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DNA

BARCODING OF SELECTED AQUARIUM PLANTS, ANUBIAS SPECIES USING


CHLOROPLAST MARKER
1Mohamad

Raheimi Md Saad, 2Norhanizan Sahidin and 1Subha Bhassu.

1Department

of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science


University of Malaya, 50603 Kuala Lumpur.
2Aquatic Plant Unit, Freshwater Fisheries Research Centre, Department of Fisheries Malaysia, Glami Lemi, Titi, 71650 Jelebu,
Negeri Sembilan.

Abstract
Aquatic plants are one of the most diverse and commercially important plants. The variety of unique morphology is one of the factors why this plant is of great
interest. Anubias species is one of the aquatic plants which have been widely used as aquarium plant for decoration. DNA barcoding is an identification
approach that may be useful in conservation strategies of the Anubias species. This method could help in identification of this species as an alternative to
morphological observation. To build a DNA barcode in selected Anubias species (Anubias nana, Anubias nana golden, Anubias nana petite, Anubias glabra,
Anubias barteri, Anubias congensis, and Anubias lanceolata), chloroplast DNA was chosen as the region for study. The PCR was performed in 10l reaction
mixture containing DNA, PCR buffer, MgCl, chloroplast DNA primer and taq polymerase. PCR product were analyzed by electrophoresis on 1 % agarose gel
and detected by EtBr. One of the region in the chloroplast genome which is matK region were selected and screened to define a universal barcoding region
across all the 7 selected species. In this research, matK region was successfully amplified from all of the 7 selected species tested. DNA sequences from matK
region from each species were analyzed to differentiate each other with haplotype. The haplotype analysis for matK region showed sufficiently high resolution to
enable differentiation between the selected Anubias species. This study has shown that the chloroplast DNA (cpDNA) region has high potential to be developed
for DNA barcoding in the Anubias species. This research is a first step towards the development of a universal DNA barcoding system for all commercial aquatic
plants in Malaysia.

Materials and methods


DNA EXTRACTION

PCR
AMPLIFICATION
GEL
ELECTROPHORESIS

GEL EXTRACTION

SEQUENCING

Result and Discussion


AN

Anubias
nana

ANP

Anubias
nana
pe,te

ANG

Anubias
nana
golden

AG

Anubias
glabra

AB

Anubias
barteri

AL

Anubias
lanceolata

AC

Anubias
congensis

Figure 1: Single band of seven


selected Anubias species using
matK region from cpDNA

Figure 2: Sequences
analyzed using
Codoncode Aligner

Figure 1 show single band of matK region run on 1%


agarose gel with 100bp DNA ladder (promega).
Sequences from all seven species were screened for
variable nucleotide sites (Figure 2) and haplotype from
each species was construct for DNA barcoding.

Table 1: Selected Anubias


species

ANALYSIS OF DATA

COD
ON
ALI CODE
GNE
R
Table 2: Haplotypes using matK region in chloroplast DNA

Variable nucleotide sites in


matK region were selected
from
each
species
and
arranged
to
construct
haplotype. Result show that
matK
region
has
high
resolution for selected Anubias
species. matK region from
chloroplast DNA has high
potential to be used as marker
for DNA barcoding in selected
Anubias species.

References:
1.
Chodon Sass,(2007). DNA Barcoding in the Cycadales: Testing the Potential of Proposed
Barcoding Markers for Species Identification of Cycads. Plos One. 11, e1154.
2.
Grierson, & Covey. Plant molecular biology. USA: Chapman & Hall.
3.
Mark W.Chase, (2005). Land Plants and DNA Barcodes: Short-term and Long-term goals. Phil.
Trans. R. Soc. B.
4.
Renaud Lahaye, (2008). DNA Barcoding the Floras of Biodiversity Hotspots. PNAS, 105(8),
2923-2928.

Acknowledgements
We would like to thank University of Malaya and Department of Fisheries for the support of the project. Special thanks to lab members Azwan,
Neena and Asmida for your support. Thanks also to Puan Siti, Kak Maz and Kak Florence for your guidance.