Start Codon Targeted (SCoT) marker reveals genetic diversity of

Dendrobium nobile Lindl., an endangered medicinal orchid species

Paromik Bhattacharyya, Suman Kumaria , Shrawan Kumar, Pramod Tandon
Plant Biotechnology Laboratory, Department of Botany, Centre for Advanced Studies, North-Eastern Hill University, Shillong 793022, Meghalaya, India
a b s t r a c t a r t i c l e i n f o
Article history:
Accepted 25 July 2013
Available online xxxx
Keywords:
Dendrobium nobile
PIC
SCoT
Genetic variation
Northeast India
Genetic variability in the wild genotypes of Dendrobium nobile Lindl. collected from different parts of Northeast
India, was analyzed using a Start Codon Targeted (SCoT) marker system. A total of sixty individuals comprising
of six natural populations were investigated for the existing natural genetic diversity. One hundred and thirty
two (132) amplicons were produced by SCoT marker generating 96.21% polymorphism. The PIC value of the
SCoT marker system was 0.78 and the Rp values of the primers ranged between 4.43 and 7.50. The percentage
of polymorphic loci (Pp) ranging from 25% to 56.82%, Nei's gene diversity (h) from 0.08 to 0.15 with mean
Nei's gene diversity of 0.28, and Shannon's informationindex (I) values ranging from0.13 to 0.24with anaverage
value of 0.43 were recorded. The gene ow value (0.37) and the diversity among populations (0.57)
demonstrated higher genetic variation among the populations. Analysis of molecular variance (AMOVA)
showed 43.37% of variation within the populations, whereas 56.63% variation was recorded among the
populations. Cluster analysis also reveals high genetic variation among the genotypes. Present investigation
suggests the effectiveness of SCoT marker systemto estimate the genetic diversity of D. nobile and that it can
be seen as a preliminary point for future research on the population and evolutionary genetics of this
endangered orchid species of medicinal importance.
2013 Published by Elsevier B.V.
1. Introduction
Dendrobium nobile Lindl., an epiphytic orchid, belonging to the
family Orchidaceae is one of the 1184 species of dendrobes found
worldwide (Leitch et al., 2009). Dendrobes are found to be distributed
in tropical and subtropical Asia (e.g. Northeast India, China, Japan,
Malaysia, Philippines and Borneo) and North Australia usually growing
at cooler climatic conditions at higher altitudes. In India, 103 species of
Dendrobium occur, out of which 84 species are found to be present in
Northeast India (Singh et al., 2001). D. nobile is valued greatly for its
attractive owers and is an immensely popular ornamental orchid;
plant breeders worldwide have raised numerous cultivars with showy
inorescences (Nayak et al., 2002). In addition, it has a great use tradi-
tionally in various herbal drug preparations (Ye et al., 2002). Two
bibenzyl (Gigantol and Moscatilin) and one alkaloid (Dendrobine)
compounds have been reported in D. nobile (Miyazawa et al., 1997;
Suzuki et al., 1973; Zhao et al., 2001). Both of these compounds have
been shown to have strong antimutagenic potential and were found
to be anti carcinogenic against lung carcinoma, ovaryadenocarcinoma
and promyelocytic leukemia (Lee et al., 1995). The stems are used to
alleviate thirst, calm restlessness, accelerate convalescence and reduce
dryness of the mouth (Faria and Illg, 1995). In Meghalaya, stem paste
of this orchid is applied externally on injuries and also to set fractured
bones (Hynniewta and Kumar, 2008). Thus because of its immense
ethanobotanical, ornamental and biopharmaceutical importance it has
been overexploited for centuries and has become threatened and
endangered in its natural habitat (Singh et al., 2001). An urgent need
therefore arises for thoroughexplorationandanalysis of all the available
natural genetic variations in D. nobile so as to accomplish its sustainable
utilization. Formulating a true estimation of the level and distribution of
genetic variation in endangered species is a primary objective of conser-
vation genetics (Fritsch and Rieseberg, 1996). Analysis of genetic diver-
sity using molecular markers is a trusted and reliable approach which
could provide useful baseline information of various wild plants.
In recent years, many new marker techniques have been developed
in line with the rapid growth of genomic research (Gupta and Rustgi,
2004). Due to the tremendous growth in public biological databases,
the development of functional markers that are located in or near
the candidate genes have become considerably easy (Andersen and
Lubberstedt, 2003). Initiating a trend away from random DNA markers
towards gene-targeted markers, a novel marker system called Start
Codon Targeted (SCoT) Polymorphism (Collard and Mackill, 2009)
was developed based on the short conserved region anking the ATG
start codon in plant genes. SCoT markers are generally reproducible,
and it is suggested that primer length and annealing temperature are
Gene xxx (2013) xxxxxx
Abbreviations: PCR, Polymerase Chain Reaction; SCoT, Start Codon Targeted
Polymorphism; PIC, Polymorphic Information Content; AMOVA, analysis of molecular
variance; UPGMA, unweighted pair-group method with arithmetic average; Rp,
Resolving Power.
Corresponding author. Tel.: +91 364 272 2210; fax: +91 364 255 0150.
E-mail address: sumankhatrikumaria@hotmail.com (S. Kumaria).
GENE-38888; No. of pages: 6; 4C:
0378-1119/$ see front matter 2013 Published by Elsevier B.V.
http://dx.doi.org/10.1016/j.gene.2013.07.096
Contents lists available at ScienceDirect
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Please cite this article as: Bhattacharyya, P., et al., Start Codon Targeted (SCoT) marker reveals genetic diversity of Dendrobium nobile Lindl.,
an endangered medicinal orchid species, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.07.096
not the sole factors determining reproducibility. These are dominant
markers like random amplied polymorphic DNA (RAPD) and inter-
simple sequence repeats (ISSR) and could be used for genetic analysis,
quantitative trait loci (QTL) mapping and bulk segregation analysis
(Collard and Mackill, 2009). In principle, SCoT is similar to RAPD and
ISSR because the same single primer is used as the forward and reverse
primer (Collard and Mackill, 2009; Gupta and Rustgi, 2004). The use
of these markers in diversity analysis and diagnostic ngerprinting
has been successfully demonstrated in peanut, potato and grape
(Gorji et al., 2011; Guo et al., 2012; Xiong et al., 2011).
The main objective of the present study was to investigate the
degree of genetic diversity in the wild populations of D. nobile using
SCoT markers so as to develop strategies for its conservation and
sustainable utilization.
2. Materials and methods
2.1. Study sites and sample collection
Sixty individuals of D. nobile representing six natural populations
(Fig. 1, Table S1) of Northeast India were collected and maintained in
the greenhouse of the Plant Biotechnology Laboratory, Department of
Botany, Centre for Advanced Studies, North-Eastern Hill University,
Shillong, Meghalaya, India. Due to the rarity of the number of individ-
uals in each population, the sampling size was determined in such a
way so as not to destroy the natural distribution of the plant in the
wild. This sampling strategy also ensured the full geographical range
of D. nobile in Northeast India.
2.2. Genomic DNA extraction
Total genomic DNAwas extracted fromyoung leaves of D. nobile fol-
lowing the method described by Murray and Thompson (1980) with
some minor modications. The quantity and purity of the isolated
DNA were checked using a UV spectrophotometer (Perkin Elmer Lamb-
da 35). The ratio of absorbance at two wavelengths (A
260
and A
280
) was
compared with the standard ratio of pure DNA. The quantities of the
DNA isolated were found to be optimum for further PCR amplication.
2.3. PCR optimization and SCoT-PCR amplication
A total of 36 SCoT primers were custom synthesized from Metabion
Inc. Ltd., Germany. Varying concentrations of (i) template DNA (20, 30,
40, 50 and 60 ng), (ii) Taq DNA polymerase (0.52 U) and (iii) Mg
++
salt (15 mM) were used to optimize the reaction conditions of the
PCR. Once the optimal conditions were established, amplication reac-
tions with all the genotypes and selected primers were carried out.
All the PCR reactions were carried out in 25 L volumes containing
50 ng of template DNA, 2 M of each of the four dNTPs, 1 PCR buffer
(10 mM Tris, pH 9.0, 50 mM KCl), 1.5 mM MgCl
2
, 1 U Taq polymerase
(Bangalore Genei, India) and 20 pmol of primer. The reaction programs
were set at 94 C for 4 min, followed by 35 cycles of 30 s at 94 C, 1 min
at 50 C and 2 min at 72 C, with a nal extension at 72 C for 5 minin a
thermal cycler 2720 (Applied Biosystems, USA). After completion of the
amplication, 2.5 L of 10 blue dye was added to the samples, and
the amplied DNA was analyzed on 1.5% agarose gel in 1 TAE buffer
at 6570 V for 34 h. DNA fragments were visualized under a UV light
Fig. 1. Geographical distribution of the sampled D. nobile genotypes.
2 P. Bhattacharyya et al. / Gene xxx (2013) xxxxxx
Please cite this article as: Bhattacharyya, P., et al., Start Codon Targeted (SCoT) marker reveals genetic diversity of Dendrobium nobile Lindl.,
an endangered medicinal orchid species, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.07.096
and photographed using Gel Documentation System (Biostep DH-20,
Germany).
2.4. Data analysis
Banding proles generated by SCoT primers were compiled into a
data binary matrix based on the presence (1) or absence (0) of the
selected band. Only clear, unambiguous and reproducible bands ampli-
ed in both cases were considered for the scoring and data. Smeared
and weak bands were excluded. The ability of the primers to distinguish
between genotypes was accessed by calculating the Resolving Power
(Rp) (Prevost and Wilkinson, 1999) for SCoT primers. This function
has been found to correlate strongly with the ability to distinguish
between genotypes, and the formula Rp = I
b
where band informa-
tiveness, I
b
= 1 (2 |0.5 p|) and p is the proportion of genotypes
containing band I. Polymorphic Information Content (PIC) value was
calculated using the formula PIC = 1 pi2, where pi is the frequency
of the ith allele (Smith et al., 1997). Genetic diversity parameters includ-
ing the percentages of polymorphic loci (Pp), Nei's gene diversity (h),
and Shannon index (I) were calculated with POPGENE version 1.31
(Yeh et al., 1999) to estimate the genetic variation level. Gene ow
(Nm) was estimated from Nm = 0.25 (1 Gst) / Gst. Analysis of
molecular variance (AMOVA) was performed using Arlequin version
3.01 (Excofer et al., 2005) at two hierarchical levels to examine differ-
ences among and within populations. The Fixation index or F statistics
(F
ST
) was also calculated with Arlequin v. 3.01. The signicance of this
analog was evaluated by 1000 random permutations of sequences
among populations (Miller, 1998). Pair-wise similarity matrices were
generated by Jaccard's coefcient of similarity, using the SIMQUAL
format of NTSYSpc (Rohlf, 1998). A dendrogram was constructed by
using the unweighted pair group method with arithmetic average
(UPGMA) with the SAHNmodule of NTSYSpc to showa phenetic repre-
sentation of genetic relationships as revealed by the similarity coef-
cient (Sneath and Sokal, 1973).
3. Results
3.1. SCoT-PCR
For analysis of variability of D. nobile, 16 primers were used for
studying the SCoT banding patterns across the entire samples. A total
of 132 amplication products were scored of which 127 were poly-
morphic, exhibiting 96.15% polymorphism. The amplication products
using 16 primers ranged from 75 to 100% in producing polymorphic
bands (Table 1). The primers S4, S5, S6, S7, S9, S10, S12, S17, S25, S26,
S32, S34, S35 and S36 exhibited the highest level of polymorphism
with the percentage of polymorphic bands to be 100% for all these
primers (Table 1; Figs. 2A and B). The average number of amplication
products was 8.25 per primer; the maximumwas 12 with S10 and S34,
whereas the minimum was 5 with S24. The PIC value for the SCoT
primers was 0.78. The Rp value of the SCoT primers ranged from 4.43
(S25) to 7.50 (S10). The genetic distance recorded using Jaccard's coef-
cients of similarity ranged from 0.32 to 0.95 (Table 1).
3.2. Population structure
The observed number of alleles (Na) and effective number of alleles
(Ne) ranged between 1.251.56 and 1.141.25, respectively. Similarly,
Nei's gene diversity (h) and Shannon's Information index (I) ranged be-
tween 0.080.15 with overall diversity of 0.28 and 0.130.24 with an
average value of 0.43, respectively. The percentage of polymorphic loci
(Pp) was estimated in the range of 25.00% to 56.82%. The gene ow
value and the diversity among populations were found to be 0.37 and
0.57, respectively (Table 2). The Fixation index or F statistics (F
ST
) was
found to be 0.56. The Nei's unbiased measures of genetic identity and
genetic distance among the population have also been calculated
(Table 3; Fig. S1). Analysis of AMOVA (P b 0.001) showed that genetic
variation (43.37%) was observed within the populations, whereas the
variance among populations was 56.63% (Table 4).
3.3. Cluster analysis
Based on UPGMA clustering algorithm generated from the obtained
SCoT dataset, the populations were grouped into three major clusters
(Fig. 2C). Cluster I, consisted of populations fromMeghalaya and Sikkim.
Cluster II, comprised of populations from Arunachal and Sikkim and
Cluster III, populations from Mizoram, Nagaland and Manipur.
4. Discussion
In spite of the immense horticultural and medicinal importance, the
available information on the genetic diversity of D. nobile is very limited.
Presently, a very large number of dendrobes are being used in oricul-
ture industry. Consequently the importance of wild germplasm for
breeding programs cannot be ignored. However, habitat destruction
and overexploitation are the two important factors threatening the
Table 1
Data of SCoT primers used in the present study and the extent of polymorphism.
Sl No. Primer
name
Primer `sequence
(5-3)
Total no.
of bands
No. of poly-morphic
bands
No. of mono-morphic
bands
% of Poly-morphisim
(P)
Resolving Power
(Rp)
PIC
a
Distance range
(Jaccard's coefcient)
1. S4 CAACAATGGCTACCACCT 8 8 0 100 7.40 0.78 0.320.95
2. S5 CAACAATGGCTACCACGA 6 6 0 100 5.26
3. S6 CAACAATGGCTACCACGC 7 6 1 85.71 5.66
4. S7 CAACAATGGCTACCACGG 9 9 0 100 6.20
5. S9 CAACAATGGCTACCACGT 6 6 0 100 6.23
6. S9 CAACAATGGCTACCAGCA 6 6 0 100 6.06
7. S10 CAACAATGGCTACCAGCC 12 12 0 100 7.50
8. S12 ACGACATGGCGACCAACG 8 6 2 75 5.26
9. S17 ACCATGGCTACCACCGAG 7 7 0 100 6.23
10. S25 ACCATGGCTACCACCGGG 5 5 0 100 4.43
11. S26 ACCATGGCTACCACCGTC 8 8 0 100 5.76
12. S32 CCATGGCTACCACCGCAC 11 11 0 100 5.50
13. S33 CCATGGCTACCACCGCAG 9 8 1 88.88 7.46
14. S34 ACCATGGCTACCACCGCA 12 12 0 100 7.13
15. S35 CATGGCTACCACCCGCCC 9 9 0 100 6.13
16. S36 GCAACAATGGCTACCACC 9 8 1 88.88 6.66
Total 132 127 5 96.15 98.47
Average 8.25 7.93 0.31
a
Polymorphic information content = PIC.
3 P. Bhattacharyya et al. / Gene xxx (2013) xxxxxx
Please cite this article as: Bhattacharyya, P., et al., Start Codon Targeted (SCoT) marker reveals genetic diversity of Dendrobium nobile Lindl.,
an endangered medicinal orchid species, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.07.096
survivability of D. nobile in India. There is an urgent need to conserve
this rare and endangered taxon. Plant population employs its genetic
system as a tool for persisting over generations in an ever changing
environment (Hatteme and Melchior, 1993). In viewof this, it is neces-
sary to assess the genetic diversity of the plant species facing threats
from over exploitation.
Fig. 2. Banding prole in D. nobile using SCoT primer (A) S10 and (B) S35. (C) UPGMA clustering of D. nobile populations based on Jaccard's similarity coefcient.
Table 2
Genetic diversity and differentiation parameters for six natural populations of D. nobile in Northeast India.
Populations Ss Na SD Ne SD h SD I SD Pp Hsp Hpop Gst Nm F
ST
Meghalaya
(ML)
14 1.52 0.50 1.24 0.33 0.15 0.18 0.23 0.26 52.27
Sikkim
(SK)
14 1.56 0.49 1.24 0.31 0.15 0.17 0.24 0.25 56.82
Mizoram
(MZ)
07 1.34 0.47 1.23 0.37 0.12 0.19 0.19 0.28 34.09
Nagaland
(NL)
05 1.36 0.48 1.25 0.38 0.13 0.20 0.20 0.28 36.36
Manipur
(MN)
04 1.27 0.44 1.14 0.28 0.08 0.15 0.13 0.23 27.27
Arunachal
(AR)
16 1.25 0.43 1.15 0.30 0.08 0.16 0.13 0.24 25.00
Total 60 1.96 0.19 1.46 0.33 0.28 0.16 0.43 0.21 96.21 0.28 0.125 0.57 0.37 0.56
h = Nei's gene diversity; I = Shannon's information index; Na = observed number of alleles; Ne = effective number of alleles; Np = number of polymorphic loci; Pp = percentage of
polymorphic loci; SD = standard deviation; Gst = diversity among populations; Nm = gene ow 0.25 (1 Gst) / Gst; Hpop = variability within population, Hsp = total variability;
Ss = no. of individuals; F
ST
= Fixation index or F statistics.
4 P. Bhattacharyya et al. / Gene xxx (2013) xxxxxx
Please cite this article as: Bhattacharyya, P., et al., Start Codon Targeted (SCoT) marker reveals genetic diversity of Dendrobium nobile Lindl.,
an endangered medicinal orchid species, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.07.096
In comparison to traditional methods, DNA ngerprinting unfolds
plant variability directly at genetic levels with reliable data required
for the estimation of genetic diversity. In contrast to the huge size of
the genus, only a small part of the Dendrobiumspecies have been exam-
ined for its genetic diversity at molecular level, primarily based on ITS
sequence polymorphisms (Burke et al., 2008; Tsai et al., 2004). Although
Wang et al. (2009) reported the phylogeny of 31 Dendrobium species
collected from the Yunan province of China, no reports exists on the
genetic diversity of Dendrobes including D. nobile found in India.
Revealing the genetic diversity among the genotypes of D. nobile
collected from Northeast India is an important step towards framing
up the signicant breeding program and conservation strategies of its
unexploited germplasm.
The SCoT technique is sensitive to low levels of genetic variations
and thus provides a very useful tool for analyzing population genetics
on a wide range of plants as well as identifying species or population
of the same species (Collard and Mackill, 2009). In the present study,
the representatives of the genus D. nobile collected from different
parts of Northeast India where the plant can still be found in its natural
habitat, were differentiated by the DNAngerprinting patterns using 16
SCoT primers. The efcacy of the SCoT technique in this study is further
supported by the obtained PIC and Rp values of the primers used in the
analysis. The PIC value of the SCoT marker system was found to be 0.78
and the Rp values ranged between 4.40 and 7.50 which are at par with
the optimal PIC and Rp values (Prevost and Wilkinson, 1999; Smith
et al., 1997). These two parameters provide a benchmark which helps
to determine the effectiveness of the primers used in the ngerprinting
process and consequently the ingenuity of the technique.
Accurate estimates of genetic diversity are important for conserving
and preserving rare plants (Nongrum et al., 2012; Zhang et al., 2005).
A lesser to higher levels of genetic diversities have been reported in
the endangered orchids, viz., Goodyera procera, Changnienia amoena,
Gastrodia elata and Vanda coerulea (Li and Ge, 2006; Manners et al.,
2013; Wong and Sun, 1999; Wu et al., 2006). In the present study,
using AMOVAit has beenrevealed that there exists a higher distribution
of genetic variation among populations (56.63%) as compared to within
populations (43.37%) of D. nobile which is also supported by Gst values.
Li and Ge (2006) also found moderate genetic differentiation among
populations of C. amoena, a rare endangered orchid of China. The distri-
bution pattern of genetic variation in plants can be inuenced by
various life-history traits, with breeding system having a particularly
signicant effect (Bussell, 1999; Nybom and Bartish, 2000).
Pollen and seed are the effective modes of gene owin other orchids
(Ding et al., 2008). In the present study the projected gene owis found
to be low (Nm = 0.37). The geographical isolation of populations is a
probable cause of low gene ow in D. nobile, as they grow primarily in
mountain forests as well as on mossy limestone rocks at elevations of
800 to 2000 m. In addition, it is difcult for pollinators to y over the
mountain barrier to enable inter-population pollen transfer (Wong
and Sun, 1999). Also, in most of Orchidaceae there is lowseed germina-
tion rate and this might as well contribute to the low gene ow. Ding
et al. (2008) also found similar results in the case of Dendrobium
ofcinale, an endangered orchid.
This is the rst study on the genetic diversity of D. nobile from
Northeast India utilizing SCoT marker system. The present study based
on SCoT method revealed higher genetic diversity among-populations
but moderate genetic diversity within-populations. Thus, identication,
collection and maintenance of such genetically diverse wild germplasm
fromNortheast India would be of great importance in the formulation of
conservation strategies for this endangered taxon. As a rare medicinal
herb and a genus which is being endangered, D. nobile deserves special
conservation attention. The ultimate goals of conservation are to ensure
the continuous survival of populations and to maintain the evolutionary
potential of the species (Wong and Sun, 1999). Most orchid species in-
cluding D. nobile have strong habitat preference and pollinator depen-
dence (IUCN/SSC Orchid Specialist Group, 1996). Therefore, habitat
protection would ensure the species' coexistence with other organisms
like fungi and pollinators on which orchids depend on for the comple-
tion of their life cycles (Li and Ge, 2006). The present study indicates
that SCoT marker system could be used for evaluation of genetic
relationships, which ultimately would be helpful in characterization of
various other endangered species belonging to the family Orchidaceae.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.gene.2013.07.096.
Conict of interest
None.
Acknowledgment
This research work was supported by the University Grants
Commission, India and a research grant to PB in the form of the
Meritorious Student Fellowship which is gratefully acknowledged.
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Within populations 54 555.129 10.280 43.37
Total 59 1246.883 23.70477 100
d.f = degrees of freedom.
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6 P. Bhattacharyya et al. / Gene xxx (2013) xxxxxx
Please cite this article as: Bhattacharyya, P., et al., Start Codon Targeted (SCoT) marker reveals genetic diversity of Dendrobium nobile Lindl.,
an endangered medicinal orchid species, Gene (2013), http://dx.doi.org/10.1016/j.gene.2013.07.096