Industrial Crops & Products 143 (2020) 111894

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Industrial Crops & Products

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Population genetic structure and diversity analysis in economically T

important Pandanus odorifer (Forssk.) Kuntze accessions employing ISSR and
SSR markers
Noohi Nasima,1, I.Sriram Sandeepa,1, Ambika Sahooa, Suryasnata Dasa, Manoj Kumar Pandab,
Laxmikanta Acharyaa, V.V. RamaRaoc, Sanghamitra Nayaka, Sujata Mohantyd,*
a
Centre for Biotechnology, Siksha ‘O’ Anusandhan (Deemed to be University), Bhubaneswar, 751003, Odisha, India
b
Department of Bioscience and Bioinformatics, Khallikote University, Berhampur, 760001, Odisha, India
c
Agro-tech & Intellectual Property Facilitation Centre, Flavour and Fragrance Development Centre, Min. of MSME, Govt. of India, Kannauj, 209726, India
d
Department of Biotechnology, Rama Devi Women's University, Bhoinagar, Bhubaneswar, 751022, Odisha, India

A R T I C LE I N FO A B S T R A C T

Keywords: Pandanus odorifer (Forssk.) Kuntze commonly known as Kewda is an economically important essential oil bearing
Population genetic structure plant belonging to the family Pandanaceae. Kewda flower distillation industry in Ganjam district (Odisha) ac-
Genetic diversity counts for nearly 90 % of the production of commercially important Kewda perfumes in the country and 50 % of
Molecular marker that of the world. Economically, it is an important natural bioresource for the perfumery industry due to the
ISSR
exquisite fragrance it possesses. Besides the extensive use of Kewda male flowers for perfume production, other
SSR
parts of the plant are also used in fibre, food, pharmaceuticals and handcraft industries. Hence, the plant con-
Breeding programme
tributes to be the livelihood of the local people of Ganjam district by providing an alternative source of income
for the poor coastal villagers. Reports on the molecular characterization of Kewda germplasm using molecular
markers are scanty. Molecular marker based assessment of genetic diversity in Kewda is very important for
germplasm characterization, conservation and future improvement of the taxon. Hence, in the present study, 43
highly polymorphic molecular markers i.e. 13 ISSRs (Inter Simple Sequence Repeats) and 30 SSRs (Simple
Sequence Repeats) were screened on 84 Kewda accessions collected from different zones of Ganjam district,
Odisha. Both ISSR and SSR markers showed moderate level of polymorphism i.e. 68.63 % and 74.36 % re-
spectively. Dendrogram was constructed by UPGMA (Unweighted Pair Group Method with Arithmetic Mean)
cluster analysis for both the ISSR and SSR markers and the result was confirmed by Principal coordinate analysis
(PCoA). Analysis of molecular variance (AMOVA) and population genetic study (POPGENE) was also done. SSR
system was found to be more effective than ISSR system in evaluating the genetic diversity in Kewda accessions.
The information obtained from genetic diversity analysis could be used for future breeding programmes for
Kewda improvement to meet its ever increasing demand in the perfumery industry.

1. Introduction
Pandanus odorifer (Forssk.) Kuntze (Synonyms P. odoratissimus
Linn.f., P fascicularis Lam.), commonly known as Kewda, is an eco-
nomically important essential oil bearing plant that belongs to the fa-
mily Pandanaceae (Jose et al., 2016; Kumar et al., 2017; Nasim et al.,
2018). It is native to South Asia including Indonesia, Philippines and
some tropical parts of Australia (Zanan and Nadaf, 2012; Adkar and
Bhaskar, 2014). In India, it is distributed in the states of Odisha, Andhra
Pradesh, Kerala, Tamil Nadu, West Bengal, some regions of Uttar
Pradesh and Gujarat (Padhy et al., 2016; Nasim et al., 2018). The
coastal areas of Ganjam district of Odisha state support luxuriant
growth of the species (Rout et al., 2015). The plant has a wide range of
medicinal properties and is reported to have anti-inflammatory and
analgesic (Londonkar et al., 2010; Udupa et al., 2011), antioxidant
(Kaewklom and Vejaratpimol, 2011; Kumar et al., 2011), anticancer
(Raj et al., 2014), cardioprotective (Sobhana et al., 2014; Kamala et al.,
2016), antidiabetic (Kumari et al., 2012) and cytotoxic activity (Jitu
et al., 2017). It is used as one of the ingredients in several Ayurvedic
formulations (Andriani et al., 2015). Studies have also demonstrated

⁎
Corresponding author.
E-mail address: sujatamohantyils@gmail.com (S. Mohanty).
1
These authors have contributed equally to this work

https://doi.org/10.1016/j.indcrop.2019.111894
Received 14 March 2019; Received in revised form 27 September 2019; Accepted 22 October 2019
0926-6690/ © 2019 Elsevier B.V. All rights reserved.
N. Nasim, et al. Industrial Crops & Products 143 (2020) 111894

Table 1
Collection of Pandanus odorifer leaf and flower samples from different geographical zones.
Zones Sample Code Locality Latitude Longitude Elevation (m)

1 RR Rushikulya River Bank 19° 4 ' 12” N 84° 0 ' 36” E 2

2 KP Kalipalli 19° 18 ' 36” N 84° 55 ' 12” E 36
3 KL Keluapalli 19° 13 ' 48” N 84° 49 ' 12” E 3
4 MR Markandi 19° 12 ' 36” N 84° 50 ' 24” E 29
5 IN Indrakhi 19° 11 ' 60” N 84° 49 ' 48” E 29
6 MN Mantridi 19° 11 ' 24” N 84° 45 ' 36” E 29
7 KB Kaliabali 19° 20 ' 24” N 84° 56 ' 24” E 36
8 BP Basanaputty 19° 8 ' 0” N 84° 55 ' 48” E 34
9 CK Chamakhandi 19° 19 ' 48” N 84° 55 ' 12” E 36
10 PP Podapdar 19° 21 ' 36” N 85° 0 ' 36” E 36
11 CH Chilika 19° 30 ' 36” N 85° 5 ' 60” E 2
12 TM Tampara 19° 21 ' 0” N 85° 0 ' 0” E 17

the role of Kewda oil in curing skin diseases, earache, headache,
rheumatoid arthritis, smallpox, syphilis, sterility, cardiac troubles, colic
infection, laxative and spasms (Udupa et al., 2011; Nasim et al., 2017b).
The leaves are used for treating leprosy, small-pox, syphilis, leucoderma
and scabies (Padhy et al., 2016). Diuretic and anti-spasmodic properties
have been reported in the extract of the plant (Adkar and Bhaskar,
2014; Nasim et al., 2017a). The plant has gained agricultural im-
portance mainly because of its unique fragrant male flowers which are
extensively used for their essential oil in the perfume industry, thereby
making it an important natural bioresource (Nasim et al., 2017a,
2017b). Different parts of the plant have multipurpose uses in fibre,
food, handcraft industries, traditional medicine, fabrication of houses,
paper and fibre industry (Eland, 2008; Abral et al., 2012; Padhy et al.,
2016; Teli and Jadhav, 2017).
Though there is an increased demand for cultivation of P. odorifer
due to its immense socio-economic value, very scanty information is
available about the genetic diversity of available Kewda germplasm.
Furthermore, the plant is vegetatively propagated, thereby highlighting
the need for proper documentation of the available germplasm for se-
lection of elite genotypes, conservation and development of agrono-
mically superior parental lines. Therefore, assessment of the genetic
diversity of P. odorifer is of prime importance.
For a sustainable and effective utilization and conservation of plant
genetic resources, analysis of genetic variation is foremost (Sun et al.,
2008). Amongst the various marker based techniques which include
morphological markers, biochemical markers and different agronomic
parameters, molecular markers are the most recent method used for
genetic diversity study because they are more efficient, reliable and
insensitive to environmental variations (Ray et al., 2019; Ismail et al.,
2019). Molecular markers have been widely used for studying variation
in different genotypes (Kaur et al., 2015), population studies (Lakshmi
et al., 2000; Ng et al., 2015), phylogeny (Acharya et al., 2004; Li et al.,
2017), stability testing (Mohanty et al., 2008; Thorat et al., 2017; Jena
et al., 2018) and genetic mapping (Yagi et al., 2017). Recently, many
molecular markers have been developed among which ISSR and SSR
being highly polymorphic have been employed for the study of genetic
variation (Xing et al., 2015; Chen et al., 2017; Andiego et al., 2019;
Ismail et al., 2019). In the present study, ISSR (Inter simple sequence
repeat) and SSR (simple sequence repeat) markers are used for genetic
diversity analysis because of their robustness, cost effectiveness, higher
reproducibility and they are not being affected by the environmental
factors (Das et al., 2017; Ray et al., 2019). The combination of both the
markers is preferred as ISSR do not require prior information about the
genome (Singh et al., 2012) and SSR markers have a good genome
coverage and show highly inter and intra specific polymorphic patterns
(Arif et al., 2010; Kaur et al., 2015).
Very limited information is provided by the previous studies on the
genetic variations in Pandanus species, mainly because of the type of
molecular marker used (Panda et al., 2007; Vinod et al., 2007; Panda
et al., 2009; Sarile and Menguito, 2010; Susanti et al., 2012; Wakte
et al., 2012; Gallaher et al., 2017). Available reports exclude any work
on assessment of genetic variation and population structure studies of
Pandanus odorifer by ISSR and SSR markers. The only report found in P.
odorifer is confined to RAPD analysis (Panda et al., 2007). Moreover,
Ganjam is considered to be the luxuriant growth centre of the plant
(Mohapatra and Sahoo, 2007; Rout et al., 2015), and due to the vege-
tative propagation same cuttings might have been used generation after
generation thereby necessitating genetic diversity studies for selection
of elite planting material.
However, the present study is the first report on the genetic di-
versity assessment of 84 Pandanus odorifer accessions collected from 12
different zones of Ganjam district of Odisha using ISSR and SSR markers
combined approach. The study was done for the characterization of
available Kewda germplasm for their genetic diversity and to meet the
ever increasing demand of Kewda in the perfumery industry.
2. Materials and methods
For the present investigation, Kewda leaves were collected from
twelve different zones of Ganjam district, Odisha (Table 1, Fig. 1). The
different zones include Rushikulya River Bank (RRB), Kalipalli, Kelua-
palli, Markandi, Indrakhi, Mantridi, Kaliabali, Basanaputty, Chama-
khandi, Podapadar, Chilika and Tampara. Randomly seven samples
from each zone were collected and used for this study. Eighty four fresh,
young and healthy leaves were sampled from all the zones, properly
cleaned followed by removal of the spines and were immediately sub-
jected for DNA extraction.
2.1. DNA extraction
Genomic DNA isolation from the young and healthy leaves of ran-
domly collected P. odorifer populations from each zone was carried out
by following the protocol of Doyle and Doyle (1990) with certain
changes. UV-VIS Spectrophotometer (Thermo fisher Scientific Evolu-
tion 220, USA) was used to measure the quantity and quality of the
extracted genomic DNA. Further, to confirm the quality of the DNA
0.8% agarose gel (Himedia, India) was used. Gel picture was taken in
Gel Doc 2000 (Bio-Rad, USA) for quality checking of the DNA. After
quantification, the DNA was diluted with T10 E1 buffer to a working
concentration of 25 ng/μl for ISSR and SSR analysis.
2.2. ISSR and SSR analysis
A total of 72 primers (15 ISSR and 57 SSR) were initially screened,
out of which 13 ISSR and 30 SSR showed polymorphic banding pattern
with clear band resolution whereas remaining primers failed to amplify
any product among the 84 accessions of Kewda.
For ISSR analysis of P. odorifer, primers were procured from IDT
(Integrated DNA Technologies, USA). For SSR analysis, SSR primers
were designed from EST sequences of Pandanus odorifer. In the present

2
N. Nasim, et al.
Fig. 1. Odisha map showing the different zones selected for this study.
study, 977 expressed sequenced tag (EST) sequences were downloaded
from the NCBI database. MISA tool was used for screening the presence
of SSR repeat motifs. The minimum number of repeated units con-
sidered for identifying SSRs were 10 for mono-, 6 for di-, 4 for tri- and 3
for tetra-, penta- and hexanucleotides. Primer 3 plus software program
was used for designing SSR primers. A total of 57 primers (containing
reverse and forward primers) were procured from IDT, USA. Each
amplification reaction mixture for ISSR was adjusted to 25 μl volume
which consisted of 25 ng of template DNA, 2.5 μl of 10X assay buffer
(Tris with 25 mM MgCl2) (Bangalore Genie, India), 200 μM of each
dNTPs (dATP, dGTP, dCTP and dTTP) (Qiagen, India), 5 pM of primer
and 0.5U Taq DNA polymerase (Bangalore Genie, India). Veriti thermal
cycler (Applied Biosystems, USA) was used for carrying out the am-
plification. Similar measurements were taken for SSR amplification
reaction mixture.
The PCR profile for ISSR included initial denaturation for 5 min at
94 °C, followed by 42 cycles at 94 °C for 1 min, primer annealing for
1 min and extension at 72 °C for 2 min. A final extension step was in-
cluded for 72 °C for 7 min. The amplified products were resolved in 1.5
% agarose gel stained in ethidium bromide (SRL, India). The electro-
phoresis was performed for 2.5 h at 60 V. For SSR, the first step con-
sisted of complete denaturation of the template DNA by a hold of 5 min.
at 94 °C. The second step consists of 36 cycles having three ranges of
temperature i.e. at 92 °C for 1 min for denaturation of template DNA, at
50–60 °C for 1 min for primer annealing and at 72 °C for 2 min for
primer extension followed by running the samples at 72 °C for 7 min for
complete polymerization. After the completion of the PCR, 2.5 μl of 6X
loading dye (MBI Fermentas, Lithuania) were added to the amplified
products and separated in 6.5–8% polyacrylamide gel electrophoresis.
3
Industrial Crops & Products 143 (2020) 111894
PAGE gel was allowed to run at 120 V for 3–4 hours. The amplified
products were documented by taking gel picture in Gel Doc 2000 (Bio-
Rad, USA).
2.3. Statistical analysis
After documentation, the gel images were observed for the absence
and presence of bands to check the amplification produced by different
primers. For ISSR and SSR analysis the presence and absence of a band
for each primer combination was scored as ‘1’ and ‘0’ respectively. The
total number of bands obtained for ISSR and SSR markers, number of
polymorphic bands and percentage of polymorphic loci were calcu-
lated. Further, different indices, such as- Polymorphic Information
Content (PIC) and Resolving Power (Rp) were calculated for finding out
the effectiveness of different primers (both ISSR and SSR). PIC was
calculated as PIC = 1-ΣPi2, where Pi is the band frequency of the ith
allele (Smith et al., 1997). Rp of all markers was calculated as Rp=ΣIb,
where Ib denotes Band informativeness of the primers with Ib = 1-[2 X
(0.5-p)] where ‘p’ is the proportion of the species containing the band
(Prevost and Wilkinson, 1999). POPGEN version 1.32 software was
used for estimating different genetic parameters such as: effective
number of alleles per locus (Ne), observed number of alleles per locus
(Na), Shannon‘s information index (I), Heterozygosity (Ht) and Nei's
Genetic diversity (H). Similarity analysis was done by Unweighted pair
group method with arithmetic averages (UPGMA) and a dendrogram
was constructed by using NTSYS-pc, version 2.11a (Rohlf, 2000).
Principal Co-ordinate Analysis (PCoA) was performed to confirm the
grouping of different accessions of Pandanus odorifer and Analysis of
Molecular Variance (AMOVA) was done for determining molecular
N. Nasim, et al. Industrial Crops & Products 143 (2020) 111894

Table 2
Details of 13 ISSR markers giving polymorphic banding patterns in Kewda accessions.
S.No. Primer Sequence of the Range of No. of Polymorphic loci Monomorphic loci % of polymorphic Resolving Power Polymorphic Information
primers amplicon in bp loci loci (RP) Content (PIC)

1 ISSR 3 (GTG)5 1200-2500 5 4 1 80 6.24 0.51

2 ISSR 4 (AC)8T 1400-3000 4 4 0 100 6.64 0.30
3 ISSR 5 (AG)8T 500-1700 5 5 1 100 7.5 0.39
4 ISSR 6 (CA)8AT 1000-2500 4 3 1 75 7.14 0.19
5 ISSR 7 (AG)8G 500-1000 3 1 2 33.33 4.67 0.3
6 ISSR 8 (GA)9T 700-2200 4 3 1 75 4.98 0.55
7 ISSR 9 T(AG)9 900-1200 3 3 0 100 4.64 0.4
8 ISSR 11 (AC)9T 1400-3000 3 2 1 66.67 4.64 0.36
9 ISSR 12 (TC)8G 700-2000 3 2 1 66.67 4.29 0.44
10 ISSR 14 (CTC)6 1000-2500 4 3 1 75 6.26 0.36
11 ISSR 15 (CA)6GG 600-1500 2 1 2 33.33 2.17 0.66
12 ISSR 19 (CT)8A 1300-2500 3 2 1 66.67 4.55 0.39
13 ISSR 20 (GA)8YC 700-1200 3 2 1 66.67 3.45 0.57
Total 46 35 11 76.08%

Table 3 Table 5
Searching results of EST-SSRs of Pandanus odorifer. Major repeat motifs in the ESTs of Pandanus odorifer.
Parameters Value Repeat type Motif Number Percent (%)

Total number of sequences examined: 977 Mononucleotide A/T 118 26.64

Total size of examined sequences (bp): 602027 C/G 16 3.61
Total number of identified SSRs: 443 Subtotal 134 30.25
Number of SSR containing sequences: 292 Dinucleotide AC/GT 1 0.23
Number of sequences containing more than 1 SSR: 92 AG/CT 134 30.25
Number of SSR present in compound formation: 127 AT/AT 3 0.68
Subtotal 138 31.15
Trinucleotide AAC/GTT 3 0.68
AAG/CTT 75 16.93
Table 4
AAT/ATT 2 0.45
Distribution of EST-SSR repeat types found in Pandanus odorifer.
ACC/GGT 2 0.45
Repeat type Number Frequency % ACG/CTG 3 0.68
ACT/ATG 3 0.68
Mononucleotide 134 30.25 AGC/CGT 6 1.35
Dinucleotide 138 31.15 AGG/CCT 17 3.84
Trinucleotide 120 27.09 AGT/ATC 2 0.45
Tetranucleotide 31 7.0 CCG/CGG 7 1.58
Pentanucleotide 12 2.71 Subtotal 120 27.09
Hexanucleotide 8 1.81 Tetranucleotide AAAC/GTTT 2 0.45
Total 443 100 AAAG/CTTT 4 0.90
AAAT/ATTT 5 1.13
AAGC/CGTT 2 0.45
AAGG/CCTT 11 2.48
diversity among and within populations by using the GenAlex version ACAG/CTGT 1 0.23
6.5 (Peakall and Smouse, 2012). ACGC/CGTG 1 0.23
ACGT/ATGC 1 0.23
AGAT/ATCT 2 0.45
3. Results
AGCG/CGCT 2 0.45
Subtotal 31 7.00
3.1. ISSR banding pattern, PIC and Rp Pentanucleotide AAGAG/CTCTT 7 1.58
AGAGG/CCTCT 5 1.13
Subtotal 12 2.71
Among the 15 tested ISSR primers, 13 primers showed polymorphic
Hexanucleotide AAAAAG/CTTTTT 1 0.23
banding pattern with clear band resolution. The amplification products ACCTCC/AGGTGG 1 0.23
ranged from 500 to 3000 bp. The number of loci per primer varied from AGAGGG/CCCTCT 4 0.90
2 (ISSR 15) to 5 (ISSR 3 and ISSR 5) with an average of 3.53 loci per AGCCGG/CCTCGG 1 0.23
primer. Out of the 46 amplified loci, 35 polymorphic loci and only 11 AGCTCC/AGGTCG 1 0.23
Subtotal 8 1.81
monomorphic loci were found. The percentage of polymorphic loci
Total 443 100
(PPL) was found to be 76.08 %, ranging from 33.33 % produced by the
primers ISSR 7 and ISSR 15–100% obtained by ISSR 4, ISSR 5, and ISSR
9. The range of resolving power (Rp) was 2.17 in primer ISSR 15 to 7.5 study (Table 3). Here, 443 SSRs in 292 ESTs were identified using the
in ISSR 5. The PIC of 13 primers was calculated and lowest PIC value MISA tool. Among these 292 ESTs, 92 ESTs had two or more SSRs. In
was found in primer ISSR 6 i.e. 0.19 and highest PIC value was found in this study, six types of repeats were found to be present and composed
ISSR 15 i.e. 0.66 (Table 2). of mononucleotide (134, 30.25 %) dinucleotide (138, 31.15 %), trinu-
cleotide (120, 27.09 %), tetranucleotide (31, 7.0 %), pentanucleotide
3.2. SSR analysis (12, 2.71 %) and hexanucleotide (8, 1.81 %) (Table 4). Whereas, 127
compound SSRs were identified. The predominant repeats were the
3.2.1. Characterization of designed SSR primers from EST sequences of dinucleotides which represented 31.15 % of the identified micro-
Pandanus odorifer satellites followed by mononucleotides (30.25 %) and trinucleotides
A set of 977 EST sequences with 602027 bp length was used for this (27.09 %). Further, the most abundant repeat motif found was (AG/CT)

4
 Table 6
Details of 30 SSR markers giving polymorphic banding patterns among Kewda accessions.
S No. SSR Marker Repeat motif Primer sequence 5'-3' Expected product Range of amplicon Total Polymor-phic Mono-morphic Percentage of polymorphic Resolving power Poly-morphic Informa-
N. Nasim, et al.

Forward/ Reverse Primer sixe (bp) (bp) alleles alleles alleles loci (%) (RP) tion content (PIC)

1 PfSSR 5 (TTTA)3 F: GGA GCC CAA ACA CCT 201 150-1000 7 7 0 100 7.36 0.65
ATC CT
R: AGT TCA GCC TTG TGA
TGG GA
2 PfSSR 6 (AGC)4 F: GGT CGG GAA GAA GGA 159 150-350 5 5 0 100 4.6 0.72
ACG A
R: GTC GAG GGT GTT GAA
GTT GG
3 PfSSR 8 (CTT)7 F: GGA GGG GAG CTT AGA 161 160-320 6 5 1 83.33 6.36 0.67
ACT CC
R: AAC GAT GCC AAA GAT
GAG CG
4 PfSSR 9 (TGTT)3 F: TGA GAG TTG TCA TTC 228 140-700 6 6 0 100 6.6 0.64
TGC AGC
R: TAC GAT CTA CCA AGC
CTG CC
5 PfSSR 11 (AGC)5 F: GGG ATG GAA GGG CAT 235 250-1200 10 7 3 70 11.45 0.55
TTT GG
R: AGG AGG AAA ATG GGA
TGG GG
6 PfSSR 12 (CTT)6 F: GGA GGG GAG CTT AGA 158 160-320 8 7 1 87.5 8.17 0.69
ACT CC
R: AAC GAT GCC AAA GAT
GAG CG

5
7 PfSSR 13 (TAAA)3 F: TGC GAT GAG ATT GTA 151 270-800 5 4 1 80 4.9 0.64
CGG GA
R: GTG ATT GAT ATG TCG
CGA CGA
8 PfSSR 14 (GAAG)3 F: AGG ATT GCA GGA AAG 172 180-200 2 1 1 50 3.67 0.15
AGG CT
R: GTA TCG ATG GCC AAA
ACC CC
9 PfSSR 15 (AAG)4 F: AGG AGA TGT TGA AGG 183 200-600 7 7 0 100 6.67 0.7
CTG CT
R: AGC TTC CTC TGC ACT
CCA TT
10 PfSSR 17 (GTT)4 F: CAT TGC TAA GGT CCT 213 150-700 5 5 0 100 5.12 0.73
GCT GG
R: TCC GTC TTG TGT GGG
ATC AT
11 PfSSR 18 (CTT)4 F: GGG GAG AGT AGG GGA 176 180-450 5 4 1 80 7.07 0.42
GCT TA
R: CAG CTC CAA ATC GAT
GCC AA
12 PfSSR 19 (TTTTCT)3 F: ACA CTG ACC GTA CAA 250 110-1200 7 5 2 71.43 11.21 0.28
GCT GA
R: GCT TGC CTT CCT TCC
TTT GT
13 PfSSR 20 (AAGG)3 F: GGG GCA CAA GTA TGG 237 150-700 5 5 0 100 6.17 0.55
TAA GAT
R: CAC TGT CGT AAT GCA
CTC GG
(continued on next page)
Industrial Crops & Products 143 (2020) 111894
 Table 6 (continued)

S No. SSR Marker Repeat motif Primer sequence 5'-3' Expected product Range of amplicon Total Polymor-phic Mono-morphic Percentage of polymorphic Resolving power Poly-morphic Informa-
Forward/ Reverse Primer sixe (bp) (bp) alleles alleles alleles loci (%) (RP) tion content (PIC)
N. Nasim, et al.

14 PfSSR 22 (TCT)4 F: GAG AGA GAG AAG GCC 162 180-800 6 6 0 100 7.19 0.58
TGA GG
R: CAC CAG ACC AAA AGG
CAT CC
15 PfSSR 24 (ACC)4 F: CAC GGA CAT AGG AGG 207 200-700 9 9 0 100 11.62 0.52
AGG TC
R: AGT CCC ATG CCA TAC
AAC CA
16 PfSSR 26 (TCC)6 F: CCC GGT TCG AGT ATA 176 180-450 7 5 2 71.43 8.64 0.53
GCA TC
R: GCT AAA CGC CGG TTC
ATT TA
17 PfSSR 27 (TCT)4 F: CTC CTC AAC TGC ATG 189 450-1500 7 6 1 85.71 9.36 0.52
CTG TG
R: CAT GAA TGC AAG CTC
CCC AG
18 PfSSR 28 (TTTC)3 F: ATA GTG TGG GTG GCA 165 180-400 3 2 1 66.67 3.17 0.6
TGG TT
R: CTT CCT TGG ATT CGC
GCT TT
19 PfSSR 30 (GCC)4 F: GGG ACG AGG GTT GGA 160 180-320 4 3 1 75 5.19 0.49
AAG AA
R: CAC TTC GCC GTC TCA
TAA GC
20 PfSSR 31 (CTC)4 F: GGG ACG AGG GTT GGA 160 150-500 5 5 0 100 5 0.66

6
AAG AA
R: CAC TTC GCC GTC TCA
TAA GC
21 PfSSR 32 (GCA)4 F: ATG AAG ATG AGC TGG 191 180-500 3 2 1 66.67 3.83 0.53
CTG GT
R: AAT GTT CTG TGG CTG
CTG TG
22 PfSSR 35 (CAG)4 F: TCA AAT CTT CCA ACT 227 240-500 2 1 1 50 2.67 0.44
GCG GC
R: ACC ATG AAC AGA CCC
AGG AG
23 PfSSR 36 (CCTCGG)3 F: GGG GAA AGC AAT TGG 203 120-700 10 10 0 100 11.71 0.6
GGA AG
R: GGT GGT GAA GAA CTT
GGT GG
24 PfSSR 39 (TCCTC)3 F: CGC CAC CCA AAA TTA 178 180-500 6 4 2 66.67 8.19 0.46
ACA AC
R: CGG ATG GAA CCC ATC
TAA TC
25 PfSSR 40 (AG)12 F: TCC TCT CCT CTC CTC 226 300-500 4 2 2 50 6.76 0.25
CTG CT
R: GGC CAG TAG ATC CGT
GAG TC
26 PfSSR 41 (CTT)5 F: GGA GAG AGG AGG GGA 191 200-1000 7 7 0 100 7.74 0.66
GCT TA
R: GAG CAA GGG TGT GGA
CAG TT
27 PfSSR 42 (GA)8 F: GGG CAG CAC AAC CAT 205 200-900 6 4 2 66.67 9.05 0.33
AGT CT
(continued on next page)
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N. Nasim, et al. Industrial Crops & Products 143 (2020) 111894

Poly-morphic Informa- n (30.25 %) which was followed by (A/T)n (26.64) and (AAG/CTT)n
tion content (PIC) (16.93 %) (Table 5).

3.2.2. SSR banding pattern, PIC and Rp

Primers were designed for 57 SSRs from the 977 EST sequences of
Kewda. Out of these 57 SSR primers, 10, 30 and 17 SSRs were resulted
0.46

0.52

0.25
in monomorphic, polymorphic and no amplifications respectively.
These 30 set of SSR primers detected 170 alleles with the amplicon size
varying from 120 to 1500 bp (Table 6). The number of alleles per
Resolving power

primer varied from 2 (PfSSR 14 and PfSSR 35) to 10 (PfSSR 11 and

PfSSR 36) with an average of 5.67 allele per primer in the 30 poly-
morphic primers used. A total of 170 alleles amplified, out of which 145
(RP)

5.83

7.21

5.14

alleles were polymorphic and only 25 alleles were monomorphic in

nature. The percentage of polymorphic loci (PPL) was found to be
Percentage of polymorphic

85.29%, ranging from 50% to 100%. The range of resolving power (RP)
varied from 2.67 in primer PfSSR 35 to 11.71 in primer PfSSR 36. The
PIC of 30 primers was calculated and lowest PIC value was found in
primer PfSSR 14 i.e. 0.15 and highest PIC value was found in PfSSR 17
i.e. 0.73. The detail of other primers is provided in the Table 6.
loci (%)

85.29%
83.33

66.67
100

3.3. Genetic variability in Kewda accessions

Mono-morphic

Genetic variability parameters like observed number of alleles per

locus (Na); effective number of alleles per locus (Ne); percentage of
polymorphic bands (PPB); total gene diversity (Ht); Nei's Genetic di-
alleles

versity (H) and Shannon‘s information index (I) for ISSR, SSR and
25
0

combined markers. Among twelve different experimental zones, zone 3

and zone 9 (Keluapalli and Chamakhandi) displayed the highest values
Polymor-phic

of effective alleles (Ne), Heterozygosity (Ht), Nei’s genetic diversity (H)

and Shannon’s information index (I), while zone 1,2,3,9,12 (Rushikulya
alleles

145

River Bank, Kalipalli, Keluapalli, Chamakhandi, Tampara) showed the

4

highest number of observed alleles (Na) for ISSR primers (Table 7).
Similarly, for SSR primers, zone 6 (Mantridi) displayed the highest
alleles

values of observed alleles (Na), effective alleles (Ne), Heterozygosity

Total

170

(Ht), Nei’s genetic diversity (H) and Shannon’s information index (I)
4

(Table 8). Analysis with combined markers also showed zone 6 (Man-
Range of amplicon

tridi) to have the highest values of observed alleles (Na), effective al-
leles (Ne), Heterozygosity (Ht), Nei’s genetic diversity (H) and Shan-
non’s information index (I) (Table 9). The results indicated that zone 6
230-500

120-700

150-400

(Mantridi) exhibited higher genetic diversity in comparison to other 11

(bp)

zones. The mean values of all the parameters revealed by SSR primers
(Na = 1.7436; Ne = 1.4790; PPB = 74.36 %; Ht = 0.2761;
Expected product

H = 0.2761; I = 0.4091) were higher as compared to ISSR primers

(Na = 1.6863; Ne = 1.4084; PPB = 68.63 %; Ht = 0.2390,
sixe (bp)

H = 0.2390, I = 0.3571).
231

229

221

3.4. AMOVA (analysis of molecular variance) analysis of Pandanus

odorifer
F: GGA GCA ACC GGA GAA

R: AAA CGA TGC CAA AGA

R: CTG GGT ATG AGC GAG

R: CGG TGG AAA GAA CTC

F: GGA AAC TCT CTG CTG
R: CGT AGA CTT CTC CGC
F: GAT ATC TCG GCT CTC
Forward/ Reverse Primer

AMOVA was done to measure variation among and within P.

Primer sequence 5'-3'

odorifer accessions (Table 10). The ISSR data indicated 42 % genetic

variation because of the variability within the Kewda accessions of 12
zones of Odisha. The remaining 58 % variations can be accounted to
GAG AG

TAA AA

TGA GC

ACA CA
AAA CC
GCA TC

CTG CT

variability among populations (Fig. 2A) AMOVA done by using SSR

data resulted with very low (18%) genetic diversity within Kewda po-
pulations while the variability among populations contributed to be
Repeat motif

higher (82 %) (Fig. 2B). AMOVA for the combined markers indicated
(ATGC)3

low genetic variation (22 %) within the Kewda populations and the
(TC)11
(AG)8

remaining 78 % variation was due to the variability among the popu-

lations (78 %) (Fig. 2C;).
Table 6 (continued)

SSR Marker

PfSSR 43

PfSSR 48

PfSSR 50

3.5. Genetic similarity, cluster analysis and principal coordinate analysis

Total

Jaccard‘s similarity coefficient calculated for ISSR and SSR markers

S No.

showed that similarity coefficient for ISSR data varied from 0.54 (zone
28

29

30

3 (Keluapalli) and zone 4 (Markandi) to 0.98 (zone 10 (Podapadar) and

7
N. Nasim, et al. Industrial Crops & Products 143 (2020) 111894

Table 7
Genetic diversity estimates as revealed through ISSR markers among 12 populations of P. odorifer.
Population code Na Ne Ht H I NPL PPB

Z1 1.2157 ± 0.4154 1.1859 ± 0.3631 0.0992 ± 0.0369 0.0992 ± 0.1920 0.1407 ± 0.2716 11 21.57
Z2 1.2157 ± 0.4154 1.1912 ± 0.3725 0.1008 ± 0.0380 0.1008 ± 0.1950 0.1423 ± 0.2747 11 21.57
Z3 1.2157 ± 0.4154 1.1965 ± 0.3815 0.1024 ± 0.0391 0.1024 ± 0.1978 0.1440 ± 0.2777 11 21.57
Z4 1.1176 ± 0.3254 1.0917 ± 0.2575 0.0512 ± 0.0202 0.0512 ± 0.1423 0.0737 ± 0.2043 6 11.76
Z5 1.1569 ± 0.3673 1.1294 ± 0.3077 0.0704 ± 0.0275 0.0704 ± 0.1657 0.1005 ± 0.2359 8 15.69
Z6 1.1961 ± 0.4010 1.1617 ± 0.3362 0.0880 ± 0.0327 0.0880 ± 0.1810 0.1256 ± 0.2576 10 19.61
Z7 1.1961 ± 0.4010 1.1776 ± 0.3665 0.0928 ± 0.0363 0.0928 ± 0.1904 0.1306 ± 0.2675 10 19.61
Z8 1.1961 ± 0.4010 1.1723 ± 0.3567 0.0912 ± 0.0351 0.0912 ± 0.1873 0.1289 ± 0.2642 10 19.61
Z9 1.2157 ± 0.4154 1.1965 ± 0.3815 0.1024 ± 0.0391 0.1024 ± 0.1978 0.1440 ± 0.2777 11 21.57
Z10 1.1961 ± 0.4010 1.1723 ± 0.3567 0.0912 ± 0.0351 0.0912 ± 0.1873 0.1289 ± 0.2642 10 19.61
Z11 1.1961 ± 0.4010 1.1723 ± 0.3567 0.0912 ± 0.0351 0.0912 ± 0.1873 0.1289 ± 0.2642 10 19.61
Z12 1.2157 ± 0.4154 1.1912 ± 0.3725 0.1008 ± 0.0380 0.1008 ± 0.1950 0.1423 ± 0.2747 11 21.57
All multi populations 1.6863 ± 0.4686 1.4084 ± 0.3740 0.2390 ± 0.0395 0.2390 ± 0.1988 0.3571 ± 0.2814 35 68.63

Values are mean ± SD.

Z1, include accessions from Rushikulya River Bank; Z2-Kalipalli; Z3-Keluapalli; Z4-Markandi; Z5-Indrakhi; Z6-Mantridi; Z7-Kaliabali; Z8-Basanaputty; Z9-
Chamakhandi; Z10-Podapdar; Z11-Chilika; Z12-Tampara.
Na, Observed number of alleles; Ne, Effective number of alleles; Ht, Heterozygosity; H, Nei’s gene diversity; I, Shannon’s Information index; NPL, Number of
polymorphic loci; PPB, Percentage of polymorphic bands.

Table 8
Genetic diversity estimates as revealed through SSR markers among 12 populations of P. odorifer.
Population code Na Ne Ht H I NPL PPB

Z1 1.1026 ± 0.3042 1.0901 ± 0.2703 0.0477 ± 0.0202 0.0477 ± 0.1420 0.0674 ± 0.2004 20 10.26
Z2 1.1077 ± 0.3108 1.0909 ± 0.2660 0.0490 ± 0.0202 0.0490 ± 0.1420 0.0696 ± 0.2014 21 10.77
Z3 1.0974 ± 0.2973 1.0811 ± 0.2509 0.0440 ± 0.0182 0.0440 ± 0.1347 0.0626 ± 0.1916 19 9.74
Z4 1.0974 ± 0.2973 1.0704 ± 0.2220 0.0402 ± 0.0155 0.0402 ± 0.1244 0.0585 ± 0.1801 19 9.74
Z5 1.1077 ± 0.3108 1.0881 ± 0.2582 0.0481 ± 0.0195 0.0481 ± 0.1396 0.0688 ± 0.1989 21 10.77
Z6 1.1231 ± 0.3294 1.1057 ± 0.2865 0.0565 ± 0.0231 0.0565 ± 0.1519 0.0801 ± 0.2150 24 12.31
Z7 1.1179 ± 0.3234 1.0980 ± 0.2726 0.0532 ± 0.0214 0.0532 ± 0.1464 0.0758 ± 0.2083 23 11.79
Z8 1.1128 ± 0.3172 1.0944 ± 0.2694 0.0511 ± 0.0208 0.0511 ± 0.1442 0.0727 ± 0.2049 22 11.28
Z9 1.1128 ± 0.3172 1.0889 ± 0.2537 0.0494 ± 0.0195 0.0494 ± 0.1395 0.0710 ± 0.2000 22 11.28
Z10 1.0974 ± 0.2973 1.0736 ± 0.2302 0.0414 ± 0.0163 0.0414 ± 0.1277 0.0599 ± 0.1839 19 9.74
Z11 1.0769 ± 0.2672 1.0614 ± 0.2163 0.0339 ± 0.0140 0.0339 ± 0.1183 0.0486 ± 0.1693 15 7.69
Z12 1.0872 ± 0.2828 1.0657 ± 0.2157 0.0373 ± 0.0147 0.0373 ± 0.1213 0.0539 ± 0.1752 17 8.72
All multi populations 1.7436 ± 0.4378 1.4790 ± 0.3754 0.2761 ± 0.0371 0.2761 ± 0.1925 0.4091 ± 0.2697 145 74.36

Values are mean ± SD.

Z1, include accessions from Rushikulya River Bank; Z2-Kalipalli; Z3-Keluapalli; Z4-Markandi; Z5-Indrakhi; Z6-Mantridi; Z7-Kaliabali; Z8-Basanaputty; Z9-
Chamakhandi; Z10-Podapdar; Z11-Chilika; Z12-Tampara.
Na, Observed number of alleles; Ne, Effective number of alleles; Ht, Heterozygosity; H, Nei’s gene diversity; I, Shannon’s Information index; NPL, Number of
polymorphic loci; PPB, Percentage of polymorphic bands.

Table 9
Genetic diversity estimates as revealed through combined markers among 12 populations of P. odorifer.
Population code Na Ne Ht H I NPL PPB

Z1 1.1260 ± 0.3325 1.1100 ± 0.2937 0.0584 ± 0.0239 0.0584 ± 0.1547 0.0826 ± 0.2185 31 12.6
Z2 1.1301 ± 0.3371 1.1117 ± 0.2933 0.0597 ± 0.0242 0.0597 ± 0.1554 0.0847 ± 0.2200 32 13.01
Z3 1.1220 ± 0.3279 1.1050 ± 0.2859 0.0561 ± 0.0229 0.0561 ± 0.1514 0.0795 ± 0.2142 30 12.2
Z4 1.1016 ± 0.3028 1.0748 ± 0.2294 0.0425 ± 0.0164 0.0425 ± 0.1281 0.0617 ± 0.1851 25 10.16
Z5 1.1179 ± 0.3231 1.0967 ± 0.2691 0.0528 ± 0.0211 0.0528 ± 0.1453 0.0753 ± 0.2070 29 11.79
Z6 1.1382 ± 0.3458 1.1173 ± 0.2977 0.0630 ± 0.0251 0.0630 ± 0.1585 0.0896 ± 0.2247 34 13.82
Z7 1.1341 ± 0.3415 1.1145 ± 0.2955 0.0614 ± 0.0246 0.0614 ± 0.1570 0.0871 ± 0.2223 33 13.41
Z8 1.1301 ± 0.3371 1.1106 ± 0.2906 0.0594 ± 0.0239 0.0594 ± 0.1546 0.0844 ± 0.2191 32 13.01
Z9 1.1341 ± 0.3415 1.1112 ± 0.2874 0.0604 ± 0.0239 0.0604 ± 0.1545 0.0861 ± 0.2198 33 13.41
Z10 1.1179 ± 0.3231 1.0941 ± 0.2637 0.0518 ± 0.0205 0.0518 ± 0.1431 0.0742 ± 0.2045 29 11.79
Z11 1.1016 ± 0.3028 1.0844 ± 0.2550 0.0458 ± 0.0188 0.0458 ± 0.1371 0.0653 ± 0.1949 25 10.16
Z12 1.1138 ± 0.3182 1.0917 ± 0.2603 0.0504 ± 0.0201 0.0504 ± 0.1417 0.0722 ± 0.2024 28 11.38
All multi populations 1.7317 ± 0.4440 1.4644 ± 0.3754 0.2684 ± 0.0376 0.2684 ± 0.1940 0.3983 ± 0.2724 180 73.17

Values are mean ± SD.

Z1, include accessions from Rushikulya River Bank; Z2-Kalipalli; Z3-Keluapalli; Z4-Markandi; Z5-Indrakhi; Z6-Mantridi; Z7-Kaliabali; Z8-Basanaputty; Z9-
Chamakhandi; Z10-Podapdar; Z11-Chilika; Z12-Tampara.
Na, Observed number of alleles; Ne, Effective number of alleles; Ht, Heterozygosity; H, Nei’s gene diversity; I, Shannon’s Information index; NPL, Number of
polymorphic loci; PPB, Percentage of polymorphic bands.

8
N. Nasim, et al. Industrial Crops & Products 143 (2020) 111894

Table 10 Fig. 4 (A)-(C). The PCoA plot obtained by ISSR, SSR and combined data
AMOVA using ISSR, SSR and combined markers. were in accordance with the results of cluster analysis.
Markers df SS MS Est. Var. Value % P value

ISSR 4. Discussion
Among Pops 11 318.702 28.973 3.756 58% 0.001
Within Pops 72 193.143 2.683 2.683 42% Assessment of genetic diversity is the key to evolution and an im-
SSR
Among Pops (AP) 11 1884.536 171.321 23.727 82% 0.001
portant approach for biodiversity conservation (Govindaraj et al.,
Within Pops (WP) 72 376.571 5.23 5.23 18% 2015). It is paramount for the breeders to have knowledge about the
Combined genetic variation on which adaptation depends for producing improved
Among Pops (AP) 11 2203.238 200.294 27.483 78% 0.001 varieties of crops without declining the yield and industrial value of the
Within Pops (WP) 72 569.714 7.913 7.913 22%
product (Deperi et al., 2018). In the present study, genetic variation was
studied among 84 accessions of Kewda collected from 12 different
zones of Ganjam district of Odisha, through study of various para-
zone 11 (Chilika) whereas similarity coefficient for SSR data ranged
meters. Two PCR-based molecular markers i.e. ISSR and SSR were used
from 0.46 (Zone 3 (Keluapalli) and Zone 11 (Chilika) to 0.88 (Zone 8
for effectively unveiling the genetic variation among the 84 Kewda
(Basanaputty) and Zone 9 (Chamakhandi) and their combined data
accessions. A total of 43 highly polymorphic molecular markers i.e. 13
varied from 0.52 (zone 3 (Keluapalli) and zone 11 (Chilika) to 0.84
ISSR and 30 SSR were screened on 84 Kewda accessions. As no SSR
(zone 1 (RRB) and zone 2 (Kalipalli).
markers were available for P. odorifer, SSR repeats were identified from
Dendrogram constructed through SAHN (Sequential Agglomerative
the EST sequences and primers were designed flanking the SSR repeat
Hierarchial and Nested) clustering and Jaccard‘s similarity coefficient
motifs. EST- SSR markers have also been developed in various other
used for UPGMA cluster analysis of ISSR, SSR and combined data are
plants (Hong et al., 2015: Sahoo et al., 2017). In the present study,
shown in Fig. 3 (A)-(C). Dendrogram generated from ISSR data sepa-
Dinucleotide repeats were found to be the predominant which is similar
rated the 84 accessions into two main clusters i.e. cluster 1 with seven
to that of coffee and tea plant (Aggarwal et al., 2007; Liu et al., 2018).
accessions of Markandi zone and cluster 2 with accessions of the re-
Dinucleotide repeats (31.15 %) were followed by mononucleotides
maining 11zones (Fig. 3A). These two clusters shared a common node
(30.25 %) and trinucleotides (27.09 %) which were in agreement with
at 68 % similarity level. The larger cluster i.e. cluster 2 was further
the report on rubber tree and citrus plant (Feng et al., 2009; Liu et al.,
divided into sub clusters, cluster 2a and cluster 2b. Cluster 2a contains
2013). In addition, (AG/CT)n were found to be the most abundant di-
only one zone i.e. seven accessions and rest 70 accessions of the re-
nucleotide repeat motif (30.25 %). Similar observation has also been
maining 10 zones were in sub cluster 2b. These two sub clusters shared
reported in Casuarina equisetifolia by Li et al., (2018). The SSR primers
a common node at 69 % similarity level. The second sub cluster i.e.
used in the present study also revealed moderate level of polymorphism
cluster 2b having 10 zones comprising of 70 accessions was further
among the 84 accessions (74.36 %).
grouped into 2 clusters of one small cluster 2b1 having 7 accessions and
In the present study, higher genetic similarity was observed with
other large cluster 2b2 with 63 accessions of the remaining 9 zones
both the marker systems i.e. 0.76 for ISSR and 0.73 for SSR. The ob-
sharing a common node at 70 % similarity level.
servations suggest low genetic variability within Kewda accessions. The
Dendrogram constructed from SSR data also separated the 84 ac-
results might be attributed to the fact that Pandanus odorifer is a ve-
cessions into two main clusters i.e. cluster 1, a smaller cluster consisting
getatively propagated plant. Low genetic variability has been reported
of 14 accessions from 2 zones (Chilika and Tampara) and a larger
earlier also in other vegetatively propagated plants (Chen et al., 2008;
cluster 2 that had 70 accessions of the remaining 10 zones (Fig. 3B).
Sun et al., 2008; Hu et al., 2012). Chen et al., (2001) and Hu et al.
These two clusters shared a common node at 55 % similarity level. The
(2012) reported low level of genetic diversity in sacred lotus (Nelumbo
larger cluster, cluster 2 was further divided into two sub clusters,
nucifera Gaertn.) by ISSR and SSR markers. Sun et al. (2008) also found
cluster 2a and cluster 2b. Cluster 2a comprised of 21 accessions and
low levels of genetic variation in Jatropha curcas L. The PCoA showed
cluster 2b consisted of 49 accessions of the remaining 7 zones. These
the differences in total number of clusters and position of accessions
two sub clusters shared a common node at 62 % similarity level. The
within the clusters in ISSR and SSR marker system. This difference
second sub cluster, cluster 2b having 7 zones was again segregated into
might be attributed to the fact that distinct regions of DNA variation
2 sub clusters. One sub cluster 2b1 with 28 accessions and second sub
were identified by different markers within the genome (Dongre et al.,
cluster 2b2 with 21 accessions. Dendrogram obtained from combined
2004; Ashraf et al., 2016).
data of ISSR and SSR showed similarity to SSR data (Fig. 3C)
AMOVA for combined marker analysis (ISSR and SSR) showed 78 %
The genetic relationship among the 84 accessions was further
variance among the populations and 22 % variance was seen within the
evaluated by PCoA for ISSR, SSR and combined data and is shown in
populations. Similar results were reported by Das et al. (2017) where

Fig. 2. Analysis of molecular variance (AMOVA) of 84 accessions of Pandanus odorifer based on (A) ISSR (B) SSR (C) Combined marker data.

9
N. Nasim, et al. Industrial Crops & Products 143 (2020) 111894

Fig. 3. Dendrogram generated from (A) ISSR, (B) SSR and (C) combined data of 84 accessions of Pandanus odorifer.

Fig. 4. PCoA analysis of Pandanus odorifer accessions based on (A) ISSR, (B) SSR and (C) combined data.

10
N. Nasim, et al.
genetic variation among the populations of ginger accessions was found
to be higher (64 %) and low genetic variation (36 %) within the po-
pulation of ginger accessions collected from different regions of Odisha.
The population genetic study was also done using the combined
data from ISSR and SSR markers and it revealed moderated level of
polymorphism i.e. 73.17 %. The mean values of Nei‘s gene diversity
(H), Shannon‘s information index (I), Percentage of polymorphic band
(PPB), Polymorphic information content (PIC) and Resolving Power
(Rp) were higher for SSR primer as compared to ISSR primers. These
results indicated the better efficiency of the SSR system as compared to
ISSR system in evaluating the genetic diversity in Kewda accessions.
Combined ISSR and SSR marker analysis have also been done in various
plants (Tantasawat et al., 2010; Babaei and Shabanimofrad, 2018;
Motha et al., 2018). SSR has been found to more effective than ISSR in
revealing the genetic diversity among accessions of many plants
(Weiguo et al., 2007; Malik et al., 2016; Das et al., 2017; Wang et al.,
2017). Out of the 84 accessions studied, the accessions of zone 6, 7 and
9 possessed comparatively higher genetic diversity (H value 0.063,
0.0614, 0.0604 respectively) proving them as conservation-worthy
populations thereby necessitating their conservation as well as large
scale cultivation.
The genetic diversity analysis in kewda using SSR marker is re-
ported for the first time. These results might be helpful for identifying
superior genotypes for commercial exploitation and breeding purpose.
Furthermore, the identified EST-SSR markers could be used for popu-
lation genetic study, genetic diversity analysis, comparative genomics
and cultivar identification in Kewda.
5. Conclusion
Molecular analysis by ISSR and SSR markers clearly showed that P.
odorifer accessions were highly polymorphic and SSR markers were
more efficient than ISSR markers in accessing genetic variation in P.
odorifer germplasm. The vegetative propagation of the plant might have
resulted in limited gene flow due to which less genetic variation was
seen among the accessions. Also, regional ecogeography of the collected
populations might have contributed significantly for the variation
among the populations. Moreover, repeated use of similar parental
material leads to similarity among P. odorifer accessions. These results
have an important implication for characterization of available germ-
plasm for their genetic diversity. The present study also suggests that
multiple marker utilization (both ISSR and SSR markers) for genetic
diversity analysis is a more reliable technique because of their highly
polymorphic nature. DNA fingerprints of Pandanus odorifer would be of
immense use in proper characterization of the species with assurance of
safety, quality and efficacy of its product. Genetic diversity study using
ISSR and SSR markers could be used for future breeding programmes
for Kewda improvement to meet its ever increasing demand in the
perfumery industry.
Acknowledgements
This research work was supported by the Department of
Biotechnology, Govt. of India [grant number BT/PR11790/PBD/17/
901/2014]. Authors are thankful to Dr S. C. Si, Dean and Mr M. R.
Nayak, President, SOA University for providing all facilities.
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