Botanical Studies (2009) 50: 21-24.
*
Corresponding author: E-mail: choumasa@mail.cmu.edu.
t w.
INTRODUCTION
The genus Ajuga (Labiatae) comprises about 40 to
50 species all over the world. Ajuga taiwanensis Nakai
ex Murata, a perennial herb, is restrictedly distributed in
the Philippines, Ryukyus, and Taiwan (Hou, 1996). Due
to rapid socio-economic development, natural habitats
for the plants have been severely disturbed, resulting in
population fragmentation. Overexploitation is another
reason for the quick population decline. Species belonging
to the genus have been used as folk medicinal plants,
as anthelmintics against intestinal disorders, and as
antifungal, hypoglycaemic, antitumor, and antimicrobial
agents (Baytop et al., 1984; Wesnner et al., 1992;
Rodriguez-Hann et al., 1994). Ajuga taiwanensis is often
used for the treatment of hepatitis and hepatoma (Hou,
1996).
Microsatellites, or Simple Sequence Repeats (SSRs),
are DNA fragments of nuclear or organellar genomes
that consist of repeating units of 1-4 base pairs in length
(Turnpenny and Ellard, 2005). SSR fingerprints are co-
dominantly inherited and are often used as molecular
markers for population studies and conservation genetics
(Weising et al., 2005). In this study, A. taiwanensis
represents some invaluable natural resources. In enforcing
the conservation of the rare species, an investigation of
genetic diversity and population structure provides vital
information. In the study, we isolated eight microsatellites
from the Labiatae species and screened its population
structure.
MATERIALS AND METHODS
Genomic DNAs were obtained from ground leaf
tissue of A. taiwanensis (AJ) using a CTAB methodology
(Doyle and Doyle, 1987). Microsatellite markers in AJ
were isolated by beginning with a random amplified
polymorphic DNA-polymerase chain reaction (RAPD-
PCR) enrichment (Hsu et al., 2004; Huang et al., 2008).
This PCR isolation of microsatellite arrays (PIMA)
approach was proposed by Lunt et al. (1999). It exploits
the fact that the RAPD fragments contain microsatellite
repeats more frequently than genomic clones (Cifarelli et
al., 1995).
The RAPD-PCR amplification was performed in a
thermal cycler (Biometra) with a reaction mixture (50 £gL)
containing 20 to 100 ng DNA, 0.2 mM of each dNTP, 2
Isolation and characterization of microsatellite loci in
Ajuga taiwanensis Nakai ex Murata using PCR-based
isolation of microsatellite arrays (PIMA)
Tsurng-Juhn HUANG
1
, Yi-Yen CHEN
1,2
, Kuo-Hsiung WANG
2
, Tsung-Hsin HSIEH
3
, and Chang-
Hung CHOU
1,
*
1
Research Center for Biodiversity and Graduate Institute of Ecology and Evolutionary Biology, China Medical University,
Taichung, Taiwan 404
2
Department of Life Sciences, Cheng Kung University, Tainan, Taiwan 552
3
National University of Tainan, Graduate Institute of Biological Resources and Technology, Tainan, Taiwan 700
(Received April 15, 2008; Accepted July 31, 2008)
ABSTRACT.
Ajuga taiwanensis is a valuable herb for traditional Chinese medical treatment. In this study,
eight microsatellite loci from A. taiwanensis were isolated. The simple sequence repeat (SSR) markers were
screened in 15 samples of wild populations of A. taiwanensis, and nine samples from its sister A. bracteosa
and A. nipponensis. In A. taiwanensis, the number of alleles ranged from 3 to 11, and values of expected (H
E
)
and observed (H
O
) heterozygosity were 0.54253- 0.90575 and 0.0000-1.0000, respectively. All loci deviated
significantly from Hardy-Weinberg expectations due to the heterozygote deficiency, indicating a dramatic
loss of genetic polymorphisms in the restrictedly distributed species. The markers amplifying well in the
three species are useful for examining genetic diversity and population genetic structure, and this can provide
information for establishing a conservation strategy for these endangered species.
Keywords: Ajuga nipponensis, Ajuga taiwanensis, Heterozygosity; Microsatellite; PIMA.
MOLECULAR BIOLOgy
pg_0002
22
Botanical Studies, Vol. 50, 2009
mM MgCl
2
, 0.5 U Ta q polymerase (Violet), and 5 pmols
of one RAPD primer. The PCR program proceeded as
follows: initial denaturing 5 min at 94oC for 1 cycles,
35 cycles of 30 s at 94oC, 1 min at 42oC, 2 min at 72
oC, followed by 10 min at 72oC for additional extension
step. Several RAPD primers were used to amplify DNA
fragments from the genome of target species. PCR
products were size-selected to obtain small fragments
(ranging from 300 to 800 bp). DNA fragments were
ligated into pGEM T-Easy Vector System (Promega), and
the plasmids were transformed into DH5£\ Escherichia coli
competent cell. Clones were screened using microsatellite-
specific primers and two vector primers (Lunt et al., 1999).
In positive clones, PCR electrophoresis would show two
DNA fragments, of which one PCR product contains
microsatellite signal. In contrast, only the whole inserted
fragment could be found in negative clones. Extraction
of the positive plasmid DNA was conducted and purified
using the plasmid purification Mini Kit (Geneaid). Plasmid
DNA was sequenced in an Applied Biosystems Model
377A automated sequencer (Applied Biosystems).
Specific-primer pairs were designed according to
the nucleotide sequences upstream and downstream of
the repetitive DNA using software Primer3 (Rozen and
Skaletsky, 2000). PCR amplification was performed in a
25 £gL volume containing 10 ng of genomic DNA, 0.2 mM
dNTP, 2 mM MgCl
2
, and 5 pmols of each primer. PCR
programs took place as follows: initial denaturing step at
94oC for 5 min; 35 cycles of 94oC for 30 s, primer-specific
annealing temperature (Table 1) for 30 s, 72oC for 30 s, and
a final extension step at 72oC for 10 min. PCR products
were electrophorezed by ethidium bromide staining in
denaturing 8% polyacrylamide gels using 25-bp molecular
size ladder (Promega) as a standard to estimate allele
sizes. Results of the allele number, size range, number of
bands per individual are listed in Table 1. Expected (H
E
),
and observed (H
O
) heterozygosities were calculated using
the Arlequin program version 3.1 (Excoffier et al., 2005).
Testes of Hardy-Weinberg equilibrium (HWE) and linkage
disequilibrium (LD) were conducted using the GENEPOP
program version 3.4 (Raymond and Rousset 1995) ( http://
genepop.curtin.edu.au/).
RESULTS AND DISCUSSION
Ajuga taiwanensis materials were collected from
Nantou, Taichung, and Pingtung County in Taiwan. From
each population, samples were collected randomly from
four to six subpopulations.
In this study, eight novel
microsatellite loci in A. taiwanensis were isolated (Table
1) and tested in 15 individuals. The number of alleles per
locus ranged from 3 to 11. The expected (H
E
) and observed
(H
O
) heterozygosities ranged from 0.54253 to 0.90575 and
0.0000 to 1.0000, respectively. Significant departures from
HWE (Table 1) were detected in all microsatellite loci.
These deviations were
due to the deficit of heterozygotes
within populations (loci 3, 6, and 7), suggesting random
losses of genetic polymorphisms by genetic drift in
Table 1. Characteristics of eight microsatellite markers isolated from Ajuga taiwanensis Nakai ex Murata.
Locus Primer sequence ( 5¡¦ to 3¡¦ )
Repeat motif Size range
(bp)
Total
number of
alleles
Ta
(¢XC) H
O
H
E
HWE
p-value
AJ01 F: TCATGCCATCATTAATCAAA
(GT)
12
135~154 8 62 1 0.85287 <0.001
R: AACCTCAATCTGTGGCTTCT
AJ02 F: GGGAGGCGGAAACTGTTTGTGT
(TC)
4
(AC)
7
229~245 11 53 0.93333 0.90575 <0.001
R: TCTTTTACTTGCCCTTGCATTTCAGA
AJ03 F: TCTCACGCATTTTGAATGCAC
(AC)
9
N
17
(AC)
9
164~170 4 55 0 0.67126 <0.001
R: TGAATTAATGTGTGGATGCATGG
AJ04 F: ATTCGATTTGGTTGCCAGTT
(AT)
4
(AC)
8
183~235 11 58 0.93333 0.88046 <0.001
R: GGCGGAGTAGTGAAACACAA
AJ05 F: AGGCTGCTTGATTTCGCAAG
(TG)
n
180~204 4 52 0.93333 0.67126 <0.001
R: GCGCCTAACAGAGCCTAGT
AJ06 F: GGCCTCCTTGGTATGTAAGTTG
(AG)
7
TG(AG)
6
156~170 4 54 0.13333 0.5977 <0.001
R: GGCATGCCTGCACCAAATTC
AJ07 F: CCGGGCTGGTGATTCTTCTT
(AC)
6
(GC)
2
(AT)
3
110~114 3 53 0 0.54253 <0.001
R: GATGATTGCAAAGAGCGGGAAT
AJ08 F: GCCAAGCACCGTCGTCTAAA (CT)
6
GAA(C)
17
(AC)
8
162~214 6 53 0.73333 0.6919 <0.001
R: CGTGTGACTGCATTTTCATGG
AJ01~AJ08, isolated microsatellite locus; F:, forward primer; R:, reverse primer.
Ta, annealing temperature of the primer pair; H
O
, observed heterozygosity; H
E
, expected heterozygosity; HWE, Hardy-Weinberg
equilibrium.
pg_0003
HUANG et al. ¡X Microsatellite DNAs of
Ajuga taiwanensis
23
fragmented populations, or possibly due to the population
structuring within specimens (Wahlund effect).
For GENEPOP analysis of linkage disequilibrium (LD),
the results revealed no significant LD was discovered
in most loci with the exception of loci AJ02 and AJ04
(data not shown). The microsatellite primer pairs were
also applied to A. bracteosa, and
A. nipponensis. A ll of
isolated primer pairs from A. taiwanesis could cross-
species amplify microsatellite fingerprints in these two
sister species. The application of these microsatellite loci
in A. taiwanensis may therefore provide a useful tool for
understanding this species demography and population
structure during environmental change.
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