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TECHNICAL NOTE
Twenty-three microsatellite loci for Styrax confusus
and Styrax japonicus (Styracaceae)
Xiao-Yan Wang •Shuo Yu •Min Liu •
Qing-Song Yang •Xiao-Yong Chen
Received: 27 November 2009 / Accepted: 8 December 2009 / Published online: 24 December 2009
ÓSpringer Science+Business Media B.V. 2009
Abstract From a genomic library enriched for AG
repeats, 23 polymorphic microsatellite loci were isolated
for Styrax confusus and Styrax japonicus. All the loci are
polymorphic in Styrax japonicus. Analysis of 23 individ-
uals from two populations revealed an average of 5.3
alleles per locus (range: 2–10), an average observed het-
erozygosity of 0.30 (range: 0–0.826) and an average
expected heterozygosity of 0.66 (range: 0.198–0.888).
Fourteen loci are polymorphic in Styrax confusus, showing
4–11 alleles per locus, an observed heterozygosity ranging
from 0.087 to 0.870 and an expected heterozygosity
ranging from 0.580 to 0.907. These polymorphic micro-
satellite loci provide useful molecular tools to study genetic
variation and phylogeography of the two Styrax species
across their ranges in eastern Asia.
Keywords Styrax confusus Styrax japonicus
Microsatellite Polymorphism Genetic variation
The family Styracaceae contains 11 genera of woody
plants, and has a widespread but disjunct distribution
(Fritsch et al. 2001). Comprising over 80% of the total
number of species in Styracaceae, Styrax distributes the
whole range of the family (Fritsch et al. 2001). Styrax was
frequently used to describe patterns and mechanisms of
disjunct distribution (for example, Fritsch 1996; Xiang
et al. 2004), and phylogeny and biogeography of the genus
had been studied using isozymes, chloroplast genes,
internal transcribed spacer region of nuclear ribosomal
DNA and morphological traits (Fritsch 1996;2001; Fritsch
et al. 2001). Genetic variation may provide important
information for biogeography. However, such information
was very limited in Styrax.S. confusus and S. japonicus are
common deciduous trees distributed in eastern Asia, and
the latter is also planted for landscape aims. Microsatellites
are powerful and effective tools for population genetic
studies (Liu et al. 2008), and may provide important
information for biogeography. We report here the isolation
and characterization of 23 polymorphic microsatellite
markers for the two Styrax species.
Due to difficulty in distinguishing S. confusus and
S. japonicus without information of flower or fruit, we
identified each leaf sample by sequencing internal tran-
scribed spacer region of nuclear rDNA. Genomic DNA was
extracted from dried leaves of S. confusus and S. japonicus as
described by Fan et al. (2004). The ITS1, 5.8S, and ITS2
regions of the ribosomal DNA were amplified together using
primers ITS-4 (50-TCCTCCGCTTATTGATATGC-30) and
ITS-5 (50-GGAAGGAGAAGTCGTAACAAGG-30) (White
et al. 1990). PCR products were visualized on 1% agarose
gels before sequenced with ABI 3730 DNA Sequence
Analyzer. After identifying the samples, a microsatellite-
enriched library of S. confusus was constructed following the
procedure of Liu et al. (2009). 903 positive clones were
chosen and tested by PCR, which was performed in a 15 ll
final volume (Liu et al. 2009) using (AG)
10
and M13?/M13
as primers, respectively. A total of 149 screened clones
contained potential repeat motifs and were sequenced with
ABI 3730 DNA Sequence Analyzer.
X.-Y. Wang S. Yu M. Liu Q.-S. Yang X.-Y. Chen (&)
Department of Environmental Sciences, Shanghai Key
Laboratory for Ecological Processes and Restoration, East China
Normal University, 200062 Shanghai, China
e-mail: xychen@des.ecnu.edu.cn
X.-Y. Chen
Tiantong National Observation Station for Forest Ecosystems,
200062 Shanghai, China
123
Conservation Genet Resour (2010) 2:51–54
DOI 10.1007/s12686-009-9159-8
Table 1 Characteristics of 23 microsatellite loci developed for Styrax confusus and Styrax japonicus, including locus name, primer sequences, repeat motif, annealing temperature (T
a
), size
range, number of alleles (N
A
), observed (H
O
) and expected (H
E
) heterozygosites, and accession number of Genbank
Locus name Primer sequences (50–30) Repeat motif Styrax japonicus Styrax confusus Genbank
accession
number
T
a
(°C) Size (bp) N
A
H
O
H
E
T
a
(°C) Size (bp) N
A
H
O
H
E
Styr 2 F: TACTGACGAAACGGAGAAAG (TG)
4
(TA)
6
(CT)
9
(AG)
18
58 278–378 6 0.476 0.726 59 286–318 7 0.870 0.828 GU220028
R: ATGGAATATGAGCTGCTGTG
Styr 5 F: ACACGCAACCCCATCTT (AG)
16
63 356–392 6 0.435 0.761 64 346–374 4 0.304 0.580 GU220029
R: GGTCTTCGGTTCAAGTATCT
Styr 6 F: AGACTGCCTTCTCCATTGT (TC)
9
54 132–146 4 0.318 0.745 59 140–148 5 0.087 0.732 GU220030
R: GCGGAAATTCAATACTT
Styr 10 F: TAACGGTGGGGATGACAATA (AG)
14
56 115–133 8 0.118 0.820 57 107–153 11 0.136 0.907 GU220031
R: CCAACCAAACTCACAAATAC
Styr 14 F: CCATAAAAGCTCAAGCTATCA (TC)
9
53 112–126 4 0.409 0.711 51.5 112 M 0.000 0.000 GU220032
R: ATTCTTACCACCCCTGTCTCT
Styr 20 F: CTTGTGTTCCATAATCTGTG (TC)
17
60 169–189 5 0.318 0.555 61.5 177–201 6 0.522 0.808 GU220033
R: TAAAAGGGCACACACTCTAC
Styr 21 F: AATAAAATGGCTGTCCCCTT (CT)
16
51.5 181–209 10 0.450 0.862 59 183–201 P – – GU220034
R: ATGGTTAGCACTGAGATGAT
Styr 22 F: TTGACACACAACAACGCTAC (AG)
4
(AG)
4
(AG)
6
62 215–229 3 0.130 0.484 63 231 M 0.000 0.000 GU220035
R: GACAACTATGGACGAAAACT
Styr 24 F: CCAGACCCAACAACCTTACA (TC)
4
(TC)
6
(TC)
10
59 189–199 5 0.409 0.712 59 193–227 7 0.435 0.777 GU220036
R: AGGAAGATCTGATAGTCA
Styr 25 F: AGGGCTAAAGTTTCAGACGA (TC)
8
TG(TC)
6
56 209–245 7 0.500 0.808 55.5 213–235 P – – GU220037
R: ATATTCCTATCATTGTGGC
Styr 26 F: ATCGCAACATCCACATCCTA (TA)
4
(TC)
4
(TC)
9
58 374–392 4 0.130 0.428 58 382–396 P – – GU220038
R: GTAACAACCATTTCCACTG
Styr 30 F: GAAGTGAGTGATAAATGTGC (TC)
23
63 201–223 5 0.091 0.720 63 189–261 P – – GU220039
R: AGTGTATCCACAGGGCAT
Styr 31 F: TGGGATTAGAAGAATAGGTA (GA)
7
(GT)
9
(GA)
10
56 284–336 5 0.238 0.744 58 264–290 4 0.364 0.610 GU220040
R: AACGCGTTCTTTATACATAT
Styr 32 F: ATACAGATGAGGTTGGTCCC (TC)
11
65 156–198 9 0.826 0.888 64 150–172 8 0.478 0.862 GU220041
R: GTCCCTAGCAGAGGTCAAAC
Styr 33 F: TCCCAACAAACAAGCCTCTG (AG)
13
65 107–117 6 0.545 0.759 65 105–115 6 0.174 0.726 GU220042
R: CAATGCCATCGGATACGACC
Styr 34 F: GGTTTTATTGGACCGTTTGG (GA)
4
(GA)
7
61 143–159 5 0.087 0.721 65 147–161 6 0.304 0.679 GU220043
R: TGACTCATTCCCCGGACAGT
52 Conservation Genet Resour (2010) 2:51–54
123
Table 1 continued
Locus name Primer sequences (50–30) Repeat motif Styrax japonicus Styrax confusus Genbank
accession
number
T
a
(°C) Size (bp) N
A
H
O
H
E
T
a
(°C) Size (bp) N
A
H
O
H
E
Styr 40 F: GTGGGGAGTGGAAAGTTGTT (TC)
22
62 118–128 2 0.217 0.198 65 122–164 9 0.391 0.835 GU220044
R: AGTGCAGATAGAGATCATCA
Styr 42 F: GAAGCGCATAAAATCACACG (TC)
10
68 222–238 7 0.227 0.674 68 222–248 6 0.478 0.767 GU220045
R: CCAAACAGGGACAGGAAACC
Styr 43 F: CAAGAAGACTAAGAAGAACA (AG)
4
(AG)
18
58 150–168 4 0.261 0.506 57.5 152–178 4 0.391 0.697 GU220046
R: AGCATCTCTTACTCAATTTC
Styr 46 F: GGTTTATCCTCATTCCTCGC (AG)
7
63 255–263 3 0.087 0.518 – – – – – GU220047
R: CTTTGGCTCTGGTATGGTCT
Styr 49 F: ATGAGATGCCAAAACACAAA (CT)
10
CCCTCT(CA)
10
55 114–202 10 0.227 0.839 56 134–168 11 0.348 0.907 GU220048
R: AAGTTTCCTTCACGCAATAA
Styr 51 F: GATGACAAAGTACCAGAAC (AG)
5
55 150–152 2 0.391 0.507 62 150 M 0.000 0.000 GU220049
R: TTTATTGTGGAAAGATGCTC
Styr 52 F: CAAGCTCTTCCATCACCACC (TC)
8
TT(TC)
6
61 103–105 2 0.000 0.394 66 103–131 P – – GU220050
R: AAAAGCATGAACGTCGCAAT
‘‘P’’ indicates polymorphic locus which was difficult to interpret, and ‘‘M’’ indicates monomorphic locus
Conservation Genet Resour (2010) 2:51–54 53
123
Forty-six of the cloned sequences were used to design
locus specific primers using program PRIMER 5.0 (
http://www.premierbiosoft.com). Variability at the loci was
tested with 23 S. confusus individuals and 23 S. japonicus
individuals. The PCR was performed in 20 ll volumes,
which included 50 ng genomic DNA, 2.0 ll109PCR
buffer, 1.875 mM MgCl
2
, 0.15 mM each dNTPs, 0.05 lM
of each primer, 1 U of DNA Taq polymerase (Sangon).
The thermal profile for PCR amplification was 94°C for
3 min, followed by 30 cycles of 94°C for 30 s, a primer-
specific annealing temperature for 30 s (Table 1), 72°C for
30 s, ending with a single extension of 72°C for 7 min.
PCR products were visualized on 1% agarose gels and then
resolved on 8% polyacrylamide denaturing gel and visu-
alized by silver staining using pUC19 DNA/Msp I (Hpa II)
(Fermentas) as the ladder.
Finally, we found 23 polymorphic loci in S. japonicus
from the 46 primer pairs (Table 1). Among the other
primers, 6 pairs produced polymorphic products but diffi-
cult to interpret, and 17 had no amplified products. The
number of alleles per locus ranged from 2 to 10, with an
average of 5.30. We calculated observed (H
O
) and expec-
ted (H
E
) heterozygosities with software GENEPOP v4.0
(Rousset 2008). Hardy–Weinberg equilibrium and linkage
disequilibrium were also tested by GENEPOP v4.0
(Rousset 2008) followed by the sequential Bonferroni
correction (Rice 1989). The H
O
and H
E
ranged from 0 to
0.826 and 0.198 to 0.888, respectively. There were 14 loci
significantly biased from Hardy–Weinberg equilibrium
(Table 1), and no locus pair exhibited significant linkage
disequilibrium.
Fourteen loci were found to be polymorphic in S. con-
fusus (Table 1). Among the other primers, 5 were mono-
morphic, 10 were polymorphic but difficult to interpret,
and 17 had no amplified products. The number of alleles
per locus ranged from 4 to 11, with an average of 6.71. 13
loci was significantly biased from Hardy–Weinberg equi-
librium (Table 1), and loci Sty2 and Sty32 exhibited
significant linkage disequilibrium (P\0.001). H
O
and
H
E
ranged from 0.087 to 0.870 and 0.580 to 0.907,
respectively.
The 23 polymorphic microsatellite loci are reliable
genetic markers and will be useful for studying the genetic
variation and phylogeography of the two Styrax species
across their ranges in eastern Asia.
Acknowledgments We thank Hong-Qing Li, Bin-Jie Ge and Jin-Jin
Hu for identifying samples, Liang Zhao, Fan-Rong Zeng and Miao-
Miao Shi for sample collections, Lin-Feng Li for data analysis. This
work was supported by Science and Technology Foundation of For-
estry (2006BAD03A15).
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