Tap chi Cdng ngheSinh hoc 11(2) 197-201, 2013
D N A B A R C O D E A N D A P P L I C A T I O N IN V I E T N A M Tran Hoang Dung', Khuat Hou Trung^, Tran Duy Quy^
' NTT Inslilule of High-Technology. Nguyen Tat Thanh University- Agricultural Genetics Institute. Vietnam academy of Agricultural Science Institute of Research for Scientific Technical Cooperation in Asia Pacific
SUMMARY
DNA barcoding was proposed as a method for recognition and identification of eukaryotic species through comparison of sequences of a standard shoit DNA fragment-DNA barcode from an unknown specimen to a library of reference sequences from known species This allows identifying an organism at any stage of development from a very smalt tissue sample, fresh or conserved many years ago. Molecular identification of plant samples can be used in various scientific and applied fields. In this review, we present an overview of DNA barcoding and its application to agriculture. We introduced an international project thai was formally initiated by the establishment of the Consortium for the Barcode of Life (CBOL) since 2004, which aimed lo develop a standard protocol for DNA barcoding and to construct a comprehensive DNA barcode library of all eukaryotes, including 5 million specimens of 500,000 species. We also outlined some research in Vietnam applied nucleotide sequences to identify species and subspecies for many biologically and economically valuable plants in which our team was one ofthe first group paid much attention to this field. Rice was the first object in our research, we used nucleotide sequences of ORPIOO m chloroplast to distinguish 31 cuitivars of glutinous rice growing in North Vietnam. Extension to native cultivated plants m Vietnam, several groups of economically valuable flower and medical plants including Cymhtdtums. Paphiopedilum. Dendrol'tiirn.
PhaenopM.s. Panax. Discoreci were used for sequencing their ITS and matK genes and companson with GenBank database to point out special differences ofthe cultivar in Vietnam and the remain of world Keywords: DNA barcode, rice glutinous cuitivars, medical plai
medical plants
:. flowers. ITS and malK genes,
OVERVIEW OF DNA BARCODING DNA barcodes, similar to that used in retail industry to identify commercial products, are molecular identifiers Ihal can be used to identify species of living organism They are short, uplo 1000 bp sequences that are different between closely related species thanks to the fast evolution. An algorithm will be used to compare a DNA barcode to a database of known species in order to identify the specie ofthe sample (Anders, 2010).
The popular form of DNA barcoding that H idely used by scientific community to distinguish over 90% of animal species is the rnitochondriat gene C O I , proposed by Hebert va dong tac g!a(2003) Since then, the DNA barcoding has grov/th significantly with the coordination of CBOL(the Consortium for the Barcode of Life) in an effort to retrieve barcode sequences of all organism worldwide (CBOL, 2009)
The Plant Kingdom, in the other hand, has seen a much slower development in DNA barcoding movement. The fact that the mitochondrial genome evolves too slow in plant has disable it as barcode provider for this group The alternative, besides being evolving fast enough, also needs locating between conserved regions to be amplifiable wilh only one single set of primers These two traits ensure the barcoding sequence to function as a molecular identifier and allow effective retrieval from over 200.000 species of plants. There is no single barcoding locus that satisfies above requirement so far and combining two or more loci, probable from plastids, is seemingly a promising approach to reach the optimal level of discrimination and universality (Kress, Erickson, 2007) USES AND GLOBAL BENEFITS Ol BARCODING
DNA Applications of DN'X barcoding are noi only for
Tran Hoang Dung et al quickly identify samples but also extending to
taxonomic studies of "cryptic" taxa or species complexes (Bums et al., 2008) In ecology, DNA barcoding is found to be very useful although there seems not much relationship at first sight. Ecologist analyses plant remains in fierce in order to understand animals' diets or migrafion direction (Valentimef a/,, 2009),
DNA barcoding is also applied at customs to assist officers specifying sources of living or death samples that imported or exported, for the prevention of illegal transportation of rare plants and animals across borders. In forensic field, only a small amount of samples, even mildly deformed, can help officer to trace the source. Recently, DNA barcode database for dog has been used by forensic departments of many countries to assist their activities (Anders, 2010)
CONTROLLING AGRICULTURAL PESTS- LESSENING POVERTY AND HUNGER
In agriculture, one of the most important concerns is to control pests for its cost of billions of USD per year. Using DNA barcoding will help quickly identify pests in latent phases (lava phase), aiding the more careful and well control pest protection program. So far, the Intemational TEphrited Barcoding Initiative has basically completed DNA barcode to identify fruit-flies in the world and is transferring the tools and technology to customs officers of many countries in order that they can identify and prevent the spread of iruit flies.
Agricultural product trading will be faster and more accurate thanks to limited time and effort in dealing with diseases (CBOL ABS Brochure, 2012) IDENTIFYING DISEASE COMBATING DISEASES
VECTORS.
DNA barcoding is also applied in identify disease transmitted vector species. Scientists that are not taxonomist or parasite researchers are enabled to quickly identify disease-carrying species that Transmit the disease between human and animals More understanding in disease transmition and treatment can be gained quicker DNA barcode for vector species are actively building and shared to community This provides institutions and community health offices with effective tools and methods to prevent diseases and limit the extended uses of mosquitoes and insect killers (CBOL ABS Brochure. 2012)
PROTECTING ENDANGERED SPECIES An alarming fact is that worldwide biological diversity is continuously decrease, in which many species have extinct. Uncontrollable hunting in some areas of A&ica has caused the decrease of 90% of primates. It is difficuh to distinguish bush-meat obtaining fi"om endangered species or not. Therefore, DNA barcode can help authority to quickly point out the meat from endangered species, preventing illegal hunting and help preserve the biological diversify. A worldwide strategic project of DNA barcodes has been announced recently aiming to building a barcode library of endangered species (CBOL ABS Brochure, 2012).
SUSTAINING NATURAL RESOURCES DNA barcode, aside from helping prevent diseases, control illegal hunting to save the endangered species, is also applied in agricultural and forestry harvesting. Some countries which the economy depends on natural resources have faced the over-harvesting of agncultural, marine and forestry resources, causing depletion or even extinction of many species To control harvesting, policy makers need to set up effective management system to control the trading of agricultural, marine and forestry products. There are at least two Barcoding projects building for fish (Fish-BOL) and Hardwood trees (Tree-BOL) aiming to increase the management and protection of such natural resources (CBOL ABS Brochure, 2012).
MONITORING WATER QUALITY
Our life depends on water. Freshwater is now becoming precious resource need protection from each country. Water pollution needs quickly identifying for efficient prevention and treatment.
Some simple organisms are considered indicators for the pollution rate of water (such as mosquitoes lava).
However, in higher rate of pollufion, indicators are more difficult to identify. Therefore, scientists are now trying to build DNA barcode library for
"obscured indicators". This will help environment management officers to recreate standards of water assessment and form better tools to control water quality in each country (CBOL ABS Brochure, 2012).
THE MAIN DNA BARCODE PROJECTS IN THE WORLD
Leader o f t h e DNA barcode projects is Barcode of Life project with the ambitious objective to 198
Tgp chi Cdng ngh? Sinh hgc 11(2): 197-201, 2013 successfully build library as well as standardize at Intemational level the identification of eukaryote based on DNA sequences. This project was started in 2004 as the Consortium for the Barcode of Life (CBOL) with ininal target to collect, sequence, analyses and document popular eukaryote in order to set up a standard protocol to identify these species.
So far, the Project has come to a new phase to upgrade to Intemational Barcode of Life project (iBOL), collaborating 26 countries aiming at creating an automatic identification system based on DNA barcode library of all eukaryotes. In the initial stage, iBOL will focus in 500,000 popular eukaryotes of which the number of samples to be collected is up to5 million (10 samples per specie). iBOL also introduces new technologies wilh higher capacity in DNA extraction, sequencing, samples management software, sequence analyzingsoftwares as well as Internet infrastmcture for information sharing(Tedeschi, 2012),
As this IS the huge project, CBOL and iBOL has separated it into several smaller projects according to groups of organisms or countries For example, for fish, there is Fish-BOL, birds with BBI, mammals with Mammalian barcode of Life, marine organisms with MarBOL and insects. Canada is the first country to establish its own Barcode project (BOLNET.ca). After that, many countries in Europe, America and Asia have launched DNA barcode projects of its own while still be part of iBOL, such as Europe (ECBOL;http://www ecbol.org/), Norway (NorBOL; http://dnabarcoding no/en/), Mexico (MexBOL; http://www.mexbol.org/) and Japan (JBOLI; http7/www,jboli.org/). (Tedeschi, 2012).
In the other hand, there are also thematic programs, such as polar life (PolarBOL), quarantine and plant pathogens {QBOL, as a part of the ECBOL) and human health (HealthBOL) (Tedeschi, 2012).
It is necessary for Vietnam to participate and establish DNA barcode projecl for our own, APPLICATION OF DNA BARCODE IN VIETNAM TO IDENTIFY NATIVE CULTIVATED PLANTS IN VIETNAM
Vietnam is the source of many biologically and economically valuable plants and animals species Since 2008, our team is one of the first group paid much attention lo apply nucleotide sequences to identify species and subspecies
Rjce IS the first object in our research, we used nucleotide sequences of ORFIOO in chloroplast to disfinguish 31 cullivars of glutinous rice growing in North Vietnam (Khuat Huu Trung),
Extension to cultivated native plants in Vietnam, we documentated the genetic pools ofthe cultivated native plants at the molecular level to conserve and use them effecfively (Khuat Huu Trung, 2009). Fox example, the ITS (internal transcribed spacer) region and malK (MaturaseK) gene of 7 species of Cymbidiums and 16 species of Paphiopedilum were sequenced and compared with GenBank database to point out special differences ofthe Vietnam orchids and remain of world.
Beside Cymbidums and Paphiopedilum, we alsoidentified two confusing orchids Dendrobium parishii and D. anosmum Several of the orchid species with a powerful fragrance such as D. panshii and D anosmum have attracted the attention of botanists because of their unique biological characters The identification of species based on morphology was difficult even though they are flowering Ten samples of D. parishii and D, anosmum were collected and differenced by the sequences of partial rDNA (ribosomal DNA). Nine of them were able to amplify and sequence ITSl, ITS2 and 5,8S regionwith a length of PCR products from 750 to 850 bp. The phylogenetic analysis showed that the ITS sequences helped to resolve the position of D. parisiui and D. anosmum undoubtedly in the trees. Several samples were revised from D.
parishii lo D ano.smum according to their ITS sequences identify and position in the evolutionary trees (Tran Hoang Dung el al.. 20! 2b),
Medical plants also attracted our attention for using nucleotides to identify the unkown specimens, such as Ngoc Linh ginseng (Panax vietnamensis), the most valuable medical plants in Vietnam. We reconstructed an evolutionary Iree based on nucleotide sequences of ITS region from 7 samples of Ngoc Linh ginseng. The evolutionary geomorphology of Ngoc Linh ginseng was compared with other Panax species. Basically, there is a genetically conserved Ngoc Linh ginseng growing in the South. But there were differences belween the North and the South samples The mitial evidence suggested that Chinese ginseng cultivating in Vietnam was adulterated in Vietnamese ginseng as Ngoc Lmh ginseng so il is necessary to take an attention lo protect precious resources (Tran Hoang Dung. 2012)
Tran Hoang Dung et ai Dioscorea is an important planl genus for food
supply and pharmaceutical applicaUons in many countries including Vietnam. However, its classification and identification are controversial.
Many cuhivated Dioscorea species in Vietnam could not be separated by morphology, DNA barcoding is a recent tool for taxonomic identification and uses a short standardized DNA region lo discnminale plant species. In this study, nucleotide sequences of maiK regions in chloroplast DNA of 31 cultivated Dioscorea were determined lo identify and assess genetic diversity The sequence analysis of malK nucleotide confirmed that these species were D.
alata or D. persimilis Phylogenetic reconslmction based on matK nucleotides and amino acid sequences aisoshowed the evolutionary geomorphologyof D alata and D. persimihs in comparison with other cultivated Dioscorea in Vietnam D. persimihs presents a highly genetic convergent while D. alata is on the way of divergent (Tran Hoang Dung ^f a/.. 2012a)
CONCLUSIONS
DNA barcode has been proved in biological research and its applications since 2003. There are more than I million short nucleotide sequences otlicially deposited in BOLD system as a DNA barcode Intemational large-scale projects will allow expanding this database and promoting the application of this simple method for identifying samples in species and sub-species In the near future, DNA barcode will become standard protocol to identify organisms Furthermore, the DNA sequence obtained can be used in ta.xonomy.
phylogenetic, ecological. conservational and agricultural research. The study of DNA barcode is strongly related to genetic biodiversity database project. Therefore, DNA barcode will become a new keyword to explore biodiversity and will serve as a bridge between the research m the field of biodiversity and genomics. In Vietnam, many scientists have paid attention this area using DNA sequencing to identify cultivated plants. This is the first step for Vietnamese scientists join the DNA barcode projects in the world
REFERENCES
Anders R (2030) DNA barcoding as a tool for the identification of unknown plant material- A case .iiudy on
medicinal roots traded in ifie medina of Marrakech, M.SC thesis, Uppsala University.
BumsJM, JanzenDH, Hajibabaei M,Hallwachs W, HebertPDN (2008) DNA barcodes and cryptics peciesof skipper butterflies m thegenus Perichares in Area Conservacion Guanacaste, Costa Rica. Proc. Natl. Acad.
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CBOLPlant Working Group(2009) AND A barcode for land plants.Proc. Natl. Acad. Sci. USA 106: 12794-12797, CBOL ABS Brochure (2012) DNA Barcoding: A new tool foridentifying biological specimens and managing species diversity, www.dnabarcodes.org.
Hebert PDN (2003) Biological identifications through DNA barcodes. Philos Trans Royal Soc B 270.313-321.
Khuat Huu Tmng{2009) Evaluation studies, document edin digenous plant genetic resources at the molecular levelto conserveand use themeffeciively. Project Grant from Ministry of Scienceand Technology. KC.04 16/06-10 {reported m Vietnamese).
KressJW, Enckson DL (2007) Atwo-locus global DNA barcode for land plants: The coding rbcL gene complements the Non-CodingtmH-psb Aspacerregion PLoSONE, doi:IO !37l/joumal pDne,0000508.
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Tran Hoang Dung, Do Thanh Tn, Tran Le Tmc Ha, Vu Thi Huyen Trang. Truong Quoc Anh, Le Quan, Nguyen Thi Van Anh (2012a) Using maiKgeneto idenlily, assess the genetic diversityan devolution ary geomorphology of Dioscoreaalatazt\d DioscoreapersimiH.K\i\t\vatmg in Vietnam.r/if Central Conferenceof BioiechnologyX^, Hue, 2012 {in Vielnamese)
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Tap chi Cdng ngh4 Sinh hgc 11(2)- 197-201, 2013
DNA BARCODE VA LTNG DUNG 6 VIET NAM Trfin Hoang Dung', Khu3t Hfru Trung^, T r i n Duy Quy'*
'Vien Cong nghe cao NTT, Trudng Dgt hgc Nguyin Tdt Thdnh
^Vien Di truyen Ndng nghiep, Vien Khoa hgc Ndng nghiep Viel Nam Wien Nghiin cuu hap tdc khoa hoc ky thuat chdu A Thai Binh Ducmg
TOM TAT
DNA ma vach da dugc de xuat nhu mot phuong phap de nhan dang va xac dinh cac loai nhan that, thong qua viec so sanh cac trinh tu mpt doan DNA ngan (DNA barcode) tiJ mgt miu chua biei voi mot Ibu vien cac trinh tu ciia cac loai d3 biet. Dieu nay cho phep xac dinh mgt sinh vat tai bat ky giai doan phat trien tir mot m^u mo rat nho, dang tiroi hoac/hay dang bao ton tir nhieu nam truoc. Viec dinh dang mau thuc vat 6 miic phan tir xac dinh cac mau thuc vat co the dirge ap dung trong nhieu linh vuc khoa hgc va iing dung khac nhau. Trong bai tong quan nay, chimg toi trinh bay vai tro ciia ma vach DNA va img dung ciia no ttong linh virc nong nghiep. Chiing toi cung giai thieu mgt so dir an quoc te da dugc chinh thirc khoi xucmg bang viec thanh lap Consortium for the Barcode of Life (CBOL) tir nam 2004 Muc dich ciia dir an giiip phat tnen mot quy trinh chuan cho ma vach DNA va de xay dung mgt thu vien ma vach DNA toan dien cho tit ca cac sinh vat nhan chuan, trong do co 5 trieu mau vat ciia 500,000 loai. Chimg toi cung neu ra mot so nghien cim tai Viet Nam ap dung trinh ttr nuclecotide de xac dinh loai va dudi loai cho nhiing ioai thuc vat co gia tri sinh hgc va kinh le, trong do, nhom nghien ciia chung toi la mot trong nhimg nhom dau tien rat quan tam den lmh vuc nay. Cay liia la doi tugng dau tien trong nghien ciiu ciia chiing toi, nhom nghien ciru da sir dung trinh tir nucleotide ciia ORFIOO trong luc lap de phan biet 31 giong liia nep a mien Bac Viet Nam, Mo rgng cho nhom ban dia nuoi trong d Viet Nam, trong do, chii yeu la nhom hoa va cay thuoc co gia tn nhir Cymbidium. Paphiopedilum.
Dendrobium, Phaelenopsis, Panax, Discorea. Nh6m cay trong nay da dugc giai trinh tu ITS va gen matK de so sdnh vol CO sd dir lieu GenBank, giup chi ra sir khac biet dSc hieu ciia cac giong cay trong tai Viet Nam voi th^ gicti.
Tu-lchda: ITS. ma vach DNA, matK, nhdn dang phdn tit, cav duoc lieu
' Aulhor for correspondence: E-mail duvquvvaas@smail. com
