TECHNICAL NOTE

Isolation and characterization of microsatellite lociin Peganum harmala (Peganaceae), an importantresist-drought and medicinal plant

Jin Han Æ Li-Zhe An

Received: 22 January 2009 / Accepted: 4 February 2009 / Published online: 21 February 2009

� Springer Science+Business Media B.V. 2009

Abstract Peganum harmala is a herb grows spontane-

ously in arid and rocky areas. From ancient time, it has

been claimed to be an important medicinal plant in rough

environment. In this study, we developed 12 microsatellite

loci from P. harmala by the combining biotin capture

method for the first time. A total of 31 microsatellite

sequences were recovered through screening the library

and 12 of them are polymorphic. The number of alleles per

locus in 36 sampled individuals ranged from 3 to 8,

expected heterozygosity and observed heterozygosity ran-

ged from 0.1381 to 0.6821 and from 0.3573 to 0.8739,

respectively. In addition, all markers have been crossly

checked in the other congeneric species. These microsat-

ellite markers would provide a useful tool for investigating

the genetic diversity and study the population genetic

structure in detail.

Keywords Peganum harmala � Microsatellite

markers � Genetic diversity

Peganum harmala (Peganaceae), also known as Harmal or

Syrian rue, is a perennial herbaceous, glabrous plant, which

grows in arid conditions, sandy soils and rocky areas,

native to eastern Mediterranean region and widely dis-

tributed in Middle East, India, Mongolia and China

(Decaraene et al. 1996; Frison et al. 2008). P. harmala is

used in traditional medicine and is rich in alkaloids that

have a wide spectrum of pharmacological actions in vari-

ous areas. These include antispasmodic, antipyretic,

anticancerous, central nervous system effects and so on

(Bruinvels and Sourkes 1968; Fan et al. 1997). In addition,

P. harmala play an important role in restoring the local

ecosystem as resist-drought species. Therefore, there were

valuable applications for P. harmala as a medicinal plant

inhabited in rough environment. However, little is known

about its genetic diversity of this important medicinal plant

until now. In this study, we developed 12 microsatellite

markers on P. harmala and tested them on the congeneric

species P. nigellastrum for the first time. These markers

provide powerful tools for the conservation genetics studies

and precise estimation of population genetic structure.

The total genomic DNA was extracted from the silica gel

dried leaves of a single individual of L. chinense using the

CTAB method (Doyle and Doyle 1987). Enrichment of the

genomic DNA for microsatellite motifs was performed

using magnetic bead capture in accordance to the method

described by (Glenn and Schable 2005). About 500 ng

genomic DNA was digested into approximately 500 bp

fragments with a restriction enzyme RsaI (NEB) and XmnI

(NEB), then ligated to SuperSNX24 double-stranded

adaptors (mixation of equal volumes of equal molar amounts

of SuperSNX24-F:50-GTTTAAGGCCTAGCTAGCAGAA

TC-30 ? SuperSNX24 ? 4P-R:50-GATTCTGCTAGCTAG

GCCTTAAACA AAA-30). For enrichment, the ligation

products were hybridized with an oligonucleotide combina-

tion of 50-biotinylated probes, (AG)15, (CT)12, (AC)15,

(GT)15, (CG)15, (AG)12. The hybridization in the 50 ll

solution (29 SSC, 1 lmol/lprobe and 10 ll ligation prod-

ucts) was as follows: an initial 5 min at 95�C, then a rapid

cooling to 70�C followed by 0.2�C incremental decreases

every 5 s for 99 cycles, and maintenance at 50�C for 10 min;

then decreases of 0.5�C every 5 s for 20 cycles, and finally

rapid cooling to 15�C. The DNA hybridized to the probe was

captured by streptavidin-coated magnetic beads at 37�C for

J. Han � L.-Z. An (&)

Key Laboratory of Arid and Grassland Ecology, School of Life

Sciences, Lanzhou University, 730000 Lanzhou, China

e-mail: lizhean@lzu.edu.cn

123

Conserv Genet (2009) 10:1899–1901

DOI 10.1007/s10592-009-9849-5

1 h and then washed by the solution I (29 SSC, 0.1% SDS)

and solution II (19 SSC, 0.1% SDS). The captured DNA

was recovered by polymerase chain reactions (PCR) with

SuperSNX-F (50-GTTTAAGGCCTAGCTAGCAGAA TC-

30) and PCR product was purified with TIANquick midi

purification kit (TIANGEN). These fragments enriched with

microsatellite loci were cloned using pMD18-T vector (Ta-

kara) and transformed into the E. coli competent cell (JM109,

Takara). Transformants were identified by blue/white

screening on LB agar plates containing ampicillin, X-gal and

IPTG. Positive colonies were amplified using M13 forward

and reverse primers. PCR products of 300–600 bp were

sequenced using ABI 3130xl Genetic Analyzer. The

sequences containing motifs repeating more than five times

were regarded as microsatellites. A total of 31 sequences

were identified out of the sequenced 132 sequences and pri-

mer pairs for amplification of the microsatellite regions were

designed using the Primer 5.0 (Clarke and Gorley 2001).

In order to check polymorphisms of the identified

microsatellite loci, 36 individuals from eight distantly

populations were selected for test. The PCR reactions were

performed in 25 ll reaction mixtures with 10–40 ng

template DNA, containing 19 ll of sterile double-distilled

water; 2.5 ll of 109 Taq polymerase reaction buffer; 1 ll

each of the primers; 1 unit TaqDNA polymerase. The

amplifications used an initial denaturation of 5 min at

94�C, and then followed by 38 cycles of 94�C for 40 s,

annealing for 40 s at 46–50�C, 72�C for 45 s plus a final

extension of 72�C for 10 min. PCR products were initially

checked for PCR amplification on 1.5% agarose gels. Of

the 31 microsatellite loci tested on all 36 individuals, 19

were excluded as they were either not amplifiable or were

monomorphic, while the remaining 12 revealed microsat-

ellite polymorphism.

For better resolution of the 12 polymorphic microsat-

ellite loci, capillary electrophoresis was carried out using

an automated sequencer ABI 3130xl Genetic analyser (PE

Applied Biosystems), and fragment lengths were deter-

mined with the help of internal size standards GeneScan

600 LIZ Size Standard (PE Applied Biosystems). The

number of alleles per locus (A), observed heterozygosity

(HO) and expected heterozygosity (HE), Hardy–Weinberg

equilibrium as well as linkage disequilibrium were esti-

mated using the program Cervus 3.0 (Marshall et al. 1998)

Table 1 Characteristics of 10 polymorphic microsatellite loci for Peganum harmala

Locus Primers sequence (50–30) Repeat Ta(�C) N Size

range (bp)

No.

alleles

HO HE GenBank

accession No.

Pe17 F: AAAATCATTTCAGGGTGC

R: TACTTTGAGCCAGGTGCC

(CA)9–(TG)5 50 36 197–228 7 0.6521 0.7559 FJ628149

Pe39 F: TAGCAGAATCACAGAGTT

R: CTAGAAATCCCACCAAAA

(TC) 12 46 36 175–213 3 0.1381 0.3573 FJ628150

Pe42 F: TATTACAATGGGAACAAG

R: AGAAATATCATTACAACCC

(TG)7 48 36 136–162 6 0.4509 0.6322 FJ628151

Pe68 F: CTTGGCACTGGGCAACAT

R: TTGGCTGTCCCGGTCTTG

(GT)9 55 36 310–355 5 0.5517 0.7780 FJ628152

Pe73 F: TCTGTTCAATGAGAAC

R: AAAGCACTTACAAAAT

(GT)11–(GT)10 46 36 226–244 4 0.6471 0.7328 FJ628153

Pe86 F: CTTGGCACTGGGCAACAT

R: AGAGCCTTTAGCAGCATA

(GT)8 50 36 122–157 4 0.3121 0.5069 FJ628154

Pe88 F: AGCTTCACAGCTACGCTT

R: TCAGAATTTGAAACCCAC

(CT)12–(TG)5 50 36 93–131 8 0.5089 0.6222 FJ628155

Pe145 F: AATGGGGACGTGTTGTTA

R: TGCAGATGGACGATGTTT

(CT)9–(TC)9 50 36 189–234 6 0.4789 0.6922 FJ628156

Pe192 F: GGACTGGAAATGGGTCTC

R: GATGTTTAAGGCCTAGCT

(GA)10–(GT)5 50 36 102–155 5 0.4351 0.5899 FJ628157

Pe211 F: GTGGATTTCACTGTCTAT

R: GCATCTCGTCTAACTGTA

(TG)18 46 36 124–144 4 0.3049 0.4092 FJ628158

Pe217 F: AAAAGCAGAACGCTCCCC

R: CGGTGCCACGAAATAGTA

(CT)7 50 36 135–162 4 0.6821 0.8739 FJ628159

Pe255 F: ATCTGCTTTCTTCACGTA

R: TGTTGTCATGGAATCTTT

(AC)8 46 36 87–119 6 0.5089 0.6772 FJ628160

Ta annealing temperature of primer pair, N number of individuals genotyped, Ho observed heterozygosity, HE expected heterozygosity

1900 Conserv Genet (2009) 10:1899–1901

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and GENEPOP version 3.4 (http://wbiomed.curtin.edu.au/

genepop; Raymond and Rousset 1995).

Characterization of the 12 microsatellite markers by

capillary electrophoresis confirmed all loci to be poly-

morphic. The number of alleles per locus ranged from 3 to

8, the observed heterozygosity and expected heterozygosity

ranged from 0.1381 to 0.6821 and from 0.3573 to 0.8739,

respectively (Table 1). For each locus, the expected het-

erozygosity was always significantly bigger than the

observed heterozygosity (P \ 0.05). No loci showed sig-

nificant deviations from HWE after Bonferroni correction

for multiple comparisons. No linkage disequilibrium was

detected between paired loci comparisons.

We further performed cross-priming tests in the conge-

neric species P. nigellastrum, all of the loci were success-

fully amplified in P. nigellastrum except Pe42 and Pe 211.

These 12 polymorphic microsatellite loci presented here

are the first set of microsatellite markers for P. harmala,

and they will be useful for investigating population

genetics and morphological divergence between this spe-

cies and the closely related species P. nigellastrum.

Overall, the high number of alleles per locus, high

polymorphic and expected heterozygosity demonstrate

the potential use of these polymorphic microsatellites for

population differentiation. The microsatellite markers

developed here can be used as an alternative or addition to

morphological characters for species identification. In

addition, they can also be used to deduce the spatial–tem-

poral population genetic structure and gene flow dynamics,

which should provide us with a better understanding of the

evolutionary history and vital information for the develop-

ment of conservation strategies of this species.

Acknowledgments The authors thank CNSF (90302010) and

Gansu Agricultural Bio-technology Research & Technology Basic

Data Platform Project (505016) supporting the primary author. Key

Laboratory of Arid and Grassland Agroecology of Ministry of

Education.

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