TECHNICAL NOTE

Isolation and characterization of polymorphic microsatellite lociin Ardisia crenata (Myrsinaceae)

Lan Hong Æ Hongyu Niu Æ Hao Shen Æ Wanhui Ye ÆHonglin Cao

Received: 3 June 2007 / Accepted: 8 June 2007 / Published online: 7 July 2007

� Springer Science+Business Media B.V. 2007

Abstract Ardisia crenata, an evergreen shrub native to

East Asia, has been a serious invasive plant to the south-

eastern USA. Here 13 polymorphic microsatellite loci were

isolated and characterized from an enrichment genomic

library of A. crenata. The average allele number of these

microsatellites was four per locus, ranging from two to

seven. The ranges of observed and expected heterozygosity

were 0.000–1.000 and 0.239–0.789, respectively. These

microsatellite markers will be useful for investigating

population genetics and reproductive ecology of A. crenata.

Keywords Microsatellite � Ardisia crenata � Population

genetics

Ardisia crenata Sims (Myrsinaceae), a woody evergreen

shrub, is naturally distributed in East Asia including China,

Japan, Korea and India. Ardisia crenata is an important

medicinal and ornamental resource throughout its distri-

bution range, enduring high levels of exploitation. How-

ever, the population biology and genetics of A. crenata

have not been studied extensively. We developed 13 mi-

crosatellite markers in order to analyze population genetics

and reproductive ecology of A. crenata.

Genomic DNA was extracted from leaf tissue of a single

fresh individual of A. crenata using the cetyltrimethyl

ammonium bromide (CTAB) method (Doyle and Doyle

1987). An enriched partial genomic library was constructed

using the protocol of fast isolation by AFLP of sequences

containing repeats (FIASCO) (Zane et al. 2002) with minor

modifications.

Approximately 250 ng of DNA was digested with

restriction enzymes MseI (New England BioLabs) and

fragments (400–1000 bp) were ligated to adapters MseI

restriction site. This was followed by ligation with T4 DNA

ligase and double stranded MseI linker (MseI F: 5¢-TACTCAGGACTCAT-3¢ and MseI R: 5¢-GACGATG-

AGTCCTGAG-3¢) to each end of the DNA fragment. The

digestion-ligation mixture was subsequently diluted ten

times and polymerase chain reaction (PCR)-amplified

using MseI-adaptor specific primers (5¢-GAT-

GAGTCCTGAGTAAN-3¢ i.e. MseI-N) for 19 cycles in a

total volume of 20 ll. Approximately 500 ng of amplified

product was mixed with 150 pmol of biotinylated (AC)15

and (AAG)8 probe in a total volume of 250 ll of SSC 4.2·and 0.07% SDS. The DNA was denatured for 5 min at

95�C, and incubated at 48�C for 2 h. Hybridized DNA was

then mixed with Streptavidin MagneSphere Paramagnetic

Particles (Promega), and magnetism was used to selec-

tively retain microsatellite-containing fragments. First

hybridized DNA allowed a selective binding at room

temperature for 30 min, and afterwards non-repetitive

DNA was removed by non-stringent washes in 500 ll of

TEN1000 (10 mM Tris-HCl, 1 mM EDTA, 1 M NaCl, pH

7.5) for three times and then by three additional stringent

washes in 500 ll high stringency buffer (SSC 0.2· and

0.1% SDS). After removing nonspecific DNA fragments,

DNA was eluted from the bead-probes with TE 1· buffer at

95�C for 5 min. Recovered DNA fragments were amplified

again with MseI-N primers. PCR products were purified

using E.Z.N.A� Gel Extraction Kit (OmegaBiotek,

L. Hong � H. Niu � H. Shen � W. Ye (&) �H. Cao

South China Botanical Garden, The Chinese Academy of

Sciences, Guangzhou 510650, China

e-mail: why@scbg.ac.cn

L. Hong

Graduate School of the Chinese Academy of Sciences, Beijing

100049, China

123

Conserv Genet (2008) 9:491–494

DOI 10.1007/s10592-007-9360-9

Ta

ble

1C

har

acte

rist

ics

of

13

mic

rosa

tell

ite

loci

inA

rdis

iacr

ena

tate

sted

on

two

po

pu

lati

on

s:F

anji

ng

shan

po

pu

lati

on

(FJS

)in

Gu

izh

ou

Pro

vin

cean

dH

eng

shan

po

pu

lati

on

(HS

)in

Hu

nan

Pro

vin

ce,

Ch

ina

Lo

cus

Gen

Ban

k

acce

ssio

nn

o.

Rep

eat

mo

tif

Pri

mer

seq

uen

ce(5

¢–3¢)

Tm

(�C

)N

at

To

tal

alle

lesi

ze(b

p)

Po

pN

aH

oH

EF

ISP

-HW

E

Ac0

4(A

C) 2

TC

(AC

) 1A

(AC

) 2T

C(A

C) 3

GC

TC

AC

CC

TC

AA

CT

CA

CG

AT

60

21

67

–1

72

FJS

21

.00

00

.51

7–

1.0

00

0.0

00

2*

EF

61

41

38

TC

GT

GT

TG

TG

GG

TT

TG

AG

AA

HS

21

.00

00

.51

7–

1.0

00

0.0

00

2*

Ac0

7(C

A) 1

1(T

A) 5

GG

GA

TT

GA

AA

TG

TA

GG

GG

TT

G6

23

23

7–

25

1F

JS3

0.0

67

0.5

91

+0

.89

10

.00

00

*

EF

61

41

39

TA

TC

CG

AA

TG

GC

AA

GA

AA

GG

HS

1–

––

–

Ac1

0(C

A) 4

(CC

CA

) 2(C

A) 5

GG

CA

GA

GT

AT

CC

GA

CC

CT

TA

62

41

74

–1

84

FJS

21

.00

00

.51

7-1

.00

00

.00

03

*

EF

61

41

40

CC

TT

TC

TT

TT

GT

GG

TA

GT

TC

GA

G

HS

21

.00

00

.51

7-1

.00

00

.00

02

*

Ac2

6(T

G) 8

CT

AC

GG

GG

AT

CA

CA

AG

TC

GT

60

32

46

–2

56

FJS

20

.00

00

.23

91

.00

00

.00

44

EF

61

41

41

GG

AA

AC

GA

AA

TC

GA

GT

GA

AA

TC

HS

1–

––

–

Ac2

7(T

C) 1

0T

T(T

C) 3

TG

TC

CA

AA

AA

TG

GA

GG

GC

TA

62

51

90

–1

99

FJS

40

.53

30

.68

3+

0.2

25

0.0

45

0

EF

61

41

42

GA

GG

GA

TG

AC

AG

CG

AC

TT

GT

HS

1–

––

–

Ac2

9(T

A) 3

(TG

) 3A

(TG

) 6C

GA

TT

GT

AG

CC

TC

CG

GT

TT

59

21

57

–1

60

FJS

1–

––

–

EF

61

41

43

TC

AC

AG

AA

GC

TC

TC

AA

TT

TT

GG

HS

21

.00

00

.51

7–

1.0

00

0.0

00

2*

Ac3

0(A

C) 6

GC

(AC

) 4G

CG

CC

AA

CA

AT

GA

AG

AT

AT

G6

23

18

3–

20

6F

JS2

0.2

67

0.2

39

–0

.12

01

.00

00

EF

61

41

44

TG

TT

GG

GG

TG

TG

TG

TA

GG

TG

HS

1–

––

–

Ac4

6(T

C) 3

AC

(TC

) 2C

C(T

C) 4

AG

TC

AC

CT

TT

TT

GG

GC

TG

TG

62

21

73

–1

76

FJS

21

.00

00

.51

7–

1.0

00

0.0

00

1*

EF

61

41

45

AG

AA

CG

AG

AG

TG

AG

GG

AG

CA

HS

1–

––

–

Ac4

9(G

A) 1

4G

CC

GA

TT

TT

CC

CA

AT

CC

TT

A6

27

18

8–

22

2F

JS7

0.3

33

0.7

82

+0

.58

20

.00

00

*

EF

61

41

46

AC

TG

CC

CT

TC

TG

TT

TG

AA

CG

HS

1–

––

–

Ac5

2(G

T) 9

(GA

) 9T

CA

CT

TT

TT

GG

CC

AT

CT

TC

C6

24

14

8–

17

2F

JS4

0.4

00

0.3

56

–0

.12

81

.00

00

EF

61

41

47

TC

AA

AA

GG

AT

CT

CC

GT

CG

AA

HS

30

.93

30

.66

0–

0.4

36

0.0

02

1*

Ac5

3(T

C) 1

5C

CA

CA

GT

AC

CA

AT

AT

GC

AC

GA

62

71

71

–2

00

FJS

30

.13

30

.24

8+

0.4

72

0.0

66

7

EF

61

41

48

AC

AG

GC

AT

CT

TT

GG

TG

AT

CG

HS

40

.73

30

.60

5–

0.2

22

0.3

76

8

Ac6

1(A

TC

) 8(T

TC

) 26

GG

GG

CT

GG

TT

TT

GA

AG

AA

GA

54

71

78

–2

39

FJS

50

.46

70

.78

9+

0.4

17

0.0

08

6

492 Conserv Genet (2008) 9:491–494

123

Lilburn, USA). The purified fragments were ligated into

the pMD 18-T plasmid vector (TaKaRa) and transformed

into Escherichia coli strain (JM109, Promega). The cells

were plated on a selective medium and recombinants were

identified using blue/white screening on Luria-Bertani agar

plates containing ampicillin, Xgal and IPTG. Insert posi-

tive bacterial clones were amplified using M13 primers and

visualized by agarose gel electrophoresis.

About 128 positive clones were isolated and sequenced

using M13 primers on an ABI 3730 automated sequencer

(PE Applied Biosystems) using BigDye Terminator Cycle

Sequencing Kit (Applied Biosystems). About 61 (47.65%)

clones yielded repeat motifs and 36 primer pairs were de-

signed using the PRIMER 3 web interface program (Rozen

and Skaletsky 2000) and then used for further analyses.

Polymorphic loci were used to characterize 15 individuals

each from two natural A. crenata populations of Fanjing-

shan (FJS) in Guizhou Province and Hengshan (HS) in

Hunan Province (Table 1). PCR amplifications were per-

formed in a final volume of 10 ll containing 10 mM Tris-

HCl (pH 8.4), 50 mM (NH4)2SO4, 1.5 mM MgCl2,

0.2 mM dNTPs, 0.2 lM each primer, 50 ng of genomic

DNA, and 1 U Taq polymerase (TaKaRa). The amplifica-

tion profiles included an initial denaturing at 94�C for

5 min, followed by 35 cycles of 50 s at 94�C, 50 s at the

locus-specific annealing temperature (Tm in Table 1) and

90 s at 72�C, and then a final extension step for 10 min at

72�C. Amplified products were separated on a 6% dena-

turing polyacrylamide gel using silver staining and sized by

comparison with a 25-bp DNA ladder standard (Promega).

Population genetics analyses were tested using GENE-

POP version 3.4 software (Raymond and Rousset 1995),

and Rice’s method of sequential Bonferroni (Rice 1989)

was applied to correct the P values for multiple testing.

About 17 of the 36 primer pairs successfully amplified

DNA fragments, and 13 loci showed polymorphism, while

the other four loci were monomorphic. The characteristics

of the 13 polymorphic loci are summarized in Table 1.

All loci were polymorphic in at least one population. Six

loci, Loc Ac07, Ac26, Ac27, Ac30, Ac46 and Ac49, were

monomorphic in HS population and one locus, Loc Ac29,

in FJS population. Although Loc Ac26 was monomorphic

in both populations, homozygous genotypes (i.e. fixed al-

leles) differed. In loci Ac07, Ac27 and Ac49, HS popula-

tion did not show any variation, whereas in FJS population

they were highly polymorphic. Such allele substitution

might be used as diagnostic markers to discriminate the

populations. The expected heterozygosity (HE) ranged

from 0.239 to 0.789, and the observed heterozygosity (Ho)

varied from 0.000 to 1.000, respectively (Table 1). The

average allele number of these microsatellites was four per

locus, ranging from two to seven. In addition, there have

allele fixations in Loc Ac04. Five of the loci from FJSTa

ble

1co

nti

nu

ed

Lo

cus

Gen

Ban

k

acce

ssio

nn

o.

Rep

eat

mo

tif

Pri

mer

seq

uen

ce(5

¢–3¢)

Tm

(�C

)N

at

To

tal

alle

lesi

ze(b

p)

Po

pN

aH

oH

EF

ISP

-HW

E

EF

61

41

49

TC

CT

TT

TT

CC

AA

GC

TT

TC

CA

HS

40

.20

00

.70

6+

0.7

24

0.0

00

0*

Ac6

3(A

AG

) 13

GA

GA

AG

CT

AC

AC

GT

GC

GT

TT

T6

23

20

9–

23

5F

JS2

0.1

33

0.4

05

+0

.67

80

.01

98

EF

61

41

50

CG

TC

AC

AC

GA

CA

CG

AA

AG

TC

HS

20

.06

70

.50

8+

0.8

73

0.0

00

7*

Tm

,an

nea

lin

gte

mp

erat

ure

;N

at,

tota

ln

um

ber

of

alle

les

ov

eral

lp

op

ula

tio

ns;

Na,

nu

mb

ero

fal

lele

sw

ith

inp

op

ula

tio

ns;

HO

,o

bse

rved

het

ero

zyg

osi

ty;

HE,

exp

ecte

dh

eter

ozy

go

sity

;F

IS,

inb

reed

ing

coef

fici

ent;

P-H

WE

,p

rob

abil

ity

of

Har

dy

–W

ein

ber

geq

uil

ibri

um

.S

equ

ence

sar

eli

sted

inG

enB

ank

un

der

Acc

essi

on

no

sin

ord

er,

EF

61

41

38

–E

F6

14

15

0

*D

eno

tes

sig

nifi

can

td

evia

tio

nfr

om

Har

dy

–W

ein

ber

geq

uil

ibri

um

corr

ecte

dfo

rm

ult

iple

com

par

iso

ns

usi

ng

the

Bo

nfe

rro

ni

corr

ecti

on

(P<

0.0

03

8)

Conserv Genet (2008) 9:491–494 493

123

population and six of the loci from HS population exhibited

significant deviation from Hardy–Weinberg equilibrium

corrected for multiple comparisons (adjusted P va-

lue = 0.0038; Table 1), No evidence of linkage disequi-

librium between pairs of loci was found in any combination

of loci pairs, which indicates physical independence of the

13 loci. The observed departures from HWE could be due

to the limited sample size or the occurrence of null alleles

and/or inbreeding effects. These 13 polymorphic micro-

satellite loci presented here are the first set of microsatellite

markers for A. crenata, and they will be useful for inves-

tigating population genetics and reproductive ecology of

this species.

Acknowledgements This work was supported by the National

Natural Science Foundation of China (30530160) and the Natural

Science Foundation of Guangdong Province, China (05200701).

References

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Rice WR (1989) Analyzing tables of statistical tests. Evolution

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Rozen S, Skaletsky HJ (2000) Primer 3 on the www for general users

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123