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.  

DNA  BARCODING  OF  SELECTED  AQUARIUM  PLANTS,  ANUBIAS  SPECIES  USING  CHLOROPLAST  MARKER  

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 10µl 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

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.

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.

Result and Discussion

DNA EXTRACTION

PCR AMPLIFICATION

GEL ELECTROPHORESIS

GEL EXTRACTION

ANALYSIS OF DATA

CODONCODE ALIGNER

SEQUENCING

Figure 1: Single band of seven selected Anubias species using matK region from cpDNA

Figure 2: Sequences analyzed using Codoncode Aligner

Table 1: Selected Anubias species

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 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.

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.

Table 2: Haplotypes using matK region in chloroplast DNA