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

Conservation Genetics Resources

ISSN 1877-7252

Conservation Genet Resour

DOI 10.1007/s12686-014-0361-y

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 Author's personal copy
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
Ó 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. hatagi-
rea. 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
Hardy–Weinberg 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 (2,500–5,000 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 Conserva-
tion 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 devel-
oped 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, micro-
satellite 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
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 Table 1 Characteristics of 15 microsatellite markers developed in the present study
Marker name Primer sequence (50 –30 ) Repeat motif Ta (°C) Size range (bp) No. of Ho He P PIC Gene bank
alleles accession no.£

123
KSSR-02 F-GGTCCAGGGGGATAAGTTCT (TTC)4 53 200–290 8 0.800 0.821 0.001* 0.751 AY283535
R-AGAAAGAACGCCAAAGACGA
KSSR-04 F-CGCGAAGTCAAGATTGAAAA (TA)6 50 150–250 2 0.500 0.394 0.354 0.305 EF594447
R-CCCGGCCAGTACTTAACCAG
KSSR-07 F-AAACAAACATGCCCCAGTTA (TA)6 51 205–220 7 0.900 0.813 0.164 0.504 DQ022918
R-GAGCCGGACATGAGAGTTTC
KSSR-11 F-TCCTCTGCAGTCTTGTTCCA (TTC)4..(TTCCTC)3 53 360–480 6 0.900 0.763 0.029* 0.680 AY283532
R-GAGAAAGAACGCCAAAGACG
KSSR-12 F-CAGGGGGATAAGTTCTCGAC (AGA)3 53 205–400 8 0.625 0.892 0.007* 0.816 AY283531
R-AGAAAGAACGCCAAAGACGA
KSSR-15 F-GGTGTTCCTAACTGCCCACT (TTC)4 53 320–380 8 0.800 0.858 0.013* 0.790 AY283526
R-GAGAAAGAACGCCAAAGACG
KSSR-18 F-CGCGAAGTCAAGATTGAAAA (TA)6 50 150–320 4 0.800 0.774 0.046* 0.685 EU884288
R-GGGAAATGAACCTTTTGCAC
KSSR-20 F-CGCCGACAAACTCTACATCG (GAA)6 52 230 1 – – – – DQ986361
R-CGATCCTCATCCTGTTTTGC
KSSR-21 F-CTGGAAGTAGGGGGAGCAAT (AGA)6 51 190–200 3 0.400 0.353 0.929 0.303 DQ986360
R-CTCAATCATCCAAAGGGACAA
KSSR-22 F-AAGGTACCACGCTTCGTCAG (TCT)8 56 130–150 2 0.400 0.337 0.502 0.269 DQ986357
R-GACTGCAGGTAAGGGCTCAG
KSSR-30 F-GCCCGCGAACACTTTATTTA (TA)8 54 550–570 2 0.600 0.442 0.223 0.332 EU176076
R-CTCCTCGCGAATGAAATGAT
Author's personal copy

KSSR-32 F-CGATGGAAGCTGTTCTAACGA (CAA)3 53 390–405 2 1.00 0.611 0.029* 0.492 GQ244815

R-TGGGACTCTCTCTTTATTCTCGTC
KSSR-35 F-TCAGCGGAGGAGAGGTAGAA (GAA)6 56 230–360 2 0.400 0.337 0.502 0.269 JQ229975
R-TGGCCACTTGTAGTGAGCTG
KSSR-37 F-CATGCCCCAGTTATCCACTT (TA)8 53 210–230 4 0.100 0.658 0.000* 0.551 DQ022926
R-GAGCCGGACATGAGAGTTTC
KSSR-39 F-TAAACAAACATGCCCCAGTT (TA)15 50 190–205 5 0.500 0.789 0.051 0.709 DQ022920
R-CTCCTCGCGAATGAAATGAT
Mean 4.2 0.623 0.631 0.532
Ta annealing temperature, Ho observed heterozygosity, He expected heterozygosity, P probability that genotype proportions conform to Hardy–Weinberg equilibrium, PIC polymorphism
information content
* The deviation from Hardy–Weinberg equilibrium
£
Gene bank accession no. of the sequence from which primers were designed
Conservation Genet Resour
 Author's personal copy
Conservation Genet Resour
72 °C and final extension for 7 min at 72 °C. The ampli-
cons 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 hetero-
zygosity (Ho) and Hardy–Weinberg 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 eco-
types, 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 130–570 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 maxi-
mum PIC value of 0.816 was recorded for KSSR-12 with
an overall mean PIC of 0.532. Seven markers significantly
deviated from Hardy–Weinberg 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 crit-
ically endangered rare orchid species.
Acknowledgments We thank University Grant Commission
(UGC), GOI for financial support under the scheme ‘‘Major Research
Projects’’.
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