T
C
PH
30
F:
C
T
G
T
T
T
G
G
T
T
G
G
C
A
G
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T
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A
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(T
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)1
4
56
11
16
4–
19
0
0.
82
7
0.
87
1
E
F4
23
56
1
R
:
C
A
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A
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A
C
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T
PH
78
*
F:
T
T
T
C
C
T
T
G
C
T
T
T
C
C
C
A
T
C
C
(A
C
)1
9
59
9
23
3–
25
9
0.
83
9
0.
41
7
E
F4
23
56
2
R
:
G
C
A
G
T
C
G
G
C
T
G
G
A
G
G
A
T
T
A
PE
22
F:
T
G
T
G
A
A
G
G
G
A
A
G
A
T
T
A
G
A
A
G
(G
T
)2
0
55
16
15
5–
19
3
0.
90
2
0.
91
7
E
F4
23
56
4
R
:
A
A
A
C
T
G
T
C
C
A
C
T
T
G
T
A
G
A
C
A
T
PD
3
F:
T
C
T
G
T
G
G
G
A
G
G
A
A
G
G
G
A
T
G
A
(A
C
)1
4
57
12
21
0–
26
0
0.
82
1
0.
74
2
E
F4
23
56
5
R
:
G
T
G
T
T
G
A
T
G
G
G
A
C
A
G
T
G
G
G
T
T
PD
9*
F:
C
T
C
A
T
C
T
T
C
C
C
G
T
T
C
G
C
A
T
A
A
(A
C
)1
1
59
13
26
0–
29
6
0.
88
6
0.
32
1
E
F4
23
56
6
R
:
G
A
C
A
A
C
C
G
C
A
G
A
C
A
G
G
A
C
A
A
T
PH
96
*
F:
T
T
G
G
A
C
A
C
G
C
A
G
G
G
C
A
G
A
C
(A
C
)1
3
65
15
21
3–
24
7
0.
91
8
0.
57
7
E
F4
23
56
8
R
:
G
C
T
A
G
G
A
G
C
G
G
G
C
A
G
A
A
A
C
PH
57
*
F:
G
G
C
C
A
G
T
C
T
G
T
C
T
A
A
C
G
C
T
(G
T
)1
7
61
10
24
5–
27
1
0.
83
1
0.
23
1
E
F4
23
56
9
R
:
A
A
G
T
G
C
T
G
A
T
G
C
C
C
G
A
A
C
C
H
3
F:
A
C
A
G
G
G
A
A
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A
T
A
A
A
T
A
T
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A
G
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)1
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)4
57
16
25
8–
30
8
0.
85
7
0.
87
1
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F4
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0
R
:
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G
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G
T
T
T
A
C
T
G
G
T
T
A
G
G
G
A
C
A
10
A
F:
C
A
T
A
T
A
G
A
G
C
A
C
A
C
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A
A
G
A
G
(A
C
)1
7
54
11
18
9–
21
5
0.
87
6
0.
90
3
E
U
55
92
47
R
:
T
T
C
C
T
G
C
C
T
C
G
T
G
T
C
C
A
A
E
11
A
F:
T
T
C
C
T
G
T
T
G
T
G
G
A
A
G
A
G
C
T
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(G
T
)1
7
60
9
10
6–
12
6
0.
84
4
0.
80
0
E
U
55
92
48
R
:
G
T
C
T
G
A
C
T
C
T
C
T
G
T
A
A
G
G
C
T
G
G
PH
69
A
F:
G
T
A
G
C
A
A
A
C
C
A
C
G
G
T
C
T
C
A
G
(T
G
)1
7
56
11
10
1–
13
9
0.
82
3
0.
79
2
E
U
55
92
49
R
:
A
G
G
C
G
A
A
G
G
C
A
C
T
T
A
A
T
C
A
A
PE
1A
*
F:
C
A
A
T
G
G
A
A
C
A
G
A
G
A
T
G
G
A
G
C
A
G
(T
G
)1
6(
A
G
)1
3
60
16
14
9–
18
1
0.
90
0
0.
71
4
E
U
55
92
50
R
:
A
T
T
A
C
G
G
C
T
G
C
C
T
T
G
C
T
T
G
T
F,
fo
rm
er
pr
im
er
(5
0 -
la
be
le
d
w
it
h
FA
M
,
T
A
M
R
A
,
or
H
E
X
);
R
,
re
ve
rs
e
pr
im
er
;
T
a,
an
ne
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in
g
te
m
pe
ra
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;
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O
,
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se
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te
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;
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pe
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te
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ni
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(P
\
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fr
om
H
ar
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ei
nb
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eq
ui
li
br
iu
m
598 Conserv Genet (2009) 10:597–600
123

used to transform Escherichia coli DH5a competent cells
(Tiangen).
Eighty-three positive clones were identified from 138
white colonies via PCR with Adapter A primers. After
sequencing with M13 universal primers (Invitrogen), 46
sequences contained AC/TG repeats, of which 32 were
chosen for primer design with Primer3 (Rozen and Ska-
letsky 2000). Seventeen primer pairs successfully amplified
genomic DNA of R. pusillus and 13 pairs showed high
levels of polymorphism.
To quantify levels of polymorphism, we used a panel of
31 individuals of R. pusillus. PCRs were carried out on a
PTC-220 thermal cycler (Bio-Rad) with the following
profile: 95C for 15 min; 30 cycles of 30 s at 94C, 30 s at
the annealing temperatures (Table 1) and 30 s at 72C; final
extension step of 20 min at 72C. Each PCR mix (volume
15 ll) contained *100 ng genomic DNA, 0.2 lM 50-
labeled forward primer (FAM, TAMRA, or HEX, see
Table 1), 0.2 lM unlabeled reverse primer, 0.2 mM of each
dNTP, 0.2 U Hotstar Taq DNA polymerase (Qiagen) and
19 PCR buffer containing 1.5 mM MgCl2. PCR products
were genotyped an ABI 3100 sequencer and alleles were
sized and analyzed using the software Genescan v3.7 and
Genotyper v3.6 (ABI). Estimates of expected and observed
heterozygosity and tests for deviation from both Hardy–
Weinberg equilibrium (HWE) and linkage equilibrium (LE)
were performed with either FSTAT v2.9.3.2 (Goudet 1995)
or Genetix v4.03 (Belkhir et al. 2004).
Based on 13 polymorphic loci, we recorded a total of
164 alleles with the number of alleles per locus ranging
from 9 to 16 (mean 12.6). Expected and observed values of
heterozygosity ranged from 0.82 to 0.91 and 0.23 to 0.92,
respectively. Five loci (PE1A, PH78, PD9, PH57, PH96)
showed evidence of departure from HWE (P \ 0.05), and
analysis with the program Micro-checker v2.2.3 (Van
Oosterhout et al. 2004) indicated that this is likely to be
attributable to null alleles. We found no evidence of link-
age disequilibrium between any pair of loci after
Bonferroni correction for multiple tests (Rice 1989).
The microsatellite markers reported here will be used in
our ongoing research on population and conservation
genetic studies of R. pusillus. Additionally, we assessed the
cross utility of these primers by attempting to amplify the
DNA of five congeneric species that also occur in China.
Eleven of the 13 markers successfully cross-amplified at
least one of these congeneric species, with many markers
yielding amplification products in several species (see
Table 2). We conclude that these markers are likely to
prove a useful resource in population genetic studies in
other horseshoe bats and add to the existing markers
available for this speciose genus (Rossiter et al. 1999;
Dawson et al. 2004; Puechmaille et al. 2005).
Acknowledgments We are grateful to Jinshuo Zhang, Libiao
Zhang, Guangjian Zhu, and Li Wei for assistance with sample col-
lection. This work was funded by a grant under the Key Construction
Program of the National ‘‘985’’ Project and Shanghai Priority Aca-
demic Discipline awarded to SZ. SJR was supported by a Royal
Society Research Fellowship.
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Table 2 Cross-species amplification of 13 polymorphic loci based on R. pusillus in related taxa
Species N A4 PH30 PH78 PE22 PD3 PD9 PH96 PH57 H3 A10A E11A PH69A PE1A
R. macrotis 5 5+, 0- 1+, 4- 3+, 2- 5+, 0- 5+, 0- 0+, 5- 0+, 5- 5+, 0- 5+, 0- 4+, 1- 0+, 5- 5+, 0- 4+, 1-
R. affinis 5 5+, 0- 5+, 0- 4+, 1- 0+, 5- 5+, 0- 0+, 5- 0+, 5- 4+, 1- 5+, 0- 0+, 5- 0+, 5- 5+, 0- 4+, 1-
R. pearsonii 5 4+, 1- 5+, 0- 3+, 2- 5+, 0- 5+, 0- 0+, 5- 0+, 5- 0+, 5- 5+, 0- 4+, 1- 3+, 2- 5+, 0- 3+, 2-
R. ferrumequinum 5 5+, 0- 5+, 0- 4+, 1- 0+, 5- 5+, 0- 0+, 5- 0+, 5- 4+, 1- 5+, 0- 5+, 0- 0+, 5- 4+, 1- 0+, 5-
R. sinicus 4 4+, 0- 0+, 4- 1+, 3- 4+, 0- 4+, 0- 0+, 4- 0+, 4- 0+, 4- 0+, 4- 4+, 0- 0+, 4- 4+, 0- 4+, 0-
+, exists specific PCR products; -, no PCR products or products not specific; N, number of individuals tested
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