Journal of Applied Research on Medicinal and Aromatic Plants 34 (2023) 100455

Contents lists available at ScienceDirect

Journal of Applied Research on Medicinal and

Aromatic Plants
journal homepage: www.elsevier.com/locate/jarmap

Development of genomic SSR markers in Gymnema sylvestre (Retz.) R.Br. ex

Sm. using next generation DNA sequencing and their application in genetic
diversity analysis
A.C. Polaiah a, *, Parthvee R. Damor b, R.N. Reddy a, P. Manivel c, K.T. Shivakumara d,
Manish K. Suthar a, V. Thondaiman e, G.N. Manjesh e, K.H. Bindu f, Jitendra Kumar g
a
ICAR-Directorate of Medicinal and Aromatic Plants Research, Boriavi-387 310, Anand, Gujarat, India
b
Children’s University, Gandhinagar -382 021, Gujarat, India
c
ICAR-Central Tobacco Research Institute, Research Station, Vedasandur, Dindigul-624 710, Tamil Nadu, India
d
ICAR-National Bureau of Agricultural Insect Resources, Hebbal, Bengaluru, Karnataka- 560 024, India
e
ICAR-Directorate of Cashew Research, Puttur- 574 202, Karnataka, India
f
ICAR-Indian Institute of Horticultural Research, Bengaluru- 560 089, Karnataka, India
g
Institute of Pesticide Formulation Technology, Gurugram- 122 016, Haryana, India

A R T I C L E I N F O A B S T R A C T

Keywords: Gymnema sylvestre (Retz.) R.Br. ex Sm. is one of the important medicinal plants widely used in traditional
Next-generation sequencing medicine due to its anti-diabetic properties. Due to its medicinal properties, huge pressure on its natural re­
Gymnema sylvestre sources of G. sylvestre is under severe threat of diversity loss. Further, the lack of molecular markers in G. sylvestre
Genotypes
is the limiting factor for the adoption of modern genomic studies. In the present study, DNA was isolated from
SSR
Polymorphism
G. sylvestre leaves which were used for NGS sequencing (Illumina HiSeq 2500 NGS platform). After de-novo
Genetic diversity assembly of raw reads, 77679 contigs were identified with the sum of contigs length 1, 246, 048, 78 bp.
Among all the assembled contigs, 55859 contigs with SSR motifs were identified. Based on repeat sequences, we
designed SSR markers using the MISA tool. The hexanucleotide SSR was the most prominent among other SSR
types. Among various SSR motifs identified, AT/TA, AG/CT, and AC/GT repeats were most frequently found in
dinucleotide repeats. A total of 100 SSR primer pairs were synthesized through the flanking sequences of SSR
motifs using Primer3 in which 58 primers were amplified and 42 were not amplified. Novel genomic SSR markers
were used for the genetic diversity analysis of 26 genotypes of G. sylvestre. Fifty-eight primer pairs produced
alleles ranging from 1 to 6. The maximum number of alleles (6) were generated by Xdagsm10 which showed the
highest polymorphic information content (0.602) and allelic diversity (0.662) followed by Xdagsm96 recorded
maximum PIC (0.607) and allelic diversity (0.671). According to the UPGMA dendrogram, the genotypes are
clustered into three major groups, and they are not formed according to geographical origin. Among all the
genotypes, DGS10, DGS22, DGS16, DGS5, and DGS23 were found to be distinctly divergent which can be used in
the future breeding programme. Primers Xdagsm10 and Xdagsm96 are highly efficient polymorphic markers that
can be used to distinguish the genotypes of G. sylvestre, and are highly useful in the molecular breeding
programme.

Abbreviations: DNA, Deoxyribonucleic acid; NGS, Next Generation Sequencing; MISA, MIcroSAtellite Identification tool; SSR, Simple Sequence Repeat; PIC,
Polymorphism Information Content; UPGMA, Unweighted Pair Group Method with Arithmetic Mean; RAPD, Random Amplified Polymorphic DNA; ISSR, Inter
Simple Sequence Repeat; AFLP, Amplified Fragment Length Polymorphism; RNA, Ribonucleic acid; PCR, Polymerase Chain Reaction; NTSYS-pc, Numerical Tax­
onomy and Multivariate Analysis System; EST-SSR, Expressed Sequence Tag derived Simple Sequence Repeat; QTL, Quantitative Trait Locus.
* Correspondence to: Scientist, ICAR-DMAPR, Anand, Gujarat, India.
E-mail addresses: acpolireddy.hortico@gmail.com (A.C. Polaiah), p.r.damor@gmail.com (P.R. Damor), gpbreddy@gmail.com (R.N. Reddy), manivelp@yahoo.
com (P. Manivel), manish.suthar@icar.gov.in (M.K. Suthar), thons1981@gmail.com (V. Thondaiman), gn.manjesh5@gmail.com (G.N. Manjesh), himabindu.k@
icar.gov.in (K.H. Bindu), jitendra.kumardir@ipft.gov.in (J. Kumar).

https://doi.org/10.1016/j.jarmap.2022.100455
Received 4 June 2022; Received in revised form 8 December 2022; Accepted 17 December 2022
Available online 20 December 2022
2214-7861/© 2022 Elsevier GmbH. All rights reserved.
A.C. Polaiah et al. Journal of Applied Research on Medicinal and Aromatic Plants 34 (2023) 100455

Table 1
Details of 26 Gymnema sylvestre genotypes used in the estimation of genetic diversity by SSR marker.
Genotype Place of collection District State Alt. (m) Latitude Longitude

DGS-1 Waghai Dang Gujarat 528 20º44.221 ′

73º41.835′
DGS-3 Waghai Dang Gujarat 505 20º59.568′ 73º28.250′
DGS-22 Central Region of Eastern Ghats Vishakhapatnam Andhra Pradesh 747 17053.122′ 82020.685′
DGS-21 Central Region of Eastern Ghats Vishakhapatnam Andhra Pradesh 533 17046.882′ 82030.875′
DGS-13 N.R. Puram Road Shimoga Karnataka 729 13021.809′ 75027.924′
DGS-2 Waghai Dang Gujarat 500 20º43.270′ 73º41.680′
DGS-11 Kalsa Road Shimoga Karnataka 797 13012.767′ 75020.681′
DGS-8 Dharmasthala Road Udupi Karnataka 77 12057.025′ 75022.393′
DGS-20 Central Region of Eastern Ghats Vishakhapatnam Andhra Pradesh 164 17045.537′ 82032.623′
DGS-15 Tumkur road Bengaluru Karnataka 697 13017.812′ 75015.093′
DGS-6 Anajur Hasan Karnataka 923 13004.781′ 75040.501′
DGS-14 N. R. Puram Shimoga Karnataka 697 13036.300′ 75029.640′
DGS-16 Veerappaaaiyanar Teni Tamil Nadu 326 10020.905′ 77026.972′
DGS-23 Central Region of Eastern Ghats Vishakhapatnam Andhra Pradesh 945 18004.531′ 82039.729′
DGS-9 Karkal Udupi Karnataka 110 13006.071′ 75009.995′
DGS-18 Tanipurai Virudhnagar Tamil Nadu 246 09042.360′ 77037.921′
DGS-26 Banki Sisarvula Udaipur Rajasthan 2100 24033.445′ 73039.393′
DGS-25 Rahuri Ahmednagar Maharashtra 537 19021.187′ 74038.950′
DGS-33 Behal Solan Himachal Pradesh 1176 31013.132′ 76054.344′
DGS-4 West Bengal Kalyani West Bengal 11 22◦ 58.599′ 88◦ 28. 599′
DGS-19 Central Region of Eastern Ghats Vishakhapatnam Andhra Pradesh 92 17040.565′ 82036.623′
DGS-12 Blevannure Chickmanglore Karnataka 820 13014.984′ 75024.234′
DGS-17 Anakaraipatti Madurai Tamil Nadu 262 09042.360′ 77037.921′
DGS-5 Agumbe Shimoga Karnataka 651 13030.673′ 75005.537′
DGS 32 Ujire road Udupi Karnataka 537 12056.678 75022.632
DGS-10 Kudremukha South Kanara Karnataka 791 13006.071′ 75010.081′

1. Introduction
Gymnema sylvestre (Retz.) R.Br. ex Sm. (2 n = 22) is a perennial, slow-
growing vine like climber naturally distributed in India. It is commonly
known as ‘gudmar’ or ‘madhunashini’. It is also called ‘Periploca of the
wood’ in English. It belongs to the family Apocynaceae (Rapini et al.,
2003). It grows naturally on tree stems in the forest. The stem is cylin­
drical, the root is a tap root system, the branches are pubescent, and the
leaves are opposite with 3–5 cm in length and up to 3 cm in width. The
flowers are small in size and have umbellate cyme inflorescence. The
mature pods are approximately 4–7 cm long with 10–12 seeds. In India,
its leaves are widely used in Ayurvedic medicines, particularly while
treating diabetes since 2000 BC (Wild et al., 2004). The leaves of G.
sylvestre are the natural source for the extraction of gymnemagenin. The
fresh leaves of G. sylvestre when chewed inhibit the taste of sweetness
temporarily (Warrier et al., 1995) because of which its name in Hindi is
gurmar. Shanmugasundaram and Panneerselvam (1981) reported that
intake of G. sylvestre in diabetic patients lowers blood glucose. The
herbal products sold in the Indian market for diabetes cure are made
with G. sylvestre alone or in combination with other herbs. It has a great
potential to regulate blood sugar levels which is a natural alternative
medicine (Siddhiqui et al., 2000). Agarwal et al. (2000) reported that
G. sylvestre is effective against obesity when administered as gymnemic
acid formulations. Gymnema tea is used for the treatment of obesity and
weight loss. Traditionally, Gymnema is used in the treatment of malaria
and also snakebites (Singh et al., 2008). In India, it has been documented
that some districts of Tamil Nadu, and also Gujarat have the habit of
chewing a few fresh green leaves of G. sylvestre in the morning to keep
their urine clear and to reduce glycosuria (Joseph and Jini, 2011).
Due to the medicinal properties, there is a huge demand for the
leaves of G. sylvestre from herbal and pharmaceutical industries for
herbal drugs preparation. G. sylvestre is one of the best-selling medicinal
plants in the world market ranking second position (Pandey, 2012). In
general, raw materials of G. sylvestre are collected from the wild or
forest. Gymnema has become vulnerable due to over-exploitation by
destructive and unscientific harvesting. The way forward to meet the
increasing demand besides minimizing the pressure of harvesting from
the wild is to bring the herb under large-scale cultivation. To develop
good agriculture practices for G. sylvestre, the researchers need to
identify the elite genotypes with desirable traits such as high biomass
coupled with optimum gymnemagenin. Collection and conservation of
genetic material from natural sources/wild through survey and explo­
ration is the preliminary step to identify the elite genotypes which could
be useful in the crop improvement programme (Nair and Kesha­
vachandran, 2006). Thamburaj et al. (l996) documented various
morphological traits of Gymnema genotypes which were augmented
from Tamil Nadu and Kerala. Later on, Nair and Keshavachandran
(2006) also evaluated the morphological traits and total saponin. Dha­
nani et al. (2015) also reported significant variability for leaf traits,
biomass, and gymnemagenin. In earlier studies, the variability of
G. sylvestre was assessed morphologically and these morphological
quantitative traits are highly influenced by environmental factors. Thus
the selection of phenotypic trait value may not be true (Kumar et al.,
2015). To overcome this problem, molecular markers may be deployed
(Rukhsar et al., 2017). A few reports are available on the genetic di­
versity study of G. sylvestre by using DNA markers such as RAPD and
ISSR markers (Shahnawaz et al., 2012; Rathore et al., 2016) also
dominant AFLP markers (Osman et al., 2011), RAPD (Osman et al.,
2013; Lal et al., 2013; Prashanti et al., 2017) and ISSR (Mouna et al.,
2014). These dominant markers have limitations because of their
dominant nature, and low reproducibility is the bottleneck for the mo­
lecular breeding of G. sylvestre. The SSR markers are the most informa­
tive and multipurpose genetic markers and are exploited in plant
structural and functional genomic studies. The discovery of SSRs and
development by using traditional methods are laborious,
time-consuming, and costly. Next Generation Sequencing is a deep, high
throughput DNA/RNA or methylation sequencing technology. NGS is a
robust method and it can be generating large Gigabase (Gb) sized se­
quences it took less time as compared to the Sanger sequencing where
fragment cloning was used. In recent years NGS technology has the
choice of breeders for the development of SSR markers at less cost and
effort than the traditional approaches and it has wider acceptability in
the field of crop improvement programs (Taheri et al., 2018). The NGS
based molecular markers and genomic technology in plant science is a
better understanding of plant biology and advancement in crop
improvement activities. To date, there is a lack of genomic resources
particularly genomic SSR markers for the molecular breeding pro­
gramme of G. sylvestre in India. Therefore, the objective of the present

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Table 2
Details of SSR markers used in genetic diversity analysis of Gymnema sylvestre.
Primer Primer Sequence (5′ − 3′ ) Repeat motif Tm Ta (C0) Expected product size
(C0)

Xdagsm8 F-GAGGCTGTGGAAGACTTGGA (TTC)9 59.4 55 186

R-TGGCAAAAGTTATTTTTCTTCGAAGG 58.5
Xdagsm10 F-GGGGTTGAGGGTGGGGTATA (AG)14 61.4 59 223
R-GCCCAGTCTTGCTGCTGCTTT 59.4
Xdagsm16 F-CCGTTCAAGTAGCAAATGGCA (TCT)13 57.3 56.5 226
R-TACAGCAAGTTGCCCACAGG 59.4
Xdagsm17 F-TTTCCACCACCACTCAACCC (CT)13 59.4 55.3 225
R-GCCCCGGCCTATATGTGTAT 59.4
Xdagsm26 F-TCACTTCCCAGCATGAGAAACA (GGA)10 59.89 55 222
R-CCTCAGATGGGTGCCACTTT 59.96
Xdagsm27 F-AACCGATGCTCAGCAGTAGT (CT)13 59.10 59 176
R-TCTCAACAAATCTGGCAAAAGCA 59.61
Xdagsm29 F-AGCACTGCTACTACTTTCCACT (AAT)10 59.09 59 157
R-ACTGCAATGACATGTTTGCTT 57.26
Xdagsm30 F-AGCGGTGGAGAAACTGGTTT (AG)14 59.81 55 160
R-TGTTGACCGGAAAACATGGC 59.32
Xdagsm31 F-ACAAGCAAGATCTTTCGGCT (AAAAC)5 57.80 54.5 156
R-AGCAACGACACTCTTCACCC 60.25
Xdagsm33 F-AAGCTAGCTGGCCAGGTACA (AG)16 60.91 55 173
R-TCAGCTGCAGAAGATACGGC 60.17
Xdagsm34 F-ACTGCACCCCAACAATCAAA (GA)13 58.21 57.3 158
R-TCACTGGTGCCACTGGTTTT 60.03
Xdagsm35 F-GTCCAGAAATGGGTCAGCCA (CA)13 59.96 55 213
R-CTTGCTGCCCAAAGCTAAGC 60.10
Xdagsm36 F-TGGCCCTGCAGTTTTACAGT (CT)13 59.81 55 206
R-TCTTTCTATGCTTCTAGGGTTGT 57.50
Xdagsm37 F-GGACTGGTAGATGAGAATAATGAA (TGTA)7 59.82 55 185
RACAATCCACACACTGGTTTTGTTTCT 59.53
Xdagsm38 F-TTTAGAAGAAGCAATTGTCACACA (AAAAAT)5 57.30 58.7 225
R-CCAAGTCCTCGCTCGATGAA 59.82
Xdagsm39 F-TCCGAACATTTCCAGCTATTGA (AT)13 57.78 55 180
R-TGGACACATCAACAAGACACCT 59.82
Xdagsm40 F-CGACATCGTCACCGGTACTT (TGA)11 59.83 57.3 182
R-CCGCCAAAGGTCCGAAAAAT 59.39
Xdagsm42 F-AGCAGGTCGTCTAGAGGGTT (TTTTA)5 59.96 55 162
R-ACGACCACAACGAAAGATGGA 59.93
Xdagsm43 F-TCAGGGGTAAGTGGATAAGAAGA (AT)13 58.04 55 177
R-TGCCAAACAACTACTAAACATTCTC 59.93
Xdagsm44 F-TCAAGATTCCTCCATCATAGTTGCA (AAAAG)5 60.10 55 154
R-CTGCTGCGGATGGACATTTG 59.9
Xdagsm45 F-CCACCCAAGTGTGTGCTTGA (TC)17 60.75 55 178
R-GCCAGGGTTTCTTTCTTCC 58.74
Xdagsm46 F-GGCCCAGAAGATTCTAGAAGGT (GGGCTT)5 59.22 55 182
R-ACTCTCACCAATGGCCGAAA 59.6
Xdagsm48 F-CATGACCAATTAATCATGCCTCTGA (AAT)9 59.46 55 169
R-AGAATGATAAGATTTGCCCTTGC 57.47
Xdagsm51 F-GAAGGGAAGGGATCGTGTGG (TC)14 60.10 60 181
R-ACCAAGTCTTACGAGGCACAG 59.99
Xdagsm52 F-AGCATGTAACCTCACACAGTAA (CA)16 57.38 55 160
R-AAAGGAGGTGCAGCACAGAA 59.81
Xdagsm53 F-AGCGAGAAACGAAGAGCACA (CTT)11 59.96 55 219
R-CACAAAACCAGAGATGAGATGCA 59.24
Xdagsm55 F-GCCTGTTGTTGTTGTGGCTG (GAG)9 60.52 55 217
R-ACCCCAATTGAAAACGAACCA 58.61
Xdagsm58 F-TTGCCTCGACATGAACCTCC (AAATCC)5 60.03 55 159
R-GAGATTGACCATCGGCGTCT 59.89
Xdagsm60 F-ACATGGGCTTGGGATCACAA (AG)17 59.59 55 179
R-CTGAGGAGTTCTGCAGTCGT 59.39
Xdagsm61 F-CGGGAGAACCACCGTCAAAT (CAC)9 60.32 55 150
R-CTATGGAGATTGAGGCGGCG 60.66
Xdagsm64 F-TCAACTTCCGGGAATTGGGC (TTACC)5 57.36 55 194
R-AGGGATTCTGTGGTTTGAGTT 57.12
Xdagsm65 F-ACAGCATATCTCAACAAAAACTGT (TTACC)5 57.36 55 194
R-AGGGATTCTGTGGTTTGAGTT 57.12
Xdagsm67 F-TGGAGAGAAGATAATAGAGAAAAG (AG)15 59.43 55 174
R-ACCAAACAAAGGGAGAATCCTCA 59.86
Xdagsm69 F-ATCCACACCCCGGACATTTC (AG)13 60.03 55 222
R-CCACGGCATCTCCATCTTGA 59.82
Xdagsm70 F-TGTTTTGAATCCAGGAATCCCA (AG)17 57.87 55 163
RTTTATATTTTGAGGGGTAATTGTTCC 57.22
Xdagsm71 F-GCGGTTTCTTGGGTGGTTTT (CTC)9 59.54 55 161
R-CGAGCTGAAGGGATTGTCCA 59.75
Xdagsm72 (ACCAAC)5 55 158
(continued on next page)

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Table 2 (continued )
Primer Primer Sequence (5′ − 3′ ) Repeat motif Tm Ta (C0) Expected product size
(C0)

F-TACAACTTGTCCGTGCGGTC 60.59
R-TAGGTTTCTGGTGCTGGTGC 60.25
Xdagsm73 F-AAGGAGGGGTTGGTTTCTCG (ATGGG)5 59.59 55 157
R-GCACAGTGTTGTCCCCTTCT 60.18
Xdagsm74 F-TCCGCTAGTACTGCTGTCCT (GT)15 60.03 55 189
R-CCGTTTTCTCCTACCTTCTTGC 59.81
Xdagsm75 F-TGCATTACTCATAGTTGGGACTCA (AAT)10 59.53 55 167
R-TGCCAAAATAAAGGGATCGCAC 59.82
Xdagsm77 F-CGTCCATTGGGTAC TGTGCT (AAG)9 60.04 60 200
R-TACCGGAAACCTGCAATGCT 59.96
Xdagsm78 F-GCTGGGTGAAAATGGAAGGC (CT)14 59.75 58.7 151
R-TGGCAACCCCTCATAAGCAT 59.37
Xdagsm80 F-AGTTCAAGCAACCCCTCGAG (TC)13 59.97 55 194
R-TGCAGACCATCAGATACCACA 58.81
Xdagsm81 F-GGCAGGICTGTCCCAAAAGT (CT)13 60.18 55 194
R-AGCAAAGGTCATGTGGCACA 60.47
Xdagsm82 F-TGTCTTCTCCTAGCTAGTTCAGGA (AT)14 60.02 55 189
R-GCATTCTTGTCAACAATAAACATCT 59.40
Xdagsm83 F-TGACTATGATCTTGCATCCATTTGG (AAG)9 59.47 55 199
R-TTAGGAAGGGAAGGCTTGCG 60.03
Xdagsm84 F-TGGAACATGGTTAGTGAGATTGGA (AG)15 59.71 55 215
R-TTGGTTCCAAAACGTCCCAA 58.15
Xdagsm86 F-TGCTCCTCACTTATCAGGGGA (TTTTA)5 59.99 58.7 180
R-TAGTGCCCCTTTCATCCAGC 59.74
Xdagsm87 F-ACGCATTTGTTAATGATTCTTGAAG (AAT)16 58.63 55 241
R-TGGAATGGGGTTTAAAATTAGGGT 58.33
Xdagsm88 F-TGAGAGTGAACGGAGCACTG (AG)18 59.69 55 171
R-CCGAAACCAGTCCTTTTGGG 59.04
Xdagsm89 F-TGACACCTGGCAGAAGGAAA (TCACC)5 59.16 55 179
R-CGTTTTCACCTTGTCAGTTTGC 59.15
Xdagsm90 F-TTATCCACCTGTGTTTAATATTCTTG (AG)15 57.03 55 211
R-GGTCTCACAAGCGGTCTGAA 59.97
Xdagsm92 F-GCCCAAAGAAATCCTGTGTGG (AC)15 59.73 55 180
R-ATGGTCCCTCTGTGATGCAC 59.75
Xdagsm95 F-GCTCAAGCCAGAAATGGTGC (AACCAC)5 60.10 55 218
R-TCGCTATCATTTGGAGACAGCT 59.57
Xdagsm96 F-TCATTGGATTTAAGGCCGGAT (AAT)9 57.41 55 182
R-AGGGATCTTTTGTAATAGAGTTCTG 57.85
Xdagsm98 F-TCAAGCTCCATGTATGCGTACA (AC)16 59.83 55 231
R-TTATACAGCGATGCGGCACC 60.88
Xdagsm99 F-AATGCAAATTGATGAGGCCC (AAG)9 57.01 55 191
R-AGCTACACCAATGGGCTACC 59.45
Xdagsm100 F-GCTGATTGCGAGCCAACTTC (CT)13 60.18 55 202
R-TGCCCTAACCCTCCATTTCC 59.37

study is the development of genomic SSR markers and assessment of
genetic diversity in G. sylvestre using genomic SSR markers.
2. Material and methods
2.1. Plant material and genomic DNA isolation
The plant materials representing diverse regions of India were used
for DNA isolation. Twenty-six genotypes of G. sylvestre were collected for
DNA isolation and were grown at the germplasm field gene bank (Sup­
plementary Fig. 1) of ICAR-Directorate of Medicinal and Aromatic Plants
Research, Anand, Gujarat (Table 1). The young, healthy, and tender
leaves of 26 genotypes of G. sylvestre were used for the total genomic
DNA isolation by following the CTAB method (Doyle and Doyle, 1990).
The DNA quality was checked on 0.8% agarose gel which showed clear
intact bands confirming good quality DNA. The quantification of DNA
was estimated by using Nanodrop 2000 Spectrophotometer (Thermo
Fisher Scientific).
HiSeq 2500 NGS platform (outsourced from SciGenome, www.sci
genom.com). Raw reads obtained from sequencing were used to create
a de-novo assembly using MaSuRCA. In SSRs having a flanking region of
150 bp were retained from all the identified SSRs. SSRs with a motif
length of 20 bp and above were selected for designing primers. Simple
sequence repeats were identified from assembled contigs by the MISA
tool (http://pgrc.ipk-gatersleben.de/misa/misa.html). The definition
for repeat sizes was set as unit size/minimum number of repeats): (2/6)
(3/4) (4/3) (5/3) (6/2). The SSR primers were synthesized using the
flanking sequences of SSR motifs using Primer3. The key parameters set
for primer design were: primer length 18–24 bp with 20 bp as the op­
timum; PCR product size 100–300 bp; optimum annealing temperature
at 50 ◦ C; GC content 35–60% with 50% as optimum. The canonical name
proposed for designating markers includes function [unknown (X)], lab
designator (DMAPR, Anand (da)], species [Gymnema sylvestre (gs)], type
of marker [Genomic (g)], and serial no. of a marker. Hence, the markers
developed in this study were named "Xdagsm" markers.

2.3. PCR amplification and genotyping

2.2. Development of SSR primers
Genomic DNA was isolated from young leaves of genotype DGS-22
(INGR13041) of G. sylvestre. The purified DNA was used to create
paired-end and mate paired libraries; and sequenced using Illumina
The amplification of DNA was performed in a thermocycler (S1000
Thermal Cycler, Biorad). The annealing temperature of each primer was
standardized using a gradient thermal cycler. The PCR reaction condi­
tions were followed with an initial denaturation at 94 ◦ C for 4 min,

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A.C. Polaiah et al. Journal of Applied Research on Medicinal and Aromatic Plants 34 (2023) 100455

Table 3 was performed twice to confirm its reproducibility.

Throughput and quality of Illumina sequencing of Gymnema sylvestre genome.
Paired-end 1 Paired-end 2 Paired-end 3 Mate- 2.4. Data analysis and molecular diversity
[300 bp] [500 bp] [800 bp] paired

Total 96,531,726 105,858,170 28,371,358 52,548,322 The details of 58 SSR primer pairs used in the genetic diversity
number of analysis of G. sylvestre genotypes are presented in Table 2. The genetic
reads diversity was assessed among 26 genotypes of G. sylvestre which were
(R1 +R2) collected from different parts of India. The allelic data obtained from 58
Total 9653.18 10,585.82 2837.14 13,137.08
number of
SSR primers using 26 genotypes were scored using a binary matrix as
bases input format. Polymorphic Information Content (PIC) was estimated
(Mb) according to the formula i.e., 1-(Σ PI)2. The clear, reproducible, and
GC % 41.22 39.11 36.3 34.02 consistent SSR bands were scored based on the size of allele as one (1)
for present and zero (0) for absent for the amplified fragments in each
microsatellite loci and data matrixes were constructed accordingly. The
Table 4 allele sizes below 100 bp and above 500 bp are not included while
Characteristics of Gymnema sylvestre genome sequences. scoring SSR molecular data because these alleles are considered non-
Characteristics Numbers specific alleles. SSR molecular data were analyzed using the software
SAS version 9.3. The dendrogram was constructed based on the un­
Total number of sequences examined 77679
Total size of examined sequences (bp) 1246048789 weighted pair group method with an arithmetic mean (UPGMA) using
Total number of identified SSRs 221158 Jaccard’s similarity coefficient with tool NTSYS-pc version 2.02 (Rohlf,
Number of SSR containing sequences 55859 1998).
Number of sequences containing more than 1 SSR 38089
Number of SSRs present in compound formation 55355
3. Results and discussion

followed by 35 cycles of denaturation at 94 ◦ C for 30 s. The annealing
temperature is specific to the primer (as depicted in Table 2) for 45 s, an
extension at 72 ◦ C for 30 s followed by a final extension at 72 ◦ C for 10
min. Then, the PCR products were held at 4 ºC till further use. About 15
µL volume of the reaction mixture was carried out for PCR amplification.
The single reaction mixture consists of 7.5 µL of PCR master mix
(Thermo Scientific, Dream Taq™, Green PCR Master mix 2x), 1.5 µL of
nuclease-free water, 2 µL of forwarding SSR primer, 2 µL of reverse SSR
primer, and 2 µL of DNA template (25 ng/µL). The amplified PCR
products with 100 bp DNA ladder as the marker (Pure Gene) were
separated in the electrophoresis using 2 % agarose (Pure Gene) gel in 0.5
X TBE buffer and stained with ethidium bromide (10 µg/ml) at a con­
stant voltage of 80 V (Kaur et al., 2015). After electrophoresis, the gels
containing DNA fragments were visualized under UV light using a Gel
documentation system (Molecular Imager®, Biorad), and gel photo­
graphs were taken for scoring molecular data. The PCR amplification
3.1. Genome sequencing and SSR markers development
In the present study, raw read summaries of paired-end and mate
paired libraries are given in Table 3. After de-novo assembly of raw
reads, 77679 contigs were identified with a sum of contigs length
1246,048,78 bp. The maximum length of assembled contigs was
8108,957 bp whereas the length of the minimum contigs length was 500
bp. The average length of assembled contigs was 16,041 bp whereas N50
was 40,987 bp, and this value is very high when compared to dill seed
where the N50 value was 1020 bp (Kumar et al., 2020) and for cluster
bean it was 345 bp (Kumar et al., 2020). Around 55859 contigs were
identified containing SSR motifs among all assembled contigs (Table 4).
The distribution of SSR motifs in G. sylvestre genome is depicted in
Fig. 1A. The hexanucleotide SSR was most prominent over other SSR
types (Fig. 1A). The study results are similar to Yuan et al. (2015)
findings on trinucleotide and hexanucleotide repeat motifs predomi­
nance when they used EST-SSR markers in Scutellaria baicalensis. The

Fig. 1. Distribution of simple sequence repeat (SSR) motifs (A) and various simple sequence repeat (SSR) motifs identified (B) in the genome of Gymnema sylvestre.

5
A.C. Polaiah et al. Journal of Applied Research on Medicinal and Aromatic Plants 34 (2023) 100455

Table 5 repeat motifs were AAT/ATT and AAG/CTT. AAAT/ATTT was the most
Number of alleles, frequency, allele diversity and polymorphic information frequent tetranucleotide SSR motif accounting for more than 40 % of
content (PIC) value of each SSR primers. tetranucleotide motifs identified.
Primer Number of Major allele allele PIC Observed
code alleles frequency diversity Band Size 3.2. Validation of SSR markers, genotyping, polymorphism, and diversity
(%) (bp)
analysis
Xdagsm8 1 1.000 0.000 0.000 180
Xdagsm10 6 0.423 0.662 0.602 190–290 Initially, 25 SSR primers were synthesized from the assembled
Xdagsm16 3 0.500 0.588 0.504 190–500
genome and tested for amplification in 26 G. sylvestre genotypes. Out of
Xdagsm17 3 0.576 0.517 0.417 190–500
Xdagsm26 3 0.500 0.565 0.470 180–190 these 25 markers tested, 20 markers (80 %) were amplified successfully
Xdagsm27 2 0.769 0.355 0.292 100–105 indicating good quality of primers. The experiment results revealed that
Xdagsm29 2 0.884 0.204 0.183 105–110 a total of 121 alleles were obtained by the amplification of 58 SSR
Xdagsm30 2 0.846 0.260 0.226 130–140
primers. The range of alleles detected by polymorphic primers was 2–6
Xdagsm31 3 0.807 0.328 0.303 100–500
Xdagsm33 3 0.769 0.369 0.324 100–110
alleles per primer with a mean value of 2.22 alleles per primer. Out of
Xdagsm34 2 0.769 0.355 0.292 120–130 121 alleles, 12 alleles were monomorphic and 109 alleles were poly­
Xdagsm35 2 0.884 0.204 0.183 190–200 morphic. Hence, the average polymorphism of 90.08 % was recorded
Xdagsm36 2 0.884 0.204 0.183 190–200 among the genotypes of G. sylvestre. Jinu et al. (2019) observed the
Xdagsm37 1 1.000 0.000 0.000 180
average polymorphism (54.2%) in Tamil Nadu landraces of G. sylvestre
Xdagsm38 2 0.923 0.142 0.131 180–300
Xdagsm39 2 0.961 0.074 0.071 180–190 through RAPD markers which are of less value when compared to the
Xdagsm40 3 0.538 0.556 0.465 100–110 present study. Similarly, high genetic variation (85%) was reported
Xdagsm42 2 0.653 0.452 0.350 110–200 among the populations of G. sylvestre accessions collected from Karna­
Xdagsm43 2 0.961 0.074 0.071 180–190
taka using ISSR markers (Mouna et al., 2014). Whereas (Prashanti et al.,
Xdagsm44 2 0.884 0.204 0.183 150–160
Xdagsm45 3 0.846 0.272 0.255 180–200
2017) reported high polymorphism (72.7%) in 24 accessions of Gym­
Xdagsm46 1 1.000 0.000 0.000 180 nema sylvestre while using RAPD markers. It is indicated that the
Xdagsm48 3 0.846 0.269 0.248 100–110 dominant markers showed low polymorphism in Gymnema germplasm
Xdagsm51 3 0.538 0.576 0.496 140–160 as compared to the present findings with genomic SSR markers. The
Xdagsm52 1 1.000 0.000 0.000 180
results are similar to Idrees et al. (2018) reported 92.3% polymorphism
Xdagsm53 2 0.923 0.142 0.131 180–190
Xdagsm55 2 0.730 0.393 0.316 180–190 in Asparagus species while using SSR markers. The number of alleles
Xdagsm58 2 0.846 0.260 0.226 100–110 produced among Gymnema genotypes ranges from 1 to 6 (Table 5). The
Xdagsm60 2 0.923 0.142 0.131 95–100 present results are in tune with the findings of Paliwal et al. (2016) in
Xdagsm61 1 1.000 0.000 0.000 140 Tinospora cordifolia where the alleles number varied from 1 to 4, and the
Xdagsm64 1 1.000 0.000 0.000 140
Xdagsm65 2 0.923 0.142 0.131 140–150
highest number of alleles were amplified by TCg-SSR12. The number of
Xdagsm67 1 1.000 0.000 0.000 110 alleles produced per locus was 2–5 in Paris polyphylla, a medicinal plant,
Xdagsm69 2 0.692 0.426 0.335 190–200 by using SSR markers (Zheng et al., 2012). Whereas Addisalem et al.
Xdagsm70 4 0.576 0.594 0.544 200–230 (2015) reported the maximum number of alleles ranging from 2 to 12
Xdagsm71 1 1.000 0.000 0.000 240
with a mean value of 4.8 in Boswellia papyrifera using SSR markers, and
Xdagsm72 2 0.923 0.142 0.131 100–110
Xdagsm73 1 1.000 0.000 0.000 100 those values are higher as compared to the present study results. Li et al.
Xdagsm74 2 0.576 0.488 0.369 140–150 (2012) reported that the number of alleles per locus in Launaea arbor­
Xdagsm75 2 0.807 0.310 0.262 110 escens (endangered medicinal plant) and Lancea tibetica (Tian et al.,
Xdagsm77 4 0.807 0.334 0.316 150–200 2016) was close to the present study findings. The maximum numbers of
Xdagsm78 1 1.000 0.000 0.000 100
Xdagsm80 2 0.807 0.310 0.262 180–190
alleles recorded in Xdagsm10 were six followed by four alleles produced
Xdagsm81 4 0.807 0.331 0.310 135–150 by Xdagsm70, Xdagsm77, Xdagsm81, Xdagsm87, and Xdagsm96; and
Xdagsm82 1 1.000 0.000 0.000 110 the least number of alleles by Xdagsm8, Xdagsm37, Xdagsm46,
Xdagsm83 1 1.000 0.000 0.000 100 Xdagsm52, Xdagsm61, Xdagsm64, Xdagsm67, Xdagsm78, Xdagsm82,
Xdagsm84 3 0.615 0.544 0.483 200–210
Xdagsm83 and Xdagsm86. Among 58 primers, 12 monomorphic
Xdagsm86 1 1.000 0.000 0.000 110
Xdagsm87 4 0.576 0.544 0.461 190–490 markers exhibited a single allele and gel pictures of SSR marker analysis
Xdagsm88 2 0.576 0.488 0.369 180–190 has given in the Supplementary material. Major allele frequencies were
Xdagsm89 2 0.500 0.500 0.375 120–130 observed maximum in the primers Xdagsm38 (0.9231), Xdagsm39
Xdagsm90 2 0.538 0.497 0.373 120–130 (0.9615), Xdagsm43 (0.9615), Xdagsm53 (0.9231), Xdagsm60 (0.9231)
Xdagsm92 3 0.576 0.541 0.456 120–130
Xdagsm95 2 0.500 0.500 0.375 180–190
Xdagsm65 (0.9231), Xdagsm72 (0.9231), and minimum values of major
Xdagsm96 4 0.423 0.671 0.607 140–160 allele frequencies were observed in Xdagsm10 (0.4231) and Xdagsm96
Xdagsm98 2 0.576 0.488 0.369 150–160 (0.4231). Similar results were reported in Glycyrrhizzia uralensis as
Xdagsm99 3 0.538 0.529 0.424 60–120 major allele frequency varied from 0.28 for Gly SSR2 to 0.88 for Gly
Xdagsm100 2 0.807 0.310 0.262 150–200
SSR3 (Um et al., 2016), and these values were slightly lower as
Mean 2.22 0.782 0.291 0.246 –
compared to the findings of the present study. In SSR molecular analysis,
maximum diverse alleles were found in the primers Xdagsm10 (0.6627)
present results are also in agreement with Patil et al. (2021) findings on and Xdagsm96 (0.6716), followed by Xdagsm16 (0.5888), Xdagsm70
the abundance of hexanucleotide repeats followed by the dinucleotides (0.5947) Xdagsm51 (0.5769), and minimum diverse alleles were found
in the whole genome features of ‘Tunisia’ cv of Pomegranate. However, in the markers Xdagsm38 (0.1420), Xdagsm39 (0.0740), Xdagsm43
it is contradicting Zhu et al. (2016) findings on dinucleotides which are (0.0740), Xdagsm53 (0.1420), Xdagsm60 (0.1420), Xdagsm65 (0.1420)
the most common type of repeat motif, and octonucleotides were the and Xdagsm72 (0.1420) and zero diverse allele was found in mono­
least repeat motif type in melon. Various SSR motifs representing morphic markers such as Xdagsm8, Xdagsm37, Xdagsm46, Xdagsm61,
different types of SSR are shown in Fig. 1B. Among various SSR motifs Xdagsm64, Xdagsm67, Xdagsm71, Xdagsm73, Xdagsm78, Xdagsm82,
identified, the AT/TA, AG/CT, and AC/GT repeats were most frequently Xdagsm83, and Xdagsm86. Polymorphic Information Content (PIC) is an
found in dinucleotide repeats. Similarly, the most frequent trinucleotide important trait to distinguish the genotypes of G. sylvestre. The PIC
values varied from 0.171 to 0.607 among the primers across the

6
A.C. Polaiah et al.
Fig. 2. UPGMA dendrogram of Gymnema sylvestre genotypes based on SSR markers.
genotypes. The PIC values reveal high polymorphism as compared to
(Prashanti et al., 2017) reports wherein PIC values ranged from 0.08 to
0.21 indicating that genomic SSR markers exhibited high PIC and
polymorphism compared to dominant markers. The results were in
agreement with the findings of Kaur et al. (2016) in Tribulus terrestris
with SSR markers where the value ranged from 0.142 to 0.499. In a
similar study, Bakatoushi and Ahmed (2018) reported genetic diversity
in Peganum harmala with the highest PIC value for the primer SSR 17
(0.444), and Khanna et al. (2016) in Withania somnifera (PIC 0.40) which
were quite low values as compared to the present outcome on Gym­
nema. The results are similar to Zhao et al. (2010) who reported a
maximum PIC value of 0.773 in the SSR markers study in Lycium ac­
cessions. The number of alleles per locus (3− 15) and PIC values
(0.335–0.910) reported by Sraphet et al. (2018) in Bauhinia strychnifolia
was higher, whereas in Tetrastigma hemsleyanum Wang et al. (2014)
reported PIC values varying from 0.215 to 0.760 by microsatellite
markers was close to present study findings. The PIC value and the
number of alleles per primer depend on the variability of the materials
studied (Ramu et al., 2013). The maximum PIC values were found in the
primers Xdagsm96 (0.607) and Xdagsm10 (0.602), followed by
Xdagsm16 (0.504) and Xdagsm70 (0.544) which are more than 0.500
indicating high polymorphism and least PIC value (0.071) as recorded in
Xdagsm39, Xdagsm43 and followed by 0.131 in Xdagsm38, Xdagsm53,
Xdagsm60, Xdagsm65, andXdagsm72. Among 58 primers, Xdagsm10
and Xdagsm96 produced more alleles, as well as high PIC values. Hence,
7
Journal of Applied Research on Medicinal and Aromatic Plants 34 (2023) 100455
these two primers are considered highly informative and polymorphic
markers to distinguish the genotypes of G. sylvestre. Zero PIC values were
recorded in 12 monomorphic markers. The molecular weight of the
observed band size ranged from 100 bp (Xdagsm73, Xdagsm78, and
Xdagsm83) to 500 bp (Xdagsm16, Xdagsm17, and Xdagsm31) (Table 5)
which indicated the significant variation in the number of repeats
generated by different alleles. Most of the observed allele sizes generated
by SSR markers ranged from 100 bp to 200 bp, which is the same as the
expected allele size. The results are similar to Bharti et al. (2018) on
cumin using genomic SSR markers. The present results are in agreement
with the reports of Kaur et al. (2016) in the study of Tribulus terrestris.
3.3. Genetic similarity and cluster analysis
Pooled data of 58 SSR primer pairs generated pair-wise allelic sim­
ilarities for 26 genotypes of G. sylvestre, the simple matching coefficients
between each pair of genotypes which were used to construct the
dendrogram. Genetic similarity matrix values ranged from 0.38 to 0.97
indicating high to moderate diversity among the genotypes of
G. sylvestre. The present study results are similar to (Prashanti et al.,
2017) on similarity coefficient values which ranges from 0.12 to 0.95 in
24 accessions of Gymnema while using RAPD markers. Osman et al.
(2011) found similarity coefficient values ranging from 0.212 to 0.731 is
less value in the polymorphism among parent and progenies of Gym­
nema using AFLP markers. The least genetic similarity value of 0.38 was
A.C. Polaiah et al.
recorded between DGS26 and DGS12. Therefore, DGS26 and DGS12
genotypes are the more divergent type that can be used to construct the
core germplasm and are also useful in the future crop improvement
programme. A high genetic similarity value of 0.97 (data not shown)
was recorded between DGS17 and DGS21 indicating less divergent and
more similar types were collected from Tamil Nadu and Andhra Pradesh,
respectively. Therefore, the selection of these genotypes is not effective.
Kaur et al. (2016) reported the genetic diversity study of Tribulus ter­
restris using SSR makers wherein the genetic similarity values range
from 0.270 to 0.870, but the present study results are a little higher
value than those findings. Tan et al. (2014) reported similarity coeffi­
cient values were between 0.53 and 0.95 in Cynodon transvaalensis using
genomic SSR markers, and the present study results are also similar.
Khanna et al. (2016) also reported comparable results in Withania
somnifera where similarity coefficient values were found between 0.125
and 1.000.
Dendrogram developed according to Jaccard’s similarity coefficient
and UPGMA method broadly categorized twenty-six genotypes of
G. sylvestre into three groups (I, II, III) as shown in Fig. 2. Group-I con­
sisted of 18 genotypes, and formed a large group due to high genetic
similarity among genotypes. Hence, Group-I is divided into 4 sub-groups
(1a, 1b, 1c, and 1d). In sub-group 1a consists of genotypes collected from
Gujarat, Karnataka, and Andhra Pradesh but they are not based on
geographical origin. The sub-group 1b includes 5 genotypes with ac­
cessions belonging to Andhra Pradesh, Karnataka, Tamil Nadu, Rajas­
than, and Maharashtra. The genotypes DGS9 and DGS18 have shown
91% similarity collected from Karnataka and Tamil Nadu, respectively.
Genotype DGS26 collected from Udaipur (Rajasthan) has formed a
distinct one in the sub-group 1b which is having 4.294 mg/g gymne­
magenin (Dhanani et al., 2015). The sub-group 1c was grouped with 6
genotypes, and each one was collected from Himachal Pradesh, West
Bengal, Andhra Pradesh, Tamil Nadu, and two genotypes from Karna­
taka. In sub-group 1d, DGS5 is grouped separately as a distinct type with
a 0.80% coefficient value and which was collected from Agumbe, Shi­
moga district of Karnataka. Genotype DGS4 and DGS19 showed 100%
similarity with a coefficient value of 0.92. Group II is divided into two
sub-groups (2a and 2b). The sub-group 2a includes four genotypes
collected from Andhra Pradesh, Karnataka, and Tamil Nadu. Genotypes
DGS15 and DGS14 showed similarity coefficient values of 0.86, and are
collected from Karnataka. The sub-group 2b consisted of a single entity
DGS22 (INGR13041) with profuse flowering with higher fruit yield and
biomass. It was collected from the Eastern Ghats of Andhra Pradesh, and
contains moderate gymnemagenin content (5.11 mg/g dry weight).
Group III consists of three genotypes. Group III was formed into two
sub-groups (3a and 3b). The sub-group 3a includes two genotypes
collected from Karnataka. Sub-group 3b formed as a distinct sub-group
with a single accession which is also collected from Karnataka. Kaur
et al. (2016) observed a similar grouping pattern in Tribulus terrestis
while studying the genetic diversity using SSR markers.
4. Conclusions
Genetic diversity studies in G. sylvestre by molecular markers are
limited to dominant markers. The present investigation addressed the
gap of the non-availability of genomic SSR markers in G. sylvestre.
Simple sequence repeats were identified from assembled contigs by the
MISA tool. SSR primer pairs were synthesized by flanking sequences of
SSR motifs using Primer3. Genetic diversity among 26 genotypes of
G. sylvestre with 58 SSR markers revealed moderate to high genetic di­
versity. The primers Xdagsm10 and Xdagsm96 are highly polymorphic
markers and are valuable in distinguishing the genotypes of G. sylvestre.
All 26 genotypes are broadly categorized into three groups. However,
the grouping pattern was not in tune with the geographical origin.
Among genotypes DGS10, DGS22, DGS16, DGS5, and DGS23 were
found to be divergent and can be used in the future breeding pro­
grammes. This is the first report on the design and development of novel
8
Journal of Applied Research on Medicinal and Aromatic Plants 34 (2023) 100455
genomic SSR markers and assessed genetic diversity using the large
number of genomic SSR markers in G. sylvestre. These novel genomic
SSR markers serve as a valuable tool for genetic diversity and population
structure analysis, genetic mapping, molecular assisted breeding, link­
age mapping, quantitative trait loci (QTL) mapping and DNA finger­
printing, and also conservation biology of G. sylvestre and related
species.
Declaration of Competing Interest
The authors declare that they have no conflict of interest.
Acknowledgment
We are thankful to National Medicinal Plants Board, New Delhi
(India) for funding the present investigation. The authors are grateful to
the Director, ICAR-DMAPR, Anand for providing the facilities to carry
out the present study.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.jarmap.2022.100455.
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