Available via license: CC BY 4.0
Content may be subject to copyright.
DNA Proling of the Threatened Himalayan Herb Polygonatum
Verticillatum L. using Cross-Transferred Betula SSR Markers
PITAMBER DUTT SHARMA1 and VIKAS SHARMA2*
1Department of Life Sciences Sant Baba Bhag Singh University Khiala, Jalandhar, Punjab, India.
2Department of Agriculture, Sant Baba Bhag Singh University Khiala, Jalandhar, Punjab, India.
Abstract
Polygonatum verticillatum is an important Himalayan herb that is used
in different medicine systems for improving health and curing many
diseases. Herein, simple sequence repeat (SSR) marker characterization
of this plant species was performed using cross-transferred SSR markers
of a distantly related species Betula utilis. Among the 25 SSR markers
tested, 13 generated clearly distinguishable alleles. Of these, 12 SSR
primers were polymorphic and 1 was monomorphic. All the 12 markers
collectively amplied 42 alleles. The average value of 3.5 alleles was
observed. The size of alleles ranged from 100 - 600 bp. The mean
polymorphism information content (PIC) was 0.459, and mean marker
index was 1.61. The dendrogram clustered all the studied accessions into
three groups according to geographical locations. The results showed high
genetic diversity in the populations of P. verticillatum in Indian Himalayan
region. SSR marker exhibited good amplication in distantly related species.
The SSR markers used in the present work can help diversity and breeding
research of P. verticillatum in coming days. The results of present work will
be helpful for characterization, conservation, management and improvement
of the germplasm of this plant in the future.
CONTACT Vikas Sharma vikasam@gmail.com Department of Agriculture, Sant Baba Bhag Singh University Khiala, Jalandhar,
Punjab, India.
© 2024 The Author(s). Published by Enviro Research Publishers.
This is an Open Access article licensed under a Creative Commons license: Attribution 4.0 International (CC-BY).
Doi: http://dx.doi.org/10.12944/CARJ.12.2.36
Article History
Received: 29 May 2024
Accepted: 20 June 2024
Keywords
Cross-Transferability;
Genetic Diversity;
Polygonatum verticillatum;
Simple Sequence Repeat
(SSR);
Betula Utilis.
Current Agriculture Research Journal
www.agriculturejournal.org
ISSN: 2347-4688, Vol. 12, No.(2) 2024, pg. 958-966
Introduction
Polygonatum verticillatum L. is a highly valued
medicinal herb of Indian Himalayan region (IHR)
and occurs from 2000 -3000 m asl. It is a member
of family Asparagaceae. It is known as “Meda” in
Sanskrit and “Salam Mishri” in Hindi. This herb yield
important metabolites and is one of the constituents
of many ayurvedic formulations.1-3 Morphologically,
plants are slender and generally, unbranched with
leaves arranged in verticillaster manner. Leaves are
lanceolate, and stem bear owers near the base
of leaves. Flower colour is generally pale yellow
and greenish. The fruits are round berries that are
initially green in colour and become red when ripe.
It is a plant of high importance. However, unscientic
exploitation by local traders and anthropogenic
959SHARMA & SHARMA., Curr. Agri. Res., Vol. 12(2) 958-966 (2024)
activities has resulted in habitat destruction of its
natural populations, which has resulted in the loss
of the dierent wild genetic stocks of this valuable plant
species. It has been reported from Uttarakhand that
plant is vulnerable due to overuse by pahemaceutical
companies, less awareness about the plant among
local people, habitat destruction and habitat
fragmentation in addition to unscientic harvesting
and other anthropogenic activities.4,5 Hence, it is
also listed in threatened plants category.6,7 In this
situation, supreme importance should be given for
characterizing the existing germplasm at molecular
level for its genetic diversity and conserving its
diverse germplasm. Genetic diversity data can
be useful in identifying the diverse accessions
which can be selected for conservation and for
breeding experiments on priority basis. Diversity
data can also be used for better management
of the germplasm. Further, molecular characterization
should be carried out at genetic level to identify elite
germplasm stocks. This will help maintain diverse
germplasms in the future and consequently will help
in their conservation and management. Molecular
markers, specically DNA markers, are the highly
preferred for the genetic characterization of any
type of germplasm and plant collection. There
are various types of DNA markers such as RFLP,
RAPD, AFLP, ISSR, SSR, and SNP. Of these, SSR
markers are currently the most commonly used DNA
markers due to their desirable features such as the
potential to resolve heterozygosity, easy laboratory
procedures, cross-transferable nature and evenness
in genomes.8-15 Hence, these markers are widely
used in plant genetic diversity research.
Table 1: List of Accessions Characterized in the Present Study using
Cross-Transferred SSR Markers
S. No. Sample Location District State
code
1. JH Jhungi Mandi Himachal Pradesh
2. JL Jalori Kullu Himachal Pradesh
3. TU-1 Tunga Dhar Mandi Himachal Pradesh
4. TU-2 Tunga Dhar Mandi Himachal Pradesh
5. KP Kataula Mandi Himachal Pradesh
6. KS Fatehpura Anantnag Kashmir
7. TU-3 Tunga Dhar Mandi Himachal Pradesh
8. TU-4 Tunga Dhar Mandi Himachal Pradesh
9. SR-1 Sarahan Shimla Himachal Pradesh
10. SR-2 Sarahan Shimla Himachal Pradesh
11. SN Solang Nullah Kullu Himachal Pradesh
12. JL-1 Jalori Kullu Himachal Pradesh
13. KT-1 Kala Top Chamba Himachal Pradesh
14. KT-2 Kala Top Chamba Himachal Pradesh
15. TU-5 Tunga Dhar Mandi Himachal Pradesh
16. TU-6 Tunga Dhar Mandi Himachal Pradesh
17. JL-1 Jalori Kullu Himachal Pradesh
18. KTH-1 Kainthley Chamba Himachal Pradesh
19. KTH-2 Kainthley Chamba Himachal Pradesh
20. KTH-3 Kainthley Chamba Himachal Pradesh
21. KTH-4 Kainthley Chamba Himachal Pradesh
22. KTH-5 Kainthley Chamba Himachal Pradesh
Moreover, the cross-transferability of SSR markers
across species and genera is of great importance
for exploring plant species for which SSR markers
are not available. Many researchers have utilized
the SSR markers from closely related and distantly
related plant species to characterize and study the
960SHARMA & SHARMA., Curr. Agri. Res., Vol. 12(2) 958-966 (2024)
genetic diversity of germplasms of other species.7,12-15
Few biochemical and genetic studies have been
conducted in different species of Polygonatum,
and more such studies are required for proper
utilization and maintenance of this important genetic
resource. Researchers categorized Polygonatum
as an important genus with immense potential.16,17
This plant contains many vital compounds, which
exhibits curative and health promoting eects. Some
important chemical constituents have been isolated
and identied in this species. The researchers have
developed method for identifying adulterants in
mixtures of P. verticillatum.18 As it is a threatened
plant, some conservationists have tried to establish
tissue culture methods for its rapid propagation
and availability.19 However, genetic characterization
studies on this species are lacking. In other species
of the genus Polygonatum, such investigations have
been performed. In P. sibiricum, attempt has been
made to explore genes involved in biosynthetic
pathways.20 Other researchers identified some
phytochemicals and antioxidants from this species21
P. cyrtonema Hua, which is an endemic species
of this genus in China, was evaluated for its genetic
diversity and structure in Anhui with the help
of SSRs and morphological traits.22 Genetic diversity
of P. multiorum and P. odoratum was also explored
by Chinese researchers.23 Cross-species SSR
markers were evaluated for their cross- transferability
in P. verticillatum.24 A comparative study on clones
and genetic structure was also undertaken in
Polygonatum.25 Dierent DNA markers were used
for studying the genetic similarity of three species of
Polygonatum, including P. verticillatum, occurring in
Poland.26 In India only one study using ISSR markers
was conducted in P. verticillatum.27 Therefore, In the
present study, SSR markers of an alpine species,
namely, Betula utilis (Betula) were checked for cross-
transferability in P. verticillatum and unambiguously
amplied markers were utilized to characterize and
study the genetic diversity of P. verticillatum. The
rationale for using Betula utilis SSR was overlapping
habitat of both the species. Both these species occurs
in temperate and alpine regions of Himalaya and it
was assumed that at genetic level these may contain
similar genomic regions which help in adaptation
of these species in their habitats. The results of current
research work can be important for future research
pertaining to conservation, breeding and manage-
ment of this species.
Materials and Methods
Plant Sampling and DNA Extraction
Our sampling sites included state of Himachal
Pradesh and Union Territory of Kashmir, India. Leaf
samples from twenty two accessions of P.
verticillatum were collected from dierent regions
of four districts of Himachal Pradesh and one district
of Kashmir. Young and fresh leaf samples were
collected in plastic bags containing silica gel and
transported to the laboratory at room temperature.
DNA isolation was performed according to the CTAB
method28 using liquid nitrogen. A detailed description
of accessions is given in Table 1.
PCR Reactions
In total, twenty-ve SSR primers were checked for
amplication in a pooled DNA sample. Among the
25 SSR primers of B. utilis,29 13 SSR primers were
clearly amplied. Finally, these 13 unambiguously
amplied primers were chosen for the SSR diversity
study. The nal volume of SSR reaction mixture was
consisted of 10 µl. The composition of this included
4.8 µl deionized water, 2.0 µl template DNA of 13 ng/
µl quantity, 0.5 µl of each forward and reverse primer
with 5 µM concentration, 0.5 µl MgCl2 (25 mM),
1.0 µl 10 X buer containing 10 mMTris-Hcl, 50
mMKcl having pH 8.3, 0.5 µl dNTP mix consisting
of 0.2 mM each dATP, dGTP, dCTP and dTTP, and
lastly 0.2 µl Taq polymerase with 5U/ µl. The PCR
conditions were set as: First stage- 1 cycle of 5 min
at 94 ºC, Scond stage- 35 cycles of 1 min at 94 ºC, 1
min at annealing temperature of each primer, 1 min
at 72 ºC and third stage- 7 min at 72ºC. PCR amplied
products were run on 3% agarose gel in 1 X TBE
buer for visualization of fragments, and size of each
fragment was determined with 100 bp DNA ladder
(Genei, Bangalore). Ethidium bromide dye was used
for detecting DNA fragments. Photo of gel was taken
with the help of gel documentation system (Bio-print,
Vilber Eppendorf, France).
Data Analysis
For analysis, DNA bands detected in the agarose
gel were manually scored. The clearly amplied
alleles were considered for scoring. After scoring,
a binary data le was created in an Excel sheet,
and all downstream analyses were performed
using this binary le. Polymorphism Information
Content (PIC) was determined with the formula as
per Botstein and his group.30 PIC is the indicatore
961SHARMA & SHARMA., Curr. Agri. Res., Vol. 12(2) 958-966 (2024)
of the level of polymorphism which the samples
exhibit and can be employed to select polymorphic
samples and primers. Similarly, Marker Index (MI)
is the indicator of the eciency of a marker and can
be used to dierentiate most informative versus
least informative markers. Cluster analysis was
done using distance based method and Jaccards
similarity coefficient with UPGMA was used
to generate dendrogram with DARwin.31
The Groupings shown in the dendrogram were
observed, and inferences and interpretations were
made to reach conclusions.
Results
SSR Data and Diversity Indices
Thirteen SSR primers generated clear prominant
alleles. In total, 12 primers were polymorphic, and
generated 42 alleles. The average value of allele was
3.5. The size of alleles ranged from 100 bp to 600
bp. A representative gel image is given in Figure 1.
Fig. 1: Representative gel showing the amplication the of SSR primer BUMS-5
in twenty-two accessions of Polygonatum verticillatum.
Table 2: Features of Primers used to Characerize Polygonatum Verticillatum Accessions
PIC: Polymorphism Information Content, MI: Marker Index, *BUMS-14 was not included for
S.No. Name of Amlied or No. of Sige PIC MI
SSR primer not (Yes/ No) Alleles range
1. BUMS-03 Yes 5 160-500 0.404 2.02
2. BUMS-05 Yes 4 150-700 0.490 1.96
3. BUMS-06 Yes 2 200-330 0.5 1
4. BUMS-08 Yes 4 170-500 0.456 1.82
5. BUMS-10 Yes 4 180-420 0.493 1.97
6. BUMS-14 Yes 1 400 - -
7. BUMS-15 Yes 5 150-500 0.486 2.43
8. BUMS-16 Yes 3 150-500 0.444 1.33
9. BUMS-18 Yes 3 200-500 0.454 1.36
10. BUMS-21 Yes 4 100-400 0.479 1.91
11. BUMS-22 Yes 4 190-600 0.5 2
12. BUMS-24 Yes 2 160-250 0.375 0.75
13. BUMS-25 Yes 2 220-370 0.433 0.867
Mean 3.5* - 0.459 1.61
nding the mean value of No. of Alleles as it was monomorphic.
962SHARMA & SHARMA., Curr. Agri. Res., Vol. 12(2) 958-966 (2024)
The lowest number (2) of alleles were amplied
by two primer pairs, i.e. BUMS-24 and BUMS-25
while the maximum number of alleles was 5 and
amplied by two primers i.e. BUMS-03 and BUMS-
15, as shown in Table 2. Maximum value of PIC
was 0.500, exhibited by the primer BUMS-6 and the
primer BUMS-22. Minimum value of PIC (0.375) was
observed in case of primer BUMS-24. The mean PIC
was 0.459. Maximum marker index was detected by
the primer BUMS-15 (2.43), and lowest marker index
was observed in case of the primer BUMS-24 (0.75).
The mean marker index was 1.61. The dendrogram
clustered studied accessions into three groups
(Figure 2). Group-I consisted of two accessions
i.e. SR-2 and KP. Group-II consisted of JA, JH, KS,
SN, SR-1, TU-1, TU-2, TU-3, TU-4, TU-5 and TU-6.
Group-III contained accessions KTH-1, KTH-2, KTH-
3, KTH-4, KTH-5, JL, JL-1, KT-1 and KT-2.
Discussion
Genetic diversity indicates allele polymorphism in an
observed plant species and can be an excellent
measure for estimating the dynamics of alleles
through time. Change in alleles of the existing
populations may be driven by forces such as
selection, habitat destruction, overexploitation,
anthropogenic and geographical disturbances. On
the other hand, the detected polymorphisms can
be utilized to manage and manipulate the existing
germplasm for improvement through different
plant breeding approaches. Hence, detecting
DNA diversity is a prerequisite for dierent types
of research. Previously, Suyal and her co-authors
investigated the morphological, phytochemical and
genetic diversity of P. verticillatum from Uttarakhand
using ISSR markers.26 However, there study did
not include any sample from Kashmir or Himachal
Pradesh. In addition, ISSR markers are supposed
to be dominant markers and may not reveal genetic
aspects as the SSR markers can. Furthermore,
they also suggested a high genetic diversity in the
studied samples. In the present study, SSR markers
of a distantly related plants species namely, B. utilis
were successfully employed. Among 12 markers, 11
had PIC value greater than 4. Hence, it is suggested
that the markers used in the present study can also
be helpful for proling and assessing population
structure at larger levels with more samples. In the
past, several authors have studied dierent species
of Polygonatom, but the lines of work and methods
used differed. Sheng and his group analyzed
P. cyrtonema and showed normal levels of genetic
diversity, and accessions clustered into three
distinct genetic groups.21 Other workers evaluated
the phenotypic variation and population genetic
structure of P. multiorum and P. odoratum and
their results revealed the eect of light availability
to owering intensity and population structure.22
Sharma with his co-authors checked the cross-
transferability of SSR markers of T. govanianum in
P. verticillatum and oberseved that 10 SSRs showed
reliable amplication.23 Chung and his team used
twenty-one allozyme loci in P. stenophyllum and
P. inatum.24 Their data suggest that populations of
P. stenophyllum have been mainly founded by a single
seed or rhizome by river water or by few seeds,
whereas populations of P. inflatum would have
been established through multiple, repeated
seedling recruitment. Szczecińska with his group
determined the genetic similarity of three species
i.e. P. multiorum, P. odoratum and P. verticillatum,
and concluded that P. verticillatum showed no
considerable diversity.25 When compared to some
earlier studies which were done on dierent Hima-
layan herbs by various authors, it was found that
mean value (0.459) of PIC and mean MI value (1.61)
was higher than observed in Trillium govanianum by
Dhyani and his co-workers.32 On the other, Rana et al.
observed 0.513 value of PIC which was higher
than observed by us in current study.33 A high
level of genetic diversity was reported in another
Himalayan herb Rheum australefrom western
Himalayan region.34 Pant et al. also recorded 0.46
value of PIC which was almost equal to the value
detected by us herein.35 These diversity values
indicated that considerable genetic diversity exists in
P. verticillatum which is comparable to diversity
detected in other Himalayan herbs.
The diversity detected in the present study was high,
and the dendrogram clustered all the twenty-two
accessions into three groups. The samples were
grouped according to geographical locations;
however, few samples were mixed within dierent
geographical locations. Furthermore, groupings of the
dendrogram revealed that the samples from Chamba
district (KTH-1, KTH-2, KTH-3, KTH-4, KTH-5, KT-1
and KT-2) of Himachal Pradesh were conserved and
grouped into a single group with two accessions
(JL and JL-1) from Kullu district. Other accessions
963SHARMA & SHARMA., Curr. Agri. Res., Vol. 12(2) 958-966 (2024)
seemed to be mixed within the three groups; however,
the accessions of disctrict Mandi (JH, KS, SN, SR-1,
TU-1, TU-2, TU-3, TU-4, TU-5 and TU-6) remained
in one group except for KP. These exceptions
in clustering may be attributed to low number of
markers used. However, it is interesting that the
distantly related SSR markers have the ability to
distinguish and generate enough polymorphisms for
genetic diversity studies in P. verticillatum. Group-II
also showed subclustering to some extent, and three
sub-groups were detected. Except for one accession
from Kashmir and one accession from Shimla, the
other accessions from district Mandi and district
Kullu almost grouped in single groups, which indicate
that every population has some conserved alleles
which resulted in such clustering. Based on this
diversity data, the populations belonging to diverse
accessions such as TU4, SR2, KP and KS can be
selected for conservation in their natural strands.
Moreover, the germplasm of identied populations
can be raised through tissue culture techniques for
its large scale propagation for even commercial
cultivation. Furthermore, a detailed study with a
large sample size could be helpful. The limited
number of SSR markers and small sample size can
be the limitation of the present study, however, the
results are encouraging. In the future, studies with
large number of samples and more SSR markers
are needed for the clarity of allele arrangements in
dierent populations of this species.
Fig. 2: Dendrogram showing twenty-two accessions of Polygonatum verticillatum clustered
based on thirteen SSRs marker data.
Prospective Conservation and Management
Plans
At present, natural populations of P. verticillatum are
found in patches of Indian Himalayan regions and
extracted from these natural populations to meet the
demand for the dierent purposes, such as medicine
production and other dietary items by folklore and
industrial systems. Hence, this threatened plant
species need conservation and management for
its proper utilization. Urgent attention and steps are
required in this direction to safeguard this important
germplasm resource. For in-situ conservation,
cultivation in native regions with the help of neigh-
boring dwellers and forest departments can be highly
benecial. This can help increase its population size
and be utilized economically in a sustainable way.
For ex-situ conservation, introduction and propa-
gation at dierent locations with the help of scientic
964SHARMA & SHARMA., Curr. Agri. Res., Vol. 12(2) 958-966 (2024)
advisors from nearby organizations is necessary.
Tissue culture strategies should be employed for
large scale plantations. Furthermore, it can also
be introduced in those Himalayan regions that are
suitable for its natural growth so that its distribution
may be widened. Natural extraction should be
monitored scientifically to ensure the collection
of roots and plant parts at appropriate stage
of growth. At least extraction should not be done
before the seed set. Government bodies and other
organizations involved in the conservation of ora
and environment needs to take care of these types
of activities. Proper documentation of the quantity
of the plant material harvested and supplied should
be maintained at the native production site.
Conclusion
In conclusion, the present study reported the diversity
of P. verticillatum using cross-transferred SSR
markers of B.utilis. The results of this study showed
that considerably high genetic diversity prevails
in the populations of P. verticillatum in IHR. However,
record of the population distribution and spreading
trends in this region are not available. Proper conser-
vation and management of this plant species is
urgently needed for sustainable utilization. Large-
scale studies involving maximum populations of IHR
should be initiated for the identication of diverse
accessions for improvement in the future. The SSRs
used in this study can help exploring genetic
variations and identifying important alleles in
P. verticillatum.
Acknowledgement
N-PDF to Dr. Vikas Sharma from the SERB (DST)
Government of India is highly acknowledged.
Authors are thankful to Chancellor, Vice Chancellor
and Registrar of Sant Baba Bhag Singh University
for the creation of necessary laboratory facility in
the University.
Funding Source
The author(s) received no nancial support for the
research, authorship, and/or publication of this article.
Conict of Interest
The authors declare that they have no conicts
of interest.
Data Availability Statement
This statement does not apply to this article.
Ethics Statements
This research did not involve human participants,
animal subjects, or any material that requires ethical
approval.
Authors' Contribution
PDS done sampling, experimental work and VS
analyzed the data and wrote manuscript.
References
1. Balkrishna A., Srivastava A., Mishra R.K.,
Patel S.P., Vashistha R.K., Singh A. Astavarga
plants- threatened medicinal herbs of
the North-West Himalaya. Int J Medicinal
Aromatic Plants, 2012; (2) 661-76.
2. Khan H., Saeed M., Khan M.A., Dar A., Khan
I. The antinociceptive activity of Polygonatum
verticillatum rhizomes in pain models.
J Ethnopharmacology. 2010;127(2):521-7.
3. Virk J.K., Bansal P., Gupta V., Kumar S.,
Singh R. Lack of pharmacological basis of
substitution of an endangered plant group
Ashtawarga–A significant ingredient of
polyherbal formulations. Am J Phytomed Clin
Ther, 2015; 2:690-712.
4. Bhatt D., Kumar R., Tewari L.M., Joshi
G. C. Polygonatum cirrhifolium Royle
and Polygonatum verticillatum (L.) Allioni:
Status assessment and medicinal uses in
Uttarakhand, India. Journal of Medicinal Plant
Research, 2014; 8(5): 253-259.
5. Lohani N., Tewari1 L.M., Kumar R, Joshi G.C.,
Chandra J., Kishore K., Kumar S., Upreti
B.M. Population studies, habitat assessment
and threat categorization of Polygonatum
verticillatum (L.) Allioni in Kumaun Himalaya.
J Ecol Nat Environ, 2013; 5(5): 74-82.
6. Tiwari T., Chaturvedi P. Polygonatum
verticillatum: A Highly Valued “Astavarga’’
Medicinal Herb of North West Himalaya. Med
Plants, 2016; 8 (2): 97-103.
7. Suyal R., Jugran A.K., Rawal R.S., Bhatt
I.D. Morphological, phytochemical and
genetic diversity of threatened Polygonatum
965SHARMA & SHARMA., Curr. Agri. Res., Vol. 12(2) 958-966 (2024)
verticillatum (L.) All. populations of dierent
altitudes and habitat types in Himalayan
region. Physiol Mol Biol Plants, 2021;
27(8):1795-809.
8. Sharma R.K., Gupta P., Sharma V., Sood A.,
Mohapatra T., Ahuja P.S. Evaluation of rice
and sugarcane SSR markers for phylogenetic
and genetic diversity analyses in bamboo.
Genome, 2008; 51:91–103.
9. Zhou Q., Cai H., Liu Z., Yuan L., Yang L.,
Yang T., Li B., Li P. Development of genomic
resources for Wenchengia alternifolia
(Lamiaceae) based on genome skimming
data. Plant Diversity. 2022; 44:542-551.
10. Kaur K., Sharma V., Singh V., Wani M.S.,
Gupta R.C. Development of novel SSR
markers for evaluation of genetic diversity
and population structure in Tribulus terrestris
L. (Zygophyllaceae). 3Biotech, 2016; 6:156.
11. Koul P.M., Sharma V., Rana M., Chahota R.K.,
Kumar S., Sharma T.R. Genetic Structure
and Interrelationships in Lentil Species Using
Morphological and SSR Markers. 3Biotech,
2017; 7:83.
12. Sharma H., Kumar P., Singh A., Aggarwal K.,
Roy J., Sharma V., Rawat S. Development
of polymorphic EST-SSR markers and their
applicability in genetic diversity evaluation in
Rhododendron arboreum. Mol Biol Reports,
2020; 47(4):2447-2457 http://doi.org/10.1007/
s11033-020-05300-1.
13. Sharma H., Kumar R., Sharma V., Kumar
V., Bhardwaj P., Ahuja P.S., Sharma R.K.
Identication and cross-species transferability
of 112 novel unigene-derived microsatellite
markers in tea (Camellia sinensis) Am J Bot,
2011; 98:e133-e138.
14. Aiello D., Ferradini N., Torelli L., Volpi C.,
Lambalk J., Russi L., Albertini E. Evaluation
of Cross-Species Transferability of SSR
Markers in Foeniculum vulgare. Plants,
2020;9:175.
15. Bhandawat A., Sharma V., Sharma H.,
Sood A., Sharma R.K. Development and
cross transferability of functionally relevant
microsatellite markers in D. latiorus and
related bamboo species. J Genetics, 2014;
DOI 10.1007/s12041-014-0377-9.
16. Rana A., Kumar A., Savita. Polygonatum
species and strategies for sustainable
harvesting. The Indian Forester, 2017; 143(3).
17. Zhao X., Li J. Chemical constituents of
the genus Polygonatum and their role in
medicinal treatment. Nat Prod Comm, 2015;
10(4):1934578X1501000439.
18. Virk J.K., Kumar S., Singh R., Tripathi A.C.,
Saraf S.K., Gupta V., Bansal P. Isolation and
characterization of quinine from Polygonatum
verticillatum: A new marker approach to
identify substitution and adulteration.
J Advanced Pharma Tech Res, 2016;
7(4):153.
19. Bisht S., Bisht N.S., Bhandari S. In
vitro micropropagation in Polygonatum
verticillatum (L.) All. an important threatened
medicinal herb of Northern India. Physiol Mol
Bio Plants, 2012; 18:89-93.
20. Wang S., Wang B., Hua W., Niu J., Dang
K., Qiang Y., Wang Z. De novo assembly
and analysis of Polygonatum sibiricum
transcriptome and identification of genes
involved in polysaccharide biosynthesis. Int
J Mol Sciences, 2017; 18(9):1950.
21. Suyal R., Rawat S., Rawal R.S., Bhatt I.D.
Variability in morphology, phytochemicals,
and antioxidants in Polygonatum verticillatum
(L.) All. populations under dierent altitudes
and habitat conditions in Western Himalaya,
India. Environ Monit and Assess, 2019;
191:1-8.
22. Sheng Y., Chen L., Li X., Shao J. Analysis
of the genetic structure and morphology of
Polygonatum cyrtonema in Anhui Province,
eastern China revealed three distinct genetic
groups. Nordic J Bot, 2020; 38(2).
23. Karpavičienė B., Mlečkaitė E.G. Response
of Polygonatum multiorum and P. odoratum
morphological characteristics and population
structure to variation in environmental factors.
Botanica, 2019; 25(2):111-20.
24. Sharma V., Wani M.S., Singh V., Kaur K., Gupta
R.C. Development and Characterization
of Novel Microsatellite Markers in Trillium
govanianum- a Threatened Plant Species
from North-Western Himalaya. 3Biotech,
2017; 7:190.
25. Chung M.Y., López-Pujol J., Chung J.M.,
Kim K.J., Chung M.G. Contrasting levels
of clonal and within-population genetic
diversity between the 2 ecologically dierent
966SHARMA & SHARMA., Curr. Agri. Res., Vol. 12(2) 958-966 (2024)
herbs Polygonatum stenophyllum and
Polygonatum inatum (Liliaceae). J heredity,
2014; 105(5):690-701.
26. Szczecińska M., Sawicki J., Polok K.,
Hołdyński C., Zieliński R. Comparison of three
polygonatum species from poland based on
dna markers. Annales Bot Fennici, 2006; 43:
379–388.
27. Suyal R., Bahukhandi A., Bhatt I.D., Rawal
R.S. Comparative Analysis of Biochemical
Attributes of Genus Polygonatum in Western
Himalaya. Nat Acad Sc letters, 2021; 457-
460. doi:https://doi.org/10.1007/s40009-020-
01028-5
28. Doyle J.J., Doyle J.L. Isolation of plant DNA
from fresh tissue. Focus, 1990; 12(1):13.
29. Wani M.S., Gupta R.C., Munshi A.H., Sharma
V. Development and Characterization of SSR
markers in Himalayan tree species Betula
utilis D. Don. J Forestry Res, 2018; 31: 1453-
1460 doi.org/10.1007/s11676-019-00932-x.
30. Botstein D., White K.L., Skolnick M., Davis
R.W. Construction of a genetic linkage
map in man using restriction fragment
length polymorphisms. Am J Human Genet,
1980;32: 314–33l.
31. Perrier X., Jacquemoud-Collet, J.P. DARwin
software http://darwin.cirad.fr/darwin, 2006.
32. Dhyani P., Sharma B., Singh P., Masand
M., Seth R., Sharma R.K. Genome-wide
discovery of microsatellite markers and,
population genetic diversity inferences
revealed high anthropogenic pressure on
endemic populations of Trillium govanianum.
Scientic Reports, 2020; 15: 11269.
33. Singh H.C., Tiwari V., Tiwari A., Rana T.S.
Development of EST-SSR markers in
Bergenia ciliata using de novo transcriptome
sequencing. Genome, 2024; 67(4), doi.
org/10.1139/gen-2023-0059.
34. Kumar A., Bisht Y., Rautela K., Arun K. Jugran
A.K., Bhatt I.D., Bargali S.S.Morphological
and genetic diversity assessment of Rheum
australe D. Don – A high value medicinal
herb from the Himalaya, and implications
for conservation strategies. South African
Journal of Botany, 2023; 163:620-629.
35. Oliya B.K., Maharjan L., Pant B. Genetic
diversity and population structure analysis of
Paris polyphylla Sm. revealed by SSR marker.
Heliyon, 2023; DOI10.1016/i.heliyon.2023.
e18230.
