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Conserv Genet (2009) 10:721724

DOI 10.1007/s10592-008-9630-1

TECHNICAL NOTE

Identification and cross-species amplification of EST derived SSR


markers in different bamboo species
Vikas Sharma Pankaj Bhardwaj Rahul Kumar
Ram Kumar Sharma Anil Sood Paramvir Singh Ahuja

Received: 20 May 2008 / Accepted: 15 June 2008 / Published online: 4 July 2008
Springer Science+Business Media B.V. 2008

Abstract The availability of expressed sequence data


derived from gene discovery programs became an alternative source of mining simple sequence repeat (SSR)
and developing inexpensive genetic markers for the
crop improvements. In present study, 10 express sequence
tags (EST)-SSR markers were identified from Bambusa
oldhamii public sequence data base. Transferability to 25
species of Bambusoideae ranged from 30% to 100%. The
number of alleles detected per locus ranged from 2 to 10.
All the newly identified SSR markers were found to be
moderately to highly polymorphic with an average Polymorphic Information Content (PIC) value of 0.54. As these
loci represents transcribed region and recorded high level
of cross transferability and reliable amplification across the
species, demonstrating the utility of these markers for
functional and genetic analyses of bamboo species.
Keywords Bambusa oldhamii  Bamboo 
Cross-amplification  EST-SSR

The Bamboo subfamily; Bambusoideae, comprising almost


110 genera and 1,200 species (Kumar 2004) and occurs
naturally in all parts of the world except Europe. Multiple
uses ranging from domestic to housekeeping, building
materials and raw materials for the industries makes the
bamboo important genetic resource world over. Current

V. Sharma  P. Bhardwaj  R. Kumar  R. K. Sharma (&) 


A. Sood  P. S. Ahuja
Division of Biotechnology, Institute of Himalayan Bioresource
Technology, IHBT (CSIR), Post Box 6, Palampur, H. P. 176061,
India
e-mail: mrk_sharma@yahoo.com

understanding of bamboo genetic resources are largely


based on genetic diversity studies by Ding (1998), Loh
et al. (2000), and more recently by Sharma et al. (2008).
Simple sequence repeat (SSR) markers have become the
marker class of choice for genetic mapping, genotype fingerprinting and genetic diversity studies because they are
mostly co-dominant, abundant in the genome, highly
reproducible and some have high rate of transferability
across species (Gaitan-Sols et al. 2002; Saha et al. 2004).
The availability of SSR markers in bamboo is very limited
and few genomic simple sequences repeat (SSR) markers
have been developed for bamboo in recent years (Nayak
and Rout 2005; Kaneko et al. 2007). Nevertheless, the
development of SSR markers has traditionally been limited
by the time consuming and labor intensive methods of
SSRs development (Edwards et al. 1996). Therefore,
identification of SSRs from expressed genome provides a
rich source of valuable markers. EST-SSRs associated with
known function genes can be more useful in comparative
gene mapping (Liu et al. 1999) and tend to be more widely
transferred at the species level and even at the genera
(Bouck and Vision 2006). The first set of ten EST-SSRs in
bamboo reported in our study, would be valuable for
genetic diversity and phylogenetic studies in bamboo.
In total 329 ESTs of B. oldhamii and Phyllostachys
edulis were mined from public sequence data base. These
ESTs were subsequently clustered into 55 unique clusters
(contigs/unigenes) using SeqMan DNA Star lasergene
version 7.1. Non-redundant EST sequences were screened
for identification of SSRs containing sequences using repeatmasker software (http://www.repeatmasker.org/) with
search criteria to mask C20 bp SSRs. In total 16 such SSR
containing contigs were identified and 13 of which can be
utilized successfully for primer designing using Generunner 3.05 version software.

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Conserv Genet (2009) 10:721724

Total genomic DNA was extracted from fresh young


leaves of single plant of each species using CTAB method
with minor modifications (Doyle and Doyle 1990). PCR
was performed in a10 lL reaction volume containing 20 ng
of template DNA, 15 ng of each primer, 200 lM of each
dNTP, 10 mMTris-HCL (pH 8.3), 50 mM KCL, 1.5 mM
MgCl2, 0.01% gelatin and 0.5U of Taq DNA polymerase
(Bangalore Genei Pvt. Ltd., Bangalore, India). All PCR
reactions were performed on I-cycler PCR system (BioRad, Australia). The PCR Conditions were: 1 cycle of 5
min at 94 C, 35 cycles of 1 min at 94 C, 1 min at
respective annealing temperature for each primer (Table 1),
2 min at 72 C and final extension for 7 min at 72 C.
Amplification products were electrophoresed on 6%
denaturing polyacrylamide gel in 1X TBE buffer. Fragments were then visualized by silver staining (Silver
sequence staining reagents, Promega, USA) and sized with
50 bp DNA ladder (MBI fermentas, Lithuania). The PIC of
each marker was calculated according to Anderson et al.
(1993).
Each primer pair was used to amplify DNA from
accessions representing 25 different species of Bambusoideae. Of the 13 newly identified EST-SSRs primers, 10
(76.9%) could produce repeatable amplifications in most of
the tested species of bamboo. All the EST-SSR markers
tested proved to be moderately to highly polymorphic

(Table 1). The number of alleles amplified ranged from


two to ten alleles per locus. A high range of genetic variability was observed between species with polymorphism
information content (PIC) values ranging from 0.07 to 0.84
with an average of 0.54. Five primers (Boes-3, Boes-10,
Boes-11, Boes-12, and Boes-13) with PIC values C0.70
were identified more informative and thus would be useful
for genetic characterization of bamboo germplasm. The
marker wise transferability rate varied from 44% to 100%
in 25 bamboo species (Table 2). Three (30%) locus namely
Boes-3, Boes-4, Boes-7 found to be conserved in Bambusoideae. Recently, Sharma et al (2008) reported 7% of rice
(genomic) and 15% of EST derived sugarcane SSR primers
recorded well conserved loci across the taxa. At the species level high transferability rate ranging from 30% in
P. pubescens to 100 % in four species namely D. hamiltonii,
D. membranaceus, B. pallida and P. aurea was reported with
newly identified EST-SSR markers and hence suggested
their suitability for large scale bamboo germplasm characterization, irrespective to the species types.
In summary, it is concluded that the SSRs reported in
the present study derived from the functional portion of
genome of bamboo will facilitate the understanding of intra
and inter species gene flow, genetic structure of wild and
cultivated bamboo species, genetic diversity and evolutionary relationships in bamboos.

Table 1 Characteristics of 10 polymorphic EST-SSR markers of bamboo identified in present study


Locus name

Primer sequence

Ta (C)

Repeat motif

Boes-3

F50 AAACCTGTCGTGCCAGC30

58

(ATG)23

60

No. of
alleles

Size range
(bp)

PIC

Gene bank
accession no.

140180

0.8

EE-661530

(TAA)8

240280

0.07

EE-661510

60

(CTCG)12

310350

0.33

EE-661498

R50 ATTACCGCCTTTGAGTGAG30
Boes-4

F50 GCGAATGAGAGACCTGCA30
0

R5 CTCGCATAAGCCACATCTG3
Boes-5

F50 ACGGTCGGGAACTTGAAG30
R50 CTCTCGCATAAGCCACATCT30

Boes-6

F50 GGAGGAGACTGAATGATGAC30
R50 GTTTACCACTTCCTTTGCG30

58

(CGG)29

10

210250

0.68

EE-661496

Boes-7

F50 GCCCCAACTGATCCCAAT30

62

(CGG)29

280300

0.48

EE-661496

62

(TG)12

9095

0.53

EE-661488

57

(CTGTG)5

200260

0.71

EE-661477; EE-661555

57

(TC)16

300340

0.84

EE-661410

61

(GA)23

240260

0.80

EE-661366

58

(GA)85

150170

0.77

EE-661316

R50 GCAAACCAAGCTCACATGC30
Boes-8

F50 GCATGTGAGCTTGGTTTGC30
R50 CAACGGTACATCAGTTCACCA30

Boes-10

F50 GCAATAGCATAGCACCAAC30
0

R5 CAAGAACAGAGATGGAGCAG3
Boes-11

F50 GGTACTGTTGTTGAGGAAGG30

R50 TCCCACAACTTCATTCTGC30
Boes-12

F50 GGCAAACAACGGTACATCA30
R50 AGTTCATCTCCCGACATGC30

Boes-13

F50 ACTGATCCCTTGTGTGTACG30
0

R5 GTTCATCTCCCGACATGC3

Ta: annealing temperature, PIC: polymorphism information content

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Bamboo Species

Dendrocalamus hamiltonii

Dendrocalamus asper

Dendrocalamus giganteus

Dendrocalamus strictus

Dendrocalamus hookeri

Dendrocalamus
membranaceous

Dendrocalamus longispathus

Bambusa bambos

Bambusa polymorpha

Bambusa balcooa

Bambusa nutans

Bambusa vulgaris

Bambusa multiplex

Bambusa ventricosa

Bambusa nana

Bambusa tulda

Bambusa pallida

Phyllostachys nigra

Phyllostachys aurea

Phyllostachys pubescens

Phyllostachys heteroclada

Phyllostachys bambusoides

Ochlandra travancorica

Ochlandra scriptoria

Melocanna baccifera

S.No.

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

Boes-3

Boes-4

Name of locus

Boes-5

Boes-6

Boes-7

Boes-8

Boes10

Boes11

Boes12

Boes13

70

80

80

40
70

30

100

70

100

80

70

90

80

80

70

90

70

80

90

100

80

70

80

80

100

Transferability
(%)

Table 2 Amplification pattern across 25 bamboo species utilized in this study, (+) indicates clear amplification and (-) indicates no amplification/ambiguous amplification or null allele

Conserv Genet (2009) 10:721724


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Acknowledgement We gratefully acknowledge Department of
Biotechnology (DBT), Ministry of Science and Technology,
Government of India, New Delhi for financial support.

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