Indian Journal of Biotechnology
Vol 10, July 2011, pp 285-293
Genetic diversity among three Morinda species using RAPD and ISSR markers
D R Singh*, Abhay K Srivastava, Amit Srivastava and R C Srivastava
Division of Horticulture and Forestry, Central Agricultural Research Institute
Port Blair 744 101, Andaman and Nicobar Island, India
Received 8 January 2010: revised 27 August 2010; accepted 2 November 2010
A total of 22 accessions of 3 Morinda spp. (Family: Rubiaceae), namely, M. citrifolia, M. tinctoria and M. pubescens
were collected from three geographical locations of India, viz., Andaman and Nicobar Islands, Tamil Nadu and Karnataka.
RAPD (52) and ISSR (60) primers were employed as genetic marker to study the genotypic variation within and between
Morinda spp. In RAPD, only 26 primers amplified and gave reproducible fragments, of which 11 primers were
polymorphic. Among 1767 amplified DNA fragments obtained, 953 (54.33%) were polymorphic. The accessions AHD and
SHE-1 were the most closely related cultivars with the highest similarity index (0.943) and BBD and MAA-1 were the most
distantly related cultivar with lowest similarity index (0.387). In ISSR, of 60 primers tested, 22 gave clear and reproducible
fragments. In total, 1892 fragments of different lengths were amplified, of which 1052 bands (56.02%) were polymorphic.
According to ISSR results, the two most closely related cultivars were MTC and JGH with the highest similarity index
(0.944) and the most distantly related cultivars were BMN with MEM and BGL-2 with lowest similarity index (0.248).
Polymorphism and similarity index values for both RAPD and ISSR systems showed that both marker systems are equally
effective for diversity analysis in Morinda species.
Keywords: Diversity analysis, ISSR, Morinda spp., RAPD
Introduction
The genus Morinda (Family: Rubiaceae) is
originated in India and has 80 different species1.
Later, it has been naturalized in many parts of Asia,
South America, Caribbean and Polynesian Islands.
The adaptation features of its seed have enhanced its
natural dispersion in land through streams and rivers,
using ocean currents2, by fruit-eating birds and other
animals, or migrating human beings colonized the
Pacific Islands for its medicinal uses3-4. It has a broad
range of therapeutic and nutritional values.
Interestingly, it has been found that Morinda spp. are
tolerant to saline soils and can have a good
sustainability for cultivation in the saline affected
waste lands5. M. citrifolia (Fig. 1a), known as Noni,
grows predominantly along the tropical coasts. It is an
economically important species being used as a folk
medicine by the tribal and aboriginals in Andaman
and Nicobar Islands6. Its fruit is used worldwide for
making different products. More than 200 commercial
entities sell Noni products, which are distributed
across the globe and enjoy an enormous market
share7. It has been reported that Morinda extract has
_____________
*Author for correspondence:
Tel: 91-3192-250236; Fax: 91-3192-251068/233281
E-mail: [email protected]
antibacterial, antiviral, antifungal, antitumour,
antihelminthic,
analgesic,
hypotensive,
antiinflammatory and immune enhancing effects8-11.
Reviews on its medicinal uses9-12 summarized that its
popularity seems to hinge on a combination of its
traditional uses, development and distribution of
modern products and a mixture of factual and fanciful
information provided directly by manufacturers and
indirectly by academic researchers13. Another related
species, M. pubescens (Fig. 1b) is a small tree grown
in tropical regions of vacant agricultural land and
comparable to M. citrifolia in its medicinal and other
properties14. Decoction of stem bark of this plant used
in rheumatic diseases15 and root is given in
dysentery16. In the traditional system of medicine,
leaves and roots of M. tinctoria (Fig. 1c) are used as
astringent, deobstrent, emmengogue and to relive pain
in the gout17.
In recent years, a number of PCR-based DNA
markers, such as RAPD, ISSR, AFLP (amplified
fragments length polymorphism) and SSR (simple
sequence repeat) have been widely used to investigate
the genetic structure of a population18-19. The
selections of RAPD and ISSR were based on their
relative technical simplicity, level of polymorphism
they detect, cost effectiveness, easily applicable to
INDIAN J BIOTECHNOL, JULY 2011
286
Fig. 1—Showing fruits of M. citrifolia (A), M. pubescence (B) and M. tinctoria (C)
any plant species and target those sequences which
are abundant throughout the eukaryotic genome and
are rapidly evolved20-21. RAPD has been the most
employed technique because it is simple and fast.
Despite questions about its reproducibility, its utility
in diversity analysis, mapping and genotype
identification has been exploited in many plant
species22-23. A series of studies have indicated that
ISSR could be able to produce more reliable and
reproducible bands because of the higher annealing
temperature and longer sequence of ISSR primers
considered superior to RAPD24-25. ISSR has proved to
be useful to the study of population genetic
studies18,26-28,
gene
mapping29,
germplasm
identification and characterize gene bank accessions30
as well as to identify closely related cultivars31. Since,
no attempt have been made to characterize the genetic
diversity of Morinda cultivars through molecular
markers, the present study was under taken to analyze
DNA marker based genetic diversity analysis using
the RAPD and ISSR primers within and between
Morinda spp. for identifying elite genotype for its
improvement
Material and Methods
Collection of Plant material
A total of 22 accessions of Morinda spp. shown
in Table 1 were colleted. M. citrifolia samples contain
8 different accessions of Andaman and Nicobar
Islands (A & N) and 1 accession each from Chennai
(Tamil Nadu) and Bangalore (Karnataka); M. pubescens
samples contain 10 accessions from Andaman and
Nicobar Islands and one from Chennai; and M. tinctoria
samples contain only 1 accession from Chennai. All
these plant samples were planted in Horticulture Field
and maintained in Germplasm Collection Block of
Central Agricultural Research Institute, Port Blair,
Andaman and Nicobar Islands, India.
Table 1—The collection sites of Morinda spp.
Morinda
spp.
1
M. citrifolia
2
3
4
“
“
“
5
6
7
8
Locations
Accessions
State
name
Memeo
MEM
MNJ
ABH-1
SEH-1
“
“
“
M. tinctoria
Manjeri
Bahai House
Seamen
Hostel
Hadoo
Jungli Ghat
Sippighat
Chennai
AHD
JGH
SGH
MTC
9
M. citrifolia
Bambooflat
BFT
10
‘’
Chennai
MAA-1
11
12
Bangalore
Brich Ganj
BGL-2
BCG
13
‘’
M.
pubescens
“
SEH-2
14
“
15
16
17
18
19
20
“
“
“
“
“
“
Seamen
Hostel
Hut Bay
Island
Bahai House
Barma Nala
Phoneix Bay
Calicut
Bidnabad
Chennai
21
“
22
“
Campel Bay
Island
Nicobar
Island
HBY
ABH-2
BMN
PBY
CCT
BBD
MAA-2
KBY
NCB
A&N
Islands
“
“
“
“
“
“
Tamil
Nadu
A&N
Islands
Tamil
Nadu
Karnataka
A&N
Islands
“
“
“
“
“
“
“
Tamil
Nadu
A&N
Islands
“
DNA Isolation and PCR Amplification
Genomic DNA of 22 genotypes was extracted from
3 g fresh leaflets following the CTAB method with
slight modifications32. After purification, DNA was
quantified by both spectrophotometricaly and
visualized under UV light after electrophoresis on
0.8% (w/v) agarose gel. The resuspended DNA was
SINGH et al: GENETIC DIVERSITY AMONG MORINDA SPECIES
287
diluted in autoclaved ddH2O. A total of 52 RAPD
(Bangalore Genei Pvt. Ltd., Bangalore, India) and
60 ISSR primers (Sigma, St. Louis, Mo.) were
screened. PCR amplification were carried out in a
thermal cycler in a final volume of 25 µL, containing
25 ng template DNA, 100 µM of each of the four
deoxynucleotide
triphosphate,
20
ng
of
decanucleotide primer, 1.5 mM MgCl2, 10× Taq
buffer (10 mM Tris HCl pH 9.0, 50 mM KCl ) and
0.5 U Taq DNA polymerase (Bangalore Genei Pvt.
Ltd., Bangalore, India). The samples were subjected
to initial denaturation for 5 min at 94°C, followed by
39 cycles of 1 min at 94°C, 1 min at 36°C for RAPD
and 42-64°C for ISSR, and extension for 1 min at
72°C with a final extension of 7 min at 72°C. 10 µL
of amplified PCR product was separated through gel
electrophoresis on 2% agarose gel stained with
ethidium bromide and photographed with gel
documentation system (Vilber Loubmet, France,
Cat.No. Bio ID++ver.99.04)33.
Analysis of RAPD and ISSR Profiles
DNA fragment sizes on agarose gel were estimated
by comparing with 500 bp to 1 kb DNA size markers.
The bands were scored ‘1’ for presence and ‘0’ for
absence in DNA samples amplified to create a binary
data matrix. The data obtained by scoring the RAPD
and ISSR profiles with different primers individually
as well as collectively were subjected to the
construction of similarity matrix using Jaccard’s
coefficients34. The similarity values were used for
cluster analysis. Sequential hierarchical agglomerative
non-overlapping (SHAN) clustering was done using
unweighted pair group method with arithmetic
averages (UPGMA) method. Data analysis was done
using NTSYS-PC, version 2.0235. The similarity
matrix was obtained after multivariate analysis using
the Dice coefficient similarity36.
Results
RAPD Analysis
Amplified products were observed for each
accession of M. citrifolia, M. tinctoria and
M. pubescens using RAPD-PCR analysis with
52 arbitrary random primers (Figs 2a & b). A total of
26 RAPD primers were amplified and 11 showed
polymorphic banding pattern. The codes and
sequences of the amplified primers are shown in
(Table 2). A total number of 1767 DNA fragments
were amplified from 26 primers, among them 953
(54.33%) were polymorphic. The results of the
Fig. 2 (a & b)—Amplification with RAPD primers OPH 11
(a) and OPH-31 (b): Mx & My, 500 bp ladder (a); Mx, 1 kb &
My, 500 bp ladder (b); lane 1, MEM; 2, MNJ; 3, ABH-1; 4,
SEH-1; 5, AHD; 6, JGH; 7, SGH; 8, MTC; 9, BFT; 10, MAA-1;
11, BGL-2; 12, BCG; 13, SEH-2; 14, HBY; 15, ABH-2; 16,
BMN; 17, PBY; 18, CCT; 19, BBD; 20, MAA-2; 21, KBY; & 22,
NCB (a & b).
consensus tree indicated that tree was divided into
two major clusters, each having 11 accessions
showing 45% similarity (Fig. 3). The cluster I
sub-divided in two sub-clusters with 82% similarity.
The first sub-cluster had 8 collections including
1 accession of M. tinctoria and 7 accessions of
M. citrifolia from A & N, and second subcluster had
3 collections each from A & N, Bangalore and
Chennai. The second major cluster divided in two
sub-sub-clusters with 76% similarity. The first
sub-cluster had all 5 accessions of M. pubescens from
South Andaman and second had 6 accessions, 3 from
Andaman and 1 each from Chennai, Kambel Bay
and Nicobar.
The similarity coefficient based on the 1767 DNA
products ranged from 0.387 to 0.943. In RAPD
analysis two most closely related genotypes were
AHD and SHE-1 with the highest similarity index
INDIAN J BIOTECHNOL, JULY 2011
288
Table 2—Amplified primers and sequences for RAPD
profiling
No.
Primer code
Sequance 5'-3'
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
OPH-1
OPH-2
OPH-3
OPH-4
OPH-5
OPH-6
OPH-7
OPH-8
OPH-9
OPH-10
OPH-11
OPH-13
OPH-14
OPH-15
OPH-17
OPH-19
OPH-20
OPH-21
OPH-31
OPH-33
OPH-34
OPH-36
OPH-39
OPH-41
OPH-42
OPH-43
OPH-44
OPH-45
AATCGGGCTG
CAATCGCCGT
TCTGTGCTGG
GACCGCTTGT
GTTGCGATCC
TCGCCGCAAA
AGCGTCACTC
GTCCGTACTG
GGTGCTCCGT
GACCGACCCA
CGGTTCCCCC
GTCTGACGGT
CAGCTCAAGT
CGATCGAGGA
AAGCAGCAAG
AGCATTCGGT
CACCGTTCTG
ACTCCGCAGT
TAGACAGTCG
AGGCCGTATC
ATGAGTCCAC
TCAAACTCGG
TAGCCGTCAA
ATTTGATCGC
ACGCTGATCA
AACCGACGGG
TGCCCTGCCT
CTTGCCTCCC
Fig. 3—Dendrogram showing similarity coefficient of Morinda
accessions based on RAPD analysis
(0.943) and the two most distantly related cultivar
were BBD and MAA-1 with the lowest similarity
index (0.387). Within species diversity, the two most
closely related accessions were AHD and SHE-1
(0.943) and the two distant accessions were MEM and
MAA-1 (0.750) for M. citrifolia; while for M.
pubescens, the two most closely related accessions
were BCG and ABH-2 (0.906) and the distant
accessions were BBD and BCG (0.700) (Table 3).
ISSR Analysis
A total of 60 ISSR primers based on dinucleotide,
tetranucleotide or pentanucleotide repeats were used
to amplify each genotype of M. citrifolia, M. tinctoria
and M. pubescens (Figs 4a & b). Among the primers
tested, 38 primers amplified but only 22 primers
(Table 4) those produced clear bands and had
reproducibility were selected for further analysis. A
total number of 1892 fragments with different lengths
were clearly amplified from 22 accessions, among
which 1052 bands (56.02%) were polymorphic. The
results showed that 10 amplified primers were
monomorphic and 12 were polymorphic. Among the
Fig. 4 (a & b)—Amplification with ISSR primer UBC 7 (a) and
UBC 34 (b): Mx, 1 kb & My, 500 bp ladder (a); Mx, 500 bp &
My, 1 kb ladder (b); lane 1, MEM; 2, MNJ; 3, ABH-1; 4, SEH-1;
5, AHD; 6, JGH; 7, SGH; 8, MTC; 9, BFT; 10, MAA-1;
11, BGL-2; 12, BCG; 13, SEH-2; 14, HBY; 15, ABH-2; 16,
BMN; 17, PBY; 18, CCT; 19, BBD; 20, MAA-2; 21, KBY; & 22,
NCB (a & b).
primers studied, the highest number of bands were
generated with primer UBC-10 (132 bands), while the
lowest number of 34 bands were generated with
primer UBC-54. The results of the consensus tree
from ISSR data indicated that tree was divided into
SINGH et al: GENETIC DIVERSITY AMONG MORINDA SPECIES
289
Table 3—Similarity index for RAPD
two major clusters each having 11 accessions with
38% similarity (Fig. 5). The first major cluster
divided into 2 sub-clusters with 85% similarity; first
sub-cluster had 8 collections including M. tinctoria
and second had 3 collections, 1 each from Andaman,
Chennai and Bangalore. The second major cluster
again divided into 2 sub-clusters, first having
5 accessions and second having 6 accessions with
67% similarity. According to the ISSR results, the
similarity coefficient based on the 1892 DNA
products ranged from 0.248 to 0.944. The most
closely related genotypes were MTC and JGH
with the highest similarity index (0.944) and
the most distantly related genotypes were BMN,
MEM and BGL-2 with lowest similarity index
(0.248) (Table 5). The ISSR results showed the
same pattern of clustering as in RAPD. Within
species diversity studies by ISSR, the two most
close accessions were AHD and SHE-1
(0.95) and the most distant accessions were BGL-2
and MEM (0.77) for M. citrifolia; while for
M. pubescens, the two most close accessions
were NCB and KBY (0.86) and the most distant
accessions were BCG and CCT (0.58).
Table 4—Amplified primers and sequences for ISSR profiling
No.
Primer code
Sequance 5'-3'
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
UBC-1
UBC-2
UBC-3
UBC-7
UBC-8
UBC-9
UBC-10
UBC-14
UBC-15
UBC-18
UBC-19
UBC-20
UBC-34
UBC-35
UBC-36
UBC-45
UBC-46
UBC-47
UBC-54
UBC-55
UBC-56
UBC-57
ATATATATATATATATT
ATATATATATATATATG
ATATATATATATATATC
AGAGAGAGAGAGAGAGT
AGAGAGAGAGAGAGAGC
AGAGAGAGAGAGAGAGG
GAGAGAGAGAGAGAGAT
CTCTCTCTCTCTCTCTA
CTCTCTCTCTCTCTCTG
CACACACACACACACAG
GTGTGTGTGTGTGTGTA
GTGTGTGTGTGTGTGTC
AGAGAGAGAGAGAGAGYT
AGAGAGAGAGAGAGAGYC
AGAGAGAGAGAGAGAGYA
CTCTCTCTCTCTCTCTRG
CACACACACACACACART
CACACACACACACACARC
TCTCTCTCTCTCTCTCRG
ACACACACACACACACYT
ACACACACACACACACYA
ACACACACACACACACYG
R=Purines Y=Pyrimidine
290
INDIAN J BIOTECHNOL, JULY 2011
Combined RAPD and ISSR analysis
Genetic diversity of 22 accessions was estimated
using combined RAPD and ISSR data. The results
indicated that consensus tree was divided into two
major clusters, each having 11 accessions showing
42% similarity (Fig. 6). The first major cluster
divided into 2 sub-clusters with 84% similarity, the
first su-bcluster consisted of 8 collections including
M. tinctoria and the second one with 3 collections.
The second cluster divided into 2 sub-clusters with
76% similarity and had 5 and 6 accessions,
Fig. 5—Dendrogram showing similarity coefficient of Morinda
accessions based on ISSR analysis
respectively. The combined data showed the same
pattern as clustered in RAPD and ISSR.
The combined data revealed that the two most
closely related cultivars were SEH-1 and AHD-1 with
the highest similarity index (0.948) and the most
distantly related cultivars were BMN with BGL-2
with low similarity index (0.335) (Table 6). Within
species diversity studies, the two most closely related
accessions were AHD and SHE-1 (0.95) and the
distant accessions were BGL-2 and MEM (0.76) for
Fig. 6—Dendrogram showing similarity coefficient of Morinda
accessions based on combined RAPD-ISSR analysis
Table 5—Similarity index for ISSR
SINGH et al: GENETIC DIVERSITY AMONG MORINDA SPECIES
291
Table 6—Similarity index for combined RAPD-ISSR
M. citrifolia; while for M. pubescens, the two most
closely accessions were NCB and KBY (0.86) and the
distant accessions were BCG and BBD (0.64).
Discussion
Morphological characters in plants may be affected
by environmental conditions and a species grown in
different environmental conditions may be different
morphologically. Thus, the use of morphological
characters for classification may result in discrepancy.
Efficiency of a molecular marker technique depends
on the amount of polymorphism it can detect among
the set of accessions under investigation. Genotype
distribution on the consensus tree based on the
combined banding patterns of RAPD and ISSR may
significantly differ because it is possible that each
technique amplify different parts of the genome. It is,
therefore, better to use the combination of banding
patterns of the two techniques in order to use more
segments sites of the genome that will increases the
validity of the consensus tree.
RAPD analysis has been used to study the genetic
relationship in a number of grasses like switch and
forage37-40. RAPD and ISSR has been extensively
used in many crops and a comparison of both
concluded that ISSR would be a better tool than
RAPD for phylogenetic studies41-45. Nagaoka and
Ogihara (1997) have also reported that the ISSR
primers produced several times more information than
RAPD markers in wheat. In a study on 16 barley
cultivars form different countries, higher similarity
index was observed with ISSRs in comparison to
RAPDs47. In the present study, RAPD results showed
BBD and MAA-1 as the most divergent ones, while
BMN, MEM and BGL-2 accessions were most
diverse as per the ISSR results. On the other hand, the
combined RAPD-ISSR results showed that BMN and
BGL-2 accessions were the most divergent
acccessions.
A close genetic similarity was observed in some of
the cultivars analyzed as shown by high values of
similarity index. Based on similarity matrix using
simple matching coefficient, the similarity values
between all the Morinda spp. were from 38-94% for
RAPD, 25-94% for ISSR and 33-95% for combined
RAPD-ISSR analysis. Although, the similarities
292
INDIAN J BIOTECHNOL, JULY 2011
detected with ISSRs (0.944) between MTC and JGH
were nearly same as observed with RAPDs (0.943)
between AHD and SHE-1. Also for combined
RAPD-ISSR, it was found 0.948 between SEH-1 and
AHD-1, which is similar to RAPD and ISSR result.
M. tinctoria from Chennai showed close relationship
with M. citrifolia in all cases, i.e., RAPD, ISSR and
combined RAPD-ISSR analysis. This may be due to
either seed dispersal through sea or the seedlings
transported from one place to another by migrating
people for its medicinal uses. Genetic variations
observed in some of the accessions are very narrow
like BMN and MEM and between BMN and BGL-2,
it might be because of less distance between
accessions. This study provides evidence that
RAPD and ISSR polymorphisms could be used as
efficient tools for the detection of similarities and
phylogenetic relationships between the studied
genotypes. A similar observation was made by several
other studies48-52.
8
9
10
11
12
13
14
15
Acknowledgment
The authors express their sincere thanks to Central
Instrumentation Facility (CIF), Central Agricultural
Research Institute, Port Blair, Andaman and Nicobar
Island, India for technical assistance. Authors are also
thankful to World Noni Research Foundation,
Chennai for financial assistance for the study.
16
17
18
References
1 Singh D R, Srivastava R C, Chand S & Kumar A, Morinda
citrifolia L.—An evergreen plant for diversification in
commercial horticulture, Int J Noni Res, 2 (2007) 42-58.
2 Guppy H B, Plants, seeds and currents in the West Indies
and Azores (Williams and Norgate, London, UK) 1917,
pp 531.
3 Whistler W A, Polynesian herbal medicine (National
Tropical Botanical Gardens, Hawaii, USA) 1992, pp 238.
4 Abbot I A, La’au Hawaii: Traditional Hawaiian uses of
plants (Bishop Museum Press, Honolulu, Hawaii, USA),
1992, pp 176.
5 Norman B, David H, Julie Y, James T, Fred R et al, Salt and
wind tolerance of landscape plants for Hawaii (College of
Tropical Agriculture and Human Resources (CTAHR)
Instant Information Series 19, Universityof Hawaii at Manoa,
Honolulu, Hawaii) 1996.
6 Sharief M U & Rao R R, Ethnobotanical studies of
Shompens—A critically endangered and degenerating ethnic
community in Great Nicobar Island, Curr Sci, 11 (2007)
1623-1628.
7 Etkin N L & McMillen H L, The ethnobotany of noni
(Morinda citrifolia L., Rubiaceae): Dwelling in the Land
between Lä‘au Lapa‘au and TestiNONIals, in Proc of 2002
Hawaii Noni Conf, edited by S C Nelson (College of
19
20
21
22
23
24
Tropical Agriculture and Human Resources, University of
Hawaii at Manoa, Hawaii, USA) 2003.
Duke J, Bogenschutz M & Duke P, Handbook of medicinal
plants, 2nd edn (CRC Press, Boca Raton, FL, USA) 2002, 529.
McClatchey W, From Polynesian healers to health food
stores: Changing perspectives of Morinda citrifolia
(Rubiaceae), Integr Cancer Ther, 1 (2002) 110-120.
Wang M Y & Su C, Cancer preventive effect of Morinda
citrifolia (Noni), Ann N Y Acad Sci, 952 (2001) 161-80.
Liu G, Bode A, Ma W-Y, Sang S, Ho C-T et al, Two novel
glycosides from the fruits of Morinda citrifolia (noni) inhibit
AP-1 transactivation and cell transformation in the mouse
epidermal JB6 cell line, Cancer Res, 61 (2001) 5749-56.
Chan-Blanco Y, Vaillant F, Perez A M, Reynes M, Brillouet
J-M et al, The noni fruit (Morinda citrifolia L.): A review of
agricultural research, nutritional and therapeutic properties,
J Food Compos Anal, 19 (2006) 645-654.
Morton J F, The ocean-going Noni, or Indian Mulberry
(Morinda citrifolia, Rubiaceae) and some of its colourful
relatives, Econ Bot, 46 (1992) 241-256.
Mathivanan N & Surendiran G, Chemical and biological
activities of Morinda spp., in Proc First Natl Symp Noni Res
(World Noni Research Foundation, Chennai, India) 2006,
1-8.
Sudhakar R, Reddy K N, Murthy E N & Raju V S,
Traditional medicinal plants in Seshachalam hills, Andhra
Pradesh, India, J Med Plants Res, 3 (2009) 408-412.
Rout S D, Panda T & Mishra N, Ethno-medicinal plants used
to cure different diseases by tribals of Mayurbhanj District of
North Orissa, Ethno-Med, 3 (2009) 27-32.
Nadkarni A K, Indian matria medica (Bombay Popular
Prakashan, Bombay, India) 1998, 138-139
Esselman E J, Li J Q, Crawford D J, Windus J L & Wolfe A
D, Clonal diversity in the rare Calamagrostis porteri ssp.
insperata (Poaceae): Comparative results for allozymes and
random amplified polymorphic DNA (RAPD) and inter
simple sequence repeat (ISSR) markers, Mol Ecol, 8 (1999),
443-451.
Rossetto M, Jezierski G, Hopper S D & Dixon K W,
Conservation genetics and clonality in two critically
endangered eucalypts from the highly endemic Southwestern Australian flora, Biol Conserv, 88 (1999) 321-331.
Gupta M, Chyi Y S, Romero-Severson J & Owen J L,
Amplification of DNA markers from evolutionarily diverse
genomes using single primers of simple-sequence repeats,
Theor Appl Genet, 89 (1994) 998-1006.
Zietkiewicz E, Rafalski A & Labuda D, Genome
fingerprinting by simple sequence repeats (SSR)-anchored
polymerase chain reaction amplification, Genomics, 20
(1994) 176-183.
Harris S A, RAPDs in systematics—A useful methodology?,
in Molecular systematics and plant evolution, edited by P M
Hollingsworth, R M Bateman & R J Gornall (Taylor and
Francis, London, UK) 1999, 211-228.
Lanham P G, Brennan R M, Hackett C & McNicol R J,
RAPD fingerprinting of blackurrant (Ribes nigrum L.)
cultivars, Theor Appl Genet, 90 (1995) 166-172.
Bornet B & Branchard D, Nonanchored inter simple
sequence repeat (ISSR) markers: Reproducible and specific
tools for genome fingerprinting, Plant Mol Biol Rep, 19
(2001) 209-215.
SINGH et al: GENETIC DIVERSITY AMONG MORINDA SPECIES
25 Qian W, Ge S & Hong D Y, Genetic variation within and
among populations of a wild rice Oryza granulata from
China detected by RAPD and ISSR markers, Theor Appl
Genet, 102 (2001) 440-449.
26 Hokanson S C, Szewc-McFadden A K, Lamboy W F &
McFerson J R, Microsatellite (SSR) markers reveal genetic
identities, genetic diversity and relationships in a Malus ×
domestica Borkh. core subset collection, Theor Appl Gene,
97 (1998) 671-683.
27 Luan S, Chiang T-Y & Gong X, High genetic diversity vs.
low genetic differentiation in Nouelia insignis (Asteraceae), a
narrowly distributed and endemic species in China, revealed
by ISSR fingerprinting, Ann Bot, 98 (2006) 583-589.
28 Cortesi P, Mazzoleni A, Pizzatti C & Milgroom M G,
Genetic similarity of flag shoot and ascospore
subpopulations of Erysiphe necator in Italy, Appl Environ
Microbiol, 71 (2005) 7788-7791.
29 Sankar A A & Moore G A, Evaluation of inter-simple
sequence repeat analysis for mapping in Citrus and extension
of the genetic linkage map, Theor Appl Genet, 102 (2001)
206-214.
30 Blair M W, Ponaud O & McCouch S R. Inter-simple
sequence repeats (ISSR) amplification for analysis of
microsatellite motif frequency and fingerprinting in rice
(Oryza sativa L.), Theor Appl Genet, 98 (1999) 780-790.
31 Fang D Q & Roose M Z, Identification of closely related
citrus cultivars with inter simple sequence markers, Theor
Appl Genet, 95 (1997) 408-417.
32 Khanuja S P S, Shasany A K, Darorkar M P & Kumar S,
Rapid isolation of DNA from dry and fresh samples of plants
producing large amounts of secondary metabolites and
essential oils, Plant Mol Biol Rep, 17 (1999) 1-7.
33 Sambrook J, Fritsch E F & Maniatis T, Molecular cloning: A
laboratory manual, (Cold Spring Harbor Laboratory, Press,
New York) 1989.
34 Jaccard P, Nouvelles recherches sur la distribution florale,
Bull Soc Vaudoise des Sci Nat, 44 (1908) 223-270.
35 Rohlf F J, NTSYSpc: Numerical taxonomy and multivariate
analysis system, Version 2.02, (Exeter Publications, New
York, USA) 1998.
36 Nei M & Li W H, Mathematical model for studying variation
in terms of restriction endonucleases, Proc Natl Acad Sci,
USA, 74 (1979) 5267-5273.
37 Huff D R, Peakall R & Smouse P E, RAPD variation within
and among natural populations of out crossing buffalo grass
[Buchloe dactyloides (Nutt.) Engelm.], Theor Appl Genet, 86
(1993) 927-934.
38 Gunter L E, Tuskan G A & Wullschleger S D, Diversity
among populations of switch grass based on RAPD markers,
Crop Sci, 36 (1996) 1017-1022.
39 Kolliker R, Stadelmann F J, Reidy B & Nosberger J, Genetic
variability of forage grass cultivars: A comparison of Festuca
pratensis Huds, Lolium perenne L., and Dactylis glomerata
L., Euphytica, 106 (1999) 261-270.
293
40 Nair N V, Nair S, Sreenivasan T V & Mohan M, Analysis of
genetic diversity and phylogeny in Saccharum and related
genera using RAPD markers, Genet Res Crop Evol, 46
(1999) 73-79.
41 Ravi M, Geethanjali S, Sammeyafarheen F & Maheswaran
M, Molecular marker based genetic diversity analysis in rice
(Oryza sativa L.) using RAPD and SSR markers, Euphytica,
133 (2003) 243-253.
42 Ruanet V V, Kochieva E Z & Ryzhova N N, The use of a
self-organizing feature map for the treatment of the results of
RAPD and ISSR analyses in studies on the genomic
polymorphism in the genus Capsicum L., Russian J Genet,
41 (2005) 202-210.
43 Marotti I, Bonetti A, Minelli M, Catizone P & Dinelli G,
Characterization of some Italian common bean (Phaseolus
vulgaris L.) landraces by RAPD, semi-random and ISSR
molecular markers, Genet Resour Crop Evol, 54 (2006)
175-188.
44 Ajibade S R, Weeden N F & Michite S, Inter simple
sequence repeat analysis of genetic relationships in the genus
Vigna, Euphytica, 111 (2000) 47-55.
45 Galvan M Z, Bornet B, Balatti P A & Branchard M, Inter
simple sequence repeat (ISSR) marker as a tool for the
assessment of both genetic diversity and gene pool origin in
common bean (Phaseolus vulgaris L.), Euphytica, 132
(2003) 297-301.
46 Nagaoka T & Ogihara Y, Applicability of inter simple
sequence repeat polymorphisms in wheat for use as DNA
markers in comparison to RFLP and RAPD markers, Theor
Appl Genet, 94 (1997) 597-602.
47 Fernandez M E, Figueiras A M & Benito C, The use of ISSR
and RAPD markers for detecting DNA polymorphism,
genotype identification and genetic diversity among barley
cultivars with known origin, Theor Appl Genet, 104 (2002)
845-851.
48 Seehalak W, Tomooka N, Waranyuwat A, Thipyapong P,
Laosuwan P et al, Genetic diversity of the Vigna germplasm
from Thailand and neighbouring regions revealed by AFLP
analysis, Genet Resour Crop Evol, 53 (2006) 1043-1059.
49 Li C, Fatokun C A, Ubi B, Singh B & Scoles G J,
Determining genetic similarities and relationships among
cowpea breeding lines and cultivars by microsatellite
markers, Crop Sci, 41 (2001) 189-197.
50 Abdel-Tawab F M, Abo-Doma A, Allam A I & El-Rashedy
H A, Assessment of genetic diversity for eight sweet
sorghum cultivars (Sorghum bicolar L.) using RAPD
analysis, Egypt J Genet Cytol, 30 (2001) 41-50.
51 Alexander A J, Genetic diversity of populations of
Astragalus oniciformis using inter simple sequence repeat
(ISSR) markers. M Sc Thesis, Oregon State University, USA,
2002.
52 Heikal H A, Mabrouk Y, Badawy O M, El-Shehawy A &
Badr E A, Fingerprinting Egyptian Gramineae species using
random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) markers, Res J Cell Mol Biol ,
1 (2007) 15-22.