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CURRENT SCIENCE, VOL. 100, NO. 9, 10 MAY 2011
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
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
. 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
. 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-
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,
3. Hebert, P. D. N., Penton, E. H., Burns, J.
M., Janzen, D. H. and Hallwachs, W.,
Proc. Natl. Acad. Sci. USA, 2004a, 101,
4. Hebert, P. D. N., Stoeckle, M. Y., Zemlak,
T. S. and Francis, C. M., PLoS Biol.,
2004b, 2, 1657–1663.
Head, Department of Botany, Shivaji Univer-
sity, Kolhapur for providing the facilities and
Yashwant Chavan, Genome Biotech, Pune for
Sahyadri Group Enriching Nature,
Environment and Science,
Kawla Naka,
Kolhapur 416 003, India
Department of Botany,
Shivaji University,
Kolhapur 416 004, India
Figure 1. a, The Ramkand; b, The excised rosette of Agave sisalana.
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Land plants have had the reputation of being problematic for DNA barcoding for two general reasons: (i) the standard DNA regions used in algae, animals and fungi have exceedingly low levels of variability and (ii) the typically used land plant plastid phylogenetic markers (e.g. rbcL, trnL-F, etc.) appear to have too little variation. However, no one has assessed how well current phylogenetic resources might work in the context of identification (versus phylogeny reconstruction). In this paper, we make such an assessment, particularly with two of the markers commonly sequenced in land plant phylogenetic studies, plastid rbcL and internal transcribed spacers of the large subunits of nuclear ribosomal DNA (ITS), and find that both of these DNA regions perform well even though the data currently available in GenBank/EBI were not produced to be used as barcodes and BLAST searches are not an ideal tool for this purpose. These results bode well for the use of even more variable regions of plastid DNA (such as, for example, psbA-trnH) as barcodes, once they have been widely sequenced. In the short term, efforts to bring land plant barcoding up to the standards being used now in other organisms should make swift progress. There are two categories of DNA barcode users, scientists in fields other than taxonomy and taxonomists. For the former, the use of mitochondrial and plastid DNA, the two most easily assessed genomes, is at least in the short term a useful tool that permits them to get on with their studies, which depend on knowing roughly which species or species groups they are dealing with, but these same DNA regions have important drawbacks for use in taxonomic studies (i.e. studies designed to elucidate species limits). For these purposes, DNA markers from uniparentally (usually maternally) inherited genomes can only provide half of the story required to improve taxonomic standards being used in DNA barcoding. In the long term, we will need to develop more sophisticated barcoding tools, which would be multiple, low-copy nuclear markers with sufficient genetic variability and PCR-reliability; these would permit the detection of hybrids and permit researchers to identify the 'genetic gaps' that are useful in assessing species limits.
  • 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.