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International Journal of Mol. Ecol. and Conserv. 2015, Vol.5, No.

3, 1-8
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Research Report

Open Access

Morphological and Genetic Diversity Analysis in a Germplasm Bank of


Dendrocalamus stocksii (Munro.) - Implications on Conservation
Dhavala Annapurna , Ahmed S. Muyeed, S. Viswanath
Institute of Wood Science & Technology, 18th Cross, Malleswaram, Bangalore, India
Corresponding author email: uannapurna@gmail.com
International Journal of Molecular Ecology and Conservation, 2015, Vol.5, No.3
doi: 10.5376/ijmec.2015.05.0003
Received: 25 Oct., 2014
Accepted: 17 Nov., 2014
Published: 30 Jan., 2015
Copyright 2015 Annapurna et al., This is an open access article published under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:
Annapurna et al., 2015, Morphological and Genetic Diversity Analysis in a Germplasm Bank of Dendrocalamus stocksii (Munro.) - Implications on
Conservation, International Journal of Molecular Ecology and Conservation, Vol.5, No. 3 1-8 (doi: 10.5376/ijmec.2015.05.0003)

Abstract Dendrocalamus stocksii is an economically important strong solid and thorn less bamboo species which is endemic to
Westerns Ghats of India. Increase in utilization of this species has embarked the importance of conservation, diversity study,
propagation and plantation aspects.
Out study aims to analyse the morphological and genetic diversity among the ex-situ conserved 14 Candidate Plus Clumps (CPCs)
established at Bamboo Germplasm Bank of Institute of Wood science and Technology (IWST), Bangalore. This is the first report on
diversity studies of D. stocksii. The morphological diversity data among the 14 Candidate Plus Clumps (CPCs) originated from
different regions revealed variability in terms of culm height, diameter, internodal length, no. of culms/clump and solid and
hollowness of the culms. Highest culm diameter and number of culms/clump were recorded in PS-27.
In this study, the genetic diversity existing in the fourteen CPCs was estimated using ISSRPCR. Eight ISSR primers amplified fifty
three amplicons in the size ranging from 225 to 1480. The total number of polymorphic bands varied from three to nine with 71.26 %
polymorphic banding profiles. Unweighted Pair Group Method with arithmetic mean (UPGMA) revealed two major clusters. Dice
similarity coefficient ranges from 0.48 to 1.00. Genetic diversity studies based on location divided all the 14 genotypes in to three
clusters Sirsi, Dandeli and Ponda. Existence of 60-70% genetic diversity in the species dominated with vegetative multiplication and
sporadic flowering habit is a noteworthy for a germplasm bank and its contribution for future conservation programmes.
Keywords Ex situ conservation; Candidate plus clump; Sporadic flowering

1 Introduction

with lateritic soil type, this species has a wide


adaptability and comes up well in tropical humid, sub
humid and semi-arid conditions under black and red
soils as well. Multi-location trials have shown that this
species performs well in humid, sub-humid and
semi-arid zones, which expands the scope for its
cultivation across peninsular India. This species has
great economic and ecological importance as it is well
adapted by local community for cultivation along the
bunds and in and around homesteads. It is very well
appreciated and renders bread and butter for locally
economically poor medar community. It is used in
making furniture, construction, baskets, umbrella
handles, stakes for banana and poles (Singhal and
Gangopadhyay, 1999). D. stocksii is considered as an
important agroforestry species, ideal for plantations in
watershed and coastal regions. On-farm trials have
shown success in intercropping with Ipomea

Dendrocalamus stocksii (Munro.) M. Kumar, Remesh


& Unnikrishnan (Pseudoxytenanthera stocksii Munro.,
Oxytenanthera stocksii) is locally known as Marihal
bamboo or seemae bamboo, is endemic to Western
Ghats of India. It is a medium size slender solid non
thorny bamboo species, grows to a height of 9 m and a
diameter of 2.5 to 4cm broad (Seethalakshmi et al.,
1998). It is a mid-sized bamboo species with loosely
spaced solid erect culms ranging from 30-50mm
diameter, which provides flexibility in harvesting,
easy management and steady income to farmers. It is
distributed majorly in Central Western Ghats and
spreads across Maharashtra, Karnataka, Goa and
Kerala states. It is mostly confined to the banks of
streams with a temperature range of 25-35C and
requires a well drained deep loamy soil. Though the
natural distribution of this species is in humid tropics
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International Journal of Mol. Ecol. and Conserv. 2015, Vol.5, No. 3, 1-8
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batatas, Eleusine
coracana and Curcuma
longa
(Viswanath et al., 2014). In recent times due to the
scarcity of cane/rattan this species is increasingly been
seen as a substitute in furniture industry due to its
typical anatomical characteristics like the presence of
non-predominant nodes, solid nature of culms and
good culm wall thickness (Chandramouli et al., 2014).
Because of its multifarious uses this species was
considered as one among 15 industrially important
species by National Bamboo Mission (NBM) and it is
the most preferred species by the farmers in
Peninsular India.

One of the major requirements of ex situ conservation


programme is to study the genetic diversity
information about the material conserved and use the
same for mass multiplication of the species.
Estimation of genetic diversity is also important in
designing improvement programmes, for management
of germplasm and evolving conservation strategies.
DNA based molecular marker technique have been
powerful in genetic diversity estimation (Liu et al.,
1996). Compared with the widely used RAPD
markers, ISSR has several advantages particularly in
reproducibility and informativeness (Yang et al., 1996;
Nagaoka and Ogihara, 1997).

Peculiar flowering habit in bamboo has been made it


almost impossible to breed for superior traits
particularly in woody bamboos. Clonal propagation
can encompass the traits of plus trees and clonal
forestry based on elite selected genotypes allow a
considerable improvement (Geilis et al., 2002).
Sporadic flowering in D. stocksii has been reported
during 1884 and 1889 in North Kanara twice (Singhal
and Gangopadhyay, 1999), during 1994 in silent
valley, Kerala (Seethalakshmi and Muktesh Kumar,
1998) and during 2003-2006 in northern Kerala
(Veena, 2011). Unfortunately seed setting has not been
reported in all the times. There is no natural
regeneration due to lack of fertile seed setting.
Traditionally propagated by the offset cutting and
splitting rhizome. Vegetative propagation through
culm cuttings by which plantable saplings can be
obtained (Reddy and Yekanthappa, 1989; Somashekar
et al., 2007). In vitro propagation through nodal
segments from mature tree has been reported (Sanjaya
et al., 2005, Somashekar et al., 2008; Muyeed, 2012).

Germplasm bank serves as a major repository for


conservation of germline. As such there is no
published information on genetic diversity studies of
D. stocksii. Our study aimed at evaluating the
morphological and genetic variation within these
CPCs collected for ex situ conservation programme
which can be utilized for mass multiplication.

2 Material and methods


2.1 Morphological diversity
Offsets of 14 Candidate Plus Clumps (CPCs) collected
by Forestry College, Sirsi, Uttar Kannada from
Central Western Ghats were transported to Bangalore
and planted in Bamboo Germplasm Bank, IWST,
Bangalore under DBT funded project in 2005 with a
spacing of 5 x 5 m (Figure 1). Since it is a collection
of CPCs from three regions based on morphological
parameters with an objective to use for micro
propagation studies, there was no sample size and
sampling design followed for planting.

Germplasm Bank for 21 industrially important species


of bamboos was established in 0.5ha area in Gottipura,
Hoskote, Bangalore, Karnataka during the year 2004
and 2007 under the DBT sponsored project under the
activity ex-situ germplasm conservation with an
objective to use the elite germplasm for production of
quality planting stock. D. stocksii is one among
them. Macro and micropropagation assumes
importance in this species since seed setting is very
poor. From the above Germplasm Bank, materials
have been collected for micro and macro propagation
studies.

Figure 1 Overview of Dencrocalamus stocksii germplasm bank


of IWST at Gottipura, Hoskote
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International Journal of Mol. Ecol. and Conserv. 2015, Vol.5, No. 3, 1-8
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Morphological variation was recorded in terms of
clump characters (clump height) and culm characters
(culm diameter at 5th internode, wall thickness, culm
wall thickness to culm diameter ratio, no. of
culms/clump and new culm growth and culm
internodal length) for all 14 CPCs.

(10 min, 72C). Amplification products were resolved


in a 2% agarose gel with 1X TAE buffer at 70 V for 2 h
along with 100bp plus ladder (Fermentas, USA), for
molecular weight determination. The gel profiles were
viewed under UVtransilluminator (Hero lab
technologies, Germany) and documented.

The number of culms found in the clump was counted.


The total number of culms included emerging (the
emerging culms which had not completed their
vertical growth and the culms in which branching had
not taken place), matured (culms over 3 years old
without leaf sheath) and harvested (stumps of culms
that had remained after harvesting).Since each clump
of CPC has many culms, five culms of approximately
uniform growth (extremes were not taken), were taken
to note the culm characters like height, diameter,
intermodal length and hollowness. The height of five
representative culms in each CPC was recorded using
Ravi multimeter (altimeter). The mean height of five
representative culms was computed as average height
of the clump and expressed in meters. The culm
diameter was recorded on five representative culms at
breast height (fifth internode from the base) with the
help of digital vernier calliper and expressed in mm.
The wall thickness and culm thickness was recorded
using digital vernier calliper and expressed in ratio.

2.3 Statistical analysis


2.3.1 Morphological variability
Mean, standard deviation and co efficient of variation
was calculated for various culm characters as per
standard procedures (Panse and Sukhatame, 1977).
2.3.2 Genetic variability
Amplified products were scored as present (1) or
absent (0) to form a binary matrix. The resulting
matrix was used to estimate genetic similarity (GS)
among all CPCs by Dice coefficient of similarity (Nei
and Li, 1979). Based on the similarity matrix, a
dendrogram showing the genetic relationships
between genotypes was constructed by the unweighted
pair group method with arithmetic average (UPGMA)
using the software NTSYS-pc (numerical taxonomy
and multivariate systems) Version 2.01 (Rohlf, 1998).
3 Results and Discussion
3.1 Morphological diversity
All the culms of CPCs are nearly solid and culms are
loosely packed. The culm sheath was erect, awl
shaped (narrow and gradually tapering to a sharp point)
blade with rolled margins and sharp tip with 2-3 long
auricles clothed with numerous erect and stiff bristles
in all CPCs (Fig. 1) which is s specific character used
in species identification (Seethalakshmi et al., 1998).
Diameter (>40mm) and ratio of culm wall thickness to
culm diameter (1:3) are the parameter used for
selection of CPCs in the field. Variability was
observed among all 14 genotypes in terms of culm
height, internode length, diameter at 5th internode and
culm wall thickness (Figure 2 and Figure 3) Among
the 14 CPCs, highest culm height was observed in PS
31 (8.1m) with a diameter of 34.2mm. PS 14, PS 17,
PS27, PS32, PS57 and PS58 showed 100% solidarity
with a diameter of 35.65, 34.74, 26.9, 29.97 and
33.7mm. Among the five genotypes, higher diameter
was observed in PS 27 with 65 culms/clump (Table 1,
Figure 2).

2.2 Genetic Diversity


2.2.1 DNA extraction and PCR amplification
Genomic DNA was extracted from juvenile leaf
tissues as described by Doyle and Doyle (1990) with
minor modifications. Extracted DNA was quantified
using a spectrophotometer and by comparing band
intensities with known standards of lambda DNA
(Bangalore Genei Ltd, India) on 0.8% agarose gels.
ISSRPCR amplifications were performed in a 25l
reaction volume containing 30 ng of template DNA,
2.5 l of 10X PCR buffer (Bangalore Genei Ltd,
India), 2.5 mM MgCl2, 0.4 mM dNTPs, 100nM
primer (synthesized at SigmaAldrich, USA) and
1.66U Taq DNA polymerase (Bangalore Genei Ltd,
India). A total of eight primers were used in the
present study. PCR amplifications were carried out in
a programmable thermal cycler (Eppendorf master
cycler gradient with following conditions: Initial
denaturation (3 min, 94C), followed by 35 cycles
consisting of denaturation (30 s, 94C), annealing (30 s,
50C), extension (1 min, 72C) and a final extension

Among the 14 genotypes flowering was observed in


PS57 only without seed setting and new culms.
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International Journal of Mol. Ecol. and Conserv. 2015, Vol.5, No. 3, 1-8
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Figure 3 Variation in diameter (mm) in different CPCs


diameter at 5th inter node wall thickness of CPC 57 at the age
of 6 years

Figure 2a & b Variation in diameter (mm) in different CPCs


culm at 5th inter node at the age of 6 years; a. Total solid culm
in CPC 27; b. Hollow clump in CPC 31

Table1 Details of morphological variation in the 14 CPCs at 6 years in Germplasm Bank at Gottipura, Bangalore

Sl.
No.

CPCs
No.

Source
(loca.)

1
2
3
4
5
6
7
8
9
10
11
12
13

PS-14
PS-15
PS-17
PS-18
PS-24
PS-27
PS-28
PS-31
PS-32
PS-34
PS-50
PS-57
PS-58

14

PS-102

Sirsi
Sirsi
Sirsi
Sirsi
Sirsi
Sirsi
Dandeli
Dandeli
Dandeli
Dandeli
Sirsi
Sirsi
Dandeli
Ponda
(Goa)

Mean
SD
Coeffic.
of
variat.

Aver.
culms
height
(m)

No. of
nodes/
culm

Internode
length
(cm)

Culm wall
thickness
(mm)

Culm
diameter
(mm)

Culm wall
thick.:
culm
diameter

Total
(culms/
clump)

7.0
6.5
6.0
7.2
7.1
6.3
6.2
8.1
6.3
6.5
6.3
7.0
7.5

24
20
22
22
19
23
21
22
20
22
21
21
24

39.1
39.16
34.74
30.9
37.94
38.58
32
38.16
35
20.0
36.08
34.83
35.83

35.65 (S)
11.6
34.74 (S)
12.9
10.72
37.15 (S)
34.35 (S)
8.2
26.9
36.2 (S)
10.3
29.97
33.7

35.65
32.6
34.74
34.3
36.82
37.15
34.35
34.2
26.9
36.2
32.7
29.97
33.7

1:1
1:2.81
1:1
1:2.66
1:3.44
1:1
1:1
1:4.17
1:1
1:1
1:3.17
1:1
1:1

52
77
61
72
68
65
8
69
74
12
71
51
52

No. of
new
shoots
emerged
in 6th year
8
4
3
5
7
7
9
6
3
0
11

8.0

22

36.35

7.55

29.8

1:3.95

52

6.86
0.67

21.64
1.45

34.91
4.96

23.57
12.33

33.51
2.92

0.02
0.02

56.00
21.44

5.67
2.99

9.77

6.69

14.21

52.32

8.72

87.18

38.29

52.85

3.2 Genetic diversity


Of a total of 54 ISSR primers tested 39 primers
showed amplification. Among them, eight ISSR
primers showed polymorphism with clear distinct
bands. The eight selected ISSR primers (814, 818, 826,
852, 849, 835, 843 and 864) each of which generated
3-9 bands gave a total of 53 bands. The percentage of
polymorphism across all the samples ranged from
50.0 to 100% (average 72.0). Highest polymorphism
(100%) was observed with primer UBC 864 while it
was lowest (Table 2) in UBC 818 (50%).
Amplification profile of D. stocksii 14 genotypes
using ISSR primer 835 was shown (Figure 4).

Figure 4 Amplification profile of D. stocksii 14 genotypes of


using ISSR primer 835
Note: M on either side represent molecular ladder with
molecular weights (bp);
Lanes 1-14 sequentially represent Candidate plus clumps;
PS-14, PS-15, PS-17, PS-18, PS-24, PS-27, PS-28, PS-31,
PS32, PS-34, PS-50, PS-57, PS-58 and PS-102
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Table 2 Details of ISSR primers used for the genetic analysis of Dendrocalamus stocksii (Munro.)
Primer
code
864
814
826
852
849
818
835
843

Sl. No.
1
2
3
4
5
6
7
8

Primer sequence
GGC GGCGGCGGCGGCGGC
CTC TCT CTC TCT CTC TA
ACA CAC ACA CAC ACA CC
TCT CTC TCT CTC TCT CRA
GTGTGTGTGTGTGTGTYA
CACACACACACACACAG
AGA GAG AGA GAG AGA GYC
CTC TCT CTC TCT CTC TRA

Total no.
of bands
8
3
9
6
7
6
7
7

No. of polymorphic
bands
8
2
7
2
4
3
6
6

Percent
polymorphism
100
66.67
77.78
83.33
57.14
50.00
85.71
85.71

Molecular
Weight (bp)
359-1305
416-1545
314-1580
506-1411
420-1307
574-1532
225-1029
266-1584

Table 3 Similarity matrix of D. stocksii generated from Dice estimate similarity based on the number of shared fragments
PS-14
PS-15
PS-17
PS-18
PS-24
PS-27
PS-28
PS-31
PS-32
PS-34
PS-50
PS-57
PS-58
PS-102

1
1.00
0.99
0.99
0.95
0.99
0.97
0.83
0.97
0.95
0.84
0.77
0.95
0.54
0.95

10

11

12

13

14

1.00
1.00
0.96
1.00
0.98
0.81
0.98
0.93
0.83
0.76
0.97
0.53
0.93

1.00
0.96
1.00
0.98
0.82
0.98
0.93
0.83
0.76
0.97
0.53
0.93

1.00
0.96
0.93
0.81
0.93
0.89
0.82
0.80
0.93
0.50
0.94

1.00
0.98
0.82
0.98
0.98
0.83
0.76
0.97
0.53
0.94

1.00
0.79
0.98
0.93
0.80
0.73
0.95
0.52
0.93

1.00
0.79
0.83
0.99
0.95
0.81
0.66
0.78

1.00
0.93
0.80
0.73
0.95
0.48
0.93

1.00
0.84
0.77
0.90
0.54
0.91

1.00
0.93
0.82
0.64
0.80

1.00
0.75
0.63
0.72

1.00
0.55
0.93

1.00
0.52

1.00

PS-58

Molecular diversity based on Dice similarity


coefficient among 14 CPCs ranged from 0.48 to 1.00

PS-50

PS-28

PS-34

(Table 3) in this assay. The range indicated diverse


nature of CPCs which were selected from different

PS-32

PS-102

geographical locations. Lowest similarity coefficient

PS-18

PS-57

range of 0.48 to 0.64 was observed with CPC 58


where as highest similarity coefficients were observed

PS-14

PS-24

in 6 other genotypes 0.95 to 1.00 (PS 14 15 17, 18 24

PS-15

PS-17

and 27). Of these that were polymorphic were used to


study the genetic distance and generated neighbouring

PS-27

PS-31

Figure 5 Dendrogram showing relationship among 14


Candidate Plus clumps of Dendrocalamus stocksii

joining tree.

Ponda

UPGMA tree (Figure 5) based on the values for the


genetic distance D, revealed that the 14 cpcs could be
separated into two major clusters; PS58 and other 13
clones. Among the 13 CPCs, three minor clusters were

Dandeli

observed in which PS 14, 15, 17 and 24 clustered into


one group. The three clones CPC 28, 34 and 50 were
clustered in one group and the other 6 CPCs which
were mostly from Sirsi clustered into one group.
Genetic diversity studies based on location divided all

Sirsi

Figure 6 Dendrogram showing relationship among 14


Candidate Plus clumps of Dendrocalamus stocksii as per
location

the 14 genotypes in to three clusters in which Sirsi and


Dandeli are closely related than Ponda (Figure 6).
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ISSR markers target a small segment between the two
microsatellites of the genome which possibly makes
few loci available for amplification by these primers
(Zietkiewicz et al., 1994). ISSR primers are able to
amplify highly variable but small segments (Mc
Gregor et al., 2000). Thats why few ISSR primers can
capture much more variability from genomic segment
than several RAPD primers with random coverage of
entire genome (Moreno et al., 1998).

Hartman and Kester, 1983; Panetos et al., 1987;


Haissig and Riemenschneider, 1988; Foster, 1990;
Zsuffa et al., 1993; Leakey et al., 1994). In D. stocksii,
different studies conducted to know the effect of
genotype on multiplication rate, CPC 27 and CPC 15
showed higher multiplication rate (Somashekar, 2007
and Muyeed, 2012). Among the fourteen clones, CPC
27 was used for in vitro studies and plants raised from
those studies were used for field trials in FRC,
Hyderabad, Yelawala, Mysore and Hosakote,
Bangalore.

Published information on species diversity is limited


in Bamboos (Rao, 2013). Genetic uniformity within
the species collected from distant locations with
distinct phenotypic variations was observed in
Bambusa balcooa and B. tulda (Bhattacharya et al.,
2005). Studies on B. tulda (Bhattacharya et al., 2006)
and Thamnocalamus spathiflorus (unpublished data
from S.B) at different ecogeographical regions of
eastern India indicated a low level of population
genetic diversity for these two species. Whereas low
genetic diversity was observed in the small giant
bamboo population in the Royal Botanic Garden in
Dendrocalamaus giganteous wall Ex Munro
(Ramanayake et al., 2007).
Similar trend was
identified in P. pubescens from Taiwan (Lai and Hsiao,
1997) and Guadua angustifolia from Colombia
(Marulanda et al., 2002). It is quite possible that only
a few clones of individual species acted as the genetic
donor within a particular geographic area and thus
resulted in low level among population genetic
variability.

Operational deployment of relatively few clones also


raises concerns about erosion of genetic diversity in
the species as a whole (White et al 2007). Future
studies can be focused on the optimization of
conditions for multiplication of different genotypes
since cloning the single genotype leads to reduced
diversity. Sexuality and its function in plants is an
important strategy to generate genetic variation but
many bamboos do not have a regular reproductive
cycle (Rao, 2013). In this species although many
times sporadic flowering was observed, but seed
setting was not observed. This might be because of
self incompatibility or inbreeding depression.

4 Conclusions
There exists large morphological and genetic
variability in this the germplasm bank with fourteen
CPCs.
5 Recommendations
Due to poor seed setting and non-gregarious nature of
this bamboo, genetic diversity could be highly
restricted and continuous vegetative propagation from
a narrow genetic base could have serious implication
for conservation of the species. So, to increase the
diversity in future generations, planting clones of
different origin may be encouraged. More CPCs are to
be selected from natural growing areas of D. stocksii,
and widening of the existing gemplam in Germplasm
Bank is recommended.

On the other hand, relatively higher clonal variation


was found in Sasa senanensis from Japan (Suyama et
al., 2000) and G. amplexifolia from Colombia
(Marulanda et al., 2002). Good polymorphism was
observed in twelve natural populations in Yunnan
using ISSR in D. membranaceus (Yang et al., 2012). It
indicates that the differential reproductive systems
might have influence on population genetic diversity
in different bamboo species, since it is expected that
the allogamous species are usually more diverse than
the autogamous ones. However, further studies are
required to better understand emphatically the level of
population genetic diversity and clonal structure in
bamboo.

6 Acknowledgements
The authors are grateful to the Director and the Group
Coordinator of Research of the Institute of Wood
Science and Technology for the encouragement and
for providing the facilities. Germplasm Bank,
Gottipura was established with the financial support of

It is well known that genotype plays a major role in all


phases of vegetative propagation (Brown, 1981;
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International Journal of Mol. Ecol. and Conserv. 2015, Vol.5, No. 3, 1-8
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