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

3, 1-8
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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 ISSR–PCR. 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 25-35ºC and
requires a well drained deep loamy soil. Though the
natural distribution of this species is in humid tropics
1

Hoskote. 1997).. Ecol. stocksii has been reported during 1884 and 1889 in North Kanara twice (Singhal and Gangopadhyay. 2012).. Vegetative propagation through culm cuttings by which plantable saplings can be obtained (Reddy and Yekanthappa.ca batatas. D. stocksii is one among them. 1999). Eleusine coracana and Curcuma longa (Viswanath et al. Macro and micropropagation assumes importance in this species since seed setting is very poor. 2002). Figure 1 Overview of Dencrocalamus stocksii germplasm bank of IWST at Gottipura. Compared with the widely used RAPD markers.biopublisher. 1-8 http://ijmec. No. materials have been collected for micro and macro propagation studies. Since it is a collection of CPCs from three regions based on morphological parameters with an objective to use for micro propagation studies.5.5ha area in Gottipura. Somashekar et al. From the above Germplasm Bank.International Journal of Mol. 2015. 2007). Kerala (Seethalakshmi and Muktesh Kumar. and Conserv. Nagaoka and Ogihara. 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. Bangalore. solid nature of culms and good culm wall thickness (Chandramouli et al. Hoskote 2 . during 1994 in silent valley.. ISSR has several advantages particularly in reproducibility and informativeness (Yang et al. there was no sample size and sampling design followed for planting. stocksii. Unfortunately seed setting has not been reported in all the times. 2005. 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. Sporadic flowering in D. for management of germplasm and evolving conservation strategies. 1989.. Peculiar flowering habit in bamboo has been made it almost impossible to breed for superior traits particularly in woody bamboos. IWST... In vitro propagation through nodal segments from mature tree has been reported (Sanjaya et al. Germplasm bank serves as a major repository for conservation of germline. 2014). 1998) and during 2003-2006 in northern Kerala (Veena. 2011). Bangalore under DBT funded project in 2005 with a spacing of 5 x 5 m (Figure 1). Vol. 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. Clonal propagation can encompass the traits of plus trees and clonal forestry based on elite selected genotypes allow a considerable improvement (Geilis et al. 2 Material and methods 2. DNA based molecular marker technique have been powerful in genetic diversity estimation (Liu et al. 2008. Uttar Kannada from Central Western Ghats were transported to Bangalore and planted in Bamboo Germplasm Bank. There is no natural regeneration due to lack of fertile seed setting. 1996. Sirsi. 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. 2014). 3. 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. Traditionally propagated by the offset cutting and splitting rhizome.1 Morphological diversity Offsets of 14 Candidate Plus Clumps (CPCs) collected by Forestry College. Muyeed.. Estimation of genetic diversity is also important in designing improvement programmes. Germplasm Bank for 21 industrially important species of bamboos was established in 0.. 1996). As such there is no published information on genetic diversity studies of D. Somashekar et al.

extension (1 min. and Conserv. 2. India) on 0. India). ISSR–PCR amplifications were performed in a 25µl reaction volume containing 30 ng of template DNA. 2. 94ºC). A total of eight primers were used in the present study. internode length. 2. No. The height of five representative culms in each CPC was recorded using Ravi multimeter (altimeter). 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. Germany) and documented. India). Ecol. Diameter (>40mm) and ratio of culm wall thickness to culm diameter (1:3) are the parameter used for selection of CPCs in the field. 29. 2015.66U Taq DNA polymerase (Bangalore Genei Ltd. for molecular weight determination.1 Morphological variability Mean.5.Since each clump of CPC has many culms.7mm. followed by 35 cycles consisting of denaturation (30 s. Variability was observed among all 14 genotypes in terms of culm height.2 Genetic variability Amplified products were scored as present (1) or absent (0) to form a binary matrix. 1979). wall thickness. 1977). five culms of approximately uniform growth (extremes were not taken). PCR amplifications were carried out in a programmable thermal cycler (Eppendorf master cycler gradient with following conditions: Initial denaturation (3 min. (10 min.4 mM dNTPs.5 µl of 10X PCR buffer (Bangalore Genei Ltd.biopublisher.1m) with a diameter of 34. no. 0. Among the five genotypes.2. 72ºC) and a final extension Among the 14 genotypes flowering was observed in PS57 only without seed setting and new culms. 94ºC).01 (Rohlf.1 DNA extraction and PCR amplification Genomic DNA was extracted from juvenile leaf tissues as described by Doyle and Doyle (1990) with minor modifications.9. 3. culm wall thickness to culm diameter ratio. Vol. Figure 2). The culm sheath was erect. Extracted DNA was quantified using a spectrophotometer and by comparing band intensities with known standards of lambda DNA (Bangalore Genei Ltd. 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. 2.74. PS27. PS57 and PS58 showed 100% solidarity with a diameter of 35. 34. The number of culms found in the clump was counted. 3 Results and Discussion 3. matured (culms over 3 years old without leaf sheath) and harvested (stumps of culms that had remained after harvesting). annealing (30 s. diameter. standard deviation and co efficient of variation was calculated for various culm characters as per standard procedures (Panse and Sukhatame.3. The resulting matrix was used to estimate genetic similarity (GS) among all CPCs by Dice coefficient of similarity (Nei and Li. 1) which is s specific character used in species identification (Seethalakshmi et al. 1-8 http://ijmec.65. The gel profiles were viewed under UV–transilluminator (Hero lab technologies.2 Genetic Diversity 2. PS 14. highest culm height was observed in PS 31 (8.ca Morphological variation was recorded in terms of clump characters (clump height) and culm characters (culm diameter at 5th internode. The wall thickness and culm thickness was recorded using digital vernier calliper and expressed in ratio.3 Statistical analysis 2.97 and 33.3.1 Morphological diversity All the culms of CPCs are nearly solid and culms are loosely packed.5 mM MgCl2. 1998). The mean height of five representative culms was computed as average height of the clump and expressed in meters. of culms/clump and new culm growth and culm internodal length) for all 14 CPCs. PS 17. diameter at 5th internode and culm wall thickness (Figure 2 and Figure 3) Among the 14 CPCs.. 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). Based on the similarity matrix. higher diameter was observed in PS 27 with 65 culms/clump (Table 1.International Journal of Mol. were taken to note the culm characters like height. USA) and 1. USA).8% agarose gels. 1998). 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. 2. 3 . 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. 50ºC). PS32. intermodal length and hollowness. 26. 100nM primer (synthesized at Sigma–Aldrich.2mm. 72ºC).

2 8.72 87.85 3.) 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. Source (loca. 852.2 (S) 10. Vol. 3. CPCs No. of new shoots emerged in 6th year 8 4 3 5 7 7 9 6 3 0 11 8.02 0.21 52.1 39.65 (S) 11.45 34.biopublisher.65 32. 2015.35 34. Bangalore Sl.9 36. stocksii 14 genotypes using ISSR primer 835 was shown (Figure 4). Among them. PS-24.69 14.32 8. of variat.44 5. b. PS-14. 818.15 34.15 (S) 34.1 6.9 36.2 26.35 (S) 8.7 29.2 32. 826.58 32 38.: culm diameter Total (culms/ clump) 7.0). Amplification profile of D.ca Figure 3 Variation in diameter (mm) in different CPC’s 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 CPC’s culm at 5th inter node at the age of 6 years.0 7. Total solid culm in CPC – 27.57 12.1 6.6 34. PS-27.16 34.55 29. 1-8 http://ijmec.0 36.74 (S) 12.74 30. culms height (m) No.29 52.9 10.94 38.97 33.08 34.81 1:1 1:2. Hollow clump in CPC – 31 Table1 Details of morphological variation in the 14 CPCs at 6 years in Germplasm Bank at Gottipura.96 23.0 7.International Journal of Mol. stocksii 14 genotypes of using ISSR primer 835 Note: ‘M’ on either side represent molecular ladder with molecular weights (bp).7 1:1 1:2. PS-17. The percentage of polymorphism across all the samples ranged from 50. 843 and 864) each of which generated 3-9 bands gave a total of 53 bands.0 to 100% (average 72.02 56.95 52 5 6.17 1:1 1:1 52 77 61 72 68 65 8 69 74 12 71 51 52 No.67 21.51 2. 849.35 7. PS-15.64 1.6 34. PS-34.9 37. and Conserv.74 34.2 7.86 0.3 6.5 6.3 36. PS32.3 7.67 2.83 35. Aver.0 6. PS-31. 835. Figure 4 Amplification profile of D.2 Genetic diversity Of a total of 54 ISSR primers tested 39 primers showed amplification. PS-18. PS-50.3 6.0 22 36. of nodes/ culm Internode length (cm) Culm wall thickness (mm) Culm diameter (mm) Culm wall thick.5.44 1:1 1:1 1:4.97 33. PS-57.92 0.3 29.66 1:3. PS-58 and PS-102 4 . No. The eight selected ISSR primers (814.16 35 20. No.8 1:3.00 21. Lanes 1-14 sequentially represent Candidate plus clumps.77 6.5 24 20 22 22 19 23 21 22 20 22 21 21 24 39.17 1:1 1:1 1:3.82 37. Ecol.5 6.2 26. PS-28.33 33.18 38.7 35.99 9.91 4.83 35. eight ISSR primers showed polymorphism with clear distinct bands. a. Highest polymorphism (100%) was observed with primer UBC 864 while it was lowest (Table 2) in UBC 818 (50%).72 37.

75 0.48 to 0.95 0.77 0.80 0.93 0. 15.00 0.84 0.14 50.93 0.00 0.00 0.00 0.64 0.52 0.91 1. No.95 to 1.53 0.93 1.82 0.79 0.76 0.93 0.81 0.89 0.International Journal of Mol. Among the 13 CPCs.76 0.93 0.00 0.54 0. The range indicated diverse nature of CPCs which were selected from different PS-32 PS-102 geographical locations.53 0.95 0.00 (PS 14 15 17.00 PS-50 PS-28 PS-34 (Table 3) in this assay.82 0.64 was observed with CPC 58 where as highest similarity coefficients were observed PS-14 PS-24 in 6 other genotypes 0.76 0.00 85.99 0.93 0.81 0.82 0.83 0.93 1.73 0.71 85.83 0.00 PS-58 Molecular diversity based on Dice similarity coefficient among 14 CPCs ranged from 0. The three clones CPC 28.98 0. 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.83 0.98 0.79 0.95 0.93 0. 17 and 24 clustered into one group. 3.98 0.67 77.81 0.97 0.95 2 3 4 5 6 7 8 9 10 11 12 13 14 1.93 0. No.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.66 0. three minor clusters were Dandeli observed in which PS 14. of polymorphic bands 8 2 7 2 4 3 6 6 Percent polymorphism 100 66.82 0.99 0.55 0.93 0. of bands 8 3 9 6 7 6 7 7 No.72 1.00 0. 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.ca Table 2 Details of ISSR primers used for the genetic analysis of Dendrocalamus stocksii (Munro.98 0.96 1.54 0.) Primer code 864 814 826 852 849 818 835 843 Sl.00 0.95 0.00 0.97 0.00 1.biopublisher.97 0. and Conserv.53 0.98 0.96 0.98 0. 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.33 57. PS58 and other 13 clones.98 0.84 0.73 0.96 1.48 to 1.63 0. 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. 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).78 1. 18 24 PS-15 PS-17 and 27).98 0.80 0.80 0.5.94 1.00 0.00 0.83 0.97 0.94 1. 2015.99 0.80 1.00 0. Lowest similarity coefficient PS-18 PS-57 range of 0.00 0.83 0.93 1.97 0.48 0.93 1.00 0. 34 and 50 were clustered in one group and the other 6 CPCs which were mostly from Sirsi clustered into one group.95 0.93 1. 5 .50 0.99 0.52 1. 1-8 http://ijmec.00 0. Vol. Ecol.90 0.95 0.78 83.77 0.00 0.

biopublisher. to increase the diversity in future generations.... Panetos et al. Genetic uniformity within the species collected from distant locations with distinct phenotypic variations was observed in Bambusa balcooa and B. 1994). 2013). but seed setting was not observed. ISSR primers are able to amplify highly variable but small segments (Mc Gregor et al. This might be because of self incompatibility or inbreeding depression. 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. 2007). In D. Leakey et al. CPC 27 was used for in vitro studies and plants raised from those studies were used for field trials in FRC. 2005). 2012). 5 Recommendations Due to poor seed setting and non-gregarious nature of this bamboo. Similar trend was identified in P. Good polymorphism was observed in twelve natural populations in Yunnan using ISSR in D. Mysore and Hosakote. 6 . 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.. 1988. further studies are required to better understand emphatically the level of population genetic diversity and clonal structure in bamboo. 2002). 1998). 1994). On the other hand. 2000) and G. planting clones of different origin may be encouraged. Hartman and Kester. 2007 and Muyeed.B) at different eco‐geographical regions of eastern India indicated a low level of population genetic diversity for these two species. Among the fourteen clones. relatively higher clonal variation was found in Sasa senanensis from Japan (Suyama et al. Bangalore. 2015. More CPCs are to be selected from natural growing areas of D. Zsuffa et al. 2000). 1990. 2012). Ecol. Foster. 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. Vol. 3. stocksii.. CPC 27 and CPC 15 showed higher multiplication rate (Somashekar. and widening of the existing gemplam in Germplasm Bank is recommended. 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.. 1981. pubescens from Taiwan (Lai and Hsiao. membranaceus (Yang et al. In this species although many times sporadic flowering was observed. Germplasm Bank. tulda (Bhattacharya et al. tulda (Bhattacharya et al. It indicates that the differential reproductive systems might have influence on population genetic diversity in different bamboo species. Operational deployment of relatively few clones also raises concerns about erosion of genetic diversity in the species as a whole (White et al 2007). 4 Conclusions There exists large morphological and genetic variability in this the germplasm bank with fourteen CPCs. amplexifolia from Colombia (Marulanda et al. and Conserv. So. Hyderabad. 1-8 http://ijmec. genetic diversity could be highly restricted and continuous vegetative propagation from a narrow genetic base could have serious implication for conservation of the species..International Journal of Mol. Yelawala. Studies on B. 1983. However.. 1987. 2006) and Thamnocalamus spathiflorus (unpublished data from S.. Haissig and Riemenschneider. Future studies can be focused on the optimization of conditions for multiplication of different genotypes since cloning the single genotype leads to reduced diversity..5. different studies conducted to know the effect of genotype on multiplication rate.. That’s 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. 2013). No. since it is expected that the allogamous species are usually more diverse than the autogamous ones... Published information on species diversity is limited in Bamboos (Rao. stocksii. 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.ca 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. 2002). 1997) and Guadua angustifolia from Colombia (Marulanda et al. 1993.

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