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

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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).

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