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MICROSATELLITE LETTERS
Development of polymorphic microsatellite loci in Lithocarpusharlandii (Fagaceae)
Yuan-Yuan Ding • Chang-Hong Yang •
Xin-Yun Gu • Xin Tong
Received: 7 April 2014 / Accepted: 11 April 2014
� Springer Science+Business Media Dordrecht 2014
Abstract Evergreen broadleaved forests (EBLFs) are the
most important vegetation type in subtropical China, but
suffer from rapid decline by anthropogenic disturbances
and notoriously exacerbated environment. Genetic infor-
mation of Lithocarpus harlandii (Hance) Rehd. (Fagaceae),
one of the main constructive species in subtropical EBLFs,
is crucial to the conservation and management of these
forests. We developed 12 microsatellite loci from its
nuclear genome to provide efficient markers for analyzing
the population genetic diversity and differentiation and
thereby for designing appropriate conservation strategies.
These loci were tested in three L. harlandii populations.
The numbers of alleles per locus varied from 3 to 13. The
observed and expected heterozygosities within populations
were 0.379–0.967 and 0.406–0.849, respectively. No
linkage disequilibrium was detected among loci. These
polymorphic microsatellites will be useful for biodiversity
conservation and forest restoration.
Keywords Lithocarpus harlandii � Fagaceae �Microsatellites � Genetic diversity
Evergreen broadleaved forests (EBLFs) are the main potential
zonal vegetation in subtropical China, but threatened by
habitat loss and fragmentation in the Anthropocene. Litho-
carpus harlandii (Hance) Rehd. (Fagaceae) is a main con-
structive species in subtropical EBLFs, and ipso facto, of
substantial importance to the conservation of these forests.
Dominant DNA-based makers, such as RAPDs (Li et al.
2007), have been used to unravel genetic diversity in L. har-
landii. Nevertheless, being co-dominant and hyper-polymor-
phic, microsatellites are more efficient for population genetic
studies compared to dominant DNA markers. Furthermore,
high polymorphism has always been found in microsatellite
loci for Fagaceae species (e.g., Liu et al. 2009; Tong et al.
2012). Herein, we isolated and characterized 12 polymorphic
microsatellites in L. harlandii, which could contribute to
understanding the genetic diversity in this species and thereby
to making effective conservation strategies.
Samples were collected from 30 L. harlandii individuals
in each of Tiantong, Tiantaishan and Tianmushan popula-
tions in Zhejiang province of China. The total genomic
DNA was extracted using the plant Genomic DNA Kit
(Tiangen, Beijing, China).
We developed microsatellites following the protocol of
Tong et al. (2012). About 250 ng DNA was digested with
the restriction enzyme MseI, and linked to an MseI-adapter
pair (F: 50-TACTCAGGACTCAT-30, R: 50-GACGATGAG
TCCTGAG-30) immediately. After amplified with MseI-N
primer (50-GATGAGTCCTGAGTAAN-30) were the PCR
products hybridized by using 50-biotinylated probe (AG)15,
and then captured with streptavidin-coated magnetic beads
(Promega, Madison, Wisconsin, USA). The enriched
fragments were amplified again with MseI-N primer. The
products, purified with a multifunctional DNA Extraction
Kit (Bioteke, Beijing, China), were ligated into pMD 19-T
vector, and then cloned into Escherichia coli strain JM109.
Finally, 384 positive clones were randomly chosen to be
tested by PCR using M13F/M13R and (AG)10 as primers,
Electronic supplementary material The online version of thisarticle (doi:10.1007/s12686-014-0207-7) contains supplementarymaterial, which is available to authorized users.
Y.-Y. Ding � C.-H. Yang � X.-Y. Gu � X. Tong (&)
Shanghai Key Lab of Urban Ecological Processes and Eco-
Restoration, Tiantong National Station for Forest Ecosystem,
School of Ecology and Environmental Sciences, East China
Normal University, Dongchuan Road 500, Shanghai 200241,
China
e-mail: txoinng@163.com
123
Conservation Genet Resour
DOI 10.1007/s12686-014-0207-7
Table 1 Characterization of 12 polymorphic microsatellite loci developed in Lithocarpus harlandii
Locus Primer sequence (50–30)a Repeat motif Size range (bp) Na Ta (�C) GeneBank
LH1 F: \6-FAM[ CACATACAAGAAGAAGGGAA
R: GCAACAAACCAGCATTAG
(AG)8 113–127 5 55 KJ630892
LH6 F: \ROX[ TATCACGAGGGTCTTACA
R:TCTCCTTTCTCCCATACA
(GA)5 291–305 7 52 KJ630893
LH9 F: \ROX[ ACCTCCAAATCGCCAGTA
R: TAGACAAGACCTCCCAAC
(AG)14 142–166 10 58 KJ630894
LH10 F: \HEX[ TCCAGATCACCGCCATAA
R: GCCCTCCTTCCACAGCAA
(TC)12 126–146 13 63 KJ630895
LH11 F: \TAMRA[ CCCTGGATTGCTTGGATA
R: TGGTTTTGGGCTCAGACT
(AG)5 216–238 7 58 KJ630896
LH13 F: \6-FAM[ AACTTGAAGCGAGGGAGA
R: ACCAATAAGGTTGCAGGA
(AG)7 235–259 11 53 KJ630897
LH14 F: \HEX[ TCAACTGCCACCACTACCA
R: GGTCTTGGGCTTCTGCTT
(GA)14 158–190 13 61 KJ630898
LH15 F: \6-FAM[ CCTCCATTTCCAACTGCC
R: CAATCATTTCCCGCCATC
(CT)10 170–190 12 58 KJ630899
LH18 F: \HEX[ TCCACTGAAACCGCAATA
R: CGAGGAGCCTTGATACACTA
(GA)7 275–301 11 58 KJ630900
LH19 F: \ROX[ GAAAGGGATTATGGGACG
R: CAGAAGGCTTGAGGAGTT
(AG)5 248–252 3 55 KJ630901
LH20 F: \TAMRA[ TTACAAGGGGTTGCCAGAG
R: CCCACCCATCACTACCAT
(GA)9 188–206 6 61 KJ630902
LH24 F: \6-FAM[ GTTATGGCATGGAAGGAG
R: CAGGGAGAATTGAGTGGTG
(CT)6 287–293 4 55 KJ630903
Na = Number of alleles; Ta = annealing temperaturea Fluorescent dyes (i.e., HEX, ROX, TAMRA, and 6-FAM) are presented with the forward primers
Table 2 Population genetic characteristics of the 12 polymorphic microsatellite loci in three Lithocarpus harlandii populations
Locus Tiantong population
(29�480N, 121�470E)
Tiantaishan population
(29�090N, 120�500E)
Tianmushan population
(30�180N, 119�240E)
n Na HO HE n Na HO HE n Na HO HE
LH1 29 5 0.667 0.684 30 3 0.467 0.522 30 4 0.890 0.725
LH6 30 4 0.633 0.585 29 4 0.379 0.407 30 6 0.533 0.511
LH9 29 7 0.690 0.668 30 7 0.533 0.719 29 7 0.759 0.767
LH10 30 7 0.800 0.823 29 8 0.533 0.519 29 10 0.667 0.488
LH11 29 5 0.603 0.671 30 7 0.733 0.786 30 6 0.667 0.631
LH13 30 8 0.500 0.773 30 7 0.733 0.693 30 9 0.833 0.833
LH14 30 9 0.900 0.846 30 10 0.767 0.799 30 11 0.767 0.820
LH15 30 7 0.800 0.772 30 11 0.967 0.850 30 9 0.833 0.768
LH18 29 9 0.448 0.743 30 7 0.733 0.643 30 6 0.800 0.797
LH19 30 3 0.700 0.524 30 2 0.633 0.440 30 3 0.667 0.524
LH20 29 6 0.517 0.624 30 4 0.333 0.550 27 3 0.747 0.650
LH24 30 2 0.433 0.494 30 3 0.333 0.288 30 3 0.033 0.098
Average 29.6 6 0.637 0.676 29.8 6.1 0.571 0.584 29.6 6.4 0.659 0.623
n = Number of samples genotyped; Na = number of alleles; HO = observed heterozygosity; HE = expected heterozygosity
Conservation Genet Resour
123
and 153 clones might contain microsatellites and thereby
were sequenced on an ABI 3730 DNA Sequence Analyzer
(Applied Biosystems, Foster City, California, USA). Only
24 sequences matched the design criteria and primers were
designed with Primer Premier 5.0.
Twenty-three samples were used to check the poly-
morphism. The PCR amplification were performed in a
15-ll reaction system, containing 50 ng of DNA, 1 9 PCR
buffer (without Mg2?), 1.5 mM Mg2?, 0.2 mM each
dNTP, 0.1 uM of each primer, and 1 U of DNA Taq
polymerase (Sangon, Shanghai, China), with the following
conditions: 94 �C for 5 min; 30 cycles of 30 s at 94 �C,
30 s at 45-65 �C (depending on specific annealing tem-
perature, Table 1), and 30 s at 72 �C; and a final extension
of 72 �C for 8 min. PCR products were resolved on 8 %
polyacrylamide denaturing gels and visualized by silver
staining with pUC 19 DNA/MspI (HpaII) (Thermo) as the
ladder. A total of 12 loci were found polymorphic. These
loci were further tested on all samples of the three popu-
lations. The PCR products were labeled with one of fluo-
rescent dyes HEX, ROX, TAMRA or 6-FAM, and then
scanned on an ABI 3730 automated sequencer with an
internal lane standard, and analyzed using GeneMapper 4.0
(Applied Biosysterms).
Peak patterns indicated that L. harlandii is diploid. The
numbers of alleles per locus varied from 3 to 13 (Table 1).
The observed and expected heterozygosities within popu-
lations were 0.379–0.967 and 0.406–0.849, respectively
(Table 2). After sequential Bonferroni adjustment (Rice
1989), we found no consistently significant deviation from
either Hardy–Weinberg equilibrium for each locus, or
linkage equilibrium for each pair of loci across all popu-
lations using GENEPOP v4.0 (Rousset 2008).
The twelve microsatellite loci, showing high levels of
polymorphism in L. harlandii, not only provide a useful
way in investigating the genetic diversity and population
structure, but also lend themselves to strategies for the
management of L. harlandii as well as the protection of
subtropical EBLFs.
Acknowledgments We thank Dr. Xiao-Yong Chen for revising the
manuscript. We also thank Min-Yan Cui for assistance in field work,
and Ya-Ting Wang and Lin-Yi Zhang for conduction in experiments.
This work was supported by the National Natural Science Foundation
of China (31361123001).
References
Li JH, Jin ZX, Lou WY, Li JM (2007) Genetic diversity of
Lithocarpus harlandii populations in three forest communities
with different succession stage. Chin J Ecol 26:509–514 (in
Chinese with English abstract)
Liu M, Shi MM, Liu MH, Chen XY (2009) Isolation and character-
ization of microsatellite loci in Fagus longipetiolata Seem.
(Fagaceae). Conserv Genet 10:1981–1983
Rice WR (1989) Analyzing tables of statistical tests. Evolution
43:223–225
Rousset F (2008) Genepop’007: a complete re-implementation of the
genepop software for Windows and Linux. Mol Ecol Resour
8:103–106
Tong X, Xu NN, Li L, Chen XY (2012) Development and
characterization of polymorphic microsatellite markers in Cy-
clobalanopsis glauca (Fagaceae). Am J Bot 99:e120–e122
Conservation Genet Resour
123
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