INTRODUCTION TO DNA BARCODING RUZAINAH, A. @ J. Universiti Kuala Lumpur - Malaysian Institute of Chemical and Bioengineering Technology (UniKL - MICET)

What is DNA Barcode? DNA barcode is a short genetic sequence that can be used for biological species identification (Hebert et al., 2003). Named after the black stripes used to identify products in the supermarket: Universal Product Code (UPC). One of the potential plant DNA barcode is the coding rbcL plastid region (CBOL Plant Working Group 2009). The rbcL plastid gene encodes an important photosynthetic enzyme which is the large subunit of ribulose-1, 5-bisphosphate carboxylase/ oxygenase (RUBISCO). Can be easily amplified and sequenced in most land plants. It was also mentioned that it is well suited for recovery of high-quality bidirectional sequences (Kress & Erickson, 2007) . Furthermore, some chose to focus their research on rbcL , claiming that it is the most characterised plastid coding region in GenBank and have a wide representation from all major groups of land plants (Newmaster et al. 2006). There are at least over 10,000 rbcL sequences that can be found in the GenBank (Chase et al., 2007).

Why use DNA Barcode? Explosion of worldwide application of DNA barcode

Assist species identification Identify specimens that lack morphological traits (Gregory 2005). Only a small amount of tissue sample needed for DNA extraction. Can even be performed by non-taxonomist. Cheaper types of fishes were being sold at higher price

2) Stop invasive pests from crossing borders Use DNA barcoding to determine whether a suspected pest on board is harmful. Or just a harmless pest picked up at the port of entry.

3) Prevent wildlife smuggling In Brazil, a man caught smuggling 58 eggs in 2003 said they were quails. The eggs never hatched but genetic testing showed that he was trying to smuggle parrots (Doyle, 2009). Cheaper types of fishes were being sold at higher price

4) Uncovering frauds In north America, almost one out of four fish fillets are mislabelled after referring to DNA barcode library. (Armstrong 2010). Food & Drug Administration (FDA) has officially approved DNA barcoding to prevent the mislabelling locally produced & imported seafood in the U.S (McGuirk 2011). mislabelling cheap fish as more desirable and expensive species like tuna or snapper

Explosion of worldwide application of DNA barcode Fig 1- Authorization of DNA barcoding in the U.S. by the FDA to prevent the mislabelling of both locally produced & imported seafood (McGuirk 2011).

Uncovering frauds (continued) At least 1200 species of plants in Malaysia have been found to contain pharmaceutical values (Khatijah 2006). Several species of Malaysian herbs are been used in various commercial products. DNA barcoding can be applied to detect unlisted ingredients in these products. (a) (b) (c) In 2000, the worldwide market for phytomedicines was estimated to be US$18.5 billion. In Malaysia alone, the market value of herbal products of the same year was estimated at US$0.53 billion, with an annual growth rate of 10 to 15% (Samy et al., 2009). From left: bedak talkum aromaterapi mahkota dewa, Fig 2- Various herbal products; (a) P. microcarpa talcum powder (b) A. vera gel capsule, C. asiatica tablet, and (d) O. staminues tea.

Worldwide DNA Barcoding Community Various countries: Consortium for the Barcode of Life (CBOL). China, Canada, European Union, and U.S.: International Barcode of Life Project (iBOL). Canada : Canadian Centre for DNA Barcoding (CCDB). Canadian Barcode of Life Network (BOLNET). Japan: The Japanese Barcode of Life Initiative (JBOLI) MALAYSIA - ? Cheaper types of fishes were being sold at higher 4 Central Nodes Canada, China, European Union (France, Germany, Italy, Netherlands, Portugal, Spain, United Kingdom) and the United States will have primary responsibility in funding and coordinating iBOL and in supporting its core analytical facilities and data archives. 10 Regional Nodes Argentina, Australia, Brazil, India, Korea, Mexico, New Zealand, Norway, Russia and South Africa will oversee regional barcode efforts; additionally, New Zealand and Norway will lead barcoding programs in the Antarctic and Arctic respectively. 6 National Nodes Argentina, Colombia, Costa Rica, Kenya, Madagascar and Panama will augment national biodiversity surveys via barcoding. Their researchers will assemble specimens and interpret results but will often use sequencing facilities in the Central Nodes. price

(a) Brucea javanica Commonly known as chinese sumac, java brucea, or melada pahit (Musa et al., 2009). Decoction of the roots can treat abdominal pain, coughs, and internal poisoning (Samy et al, 2009). Leaves pummeled with a bit of chalk is useful to treat skin conditions and insect bites (Musa et al., 2009). (b) Decoction: The liquor resulting from concentrating the essence of a substance by heating or boiling, esp. a medicinal preparation made from a plant.. Samy: the root contains several quassinoids. One of tjese compounds, bruceantin, is known to have strong anti-amoebic, antimalarial and anti-cancer properties (c) Fig 3- Images of B. javanica; (a) full tree (b) and seeds (Musa et al., 2009), (c) tree bought from Universiti Putra Malaysia (UPM).

Andrographis paniculata Commonly known as king of bitter or hempedu bumi. Decoction of its leaves is taken to cure diabetes and high blood pressure. Its leaf poultice is used to treat insect bites and skin rashes (Samy et al., 2009). Musa et al., (2009) also stated that decoction of its leaves can also treat sore fever, flu, throat, and chest pain. (a) (b) Fig 4- Images of A. paniculata; (a) full tree (Musa et al., 2009), and (b) tree bought from Malaysian Agricultural Research and Development Institute (MARDI),

Cymbopogon citratus Lemon grass or serai makan. Paste of the leaves is applied to relieve headache (Samy et al., 2009). The leaves & roots are boiled in water and drunk to treat urinary problems, stomach-ache and flatulence (Samy et al., 2009). (b) Flatulence: the presence of excessive gas in the digestive tract Fig 5- Images of C. aristatus; (a) aromatic stems (Musa et al., 2009), and (b) grassy clumps (Samy et al., 2009).

METHODOLOGY

Amplification of rbcL gene Sampling DNA extraction Amplification of rbcL gene PCR purification DNA sequencing Data analysis

Amplification of rbcL gene Sampling Universiti Putra Malaysia (UPM), & Malaysian Agricultural Research and Development Institute (MARDI) DNA extraction Amplification of rbcL gene DNA extracted from fresh leaves. Result viewed via agarose gel electrophoresis and UV- VIS spectrophotometer PCR purification DNA sequencing Data analysis

UV- VIS : ultra violet visible Fig 6- Agarose gel electrophoresis method to view result of DNA extraction.

Amplification of rbcL gene Sampling DNA extraction Amplification of rbcL gene PCR purification DNA sequencing Polymerase Chain Reaction (PCR) Data processing Data analysis

Fig 7 - PCR machine model I- cycler (Biorad) that was used for this study.

Amplification of rbcL gene Sampling DNA extraction Amplification of rbcL gene PCR purification DNA sequencing Compare result with online database Data processing Data analysis

Fig 8 – Nucleotide Basic Local Alignment Search Tool (nBLAST) used for comparison with online database.

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