Conservation Genet Resour

DOI 10.1007/s12686-014-0361-y

MICROSATELLITE LETTERS

Characterization of novel polymorphic microsatellite markers

in Dactylorhiza hatagirea: a critically endangered orchid species
from western Himalayas
Shilpa Sharma Vikas Sharma Meenu Chhabra
Rajeev Rathour Kamal Dev Sharma
Rakesh Kumar Kapila

Received: 27 September 2014 / Accepted: 14 October 2014

Springer Science+Business Media Dordrecht 2014

Abstract Dactylorhiza hatagirea (D. Don) Soo, family

Orchidaceae is an endangered medicinal herb inhabiting
higher altitudes of western Himalayas. Due to reckless
exploitation for its roots, it is of high conservation concern.
In the present study fifteen microsatellites were developed
and characterized across twenty collections of D. hatagirea. The total numbers of alleles amplified by these
microsatellites were 64 with an average of 4.2 alleles per
marker. Average observed and expected heterozygosity
values for polymorphic loci were 0.623 and 0.631,
respectively. Mean polymorphism information content
value of the polymorphic markers was 0.532. Of the
fourteen polymorphic microsatellites, 7 deviated from
HardyWeinberg equilibrium. Microsatellites reported
here can be utilized to address questions related to genetic
characteristics in this species.
Keywords Dactylorhiza hatagirea  Microsatellite
markers  Orchids

Introduction
Dactylorhiza hatagirea (D. Don) Soo, an orchid of high
economic importance is endemic to western Himalayan
region (2,5005,000 m asl). It is an erect herb with

Electronic supplementary material The online version of this

article (doi:10.1007/s12686-014-0361-y) contains supplementary
material, which is available to authorized users.
S. Sharma  V. Sharma  M. Chhabra  R. Rathour 
K. D. Sharma  R. K. Kapila (&)
Department of Agricultural Biotechnology, CSKHP Agricultural
University, Palampur 176 062, H.P, India
e-mail: rkkapila@gmail.com

tuberous roots, lanceolate leaves and robust stem. The

species is an important traditional medicinal plant with
uses as aphrodisiac, demulcent and nervine tonic. Due to its
high demand for therapeutic uses, unscientific exploitation
of the species is in practice. The specialized mycorrhizal
associations for growth and specific pollinator requirement
make it a slow growing and/propagating plant. Due to
sharp decline in its natural populations, D. hatagirea has
been listed as critically endangered species by Conservation Assessment and Management Plan (CAMP status),
critically rare by International Union for Conservation of
Nature and Natural Resources (IUCN) and is listed by
Convention on International Trade in Endangered Species
(CITES) under appendix II (Bhatt et al. 2005). Little effort
has been made to study its genetic diversity and population
structure at molecular level due to paucity of DNA-based
markers. Only 14 microsatellite markers have been developed so far (Lin et al. 2014) and there is need to develop
more markers.
In the present study 784 nucleotide sequences of
Dactylorhiza species available at National Centre for
Biotechnology Information database were utilized for
developing microsatellite markers. The sequences were
down loaded, processed for redundancy removal, microsatellite search and primers designing following Sharma
et al. (2009). The markers were validated across a panel of
twenty ecotypes of D. hatagirea collected from various
locations in western Himalayan region of India. The
genomic DNA was extracted and PCR performed in 10 lL
reaction volumes containing 25 ng of template DNA,
15 ng of each primer, 200 lM of each dNTP, 0.5 U of Taq
DNA polymerase and PCR buffer (HiMedia Pvt. Ltd.,
Bombay, India). The PCR conditions were: 1 cycle of
5 min at 94 C, 35 cycles of 1 min at 94 C, 1 min at
respective annealing temperature (see Table 1), 1 min at

123

123

F-AAACAAACATGCCCCAGTTA

F-GGTGTTCCTAACTGCCCACT

F-TCAGCGGAGGAGAGGTAGAA

R-CTCCTCGCGAATGAAATGAT

F-TAAACAAACATGCCCCAGTT

R-GAGCCGGACATGAGAGTTTC

F-CATGCCCCAGTTATCCACTT

R-TGGCCACTTGTAGTGAGCTG

(TA)15

(TA)8

(GAA)6

50

53

56

53

54

56

51

52

50

53

53

53

51

50

53

Ta (C)

190205

210230

230360

390405

550570

130150

190200

230

150320

320380

205400

360480

205220

150250

200290

Size range (bp)

4.2

No. of
alleles

0.623

0.500

0.100

0.400

1.00

0.600

0.400

0.400

0.800

0.800

0.625

0.900

0.900

0.500

0.800

Ho

0.631

0.789

0.658

0.337

0.611

0.442

0.337

0.353

0.774

0.858

0.892

0.763

0.813

0.394

0.821

He

0.051

0.000*

0.502

0.029*

0.223

0.502

0.929

0.046*

0.013*

0.007*

0.029*

0.164

0.354

0.001*

0.532

0.709

0.551

0.269

0.492

0.332

0.269

0.303

0.685

0.790

0.816

0.680

0.504

0.305

0.751

PIC

DQ022920

DQ022926

JQ229975

GQ244815

EU176076

DQ986357

DQ986360

DQ986361

EU884288

AY283526

AY283531

AY283532

DQ022918

EF594447

AY283535

Gene bank
accession no.

Gene bank accession no. of the sequence from which primers were designed

* The deviation from HardyWeinberg equilibrium

Ta annealing temperature, Ho observed heterozygosity, He expected heterozygosity, P probability that genotype proportions conform to HardyWeinberg equilibrium, PIC polymorphism
information content

Mean

KSSR-39

KSSR-37

KSSR-35

(CAA)3

KSSR-32

R-TGGGACTCTCTCTTTATTCTCGTC

(TA)8

F-GCCCGCGAACACTTTATTTA

(TCT)8

(AGA)6

(GAA)6

(TA)6

(TTC)4

R-CTCCTCGCGAATGAAATGAT
F-CGATGGAAGCTGTTCTAACGA

R-GACTGCAGGTAAGGGCTCAG

F-AAGGTACCACGCTTCGTCAG

R-CTCAATCATCCAAAGGGACAA

F-CTGGAAGTAGGGGGAGCAAT

R-CGATCCTCATCCTGTTTTGC

F-CGCCGACAAACTCTACATCG

R-GGGAAATGAACCTTTTGCAC

F-CGCGAAGTCAAGATTGAAAA

R-GAGAAAGAACGCCAAAGACG

(AGA)3

(TTC)4..(TTCCTC)3

(TA)6

(TA)6

(TTC)4

Repeat motif

KSSR-30

KSSR-22

KSSR-21

KSSR-20

KSSR-18

KSSR-15

F-CAGGGGGATAAGTTCTCGAC

KSSR-12

R-AGAAAGAACGCCAAAGACGA

F-TCCTCTGCAGTCTTGTTCCA
R-GAGAAAGAACGCCAAAGACG

R-GAGCCGGACATGAGAGTTTC

KSSR-11

KSSR-07

R-CCCGGCCAGTACTTAACCAG

F-CGCGAAGTCAAGATTGAAAA

R-AGAAAGAACGCCAAAGACGA

F-GGTCCAGGGGGATAAGTTCT

KSSR-02

KSSR-04

Primer sequence (50 30 )

Marker name

Table 1 Characteristics of 15 microsatellite markers developed in the present study

Conservation Genet Resour

Conservation Genet Resour

72 C and final extension for 7 min at 72 C. The amplicons were separated on 6 % denaturing polyacrylamide
gels in 19 TBE buffer and visualized by silver staining.
Polymorphic alleles for each marker were scored manually
and the data were utilized for computing various diversity
indices. Expected heterozygosity (He), Observed heterozygosity (Ho) and HardyWeinberg equilibrium were
obtained using POPGENE version 1.32 (Yeh and Boyle
1997) and polymorphism information content (PIC) was
calculated as per Botstein et al. (1980).
From the 784 nucleotide sequences, 35 primer pairs
flanking microsatellite motifs were designed. Out of the
primer pairs tested across a panel of 20 D. hatagirea ecotypes, only 15 amplified unambiguous amplicons. Of the 15
markers giving reliable amplification, 14 were polymorphic
(Table 1). These 15 primers amplified 64 alleles with a
mean value of 4.2 alleles in a size range of 130570 bp. Ho
and He values for polymorphic loci varied from 0.100
(KSSR-37) to 1.000 (KSSR-32) and 0.337 (KSSR-35) to
0.892 (KSSR-12) with average of 0.623 and 0.631,
respectively. Minimum PIC value of 0.269 was recorded
for the markers KSSR-22 and KSSR-35, whereas a maximum PIC value of 0.816 was recorded for KSSR-12 with
an overall mean PIC of 0.532. Seven markers significantly
deviated from HardyWeinberg equilibrium at P value of
0.005. These values of diversity indices clearly indicated
their potential for uncovering the genetic diversity of D.

hatagirea. The new set of microsatellite markers developed

in this study can be used to study population genetics and
the extent of genetic diversity in D. hatagirea for
addressing the conservation and related issues of this critically endangered rare orchid species.
Acknowledgments We thank University Grant Commission
(UGC), GOI for financial support under the scheme Major Research
Projects.

References
Bhatt A, Joshi SK, Gairola S (2005) Dactylorhiza hatagirea (D. Don)
Sooa west Himalayan orchid in peril. Curr Sci 89(4):610612
Botstein D, White RL, Skolnick M, Davis RW (1980) Construction of
a genetic linkage map in man using restriction fragment length
polymorphisms. Am J Human Genet 32:314331
Lin P, Zeng L, Yang Z, Liu R, Zhong Y (2014) Development and
characterization of polymorphic microsatellitemarker for Dactylorhiza hatagirea (D. Don) Soo. Conserv Genet Res. doi:10.
1007/s12686-013-0053-z
Sharma V, Bhardwaj P, Kumar R, Sharma RK, Sood A, Ahuja PS
(2009) Identification and cross-species amplification of EST
derived SSR markers in different bamboo species. Conserv
Genet 10:721724
Yeh FC, Boyle TJB (1997) Population genetic analysis of codominant and dominant markers and quantitative traits. Belg J
Bot 129:157

123