LIFE SCIENCES | BIOTECHNOLOGY regions used as barcodes should match some key criteria: i) The universality of amplification and sequencing; ii) The Review on molecular markers pattern of intraspecific vs. interspecific variation; and iii) The power to identify for identification of Orchids species [7]. Thi Huyen Trang Vu1*, Thi Ly Le2, Truong Khoa Nguyen3, Duy Duong Tran3, The selection of a barcode locus is Hoang Dung Tran4 complicated due to the trade-off that 1School of Agricultural Science and Biotechnology, Nguyen Tat Thanh University arises between the need for universal 2School of Biotechnology, Ho Chi Minh International University 3Genetic Engineering Division, Institute of Agricultural Genetics application in a wide range of taxa and 4Nguyen Tat Thanh University sequence substitution saturation [5]. Received 1 March 2017; accepted 2 June 2017 The single region 5’ end of cytochrome c-oxidase 1 (CO1) from the mitochondrial Abstract: genome is quite successfully used for the Orchidaceae is one of the most valuable plant groups all over the world, identification of animals [8, 9]. However and is also an impressively large and complex family of flowering plants. searching for DNA barcode in plants is Effective molecular tools used for the identification of orchid species should far more challenging than in animals. be developed to support traditional morphological approaches. This study Mitochondrial genes, including CO1, reviews most of the DNA fragments that have been used as taxon identifiers in plants have low rates of synonymous in researches conducted on Orchidaceae in order to assess potential molecular markers and metric measurements for the identification of orchid species. substitution [10, 11], a large structural rearrangement in the genome, and Keywords: DNA barcode Orchids, DNA fingerprinting, molecular identification import of sequences from nucleus and Orchidaceae, molecular markers, molecular phylogeny. chloroplast [12]. Because of these Classification number: 3.5 problems, they are not recommended to use for DNA barcodes for plants. So, the nuclear and chloroplast genomes are Introduction traditional classification; even those with focused to look for identifying markers fertile parts. Besides this, the species Orchidaceae is one of the largest for plants. Until now, no single sequences can be transported in a vegetative state, can be sufficient to identify all plant and most complex families of flowering as seeds or as fragments [3]. Accurate species. Even the use of a combination of plants, comprising of approximately authentication of orchid species is multi-locus barcodes also gives different 22,500 species belong to 736 currently critical for biodiversity conservation and levels of discrimination in different recognised genera [1]. Orchids have effective utilisation of orchids as plant groups of plants (Table 1). many values ranging from the beauty resources [4]. of their flowers to therapeutic properties The aim of this paper is to assess Many researchers have, therefore, in some species. However, taxons of potential molecular markers for the tended to develop genetic tests that Orchidaceae are endangered, this is can cheaply and easily determine the identification of orchids. We review most mainly because of over-collection and present species. “DNA barcodes” tools of the DNA fragments that have been habitat destruction, and all species are are promising options in providing a used as taxon identifiers in researches included in Conventions on International practical, standardised, species-level on Orchidaceae and other land plants, Trade of Endangered Species of Fauna identification approach that can be used including orchids. The capability of taxon and Flora (CITES) I and II [2]. Illegal for biodiversity assessment, life history, discrimination is often evaluated along trade and imitations have also become ecological studies, and forensic analysis with the construction of a phylogenetic increasing problems. Unfortunately, [5]. DNA barcode refers to the use of tree. So we also use phylogenetic laws banning these issues and their a single segment of DNA to identify articles as literature sources for seeking enforcement have met obstacles mostly specific coding information that offers suitable sequences. Highly evaluated due to the imperceptible difference discriminating ability of the living taxa, markers proposed by previous authors of species’ morphology. So, it is very even if only a small fragment of the will be deeply discussed to summarise difficult to identify orchid species organism at any stage of development a database of molecular candidates for and their inter-species hybrids using is available [6]. The potential DNA orchid authentication. *Corresponding author: Email: [email protected] 62 Vietnam Journal of Science, JUNE 2017 • VOL.59 NUMBER 2 Technology and Engineering LIFE SCIENCES | BIOTECHNOLOGY Table 1. Summary of advantages and disadvantages of molecular loci in plants. pseudogenes [22]. The coexistence of Loci Advantages Disadvantages variation orthologous copies of the ITS Nuclear regions - High variability - Universal across different groups of in the hybrid genomes of Paphiopedilum (such as ITS) - High number copies in cell organisms unexpected contamination leads to poor-quality sequences which - Biparental inheritance more information - Intra-genomic variability, divergent paralogous copies (multiple functional copies), consisted of multiple peaks. Thus, the pseudogenes poor-quality sequences ITS was not found to be suitable neither Chloroplast gene - High copy number - Low evolutionary rate few choices for species resolution nor for getting an - Maternal inheritance information no insight into the parentage of the hybrids reflection of complexity - Some genes are highly variable (50% species resolution for eight natural - The high variable regions used for low- species) [6]. Another disadvantage of the level identification (species, under-species) ITS was that it could not be amplified - The lower variable regions used for high- level identification (genus, family, tribe…) from some barium samples because the Mitochondrial genes - Low rate of sequence change ITS is too variable to guarantee reliable - Genome structure of mitochondrial in plant alignments and contains variable indels rapidly change (insertion/deletions) at the species level Intergenic spacer and - High variability (do not encode any - Difficult to amplify, sequence and align intron products faster evolve than coding regions) - Too variable, even intra-species [23]. Low-copy nuclear - High variability - Low copy number However, the internal transcribed genes (such as Xdh) - The present and absent of introns, the size spacer region (ITS) of ribosomal DNA of introns, the substitution rate are greatly variable and poorly studied no universal has proved to be an effective marking barcoding progress. At the genus Studied barcodes for Orchid taxa In addition, the ITS with nuclear genes level, the ITS clearly distinguishes can provide more complex information Single locus barcodes between the two genera Paphiopedilum which relates to biparental inheritance and Phragmipedium, and also the The ITS region: In plants, nuclear in comparison with plastid markers [17]. Cypripedioid genera [24]. At the genes (particularly introns) and spacers The length of the ITS is about 600 bp species level, Kress, et al. (2005) [5] exhibit the highest variability [13]. [18-20], which can satisfy the length has evaluated that the ITS has a much The internal transcribed spacers (ITS) requirements of barcoding. higher divergence value than any of of nuclear ribosomal regions were proposed as a variable molecular marker for detecting genetic variation among genera, species, and within species. The two internal transcribed spacers (ITS1 and ITS2) do not encode any product but permit it to evolve at a faster rate than the ribosomal coding regions. For example, the ITS length of the aligned sequence Fig. 1. Coding genes. in Holcoglossum (Orchidaceae) was 567 bp and it contained 26 informative The ITS spacer, although often the plastid regions studied and has sites and 27 variable sites [14]. The ITS highly variable, also reached a number of an amplification success rate of 88%. exhibits high resolution at the species limits for DNA barcoding [5]. Using the Therefore this region was proposed as level [5]. The ITS has been shown to ITS as a barcode still has its challenges a potentially usable DNA region for the have unparalleled species resolutions [21]. The high variability among intra- application of barcoding to flowering compared with candidate barcodes genomic systems, on the contrary, was a plants using the optional supplementary proposed thus far [15]. Besides that, disadvantage of species discrimination marker trnH-psbA. The ITS was also they exist in cells with high numbers of [22]. The CBOL Plant Working Group evaluated to be an effective candidate copies [5, 9]. The flanked regions 5.8S did not recognize the ITS as a suitable DNA barcode for Orchidaceae [16, 18, at the middle and 18S, 26S at the two locus for DNA barcoding because 25]. The PCR success rate of the ITS ends of the ITS fragment, are conserved there were many factors that affected was high [16], 100% in 158 wild orchid sequences which are useful to develop the quality of sequences from direct samples [18], and in Holcoglossum [6, primers [9] (Fig. 1). The high retrieval sequencing of PCR products, including 14]. Although the combination of the