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CORRESPONDENCE
CURRENT SCIENCE, VOL. 100, NO. 9, 10 MAY 2011
1277
DNA barcoding for identification of the enigmatic plant Ramkand
For several years, the exact identity of
the Ramkand had been a curiosity for
plant researchers and students. Even
though the so-called kandmool or tuber is
being sold for several years at various
places, especially at places of pilgrimage,
its source is one of the best kept secrets by
its vendors. The name and information
provided by the vendors give an impres-
sion, that the tubers were eaten by Lord
Rama during his days of exile.
The efforts made by several workers to
identify the plant proved unsuccessful.
The only material available for study is
the slice which is sold. Anatomical study
shows typical monocotyledonous vascular
bundle arrangement. But this only added
to the confusion, as monocots have adven-
titious roots and not a tap root system.
Therefore to find out the source, the
plant material was obtained from one of
the vendors from Jyothiba hill temple
at Wadi Ratnagiri, Kolhapur District,
Maharashtra. Slices of approximately 4.5
inches radius and 2–3 mm breadth were
purchased and brought to the laboratory.
DNA was extracted from these slices
using the protocol described by Doyle
and Doyle
1
and its purity was checked on
agarose gel. For identification of the
plant species, the plastid locus for matu-
rase k (matk) was selected. The plastid
and mitochondrial DNA have been
viewed as the most appropriate regions
to sequence for species identification in
plants and animals respectively
2
. The
amplification conditions included 35
cycles of denaturation at 94°C for 1 min,
annealing at 52°C for 30 s, and extension
at 72°C for 50 s, with an initial 1 min
denaturation step at 94°C and final ex-
tension at 72°C for 5 min. Partial ampli-
fication of the gene matk was achieved
and the 1 kb amplicon was sequenced.
The sequence obtained was edited and
submitted to EMBL (accession no.
FR717534). This sequence was used to
find similarity with other submitted
sequences. The similarity search showed
89% identity with the partial sequence of
the plastid locus maturase of Agave
sisalana. Further to confirm the identifi-
cation, plants of Agave were visited and
their leaves enclosing the rosette and ju-
venile inflorescence were excised, which
exposed the core of the rosette. This core
is soft and of similar dimension to that of
the Ramkand being sold (Figure 1). Even
though the source of obtained plant ma-
terial was identified as A. sisalana on the
basis of percentage of identity, it is pos-
sible that other species of Agave are also
being processed and used for the same
purpose. It is obvious that there are
several factors in the identification of a
species, but getting the field of possibili-
ties narrowed to this extent can also help
in identification of such cryptic plants.
The potential use of DNA barcodes for
plant taxonomists is to elucidate species
limits. The term ‘DNA barcode’ gives
the impression that each species is dif-
ferentiated by a unique sequence, but
there is considerable genetic variation
within each species as well as between
species. However, the variations within a
species are usually less than those
between species. This assumption forms
the premise of the above-mentioned
method of identification. This assump-
tion gave 98–100% species identification
success rate
3,4
. Development of a system
based on one or two plastid DNA regions
in land plants has been advocated to
facilitate the need of the wider scientific
community for rapid and reasonably
accurate identification of unknown spe-
cies
2
.
1. Doyle, J. J. and Doyle, J. L., Focus, 1990,
12, 13–15.
2. Chase, M. W., Salamin, N., Wilkinson, M.,
Dunwell, J. M., Kesanakurthi, R. P.,
Haider, N. and Savolainen, V., Philos.
Trans. R. Soc. London, Ser. B, 2005, 360,
1889–1895.
3. Hebert, P. D. N., Penton, E. H., Burns, J.
M., Janzen, D. H. and Hallwachs, W.,
Proc. Natl. Acad. Sci. USA, 2004a, 101,
14812–14817.
4. Hebert, P. D. N., Stoeckle, M. Y., Zemlak,
T. S. and Francis, C. M., PLoS Biol.,
2004b, 2, 1657–1663.
ACKNOWLEDGEMENTS. We thank the
Head, Department of Botany, Shivaji Univer-
sity, Kolhapur for providing the facilities and
Yashwant Chavan, Genome Biotech, Pune for
help.
MANSINGRAJ S. NIMBALKAR
1
P
ARTHRAJ R. KSHIRSAGAR
1
VINOD B. SHIMPALE
1
NILESH V. PAWAR
1
G
HANASHAM B. DIXIT
2,
*
1
Sahyadri Group Enriching Nature,
Environment and Science,
Kawla Naka,
Kolhapur 416 003, India
2
Department of Botany,
Shivaji University,
Kolhapur 416 004, India
*e-mail: g_b_dixit@yahoo.co.in
Figure 1. a, The Ramkand; b, The excised rosette of Agave sisalana.
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  • J J Doyle
  • J L Doyle
Doyle, J. J. and Doyle, J. L., Focus, 1990, 12, 13-15.
  • M W Chase
  • N Salamin
  • M Wilkinson
  • J M Dunwell
  • R P Kesanakurthi
  • N Haider
  • V Savolainen
Chase, M. W., Salamin, N., Wilkinson, M., Dunwell, J. M., Kesanakurthi, R. P., Haider, N. and Savolainen, V., Philos. Trans. R. Soc. London, Ser. B, 2005, 360, 1889-1895.
  • P D N Hebert
  • M Y Stoeckle
  • T S Zemlak
  • C M Francis
Hebert, P. D. N., Stoeckle, M. Y., Zemlak, T. S. and Francis, C. M., PLoS Biol., 2004b, 2, 1657-1663.