Molecular characterization and phylogenetic relationships of Dalbergia species of Eastern India based on RAPD and ISSR analysis

Abstract

The genetic relationships among six species of Dalbergia (D. sissoo, D. latifolia, D. volubilis, D. rubiginosa, D. paniculata and D. lanceolaria) with twelve accessions were assessed using RAPD and ISSR markers. Fourteen RAPD and thirteen ISSR primers were used for estimation of genomic variability among the species and accessions studied. High degree of polymorphism was observed with most of the primers used. All the species and accessions were related to each other with an average similarity of 0.49. Highest similarity (0.93) was observed between two accessions of Dalbergia volubilis (DV1 and DV2) and lowest (0.34) between Dalbergia rubiginosa (DR) and Dalbergia volubilis (DV3). The genetic closeness of D. latifolia, D. sissoo and D. rubiginosa was observed which is in partial agreement with the infra-generic classification of the genus Dalbergia proposed by Baker (1876), who placed all the three species under the sub-genus Sissoa. However, the genetic similarity observed between D. volubilis and D. latifolia, belonging to two separate sub-genera on the basis of molecular studies, could not be explained. In order to derive phylogenetic relationships among different species of the genus Dalbergia, more number of representative species and additional molecular markers need to be studied

Full text

Volume 3, Issue 7, July – 2018 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
IJISRT18JL193 www.ijisrt.com 417
Molecular Characterization and Phylogenetic
Relationships of Dalbergia Species of Eastern India
Based on RAPD and ISSR Analysis
Pankajini Bal, P. C. Panda
Taxonomy & Conservation Division
Regional Plant Resource Centre, Bhubaneswar 751 015, Odisha
Abstract:- The genetic relationships among six species of
Dalbergia (D. sissoo, D. latifolia, D. volubilis, D.
rubiginosa, D. paniculata and D. lanceolaria) with twelve
accessions were assessed using RAPD and ISSR
markers. Fourteen RAPD and thirteen ISSR primers
were used for estimation of genomic variability among
the species and accessions studied. High degree of
polymorphism was observed with most of the primers
used. All the species and accessions were related to each
other with an average similarity of 0.49. Highest
similarity (0.93) was observed between two accessions of
Dalbergia volubilis (DV1 and DV2) and lowest (0.34)
between Dalbergia rubiginosa (DR) and Dalbergia
volubilis (DV3). The genetic closeness of D. latifolia, D.
sissoo and D. rubiginosa was observed which is in partial
agreement with the infra-generic classification of the
genus Dalbergia proposed by Baker (1876), who placed
all the three species under the sub-genus Sissoa.
However, the genetic similarity observed between D.
volubilis and D. latifolia, belonging to two separate sub-
genera on the basis of molecular studies, could not be
explained. In order to derive phylogenetic relationships
among different species of the genus Dalbergia, more
number of representative species and additional
molecular markers need to be studied.
Keywords:- Molecular phylogeny, RAPD, ISSR, Dalbergia.
I. INTRODUCTION
The genus Dalbergia is represented by about 250
species and maximum number of species are known to
occue in Central and South America, Africa, Madagascar
and Asia (Klitgaard and Lavin, 2005). Several species of
Dalbergia such as D. latifolia, D. lanceolaria, D. sissoo are
source of quality timber used in furniture making and boat
building (Hiremath & Nagasampige, 2004a). As many as 50
species of the genus bear aeschynomenoid type root nodules
and fix nitrogen (Sprent, 2009). Indiscrimate felling of
forest trees for timber and firewood extraction in the tropical
dry and moist deciduous forests of India has been the main
cause of loss of biological diversity of commercially
important tree species including that of Dalbergia species.
The infra-generic classification and phylogeny of
Dalbergia has been dealt in a number of taxonomic
revisions, regional floras and inventories based
morphological characters (Bentham, 1860; Carvalho, 1997;
Chen et al., 2010; Niyomdham, 2002; Prain, 1904; Sunarno
and Ohashi, 1997; Thothathri, 1987). Bentham (1860) in his
infra-generic classification of Dalbergia placed all the 64
known species of Dalbergia under six series (Triptolemea
Americanae, Triptolemea, Sissoae Americanae, Sissoae
Gerontogee, Dalbergariae and Selenolobia). The South East
Asian species of Dalbergia were classified under two
subgenera, five sections and 24 series (Prain, 1904). In an
attempt to group morphologically allied species based on
androecium and fruit characteristics, Thothathri (1987) put
the forty six Dalbergia species then known to occur in the
Indian subcontinent in four sections and seven series.
Presently, the genus Dalbergia is recognized as having five
sections defined by inflorescence and fruit characters such
as: Sect. Dalbergia, Triptolemea, Selenolobium,
Pseudecastaphyllum and Ecastaphyllum (Carvalho, 1997).
The intra-specific genetic variability of some species
of Dalbergia has been assessed using RAPD and ISSR
markers in several parts of the world (Hussain et al., 2012;
Kumar et al., 2011; Amri et al., 2009; Phong et al., 2011;
Wang et al.,2011; Ashraf et al., 2010; Hien & Phong, 2012;
Juchum et al., 2007). Besides, the genetic variability and
population genetics of many species have been assessed
using markers other than RAPD and ISSR (Vatanparast et
al., 2013; Pandey et al., 2004; Ribeiro et al., 2007;
Andrianoelina et al., 2009).
Dalbergia species exhibit a wide range of
morphological variations and some of them have specific
ecological and habitat preference. These attributes pose
problems in placing the New World and the Old World
species into natural groups (Bentham, 1860; Carvalho,
1989). With regard to the molecular systematic of the genus
Dalbergia, scany information is available in published
literature. Vatanparast et al. (2013) derived the molecular
phylogeny of Dalbergia species and advocated the
monophyletic origin of the genus. For Indian species of
Dalbergia, very few studies have been undertaken till date
(Hiremath & Nagasampige, 2004b; Mohana et al., 2001;
Rout et al., 2003; Arif et al., 2009; Bakshi & Sharma, 2011;
Bhagwat et al., 2015), making it imperative to conduct
studies on genetic diversity and phylogeny of the genus
occurring in Eastern Ghat region of India.
With a view to understand the genomic variability and
molecular phylogeny of the genus Dalbergia occurring in
Eastern India, molecular characterization of 12 accessions of
six species of Dalbergia (D. sissoo, D. latifolia, D. volubilis,

Volume 3, Issue 7, July – 2018 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
IJISRT18JL193 www.ijisrt.com 418
D. rubiginosa, D. paniculata, and D. lanceolaria) were
made using RAPD and ISSR markers in the present study.
II. MATERIALS AND METHODS
A. Plant materials
Leaf samples of 12 individuals/ accessions belonging
to six species of Dalbergia were collected from different
forest areas of Odisha, Andhra Pradesh, West Bengal and
also from the arboretum of Regional Plant Resource Centre
(RPRC), Bhubaneswar. The accession number, locality of
collection and abbreviation used for each taxon is shown in
Table-1. The young tender leaves were used for genomic
DNA extraction for molecular analysis.
Sl. No.
Samples collection sites
Species
Code used in text,
tables and figures
1
Ghatikia, Bhubaneswar, Odisha
Dalbergia sissoo
DS1
2
Tirupati hills, Andhra Pradesh
D. sissoo
DS2
3
Barbara, Khurda Forest Division, Odisha
D.latifolia
DL1
4
Dhuanali, Khurda Forest Division, Odisha
D. latifolia
DL2
5
Paderu Hills, Vizag, Andhra Pradesh
D. volubilis
DV1
6
RPRC, Bhubaneswar, Odisha
D. volubilis
DV2
7
Barbara, Khurda Forest Division, Odisha
D. volubilis
DV3
8
Barunei hills, Khurda, Odisha
D. volubilis
DV4
9
RPRC, Bhubaneswar, Odisha
D. volubilis
DV5
10
Khandagiri, Bhubaneswar, Odisha
D. rubiginosa
DR1
11
Indian Botanic Garden, Howrah, West
Bengal
D. lanceolaria
DLN
12
RPRC, Bhubaneswar, Odisha
D. paniculata
DP
Table 1. Details of plant samples used for study of genetic diversity and phylogeny
B. Genomic DNA extraction
Genomic DNA was extracted from the leaf tissues
using the modified CTAB (cetyl-trimethyl-ammonium-
bromide) proto- col (Doyle and Doyle, 1990). Two grams of
leaf tissues from tender parts were ground with grinding
buffer composed of 100 mM sodium acetate (pH 4.8), 500
mM NaCl, 50 mM EDTA (pH 8.0), 50 mM Tris (pH 8.0),
2% Polyvinyl pyrollidone (PVP) and 2% CTAB.
Purification of DNA was done twice with extraction of
phenol:chloroform: Isoamyl alcohol (25:24:1). RNAse @ 40
μl from 1 mg/ ml was applied in the supernatant to get rid of
RNA. The quality and quantity of DNA were checked
through 0.8% agarose electrophoresis with standard DNA
before PCR amplification.
C. RAPD and ISSR analyses
Thirty RAPD and 30 ISSR primers (Operon
Technologies, Alameda, USA) were used for PCR analysis
based upon their performance and reproducibility. Among
them, 27 primers showed distinct polymorphism. PCR
mixture of 25 μl contained 25 ng of genomic DNA template,
0.6 μq of Taq DNA polymerase (Bangalore Genei,
Bangalore, India), 0.3 μM of decamer primers, 2.5 μl of 10 x
PCR assay buffer (50 mM KCI, 10 mM Tris-Cl), 1.5 m
MgCl2) and 0.25 μl of pooled dNTPs. The PCR condition
used for RAPD was: Initial denaturing step at 94°C for 5
minutes followed by 42 cycles of 94°C for 1 minute, 37°C
for 1 minute and 72°C for 2 minute, the last cycle, primer
extension at 72°C for 7 minutes. For ISSR amplification, the
PCR condition was: Initial denaturing step at 94°C for 5
minutes followed by 42 cycles of 94°C for 1 minute, 45° -
55°C for 1 minute and 72°C for 2 minute, the last cycle,
primer extension at 72°C for 7 minutes. The amplified
products as developed by the primers were separated by
agarose (1.5%) gel electrophoresis and documented in gel
documentation system (Bio Rad XR, Biorad, USA). O’Gene
Ruler™ 100 bp DNA Ladder plus (ladder range 3000 bp to
100 bp from Fermentas Life Sciences, USA) was used as
molecular weight marker. Bands were scored for its
presence/absence (1/0) for each primer genotypes
combination. Software NTSYS-pc, version 2.1 (Rohlf,
2000) was used for estimation of genetic relatedness among
the genotypes using Jaccard’s similarity coefficient and
clustering was done with UPGMA (unweighted pair group
method using arithmetic averages).
III. RESULTS
A. RAPD analysis
Fourteen RAPD primers reproduced well and resulted
in amplification of distinct bands. The DNA profiles
obtained from RAPD analysis are represented in Fig. 1. A
total of 92 amplified loci were generated which include 86
polymorphic, 6 monomorphic and 30 unique ones (Table-2).
The resolving power of primers ranged from 2.0(N4) to
10.75(A11), whereas the primer index varied from 0.22
(N18) to 4.78 (A11). The RAPD banding pattern revealed
that primer A11 produced highest number of amplified loci
11, followed by N7 (No. of bands=10) whereas N4 and N18
amplified least number of loci (1 and 2 respectively). Nine
of the fourteen primers produced 100% polymorphic bands,
whereas least polymorphism was observed with N18 (50%).

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The primer A3 and A10 also showed high level of
polymorphism (87.5%). The average amplified and
polymorphic band per primer was 6.57 and 6.14
respectively. The overall percentage of polymorphic band
was around 93.48%. Out of total 6 monomorphic bands
generated; N16 amplified maximum no. of monomorphic
loci (2). The primers D18, A9 and N7 produced 6, 4 and 4
unique loci respectively.
All the accessions were related to each other with an
average similarity of 40% as could be obtained from
Jaccard’s similarity co-efficient analysis. Highest similarity
(90%) was observed between Dalbergia volubilis (DV2) and
Dalbergia volubilis (DV1) and lowest of 17% between
Dalbergia volubilis (DV2) and Dalbergia latifolia (DL2).
On the basis of data obtained from RAPD analysis, a
cladogram was constructed for the 8 accessions and 4
species of Dalbergia which separated them into two distinct
clusters of 6 and 2 having a common node at 24.5%
similarity level (Fig. 2). The larger cluster of 6 was
subdivided into a cluster of a lone accession of Dalbergia
sissoo and rest of the accessions of Dalbergia volubilis and
Dalbergia latifolia. While one of the accessions of
Dalbergia latifolia formed a cluster with the four accessions
of Dalbergia volubilis; all the genotype of Dalbergia
volubilis exhibited close relationship among themselves
with varying levels of similarity.
B. ISSR analysis
The details of ISSR analysis of 12 accessions of 4
species of Dalbergia is presented in Table-3. Out of the 30
ISSR primers screened, only 13 primers produced good
amplified products. Total number of loci generated was 95;
out of which 86 were polymorphic, 9 monomorphic and 10
unique ones. The size of amplicons ranged from 100bp to
2000bp. The resolving power of primers ranges from 3.83
(Oligo 3b) to 13.83 [T(GA)9] and the primer index from
0.83 to 4.99 for Primers (AG)10 and T(GA)9 respectively.
The ISSR banding pattern is shown in Fig. 3
The primer T(GA)9 produced highest number of
amplified bands (13), whereas Oligo 2a and Oligo 3b
amplified the least number of loci (4 each). Six primers
namely (CT) 8A, Oligo2a, Olio 3b, T(GA)9, (GAC)5and
(AG)8C showed 100% polymorphism but the polymorphism
observed in case of primers (AG)10 was only 66.66%. The
average no. of amplified and polymorphic bands per primer
was 7.31 and 6.62 respectively. (GA)9T and (AG)10 were
responsible for amplification of maximum no. of
monomorphic loci (2 each) and most of the primers
(GTGC)4, yielded three unique loci during amplification.
The base sequences of these 13 primers indicate presence of
repeated di-nucleotides (AG)n, (GA)n, (CT)n, tetra-
nucleotides (GACA)n. The rate of polymorphism is highly
dependent on di-nucleotides and higher % of GA content
than other primer repeats.The rate of polymorphism is
highly dependent on di-nucleotides and higher % of GA
content than other primer repeats.
From the Jaccard’s similarity table, it could be
inferred that all the accessions were related to each other
with an average similarity of 52%. Highest similarity (0.95)
was observed between two accessions of Dalbergia volubilis
(DV1 and DV2) and lowest between Dalbergia sissoo
(DS1) and Dalbergia paniculata (DP) having similarity of
0.24. The single accession of Dalbergia paniculata got
separated in the dendrogram in the first pace with very
distinct genetic resemblance (30%) similarity. The rest 11
accessions was divided into two clusters, the smallest group
contains the single accessions of Dalbergia rubiginosa and
Dalbergia lanceolaria at 44% level of similarity Fig. 4.
Dalbergia lanceolaria and Dalbergia rubiginosa also
separated from each other in the dendrogram showing a
genetic similarity of 46.5%. The bigger cluster of Dalbergia
volubilis – Dalbergia latifolia and Dalbergia sissoo was
further subdivided into two distinct clades at 52.6% level of
similarity; one contain two genotypes of Dalbergia sissoo
and the other with accessions of Dalbergia volubilis and
Dalbergia latifolia. The two accessions of Dalbergia sissoo
had a genetic similarity of about 70%. Of the 7 accessions of
Dalbergia volubilis and Dalbergia latifolia, all the five
accessions of Dalbergia volubilis and 2 accessions of
Dalbergia latifolia got separated from each at a similarity
level of 53%. Both the genotypes of Dalbergia latifolia
exhibited about 86% similarity between them. Further, all
the 5 accessions of Dalbergia volubilis came together but
shared varying genetic similarity in the range of 76% to
86% among the accessions.
C. RAPD and ISSR combined markers
By analysing both RAPD and ISSR data, it was found
that 14 RAPD and 13 ISSR primers produced good and
reproducible amplification products. All the species and
accessions were related to each other with an average
similarity of 0.49. Highest similarity 0.93 was observed
between two accessions of Dalbergia volubilis (DV1 and
DV2) and lowest 0.34 between Dalbergia rubiginosa (DR)
and Dalbergia volubilis (DV3).
The dendrogram (Fig.- 5) generated from these data
segregated the 8 accessions to distinct cluster of 3 and 5
sharing a common node at 42.5% similarity level. The small
clade included the lone accession of Dalbergia rubiginosa
(DR) and two accessions of Dalbergia latifolia (DL1 &
DL2) and had 45.5% similarity among them. Further, the
two accessions of Dalbergia latifolia shared a node at the
67% level of similarity. The bigger clade contains 4
accessions of Dalbergia volubilis and one accession of
Dalbergia sissoo having a genetic similarity of about 43%.
The accession of Dalbergia volubilis formed a clear cluster
with varying levels of similarity among them. At the first
instance, accessions DV 1 and DV2 got separated from the
other two accessions DV3 and DV4 sharing a common node
at a genetic 77% level of closeness. While D. volubilis
(DV3 and, DV4) had a genetic relatedness of 79.8%
between them, the other two accessions of DV1 and DV2
shared similarity of about 92.5% between them.

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IV. DISCUSSION
Vatanparast et al. (2013) used ITS nuclear sequence
data and interpreted the molecular phylogeny of 64 species
of Dalbergia and compared with infrageneric classifications
suggested earlier on the basis of morphological data. In this
study, they included almost the representative species of the
various subgenera, sections and series to make the
classification relevant. The results of the study revealed that
sect. Triptolemea, with cymose inflorescences and thin
samaroid pods and sect. Ecastaphyllum, with racemose or
paniculate inflorescences and orbicular to suborbicular
fruits, are potentially monophyletic in origin. However, the
species of the sections Dalbergia and Selenolobium were
found to be non-monophyletic. These results are in
agreement with the findings of Ribeiro et al. (2007), who on
the basis of ITS and trnL sequence data suggested that types
of inflorescence and fruit may serve as sources of
synapomorphies for classifications of Dalbergia as opined
earlier by Carvalho (1997). Among the Asian species of
Dalbergia, the members of sect. Dalbergaria (Prain, 1904)
are condidered as monophyletic in origin. With reflexed
standard petals and stamens in two bundles of five each, the
species of this section are distributed throughout Southeast
Asia including India.
Baker (1876) classified the 28 species of Dalbergia
them known from British India under three sub-families
namely, Sissoa, Dalbergaria and Selenolobium. Of the
species investigated in the present study, D. sissoo, D.
latifolia and D. rubiginosa came under the sub-genus
Sissoa; and D. lanceolaria, D. volubilis and D. paniculata
under the sub-genus Dalbergaria. Asian species of
Dalbergia were placed in four sections viz. Sect. Sissoa,
Sect. Dalbergia, Sect. Selenolobia and Sect. Ecastaphylla
(Thothathri 1987)
The dendrogram constructed on the basis of RAPD
data placed one accession of D. latifolia and one of D.
rubiginosa in a single clade justifying their inclusion under
the sub-genus Sissoa but the second accession of D. latifolia
and D. sissoo were remotely placed. Similarly, two
genotypes of D. sissoo and two of D. latifolia, which are
members of the sub-genus Sissoa, came together in a
common clade in the tree constructed using ISSR data their
genetic proximity. However, closeness of D. volubilis and
D. latifolia belonging to two separate subfamilies could not
be explained from taxonomic point of view. As expected,
accessions of D. latifolia and D. rubiginosa belonging to the
same sub-genus Sissoa formed a cluster in the dendrogram
constructed using RAPD and ISSR data in combination.
However, Hiremath & Nagasampige (2004) on the basis of
RAPD analysis of 10 Indian species of Dalbergia kept D.
latifolia distinctly separate from other species. He also found
close genetic similarity among D. lanceolaria, D. volubilis,
D. rubiginosa, D. paniculata and D. sissoo. In the present
study, D. sissoo was found to form cluster with D. volubilis,
which is in agreement with the above findings of Hiremath
& Nagasampige (2004).
Based on 4C DNA content and chromosome
characteristics, Hiremath & Nagasampige (2004) detected
genetic resemblance between D. latifolia and D. sissoo.
They postulated that the species differentiation in these
closely related tree species, D. sissoo, D. latifolia and D.
sissoides have occurred through small increase in genome
size. Close genetic resemblance could also be seen in the
present study using RPD and ISSR markers.
As remarked by Carvalho (1989), sect. Dalbergia is an
assemblage of heterogeneous species with pyramidal panicle
sometimes arranged in bracteate compound panicles and
samaroid fruits. Although the results of this study are
congruent with some of the traditionally recognized sections
of Dalbergia, sampling is too limited to derive a conclusion
on phylogeny of this big genus. The pantropical distribution
of Dalbergia, with higher species concentration in
Amazonia, Indo-Asiaand Madagascar, nesessitates long
term study of molecular phylogeny involving as many
species as possible to throw light on the intra-generic
classifications proposed by Bentham (1860), Prain (1904)
and Carvalho (1989).
V. ACKNOWLEDGEMENTS
The authors wish to thank the Chief Executive,
Regional Plant Resource Centre, Bhubaneswar and Head of
the Department of Botany, North Orissa University,
Baripada for providing laboratory and field facilities and to
the State Forest Department, Odisha and Andhra Pradesh for
permission for field work.
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(1): 114-120.

Volume 3, Issue 7, July – 2018 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
IJISRT18JL193 www.ijisrt.com 422
Table 2. Details of RAPD primers and band details in species and accessions of Dalbergia
Primer
name
Sequence
Range of
amplicons
Total
no. of
bands
No. of
polymorphic
bands
No. of
monomorphic
bands
No. of
Unique
bands
%
Polymorohic
bands
Resolving
power
Primer
index
A8
5’GTGACGTAGG 3’
500-1500
5
5
0
2
100
3.5
1.69
A9
5' GGGTAACGC C 3'
800-1845
6
6
0
4
100
3
1.81
A18
5' AGGTGACCG T 3'
600-1845
6
6
0
1
100
4.25
2.41
A3
5' AGT CAG CCA C 3'
350-1000
8
7
1
3
87.5
9.5
1.69
A10
5' GTGATCGCA G 3'
100-1185
8
7
1
2
87.5
8.25
2.41
A11
5' CAATCGCCGT3'
200-1845
11
11
0
0
100
10.75
4.78
A12
5'TCGGCG ATA G 3'
450-1950
7
7
0
3
100
4.75
2.34
C2
5' GTGAGGGGTC3'
100-800
5
5
0
2
100
3
1.88
D18
5' GAG AGCCAA C 3'
100-1950
8
8
0
6
100
3.25
2.28
D20
5' ACCCGGTCA C 3'
200-1500
8
8
0
1
100
6.25
3.03
N4
5' GACCGA CCC 3'
1185
1
0
1
0
0
2
0
N7
5' CAG CCC AGA G 3'
180-1450
10
10
0
4
100
5.5
3.25
N16
5' AGGCGACCT G 3'
180-1000
7
5
2
1
71.42
11
3.25
N18
5’GGTGAGGECA3’
1185-1500
2
1
1
1
50
2.25
0.22

Volume 3, Issue 7, July – 2018 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
IJISRT18JL193 www.ijisrt.com 423
Table 3. Details of ISSR primers and bands amplified in different species and accessions of Dalbergia
Fig 1:- RAPD banding pattern of different species and accessions of Dalbergia
Primer
name
Sequence
Range of
amplicons
Total no.
of bands
No. of
poly-
morphic
bands
No. of
mono-
morphic
bands
No. of
unique
bands
% poly-
morohic
bands
Resolvin
g power
Primer
index
(CT)8A
5' CTCTCTCTCTCTCTCTA 3'
400-1950
6
5
1
0
83.33
5.17
1.65
(CT)8T
5' CTCTCTCTCTCTCTCTT 3'
600-2000
8
8
0
1
100
7
3
OLIGO 2a
5' AGAGAGAGAGAGAGAG 3'
100-400
4
3
1
0
75
6.33
1.03
OLIGO 3b
5' GACAGACAGACAGACA 3'
600-1780
4
4
0
1
100
3.83
1.54
(AGG)6
5' AGGAGGAGGAGGAGGAGG 3'
550-1500
6
6
0
0
100
6.83
2.29
T(GA)9
5' TGAGAGAGAGAGAGAGAGA 3'
345-1845
13
13
0
1
100
13.83
4.99
(GA)9T
5' GAGAGAAGAGAGAGAGAGAT 3'
200-900
7
5
2
1
71.42
10.67
1.28
(GTGC)4
5' GTGCGTGCGTGCGTGC 3'
150-1780
10
9
1
3
90
7.17
2.6
(GACA)4
5' GACAGACAGACAGACA 3'
400-1050
6
5
1
1
83.33
6
1.89
(GAC)5
5' GACGACGACGACGAC 3'
300-1500
10
10
0
0
100
11
3.81
(AG)10
5' AGAGAGAGAGAGAGAGAGAG 3'
100-800
6
4
2
2
66.66
6.67
0.83
(AG)8G
5' AGAGAGAGAGAGAGAGG 3'
300-650
5
4
1
0
80
6.83
1.07
(AG)8C
5' AGAGAGAGAGAGAGAGC 3'
200-1185
10
10
0
0
100
9.67
3.92

Volume 3, Issue 7, July – 2018 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
IJISRT18JL193 www.ijisrt.com 424
Dendrogram of Dalbergia sp. using RAPD Markers
Jaccard's Coefficient
0.21 0.39 0.5 7 0.76 0.94
DV1MW
DV1
DV2
DV3
DV4
DL1
DS
DL2
DR
Fig 2:- Dendrogram showing genetic relationship among different species
of Dalbergia as revealed from RAPD marker analysis
Fig 3:- ISSR banding pattern of different species and accessions of Dalbergia
Dendrogram of Dalbergia sp. Using ISSR Markers
Jaccard's Coefficient
0.28 0.46 0.6 4 0.81 0.99
DV1MW
DV1
DV2
DV3
DV5
DV4
DL1
DL2
DS1
DS2
DLN
DR1
DP
Fig 4:- Dendrogram representing relationship among the different species
and accessions of Dalbergia as revealed from ISSR analysis

Volume 3, Issue 7, July – 2018 International Journal of Innovative Science and Research Technology
ISSN No:-2456-2165
IJISRT18JL193 www.ijisrt.com 425
Dendrogram of Dalbergia Sp. using both RAPD and ISSR Markers
Jaccard's Coefficient
0.35 0.50 0.66 0 .81 0.96
DV1MW
DV1
DV2
DV3
DV4
DS
DL1
DL2
DR
Fig 5:- Dendrogram of different species of Dalbergia using both RAPD and ISSR markers in combination.